Tesi sul tema "Enzyme technology"

Self-immobilisation enzyme technologies, such as SphereZyme™, suffer from the lack of applicability to hydrolyse large substrates. Solid support immobilisation is usually a method of choice, to produce a stable biocatalyst for large substrates hydrolysis in the industry. In order to investigate this limitation, a commercial protease called Alcalase® was chosen as a model enzyme due to its natural activity (hydrolysis of large substrates-proteins). Prior to immobilising through the SphereZyme™ technology, Alcalase® was partially purified through dialysis followed by CM Sepharose™ FF cation exchanger. Sample contaminants, such as salts and stabilisers can inhibit protein crosslinking by reacting with glutaraldehyde. Alcalase® was successfully separated into 3 proteases with the major peak correlating to a positive control run on native PAGE, indicating that it was likely subtilisin Carlsberg. A 16% alkaline protease activity for azo-casein hydrolysis was retained when 5% v/v PEI: 25% v/v glutaraldehyde solution was used as a crosslinking agent in Alcalase® SphereZyme™ production. An increase in activity was also observed for monomeric substrates (PNPA) where the highest was 55%. The highest % activities maintained when 0.33 M EDA: 25% v/v glutaraldehyde solution was initially used as crosslinking agent were 4.5% and 1.6% for monomeric and polymeric substrates, respectively. PEI is a hydrophilic branched polymer with an abundance of amine groups compared to EDA. A comparison study of immobilisation efficiencies of SphereZyme™, Eupergit® and Dendrispheres was also performed for large substrate biocatalysis. The two latter technologies are solid-support immobilisation methods. Dendrispheres reached its maximum loading capacity in the first 5 minute of the one hour binding time. Twenty minutes was chosen as a maximum binding time since there was constant protein maintained on the solid support and no enzyme loss was observed during the 1 hour binding time. PEI at pH 11.5, its native pH, gave the highest immobilisation yield and specific activity over the PEI pH range of 11.5 to 7. SphereZyme™ had the highest ratio for azocasein hydrolysis followed by Dendrispheres and Eupergit®. The SphereZyme™ was also shown to be applicable to biosensors for phenol detection. Different modifications of glassy carbon electrode (GCE) were evaluated as a benchmark for the fabrication of SphereZyme™ modified phenol biosensor. GCE modified with laccase SphereZyme™ entrapped in cellulose membrane was the best modification due to the broad catechol range (<0.950 mM), high correlation coefficient (R2, 0.995) and relative high sensitivity factor (0.305 μA.mM-1). This type of biosensor was also shown to be electroactive at pH 7.0 for which its control, free laccase, lacked electroactivity. From the catalytic constants calculated, GCE modified with laccase SphereZyme™ entrapped in cellulose membrane also gave the highest effectiveness factor (Imax/Km app) of 1.84 μA.mM-1. The modified GCE with Alcalase® SphereZyme™ was relatively more sensitive than GCE modified with free Alcalase®.

Thesis (PhD (Process Engineering))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: There is growing interest to utilise xylan as speciality biopolymers in similar ways as high molecular weight polysaccharides such as starch and cellulose. The need to utilise xylan as alternative to cellulose and starch has increased because the cellulose and starch have many other competing uses. Unlike cellulose and starch, xylans are heteropolymers with higher degree of substitution and are of lower molecular mass and therefore, do not readily become insoluble to form hydrogels and biofilms. Consequently, xylans do not suit applications of starch and cellulose as speciality biodegradable additives and coatings in the food, pharmaceutical, pulp and paper and textile and many other industries. This study was conducted to develop an enzyme technology, based on recombinant α-L-arabinofuranosidase and purified α-D-glucuronidase with polymeric xylan substrate specificity, for controlled reduction of the solubility of water soluble polymeric xylan, leading to formation of insoluble nanohydrogels. Although xylan is available in abundance, a large proportion of it is currently wasted in lignocellulose process waste streams with little prospects for recovery and addition of value. Lignocellulosic materials including Eucalyptus grandis, Pinus patula, Bambusa balcooa (bamboo) and sugarcane (Saccharum officinarum L) bagasse (bagasse) found in South Africa were investigated as sources of water soluble xylan for enzyme modification. Two mild alkali-low temperature methods (alkali charge of < 14% and temperature of < 80ºC), one with ultrapurification denoted as the Hoije and the other with ethanol precipitation, denoted as Lopez method, were evaluated for their selective extraction of water soluble xylans from the specified lignocellulosic materials. The water soluble xylans were extracted from P. patula, bagasse, E. grandis and bamboo by the Hoije method with extraction efficiencies of 71.0, 66.0, 35.0 and 20.0% respectively. Using the Lopez method, the xylans from bagasse and E. grandis were extracted with extraction efficiencies of 28.0 and 12.0% respectively. The xylans extracted from P. patula, bamboo and bagasse were identified as arabinoglucuronoxylans, which were substituted with arabinose and 4-O-methyl-D- glucuronic acid (MeGlcA) side chains, whereas, the xylan extracted from E. grandis were identified as 4-O-methyl-β-D-glucuronoxylan (glucuronoxylan) substituted with MeGlcA groups on the main xylan chain. In addition, the glucuronoxylans contained some traces of arabinose and rhaminose sugar residues. The extracted xylan fractions had degree of polymerisation (DP) of > 10 and were water soluble, which suited the required properties of xylans for customised enzyme modification. The selective removal of the arabinose, MeGlcA and acetyl groups to create linear regions of xylose units in xylans that causes intra and inter-polymer bonding is considered to be the key process for reducing the solubility of water soluble xylans. The α-L-arabinofuranosidase of Aspergillus niger (AbfB) and α-D-glucuronidase of Schizophyllum commune (AguA) are special enzymes so far identified with the ability to selectively remove arabinose and MeGlcA side chains respectively, from water soluble xylans. Large scale application of the AbfB and AguA for reducing solubility of the water soluble xylans would require their extracellular production in large quantities and free of contamination from the xylan main chain degrading enzymes including the endo-1,4-β -xylanase. Selective production of the AbfB free of xylanase activity was achieved in recombinant A. niger D15 [abfB] strain under the transcriptional control of the glyceraldehyde-3-phosphate dehydrogenase promoter (gpdP) and glucoamylase terminator (glaAT). The recombinant AbfB was secreted extracellulary in 125 mL shake flasks and 10 L bioreactor fermentation cultures with volumetric activities of up to 10.0 and 8.0 nkat mL-1 respectively, against para-nitrophenol arabinofuranoside (pNPA). The secretion of the recombinant AbfB was growth associated and therefore, increased up to 2.5 times with addition of concentrate corn steep liquor (CCSL) as an additional source of nitrogen in the 2 x minimal standard cultivation media. The biomass specific activity of the recombinant AbfB against the pNPA substrate was approximately 366 nkat g-1 (dry weight basis). The recombinant AbfB displayed a single pure species band on 10% SDS-PAGE stained with Coomassie blue and had an estimated molecular mass of 67 kDa. In addition, the recombinant AbfB showed optimal activity at 40-55ºC and pH 3.0-5.0 and was stable under cultivation, storage and operating conditions at temperatures between 30-60ºC and pH 3.0-6.0. Furthermore, the recombinant AbfB showed broad substrate specificity selectively removing arabinose side groups from low viscosity wheat and oat spelt arabinoxylans, larchwood arabinogalactan, debranched arabinan and arabiglucuronoxylans extracted from bagasse, bamboo and P. patula found in South Africa,. The recombinant AbfB was able to precipitate xylans extracted from bagasse, bamboo and oat spelt but not from P. patula. Over 95% of the activity of the recombinant AbfB against the pNPA was recyclable after selective hydrolysis of the xylan at 40ºC for 16 h. On the other hand, the purified AguA enzyme could only precipitate the birch glucuronoxylan but not the glucuronoxylan extracted from E. grandis and arabinoglucuronoxylans extracted from bagasse, bamboo and P. patula. The synergetic action of the recombinant AbfB and the purified AguA increased the removal of the arabinose side chains from bagasse xylan by 22% and from bamboo xylan by 33%, whereas, the removal of the MeGlcA side chains from bagasse xylan increased by only 5% and that from bamboo xylan decreased by 13%. The selective removal of the arabinose side chains from oat spelt, bagasse and bamboo xylans by the recombinant AbfB had higher apparent viscosity relative the corresponding untreated xylans. However, the apparent viscosity of both the treated and untreated xylans reduced with increased shear rate. The viscosity had an overall negative correlation with arabinose side chain removal reaching a minimum of 2.03 mPa.s for hydrolysis of oat spelt xylan that was performed for 9.0 h at a temperature of 45.8ºC with recombinant AbfB xylan specific dosage of 400.0 nkat g-1substrate . The alteration of the viscosity of the xylans by the selective removal of the side chains is of special interest in the production of speciality emulsifying, thickening and antifoaming agents. The optimal values for hydrolysis time, enzyme dosage and temperature for maximum degree of removal of arabinose side chains from oat spelt xylan by the recombinant AbfB and of the removal of MeGlcA side chains from birch xylan by the purified AguA were determined by the Box-Benhken response surface method (RSM). The experimental region covered the xylan specific dosage for the recombinant AbfB between 18.0 and 540.0 nkatg-1substrate and for the purified AguA xylan between 2.0 and 18.0 μkatg-1substrate at temperatures between 30 and 50ºC and hydrolysis time between 1 and 16 h. The temperature, enzyme xylan specific dosage and hydrolysis time had significant effect (p<0.05) on both the selective removal of arabinose from oat spelt xylan by the recombinant AbfB and the selective removal of MeGlcA from birch xylan by the purified AguA. However, the interaction of these hydrolysis parameters were significant (p<0.05) on only the removal of arabinose side chains from oat spelt xylan by the recombinant AbfB. The optimal values for hydrolysis time, temperature and xylan specific dosage were estimated to be 14-16 h, 38-45ºC and 607.0 nkatg-1substrate respectively, for maximum removal of 43% of the available arabinose in oat spelt xylan by the recombinant AbfB. Whereas, the optimal values for hydrolysis time, temperature and xylan specific dosage for maximum removal of 0.5% of the available MeGlcA side chains from the birch xylan by the purified AguA were estimated to be 11 h, 38ºC and 18.0 μkatg-1substrate respectively. The optimal values of the hydrolysis parameters for both the removal of the arabinose from oat spelt xylan by the recombinant AbfB and of MeGlcA side chains from birch by the purified AguA could be predicted using quadratic models that fitted the response surface plots with regression coefficients of > 0.9. The effects of in situ selective removal of arabinose and MeGlcA side chains by AbfB and AguA respectively, from water soluble xylans, on their precipitation and adsorption onto cotton lint were investigated. The cotton lint was treated with xylans extracted from bagasse, bamboo, P. patula and E. grandis using the Hoije method in the presence of the recombinant AbfB, AguA and the cocktail of the two enzymes. The effects of in situ selective hydrolysis of model xylans including birch, oat spelt and H2O2 bleached bagasse and E. grandis xylan gel by the enzymes on their adsorption onto cotton lint were used for reference purposes. The purified AguA increased the adsorption of arabinoglucuronoxylans extracted from bagasse bamboo and P. Patula using the Hoije method onto cotton lint the most compared to the effect of the recombinant AbfB and the cocktail of the recombinant AbfB and purified AguA. The purified AguA increased the adsorption of the xylans extracted from bagasse and E. grandis xylans by 334 and 29% respectively, but decreased that of E. grandis xylan gel and H2O2 bleached bagasse xylan by 31 and 6% respectively. Similarly, the presence of the recombinant AbfB increased the adsorption of the bamboo, P. Patula and oat spelt xylans by 31, 44 and 900% respectively, but decreased the adsorption of the xylan extracted from bagasse and the H2O2 bleached bagasse xylan by 13 and 30% respectively. Furthermore, different xylan-cellulose interactions and water adsorption capacities of the cotton lint were observed with the in situ modification and adsorption of the xylans extracted from bagasse, bamboo, E. grandis and P. patula in the presence of the recombinant AbfB and purified AguA. Therefore, the enzyme aided adsorption of xylans could be used to alter or improve functional properties of cellulosic materials. The performance of enzymatically formed xylan nanohydrogels as encapsulation matrices for slow delivery of bioactive agents was evaluated. Insoluble xylan nanohydrogels formed by selective removal of arabinose side chains from water soluble oat spelt xylan by the recombinant AbfB were characterized for particle size distribution, surface charge (zeta potential), morphology stability and ability to encapsulate and slowly release the HRP. The enzymatically formed oat spelt xylan hydrogels were spherical in shape with particle sizes ranging from 18 nm to > 10 000 nm. The xylan nanohydrogels exhibited a negative zeta potential of up to -19 mV and displayed self assembling behaviour when formed at xylan concentrations of higher than 1.5% (w/v) and hydrolysis time beyond 17 h. The xylan concentration significantly (P < 0.05) influenced both the particle size and zeta potential of the oat spelt xylan nanohydrogels whereas the recombinant AbfB hydrolysis time was significant (P < 0.05) on the zeta potential. The oat spelt xylan nanohydrogels successfully encapsulated the HRP enzyme both during and after formation of the oat spelt xylan nanohydrogels and the release of the encapsulated HRP in active form, was sustained for a period of 180 min. Therefore, the xylan side chain removing enzymes have a role in preparation of biodegradable nanoencapsulation devices. Overall, the AbfB and AguA have presented a novel tool for functionalising water soluble xylans to be used as speciality additives, coating and implantation or encapsulation matrices, with reduced impact on the environment. This will advance processing and expand the product spectrum of lignocellulosic materials.
AFRIKAANSE OPSOMMING: Daar is ‘n toenemende belangstelling om spesialiteit biopolimere uit xilaan ontwikkel, en op soortgelyke wyse as hoë molekulêre massa polisakkariede soos stysel en sellulose te benut. Die behoefte om xilaan biodegradeerbare polimere as ‘n alternatief tot sellulose en stysel te gebruik neem toe omdat laasgenoemde baie ander kompeterende gebruike het. Anders as sellulose en stysel is uit xilaan heteropolimere met ‘n hoë graad van substitusie in die hoofketling met sygroepe en lae molekulêre massas, en raak daarom nie geredelik onoplosbaar om hidrojel en biofilms te vorm nie. Gevolglik is xilaan nie geskik vir toepassings van stysel en sellulose as spesialiteit biodegradeerbare bymiddels en bedekkings in die voedsel-, farmaseutiese-, pulp en papier-, tekstiel-, en vele ander industrieë nie. Hierdie studie is uitgevoer om ‘n ensiemtegnologie te ontwikkel gebaseer op rekombinante α-L-arabinofuranosidase en gesuiwerde α-D-glukuronidase met polimeriese xilaan substraat spesifisiteit, vir beheerde vermindering van die oplosbaarheid van wateroplosbare polimeriese xilaan wat lei tot die vorming van onoplosbare nanohidrojels. Alhoewel xilaan volop beskikbaar is, word ‘n groot deel daarvan tans vermors in afvalstrome uit lignosellulose prosessering, primêr verpulping, met min vooruitsigte vir herwinning en toevoeging van waarde. Lignosellulose materiaal wat in Suid-Afrika geproduseer word, insluitend Eucalyptus grandis (E. grandis), Pinus patula (P. patula), Bambusa balcooa (bamboes) en suikerriet (Saccharum officinarum L) (bagasse), is ondersoek as bronne van wateroplosbare xilaan vir ensiem modifikasie. Twee gematigde, lae temperatuur alkali-metodes (‘nalkali lading van < 14% en temperatuur van < 80°C), een met ultrasuiwering aangedui as Hoije en die ander met etanolpresipitasie aangedui as Lopez metode, is evalueer vir selektiewe ekstraksie van wateroplosbare xilaan vanuit die genoemde lignosellulose materiale. Die wateroplosbare xilaan is ge-ekshaheer vanuit P. patula, bagasse, E. grandis en bamboes met die Hoije metode met ekstraksie doeltreffendhede van 71.0, 66.0, 35.0, en 20.0%, onderskeidelik. Met die Lopez metode is xilaan vanuit bagasse en E. grandis geëkstraheer met ekstraksie doeltreffendhede van 28.0% en 12.0%, onderskeidelik. Die xilaan wat vanuit P. patula, bamboes, en bagasse ekstraheer is, is as arabinoglukuronoxilaan geïdentifiseer, wat met arabinose en 4-O-metiel-D glukuronsuur sykettings vervang is, terwyl die xilaan wat vanuit E. grandis ekstraheer is as 4-O-metiel--D-glukuronoxilaan (glukuronoxilaan), met substitusie met MeGlcA en asetiel-groepe op die hoof xilaan-ketting (ruggraat) is. Die glukuronoxilaan het verder spore van arabinose en rhaminose funksionele groepe bevat. Die geëkstraheerde xilaan fraksies het grade van polimerisasie > 10 gehad en was wateroplosbaar, wat die vereiste eienskappe van die xilaan vir doelgemaakte ensiem modifikasies bevredig het. Die selektiewe verwydering van die arabinose, MeGlcA, en asetiel-groepe om xilose eenhede sonder substitusie in polimeriese xilaan te vorm, wat intra- en inter-polimeer binding veroorsaak, word beskou as die belangrikste proses vir die vermindering van die oplosbaarheid van wateroplosbare xilaan. Die α-L-arabinofuranosidase van Aspergillus niger (AbfB) en α-D-glukuronidase van Schizophyllum commune (AguA) is spsialiteutsensieme wat tot dusver is met die vermoë om selektief die arabinose en MeGlcA sykettings, onderskeidelik, vanaf wateroplosbare xilaan te verwyder. Grootskaalse toepassing van die AbfB en AguA ensieme, vir die vermindering van die oplosbaarheid van wateroplosbare xilaan , sal ekstrasellulêre produksie deur mikrobes in groot hoeveelhede en vry van kontaminasie van die xilaan hoofketting degraderende ensieme insluitend die endo-1,4--xilanase vereis. Selektiewe produksie van die AbfB vry van xilanase aktiwiteit is verkry deur kultivering van rekombinante A. niger D15 [abfB], met transkipsie van die abfB-geen beheer deur die gliseraldehied-3-fosfaat dehidrogenase promotor (gpdp) en glukoamilase termineerder (glaAT). Die rekombinante AbfB ensiem is ekstrasellulêr geproduseer in 125 mL skudflesse en ‘n10 L bioreaktor fermentasiekulture met volumetriese aktiwiteite van tot 10.0 en 8.0 nkat mL-1, onderskeidelik, teen para-nitrofenol arabinofuranosied (pNPA). Die uitskeiding van die rekombinante AbfB was groei geassosieerd en het daarom tot 2.5 keer toegeneem met die byvoeging van gekonsentreerde mielieweekvloeistof as ‘n addisionele bron van stikstof in die 2 x minimale standaard kwekingsmedium. Die biomassa spesifieke aktiwiteit van die rekombinante AbfB teen die pNPA substraat was ongeveer 366 nkat g-1 (droë massa basis). Die rekombinante AbfB het ‘n enkele suiwer spesie band getoon op 10% SDS-PAGE gevlek met Coomassie blou en het ‘n beraamde molekulêre massa van 67 kDa gehad. Die rekombinante AbfB het verder optimale aktiwiteit by 40-55°C en pH 3.0-5.0 getoon en was stabiel onder kweking-, storing-, en bedryfstoestande by temperature tussen 30-60°C en pH 3.0-6.0. Die rekombinante AbfB het ook wye substraatspesifisiteit getoon om arabinose sy-groepe selektief te verwyder vanaf lae viskositeit koring-en hawerbiopolimere, lariks arabinogalaktaan, onvertakte arabinaan, en arabinoglukuronoxilaan biopolimere, geëkstraheer vanaf bagasse, bamboes en P.patula wat in Suid-Afrika aangetief word. Die rekombinante AbfB kon xilaan, ge-ekshaheer vanaf bagasse, bamboes en hawer onoplosbaar maak, maar die xilaan geëkstraheer vanaf P. patula nie. Meer as 95% van die aktiwiteit van die rekombinante AbfB teen die pNPA kon hersirkuleer word na selektiewe hidrolise van die xilaan by 40°C vir 16 h. Aan die ander kant kon die gesuiwerde AguA-ensiem slegs berkehout glukuronoxilaan onoplosbaar maak, maar nie glukuronoxilaan wat vanaf E. grandis geëkstraheer is of arabinoglukuronoxilaan wat vanaf bagasse, bamboes en P. patula geëkstraheer is nie. Die sinergistiese aksie van die rekombinante AbfB en die gesuiwerde AguA het die verwydering van die arabinose sykettings vanaf bagassexilaan met 22% vermeerder en met 33% in die geval van bamboesxilaan. Die verwydering van MeGlcA sykettings vanaf bagassexilaan is met slegs 5% vermeerder, terwyl dit met 13% verminder het in die geval van bamboesxilaan. Die selektiewe verwydering van die arabinose sykettings vanaf xilaan van hawer, bagasse, en bamboes deur die rekombinante AbfB het hoër skynbare viskositeit gehad relatief tot die ooreenstemmende onbehandelde xilaan . Die skynbare viskositeit van beide die behandelde en onbehandelde xilaan het egter verminder met toenemende skuiftempo. Die viskositeit het ‘n algehele negatiewe korrelasie met arabinose syketting verwydering gehad en het ‘n minimum van 2.03 mPa.s bereik vir hidrolise van hawerxilaan wat uitgevoer is vir 9.0 h by ‘n temperatuur van 45.8°C met rekombinante AbfB xilaan met ‘n spesifieke dosering van 400.0 nkat g-1substraat. Die wysiging van die viskositeit van die xilaan deur die selektiewe verwydering van die sykettings is van besondere belang in die produksie van spesialiteit emulsifisering, verdikking- en skuimweermiddels. Die optimale waardes vir hidrolisetyd, ensiemdosering en temperatuur vir maksimum graad van arabinose syketting verwydering vanaf hawerxilaan met die rekombinante AbfB, en van MeGlcA syketting verwydering vanaf berkehout xilaan met die gesuiwerde AguA, is vasgestel deur middel van die Box-Benhken responsie oppervlak metode. Die eksperimentele gebied het die xilaanspesifieke dosering met die rekombinante AbfB tussen 18.0 en 540.0 nkat g-1substraat en vir die gesuiwerde AguA xilaan tussen 2.0 en 18.0 μkat g-1substraat by temperature tussen 30 en 50°C en hidrolisetye tussen 1 en 16 h gedek. Die temperatuur, ensiem xilaan spesifieke dosering en hidrolise tyd het elk ‘n beduidende invloed (p<0.05) gehad op beide die selektiewe verwydering van arabinose vanaf hawerxilaan met die rekombinante AbfB en die selektiewe verwydering van MeGlcA vanaf berkehout xilaan met die gesuiwerde AguA. Die interaksie van hierdie hidroliseparameters was egter net beduidend (p<0.05) in die geval van arabinose syketting verwydering vanaf hawer xilaan met die rekombinante AbfB. Die optimale waardes vir die hidrolise tyd, temperatuur, en xilaan spesifieke dosering is beraam om gelyk aan 14-16 h, 38-45°C, en 607.0 nkat g-1substraat, onderskeidelik, te wees vir maksimale verwydering van 43% van die beskikbare arabinose in die hawer xilaan met die rekombinante AbfB. Die optimale waardes vir die hidrolise tyd, temperatuur en xilaan spesifieke dosering vir maksimale verwydering van 0.5% van die beskikbare MeGlcA sykettings vanaf die berkehout xilaan met die gesuiwerde AguA is beraam om gelyk aan 11 h, 38°C, en 18.0 μkat g-1substraat, onderskeidelik, te wees. Die optimale waardes van die hidrolise parameters, vir beide die verwydering van die arabinose vanaf hawer xilaan met die rekombinante AbfB en van MeGlcA sykettings vanaf berkehout met die gesuiwerde AguA, kon voorspel word deur gebruik te maak van kwadratiese modelle wat die responsie-oppervlak grafieke met regressie koeffisiënte > 0.9 gepas het. Die effek van in situ selektiewe verwydering van arabinose en MeGlcA sykettings met rekombinante AbfB en gesuiwerde AguA, onderskeidelik, vanaf wateroplosbare xilaan op hulle presipitasie en adsorpsie op katoen lint is ondersoek. Die katoenlint is behandel met xilaan ge-ekstraheer vanuit bagasse, bamboes, P. patula, en E. grandis deur gebruik te maak van die Hoije metode in die teenwoordigheid van die rekombinante AbfB, AguA, en ‘n mengsel van die twee ensieme. Die effek van in situ selektiewe hidrolise, deur die ensieme van model xilaan insluitende berkehout, hawer en H2O2-gebleikte bagasse en E. grandis xilaan jel, op hulle adsorpsie op katoen lint is gebruik vir verwysingsdoeleindes. Die gesuiwerde AguA het die adsorpsie van arabinoglukuronoxilaan , wat vanuit bagasse, bamboes en P. patula ekstraheer is deur middel van die Hoije metode, op katoenlint die meeste laat toeneem in vergelyking met die effek van die rekombinante AbfB en die mengsel van die rekombinante AbfB en die gesuiwerde AguA. Die gesuiwerde AguA het die adsorpsie van die xilaan wat vanuit bagasse en E. grandis ekstraheer is met 334 en 29%, onderskeidelik, laat toeneem, maar het die adsorpsie van E. grandis xilaanjel en H2O2 gebleikte bagasse xilaan met 31 en 6%, onderskeidelik, laat afneem. Op ‘n soortgelyke wyse het die teenwoordigheid van die rekombinante AbfB die adsorpsie van die bamboes, P. Patula en hawer xilaan met 31, 44, en 900%, onderskeidelik, laat toeneem, maar die adsorpsie van die xilaan ekstraheer vanuit bagasse en die H2O2 gebleikte bagasse xilaan met 13 en 30%, onderskeidelik, laat afneem. Verskillende xilaan-sellulose interaksies en water adsorpsie kapasiteite van die katoen lint is opgemerk met die in situ modifikasie en adsorpsie van die xilaan ekstraheer vanuit die bagasse, bamboes, E. grandis en P. patula in die teenwoordigheid van die rekombinante AbfB en gesuiwerde AguA. Die ensiem bygestaande adsorpsie van xilaan kon daarom gebruik word om die funksionele eienskappe van die sellulose materiaal aan te pas of te verbeter. Die wekverrigting van ensimaties gevormde xilaan nanohidrojels as enkapsuleringmatrikse vir stadige vrystelling van bioaktiewe middels is geevalueer. Onoplosbare xilaan nanohidrojels wat gevorm is deur selektiewe verwydering van arabinose sykettings vanaf wateroplosbare hawer xilaan met die rekombinante AfbA, is gekarakteriseer vir partikelgrootteverspreiding, oppervlaklading (zeta potensiaal), morfologiese stabiliteit, en die vermoë om die ramenas peroksidase te enkapsuleer en stadig vry te stel. Die ensimaties gevormde hawer xilaan hidrojels het ‘n sferiese vorm gehad met partikelgroottes wat gewissel het van 18 nm tot > 10 000 nm. Die xilaan nanohidrojels het ‘n negatiewe zeta potensiaal van tot -19 mV getoon, en het self-vormings gedrag vir partikels ten toon gestel indien dit by xilaankonsentrasies hoër as 1.5% (m/v) en hidrolise tye langer as 17 h gevorm is. Die xilaan konsentrasie het beide die partikelgrootte en die zeta potensiaal van die hawerxilaan nanohidrojels beduidend (P < 0.05) beïnvloed terwyl die rekombinante AbfB hidrolise tyd beduidend (P < 0.05) was op die zeta potensiaal. Die hawer xilaan nanohidrojels, het die ramenasperoksidase ensiem suksesvol enkapsuleer, beide gedurende en na die vorming van die hawer xilaan nanohidrojels en die vrystelling van die geënkapsuleerde ramenas peroksidase in aktiewe vorm is volgehou vir ‘n periode van 180 min. Die ensieme wat die syketting van die xilaan verwyder het, het dus ‘n rol in die voorbereiding van biodegadeerbare nano-enkapsulasie geedskap. In die geheel veskaf die rekombinante AbfB en gesuiwerde AguA ‘n nuwe stel manier voor om wateroplosbare xilaan te funksionaliseer om as spesialiteit bymiddels, bedekking, en inplanting of enkapsulasiematrikse gebruik te word met ‘n verminderde impak op die omgewing. Dit sal prosessering bevorder en die produkspektrum van lignosellulose materiale uitbrei.

This work studied the improvement of stability, physical properties and enzymatic inactivation in fruit beverages by applying the ultrasound technology (US). In the first part, the effect of the US application on peach juice processing was evaluated. The macroscopic changes on pulp sedimentation stability, turbidity, colour and rheological properties were evaluated. As a result, it was demonstrated that the improvement in each of the properties evidenced at the macroscopic level involves interaction of complex mechanisms which depend directly on changes at the microscopic level, such as the structure, size, composition and interaction between the continuous phase (serum) and dispersed phase (pulp) of the juice. These changes were assessed by microscopy and particle size distribution. In the second and third parts, the inactivation of the enzyme peroxidase (POD) was evaluated in coconut water. In the second part, the effect of the application of US was evaluated for first time for coconut water POD, using two types of ultrasonic equipment (US baht and US probe). It was demonstrated that the changes of enzyme activity during the US process depend on the many forms that the enzyme can adopt, mainly depending on the energy applied to the system. Subsequently, in the third part, the US was applied as a pre-treatment of subsequent thermal processing. The evaluation was carried out under non isothermal conditions, being the POD inactivation kinetics modelled using the Weibull distribution function. Finally, it was observed that the pre-treatment using ultrasound slightly decreased the enzyme activity. Furthermore, the ultrasound effects resulted in a more homogeneous population and heat-sensitive enzymes, significantly reducing the needed time of thermal processing. In conclusion, this work studied and demonstrated that the ultrasound technology is an interesting alternative to improve the physical properties and enzymatic stability in fruit beverages, reflecting the importance from both the academic and industrial point of view.
Neste trabalho estudou-se a melhoria na estabilidade, propriedades físicas e inativação enzimática em bebidas de frutas através da aplicação da tecnologia de ultrassom (US). Na primeira parte, foi avaliado o efeito do US no processamento de suco de pêssego. As alterações macroscópicas na estabilidade de sedimentação da polpa, turbidez, cor e propriedades reológicas foram analisadas. Foi demonstrado que a melhoria em cada uma das propriedades evidenciadas macroscopicamente envolve interação de mecanismos complexos que dependem diretamente de alterações microscópicas, tais como estrutura, tamanho, composição e interação entre as fases contínua (soro) e dispersa (polpa) do suco. Estas alterações foram avaliadas por microscopia e análise de distribuição de tamanho de partículas. Na segunda e terceira partes, a inativação da enzima peroxidase (POD) foi avaliada em água de coco. O efeito da aplicação do US na POD de água de coco foi estudado pela primeira vez, utilizando dois tipos de equipamentos (banho e sonda de US). Demonstrou-se que as alterações na atividade enzimática durante o processamento com US estão relacionadas às diferentes conformações que a enzima pode adotar, dependendo principalmente da energia aplicada ao sistema. Na terceira parte, o ultrassom foi então aplicado como pré-tratamento ao processamento térmico. A avaliação foi realizada sob condições não isotérmicas, sendo a cinética de inativação da POD modelada usando a função de distribuição de Weibull. Foi observado que o pré-tratamento com US diminuiu a atividade enzimática. Além disso, o efeito do US resultou em uma população de enzimas mais homogênea e termosensível, reduzindo significativamente o tempo necessário para o processamento térmico. Desta forma, este trabalho estudou e demonstrou que a tecnologia de ultrassom é uma alternativa interessante para melhorar as propriedades físicas e a estabilidade enzimática de bebidas à base de frutas, indicando sua importância tanto acadêmica quanto industrial.

Enzymes are promising stimuli for the development of responsive biomaterials for biomedical applications. Enzymes are inherently present in the biological environment thus cleverly designed materials for biomedical applications may require no external stimuli to ellicit the required material response. They have been targeted as stimuli in self assembly of bulk materials owing to the material changes in chemical composition afforded by the enzyme interaction. The first examples of autonomous self-regulated drug delivery systems have been reported via the development of reversible enzyme responsive materials that undergo a material change regulated by the enzymes in their environment. Although enzyme responsive surfaces have been reported there are no examples of reversible enzyme response surfaces. The surface is the first point of contact between the biological environment and a biomedical device/implant. Improving this interaction will improve the integration of these biomaterials in biological systems and it has been proposed that biomimetic surfaces are a promising method for full biomaterial integration in the biological environment. The body strives towards homeostasis and this is frequently achieved by enzymatic activating and deactivation of proteins in the body. This process is repeatable and reversible. Herein we address the absence of reversible and repeatable synthetic enzyme responsive surfaces towards the improvement of biomaterial integration. We aim to develop a truly autonomous system wherein enzymes present in the environment can interact with the modified surface to mediate a reversible material response. This goal was achieved by modifying surfaces with copolymers that contain the recognition sequence for Casein kinase II and Alkaline phosphatase to undergo enzymatic phosphorylation and dephosphorylation. Co and homo polymers of serine and glutamic acid were synthesised in solution and conformation/composition relationship was determined by analysis with NMR, GPC and FTIR. Polymerisation from the surface with NCA-Glu and NCA-Ser was achieved as characterised by FTIR, ToF SIMS, XPS and WCA. Enzymatic mediated phosphorylation (CKII) and dephosphorylation (AP) was monitored by surface analysis (ToF SIMS), by monitoring ATP to ADP conversion and phosphate cleavage from the surface using luminescence and colorimetric assays. Conformational changes mediated by enzymatic interactions with the surface was monitored indirectly using a FRET system incorporated in the surface modification. The modified surfaces were able to support cell culture and osteogenesis. This project has made advances in several fields, 1) The use of NCA-ROP as a method to modify surfaces with copolymers, in particular for a random/ alternating amino acid sequence. 2) The use of NCA-ROP as a method to develop stimuli responsive surfaces, specifically, this is the first report of an enzyme responsive surface prepared from NCA-amino acid derivatives. 3) The use of enzymes as stimuli, specifically, this is the first report of a reversible enzymatic responsive surface. In this system reversible phosphorlyation and dephosphorylation was monitored via changes in fluorescence output indicative of induced conformational changes.

Thesis (M.B.A.)--Massachusetts Institute of Technology, Sloan School of Management, 2000.
Also available online at the DSpace at MIT website.
Includes bibliographical references (leaves 47-50).
Undesirable pharmacokinetic properties, such as poor bioavailability and drug-drug interactions, have been one of major reasons for the failure of new pharmaceuticals in clinical trials. These dropout risks can be reduced by early knowledge of human pharmacokinetics in the drug discovery process. Although new drug candidates have been first tested in animal-based systems, the prediction of human pharmacokinetics from animal data has been unsuccessful due to species differences in the enzymes involved in drug metabolism. Recent progress in molecular biology made it possible to develop in vitro drug metabolism systems using human metabolic enzymes, such as purified microsomes or expressed cytochrome P450. These systems are useful in profiling the enzymes involved in the human metabolism and extrapolating the in vitro findings to in vivo situations. The in vitro systems may yield a rapid drug metabolism screening of numerous compounds generated through combinatorial chemistry and high-throughput pharmacological screening. The integration of these technologies into the drug development process will significantly reduce the dropout risks in clinical trials and shorten the period between the drug discovery and market introduction.
by Hiroaki Sato.
M.B.A.

Aberrant expression of short non-coding micro RNAs (miRNA) in many human diseases, along with remarkable stability in physiological media, has made them attractive clinical biomarkers. In particular, miRNA-122 is substantially elevated in plasma of patients with established drug-induced liver injury and can also be used to identify early liver injury when current markers, such as alanine aminotransferase (ALT), still show normal levels. The development of a rapid test for miRNA-122 e.g. in drug poisoning would allow earlier and more sensitive clinical diagnosis of liver injury. Nucleic acids are traditionally analysed by polymerase chain reaction (PCR), which has a high degree of sensitivity but suffers from high cost and is prone to sample contamination. The aim of this project is to develop a PCR free method to directly detect miRNA- 122 in biological samples using SMART technology. The SMART technology takes advantage of dynamic chemistry for sequence specific recognition of nucleic acids using aldehyde-modified nucleobases (SMART nucleobases), and target-complementary peptide nucleic acid (PNA) probe containing an “abasic” position (so called modified PNA probe). In this study, this unique detection method was used in a fluorescent detection with the use of light up probes, which are probes with an environmental dye as nucleobase; a FRET system was also designed to allow the discrimination between perfect match target and mismatched one. The SMART technology was also transferred onto magnetic beads to develop an ELISA like assay allowing sensitive and rapid detection of single stranded DNA mimic of the miRNA-122. With its potential PCR free approach, this easily adapted platform promises to transform and expand routine clinical diagnostic testing and screening for circulating miRNAs.

The prevalence of hypertension during the last two decades has led to an intensive search for novel angiotensin I-converting enzyme (ACE) inhibitory peptides from food protein sources. Dry beans (Phaseolus vulgaris) serve as an important protein source in the human diet. However, their physiological and functional properties are poorly understood. Therefore, this thesis was undertaken to investigate the in vitro ACE inhibitory activities of nine varieties of dry beans that are largely grown in Canada.A preliminary study was carried out to compare protein profiles, including composition, thermal properties and molecular structure for protein isolates (PIs) prepared from the nine varieties of dry beans. Results showed that two varieties, namely, cranberry and light red kidney bean PIs had significantly lower denaturation temperatures as well as different electrophoresis profiles and RP-HPLC chromatograms. In vitro ACE inhibitory activities were investigated for the nine varieties of dry beans. In the first study, protein extracts from the nine varieties of dry bean were subjected to trypsin and in vitro gastrointestinal simulation (GIS) digestion. The results demonstrated all hydrolysates exerted ACE inhibitory activities, with hydrolysates produced by in vitro GIS digestion presenting significantly higher (P<0.05) degree of hydrolysis (DH) values as well as ACE inhibitory activities than that of tryptic digests. Among all the varieties, small red bean and navy bean showed the highest and second highest ACE inhibitory activity, respectively, for both digestion treatments. The results also showed that ACE inhibitory activity of in vitro GIS digests of black bean was significantly higher (P<0.001) than that of the tryptic digest. Thus, the three varieties, namely, small red, navy and black beans were selected for subsequent studies to investigate the influence of thermal and different enzymatic treatments on their ACE inhibitory activities. The highest ACE inhibitory activities were obtained for samples hydrolyzed for 100 min, 95 min, and 95 min using Alcalase/papain digestion for heated navy, black and small red bean PIs, respectively. The corresponding half maximal inhibitory concentration (IC50) values were 68 μg protein/mL, 83 μg protein/mL, and 78 μg protein/mL, respectively. Improved ACE inhibitory activities were obtained after in vitro GIS hydrolysates in the order of small red bean > navy bean > black bean.Small red bean hydrolysate, digested by Alcalase/papain and subsequently in vitro GIS digestion was further subjected to fractionation to isolate the most active ACE inhibitory peptides. After a three step purification process, including ultrafiltration, gel filtration and preparative reverse phase high performance chromatography (RP-HPLC). Fraction 28 from RP-HPLC was identified as the most active fraction with an IC50 value of 19.3 μg protein/mL. Enzymatic kinetics conducted using this fraction demonstrated competitive inhibition with inhibitor dissociation constant (Ki) of 11.6 μg protein/mL. Peptide with sequence PVNNPQIH was identified from this fraction with IC50 value of 206.7±3.9μM. Overall, the results from the current study expand the scientific knowledge of the physicochemical and biochemical properties of the nine dry bean (Phaseolus vulgaris) varieties studied. The comparative studies of the in vitro ACE inhibitory activities, protein profiles, thermal properties and secondary structures are the first to be reported in the literature to the best of our knowledge. Results from this work provide a solid base for further research on the ACE inhibitory activities of pulses and should be useful for selecting conditions for potential future utilisation of beans as a source of protein hydrolysates and functional ingredients in novel foods targeting hypertension.
La prévalence de l'hypertension au cours des deux dernières décennies a mené à une recherche intensive de nouveaux peptides inhibiteurs de l'ACE provenant de protéines alimentaires. Les haricots secs (Phaseolus vulgaris) sont une source de protéines importante dans le régime alimentaire humain. Cependant, leurs propriétés physiologiques et fonctionnelles sont mal connues. Cette thèse a donc été entreprise pour examiner, in vitro, les activités inhibitrices de l'ACE de neuf variétés de haricots secs qui sont en grande partie cultivées au Canada.Une étude préliminaire a été effectuée pour comparer les profils protéiques, ainsi que la composition, les propriétés thermiques et la structure moléculaire d'isolats de protéines (PI) préparés à partir des neuf variétés de haricots secs. Les résultats ont montré que les PI de deux variétés, à savoir, le haricot canneberge et le haricot rouge clair, avaient des propriétés distinctement différentes dont, des températures de dénaturations significativement inférieures ainsi que des profils électrophorétiques et des chromatogrammes de RP-HPLC différents. Les extraits de protéines des neuf variétés de haricots secs ont été soumis à la trypsine et la simulation d'une digestion gastro-intestinale (GIS) in vitro. Les résultats ont démontré que les hydrolysats produits par la digestion GIS in vitro présentaient un degré d'hydrolyse (DH) et d'activités inhibitrices de l'ACE significativement plus élevés (P < 0.05) que les hydrolysats obtenus avec la trypsine seulement. Parmi toutes les variétés, le petit haricot rouge et le haricot blanc ont montré, respectivement, le plus élevé et second plus élevé taux d'activité inhibitrice d'ACE, et ce, pour les deux traitements de digestion. Les résultats ont aussi montré que l'activité inhibitrice de l'ACE des produits de digestion GIS in vitro du haricot noir était significativement plus élevée (P < 0.001) que celle de la digestion avec la trypsine.Ainsi, les trois variétés, à savoir, des petits haricots rouges, les haricots Navy et les haricots noirs, ont été choisies pour des études ultérieures pour examiner l'influence de la température et de différents traitements enzymatiques sur les activités inhibitrices de l'ACE. Les activités inhibitrices de l'ACE les plus élevées ont été obtenues pour des échantillons hydrolysés lors de la digestion avec l'alcalase suivi de la papaïne pour les PI chauffés du Haricot Navy (hydrolyse de 100 minutes) du petit haricot rouge (hydrolyse de 95 minutes) et du petit haricot noir (hydrolyse de 95 minutes). La valeur correspondant à la concentration médiane d'inhibition IC50 était de 68, 83 et 78 µg de protéine/ml, respectivement pour les trois haricots susmentionnés. Les meilleures activités inhibitrices d'hydrolysats ont été obtenues après GIS in vitro par le petit haricot rouge, puis, le haricot blanc, et puis, le haricot noir. L'hydrolysat du petit haricot rouge, digéré par alcalase/papaine et par la suite par la digestion GIS in vitro a été à nouveau soumis au fractionnement pour isoler les peptides inhibiteurs de l'ACE les plus actifs. Après trois étapes de purification comprenant l'ultrafiltration, la filtration sur gel et RP-HPLC, la fraction 28 obtenue après RP-HPLC a été identifiée comme la fraction la plus active avec une valeur d'IC50 de 24.4 µg de protéine/mL. La cinétique enzymatique réalisée avec cette fraction a démontré une inhibition compétitive avec une constante d'inhibition Ki de 14.7 µg/mL. Les études comparatives des activités inhibitrices de l'ACE, des profils de protéines, des propriétés thermiques et des structures secondaires sont les premières à être rapportées dans la littérature au mieux de notre connaissance. Les résultats de ce travail devraient être utiles pour choisir des conditions pour de potentielles utilisations futures des haricots comme source d'hydrolysats protéiques et d'ingrédients fonctionnels dans de nouveaux produits alimentaires visant à réduire l'hypertension.

The presence and impact of steroid estrogens in natural water matrices has driven development and evaluation of wastewater treatment technologies that may reduce the steroid load entering water environments. This work was undertaken to assess and predict the ability of Trametes versicolor laccase to degrade estrone (E1) in water matrices under realistic conditions to wastewater treatment plants (WWTPs) and with consideration of the complex and variable nature of the wastewater matrix. A robust experimental procedure was developed to ensure the efficiency of the enzyme laccase to degrade E1 in water matrices was not overestimated due to errors arising from poor experimental design. These experiments demonstrated that commercially-obtained laccase in concentrations above > 1 mg/ml are inhomogeneous requiring centrifugation prior to use to reduce error and provide more accurate evaluation of laccase capability. Sample filtration, which is necessary for chromatographic analysis, identified regenerated cellulose (RC) membrane filters as the optimum filters for particulates removal from E1 solutions due to their low affinity toward E1 (3.2 ±1.72 %). An optimum enzyme inactivation procedure using hydrochloric acid was also developed to ensure that the enzyme laccase was instantly inactivated without affecting the target steroid E1 itself. Using the established experimental procedure, bench-scale studies evaluating the efficiency of laccase-based treatment in a ‘clean’ water matrix were investigated. Experiments in deionised water provided a proof of concept of laccase ability to degrade E1 in water under realistic ranges of temperature [6˚C - 25˚C] and contact time [0.5 hr – 8 hrs] to the WWTP and evaluate the use of models to fit experimental data and predict within that system. Box Behnken Design (BBD) was applied to determine the number and the conditions of the performed experiments. The experimental data was then utilised to build two different models to predict E1 removal efficiency under any set of conditions and optimise the performance of laccase-based treatment system. The goodness of the fit for each model was tested using statistical indices such as coefficient of determination (R2), mean squared error (MSE) and absolute average deviation (AAD). The artificial neural network (ANN) model showed a better fit to the experimental data than the response surface methodology (RSM) model (RSM and ANN of R2 = 0.9908 and R2 = 0.9992 respectively. In addition, the predictive capabilities of RSM and ANN were tested using a set of statistically designed unseen data that was not previously used in models’ training. Both models showed limited predictive capabilities. The ability of laccase-based treatment to remove E1 in real-world wastewater was studied at bench scale. To account for the complexity and variability of the wastewater matrix, effluent samples during the period December 2014 - June 2015 were characterised for standard water quality parameters, where the temporal variation in wastewater chemical oxygen demand (COD), total suspended solids (TSS) and pH, were observed. A new water quality parameter, “Benchmark” was also developed and applied to quantify the impact of wastewater variability on laccase performance for E1 removal. The average benchmark value in the period between December 2014 and June 2015 was 79.8±3.7%. In addition, the impact of laccase inhibitors, which are likely to be present within the wastewater matrix, such as chloride, copper, iron and zinc, on laccase activity was investigated. The inhibitory effect of chloride ions increased with increasing chloride concentration above 200 mg/l. Copper and zinc ions exhibited negative effects on the enzymatic degradation of E1 at concentrations equal or above 10 mg/l and 200 mg/l. The impact of water matrix temperature, contact time and laccase concentration were studied in wastewater effluent and the experimental data was used to build RSM and ANN models. The predictive capability of the generated RSM model was relatively poor (R2 = 0.863) and even lower than the achieved predictive capability in clean matrix when tested using unseen data, this was partially attributed to the variability of wastewater matrix that could have not been addressed in this type of models. Whilst the improved ANN model showed a better predictive capability than RSM (R2=0.991) An advantage of the ANN model compared to the RSM model and reported for the first time, was the ability to include the impact of matrix complexity and variability on laccase performance, assessed via the benchmark data added as a forth factor in the ANN model. The final ANN model incorporating the matrix variability observed temporally during the sampling period had extremely high predictive capabilities (R2 > 0.99). This model approach holds the potential to help researchers evaluate and optimise laccase-based treatment (as well as other treatment technologies) and predict the removal efficiency of various bioactive chemicals under a wide range of conditions. Performing laccase-based treatment in a continuous reactor, utilising actual wastewater effluent and under realistic conditions to WWTPs, is the next stage that should be investigated in detail.

Conjugates of soybean trypsin inhibitor (SBTI) and potato serine protease inhibitor (PSPI) immobilized on metal oxide particles of ~100nm diameter were prepared. Inhibition of trypsin hydrolysis of BAPA by these conjugates was measured and enzyme kinetics constants kcat, KM, kcat/KM and ki were determined. Metal oxide particles presented an inhibitory effect similar to that of a competitive inhibitor, noticed through the increase value of the K M constant. Furthermore, PSPI conjugates had the highest inhibition of trypsin, illustrated by the significantly higher value of KM relative to the value for particles only.

Finding alternatives to petroleum-based energy sources is of interest since it could reduce the emissions of net carbon dioxide to the atmosphere by increasing the usage of renewable energy sources. To do so improvements are needed in the renewable energy production sector. Biogas production is of interest since the anaerobic digestion process can degrade many different biomolecules and is, contrary to e.g. bioethanol and biodiesel, not dependent on specific molecules. Thus, many wastes such as slaughterhouse waste, sludge from waste water treatment and lignocellulose residual material etc. can be used as substrates for biogas production. However, there are limitations in the degradation process depending on the composition of the selected substrate. To overcome these limitations such as inhibition of different microorganisms, or recalcitrant substrate, different methods can be used to increase the biogas production.  In this study different substrates were selected and analyzed/treated for remedies of early stage rate limiting problems of the anaerobic digestion process. Different analyzes and techniques were selected depending on the limitations correlated to the main problematic component of the specific substrate.  Improvements could be reached for the degradation of slaughterhouse waste by augmentation with the clay mineral zeolite. Addition of different enzymes to the substrate environment of different waste water treatment plant sludges resulted in limited life time of the selected enzymes. However, certain enzymes proved to be promising candidates with an effect of increased biogas production rate and yield for the time that the enzyme remained active. In an additional experiment, cellulolytic enzymes, naturally produced by a biogas producing microbial community, were induced, collected and added to a biogas experiment of ensiled forage ley, by which it was shown that these cellulases led to an increase in biogas production rate and yield. Thus, the studies demonstrate different techniques for improving the anaerobic digestion process of different types of substrates.

Handledare saknas

La présente étude avait pour but d'identifier de nouvelles stratégies pour la présentation sélective d'enzymes à la surface de nanoparticules virales dans le but d’une application potentielle dans la technologie des biocapteurs ou des puces à protéines. Les potyvirus ont été choisis comme nanosupports modèles. Les Potyvirus, le genre le plus large de la famille des Potyviridae, la seconde plus grande famille de virus de plante, sont responsables de très graves pertes dans les cultures. Ils forment des capsides flexibles en forme de bâtonnet entourant une seule molécule d'ARN positif simple brin. Les événements moléculaires conduisant à la sélection et à l'encapsidation spécifiques de l'ARN potyviral sont inconnus. Afin de mieux exploiter le potentiel de ces virus comme nanosupports, la première étape de ce travail a porté sur l’étude, in vivo, du processus d'encapsidation de l'ARN de particules de potyvirus. Des études précédentes ont montré que la protéine d'enveloppe (CP) du virus de la pomme de terre A (PVA) interfère avec la traduction de l'ARN viral lorsqu'elle est fournie en excès en trans suggérant que cela pourrait se produire pour initier l’encapsidation de l’ARN viral. Dans cette étude, nous avons montré que cette inhibition est médiée par des interactions CP-CP co-traductionnelles se produisant entre deux populations de CP, produites en trans et en cis et permettant très probablement le recrutement spécifique de l'ARN potyviral pour son encapsidation. En accord avec les études d'assemblage in vitro publiées précédemment nous proposons un mécanisme selon lequel l’encapsidation de l'ARN viral est initiée par des interactions CP-CP co-traductionnelles. Dans la deuxième partie de ce travail, différentes approches ont été testées afin d’organiser des enzymes sur les plateformes virales dans le but d’optimiser la canalisation des intermédiaires réactionnels. Parmi les trois stratégies testées seule celle utilisant un peptide qui se liant aux anticorps, le peptide z33 de la protéine A de Staphylococcus aureus a été couronnée de succès. Une couverture de 87 % des sites sur les particules de potyvirus avec l'enzyme a été obtenue. Cette stratégie a été utilisée pour piéger deux enzymes, la 4-coumarate: coenzyme A ligase (4Cl2) et stilbène synthase (STS), catalysant des étapes consécutives dans la voie de synthèse de resvératrol à partir de lysats cellulaires solubles d’E. coli clarifiés, à la surface de particules de potyvirus immobilisées sur les parois d'un tube en polypropylène. Cette stratégie rassemble les approches ascendante et descendante pour construire des nanomatériaux à base de virus et offre un moyen efficace et économique pour co-immobiliser et purifier des enzymes
The present study intended to identify new strategies for the selective presentation of biocatalysts on the surface of viral nanoparticles with potential application in biosensor technology or protein chips. Potyviruses were chosen as model nanoscaffolds for biocatalysts. Potyviruses are the largest genus in the family Potyviridae and cause significant plant damage. They form flexible rod-shaped capsids surrounding a single stranded positive sense RNA molecule. The molecular events leading to the specific selection and encapsidation of potyviral RNA are unknown. To better exploit the potential of these viruses as nanocarriers, the first step in this study was to look into their in vivo RNA encapsidation process. Earlier studies showed that Potato virus A (PVA) coat protein (CP) interferes with viral RNA translation when provided in excess in trans and it was suggested this could occur to initiate viral RNA encapsidation. In this follow up study, we used the agroinfiltration approach for the transient expression of full length, truncated or mutated viral RNAs with wild type CP (CPwt) and showed that this inhibition is mediated by co-translational CPCP interactions occurring between two CP populations, produced in trans and in cis. Because CP inhibited translation of the entire viral genome and virus particles were formed later than during normal infection, it was assumed that the CP acted during this inhibition process to specifically recruit viral RNA for encapsidation. In line with previously published in vitro assembly studies, we propose a mechanism through which viral RNA encapsidation is initiated through co-translational CP-CP interactions. The second part of this work entailed the investigation of novel approaches for organizing biocatalysts on virus platforms. The aim was to control the display of enzymes on virus surfaces while maximizing channelling of reaction intermediates. Three strategies were tested but only one involving an antibody binding peptide, the z33 peptide from Staphylococcus aureus was successful. An 87 % occupancy of accessible sites on the potyvirus particles by the enzyme was achieved. The same strategy was used to graft potyvirus particles with two enzymes: 4- coumarate:coenzyme A ligase (4CL2) and stilbene synthase (STS), catalysing consecutive steps in resveratrol synthetic pathway or a protein chimera, generated by the genetic fusion of both enzymes. This was achieved by trapping either the monoenzymes or the protein chimera from clarified soluble E. coli cell lysates on to the surface of potyvirus particles preimmobilized in a polypropylene tube. Resveratrol was synthesized from both mono-enzymes and the protein chimera in solution and on potyvirus particles. This strategy brings together a bottom-up and top down approach for designing virus based nano-materials and offers a cost effective and efficient way to co-immobilize and purify enzymes

High pressure (HP) processing, a novel technology, has excellent potential for non-thermal preservation of apple juice, the largest consumed fruit juice in North America. The objective of this research was to evaluate the application of HP treatment for inactivation of enzymes and destruction of microorganisms in apple juice. HP inactivation kinetics of selected enzymes (amylase, pectin methyl esterase and polyphenol oxidase) and microorganisms [Leuconostoc mesenteroides, Pichia membranaefaciens and Zygosaccharomyces badii, Escherichia coli (29055) and Escherichia coli (O157:H7)] in apple juice were evaluated under various test conditions (100--400 MPa, 0--60 min and 6--40°C) using a central composite design of experiments. The enzymes selected were of importance in apple juice preparation and/or storage stability of the processed juice. Microorganisms included those that are responsible for spoilage and/or public health concern as well as those that are indicative of unsanitary handling conditions.
Enzyme inactivation and microbial destruction due to pressure followed a dual-effect model consisting of a pressure pulse effect (PE) and a subsequent semi-logarithmic (first order) inactivation during the pressure hold-time. In general, results showed that inactivation of enzymes and destruction of microorganisms was more prominent at higher-pressure levels, higher temperature and longer treatment times, and at lower pH levels of juice. Pressure pulse effect was dependent on pressure level, with higher PE achieved at higher pressures. During the pressure-hold, as expected, the associated decimal reduction times (D values) decreased with an increase in pressure. Pressure dependency of D values was well described by the conventional death time model. The pressure resistance of enzymes and microorganisms varied, but complete inactivation of enzymes and destruction of microorganisms was possible with the combination of lower pH, higher pressure and higher temperatures.
Commercial PME from a citrus source was more pressure sensitive than PME from microbial source. Spoilage bacteria (L. mesenteroides) were more pressure resistant than the yeasts. E. coli enumerated on an enrichment media (supporting both injured and healthy cells) showed larger survivors and a greater resistance than on a more selective media. An increasing number of cells got injured than killed with the application of pressure treatment until they were all finally injured or killed. High-pressure treatment (pulse at 400 MPa or by holding about 10 min at 350 MPa and 30°C) resulted in complete destruction of the pathogenic microorganism E. coli (O157:H7) ensuring the public health safety of the process.

The search for alternatives to oil-based monomers has sparked interest for scientists to focus on the use of renewable resources for energy production, for the synthesis of polymeric materials and in other areas. With the use of renewable resources, scientists face new challenges to first isolate interesting molecules and then to process them. Enzymes are nature’s own powerful catalysts and display a variety of activities. They regulate important functions in life. They can also be used for chemical synthesis due to their efficiency, selectivity and mild reaction conditions. The selectivity of the enzyme allows specific reactions enabling the design of building blocks for polymers. In the work presented here, a lipase (Candida antarctica lipase B (CalB)) was used to produce building blocks for polymers. An efficient route was developed to selectively process epoxy-functional fatty acids into resins with a variety of functional groups (maleimide, oxetane, thiol, methacrylate). These oligoester structures, based on epoxy fatty acids from birch bark and vegetable oils, could be selectively cured to form thermosets with tailored properties. The specificity of an esterase with acyl transfer activity from Mycobacterium smegmatis (MsAcT) was altered by rational design. The produced variants increased the substrate scope and were then used to synthesize amides in water, where the wild type showed no conversion. A synthetic procedure was developed to form mixed dicarboxylic esters by selectively reacting only one side of divinyl adipate in order to introduce additional functional groups.

QC 20170823

Objetivos El objetivo general de esta Tesis Doctoral se centró en la aplicación de la tecnología enzimática para el diseño y optimización de procesos integrados de sacarificación de celulosa mediante el uso de la tecnología de líquidos iónicos y reactores de membrana. Metodología Para llevar a cabo este objetivo se realizaron tests de actividad y estabilidad del derivado enzimático inmovilizado de estudio en diferentes líquidos iónicos (ILs). En los procesos de disolución, regeneración de celulosa y recuperación del IL, se emplearon diferentes etapas de operación (mezclado, filtración y destilación) para la obtención de la celulosa regenerada y separación del IL. Se contempló también, el diseño de sistemas integrados de reacción / separación basados en la incorporación de membranas para la recuperación y reutilización de enzimas. Y por último, para los controles de calidad de las disoluciones de glucosa obtenidas, se emplearon cultivos de Saccharomyces cerevisiae. Resultados y conclusiones La estabilidad de la celulasa comercial (Celluclast®), inmovilizada en un soporte polimérico (Amberlite XAD4), se estudió en 3 líquidos iónicos diferentes [N1114] [NTf2], [Bmim] [NTf2] y [Bmim][Cl]) a 50°C. Los ILs hidrofóbicos mejoraron claramente la estabilidad térmica de la enzima: En el caso del [N1114][NTf2] se consiguió aumentar hasta 4 veces el tiempo de vida media del biocatalizador, mientras que el [Bmim][Cl] se comportó como un potente desactivador. La estabilidad de la celulasa en mezclas IL hidrofóbico/ Bmim][Cl] permitió estabilizar la enzima frente a la desactivación producida por el [Bmim][Cl]. El derivado enzimático inmovilizado se recubrió con [N1114][NTf2] utilizándose exitosamente en la sacarificación de celulosa disuelta en [Bmim][Cl] (i.e. hasta un 50% de hidrólisis en 24h) a 50°C. Así mismo, se desarrolló un proceso cíclico sostenible de sacarificación enzimática de celulosa pretratada con IL. Para ello se emplearon diferente soluciones homogéneas de celulosa en [Bmim][Cl] para la producción de celulosa amorfa mediante precipitación con agua, etanol o una mezcla equimolar agua / etanol. Varios parámetros de operación (e.g. tipo de disolvente, temperatura, ultrasonidos, etc.) se probaron para la precipitación de celulosa así como para las siguientes etapas de lavado para la total recuperación del IL (hasta un 99.7%), el cual se reusó exitosamente en un proceso cíclico de disolución / regeneración. La celulosa regenerada producida en cada ciclo resultó en un sustrato excelente para la sacarificación enzimática (i.e. hasta un 97.7% de hidrólisis en 4h a 50°C) mediante la acción combinada de celulasa y celobiasa solubles. La solución transparente de glucosa obtenida fue de excelente calidad para el crecimiento aeróbico de Saccharomyces cerevisiae. El uso de un reactor de membrana polietersulfónica, en modo discontinuo demostró excelentes rendimientos de hidrólisis (95%), manteniéndose constantes durante 9 ciclos operacionales de reutilización de la enzima soluble, sin problemas de colmatación en la membrana. Por otra parte, la sacarificación en continuo de celulosa, realizada en un reactor de membrana cerámica, requirió la optimización de parámetros como la concentración del sustrato alimentado y el tiempo de residencia en el reactor. Los perfiles de hidrólisis constantes observados durante el estado estacionario del reactor demostraron la idoneidad del mismo. En lo referente a los flujos de permeado, éstos permanecieron constantes en el tiempo, probando una eficaz acción catalítica de la enzima que evitó problemas de colmatación en la membrana. Esta Tesis Doctoral presenta por primera vez una estrategia sostenible para la sacarificación de celulosa en reactores de membrana, basada en la combinación de la tecnología de los líquidos iónicos con la tecnología enzimática, lo que permite la valorización de biomasa vegetal para la producción de bioetanol.
Aims The principal aim of this PhD Thesis was focused on the application of enzyme technology for the design and optimization of integrated processes for saccharification of cellulose using ionic liquids technology and membrane reactors. Methodology To accomplish this aim, activity and stability tests of the studied immobilized enzyme derivative were performed in different ionic liquids (ILs). Different stages of operation (mixing, filtration and distillation) were used in the dissolving, regeneration and recovery of IL processes, in order to obtain regenerated cellulose and separate the IL. The design of integrated reaction / separation systems, based on the incorporation of membranes for the recovery and reuse of enzyme, was also considered. Finally, for quality control of the glucose solutions obtained, cultures of Saccharomyces cerevisiae were used. Results and discussion The stability of commercial cellulase (Celluclast ®), immobilized on a polymer support (Amberlite XAD4) was studied in three different ionic liquids [N1114] [NTf2], [Bmim] [NTf2] and [Bmim] [Cl]) at 50 °C. The hydrophobic ILs clearly improved the thermal stability of the enzyme. For the case of [N1114] [NTf2], the half-life time of the biocatalyst was increase up to 4 times, while [Bmim][Cl] behaved as a powerful deactivating agent. Cellulase stability in mixtures hydrophobic IL / [Bmim][Cl] helped to stabilize the enzyme against inactivation produced by the [Bmim][Cl]. The immobilized enzyme derivative was coated with [N1114] [NTf2] being successfully used in the saccharification of cellulose dissolved in [Bmim] [Cl] (i.e. up to 50% hydrolysis in 24 hours) at 50 ° C. A sustainable cyclic process for enzymatic saccharification of cellulose pretreated with IL was developed. To this end, different homogeneous solutions of cellulose in [Bmim][Cl] were used for the production of amorphous cellulose by precipitation with water, ethanol or an equimolar mixture water/ethanol. Several operating parameters (e.g. type of solvent, temperature, ultrasound, etc.) were tested for the precipitation of cellulose as well as for the following washing steps for the complete recovery of IL (up to 99.7%), which was successfully reused in a cyclic process of dissolution/regeneration. Regenerated cellulose obtained from each cycle was an excellent substrate for enzymatic saccharification (i.e. up to 97.7% hydrolysis in 4 h at 50 ° C) by the combined action of soluble cellulase and cellobiase. The clear solution of glucose obtained was of excellent quality for aerobic growth of Saccharomyces cerevisiae. Enzymatic hydrolysis of cellulose was also studied in different membrane reactors in continuous and discontinuous mode. The use of polietersulfonic membranes in discontinuous mode showed excellent hydrolysis yields (95%), which remained constant for 9 cycles of soluble enzyme reuse, without fouling problems in the membrane. On the other hand, the continuous saccharification of cellulose, realized with a ceramic membrane reactor, required to optimize parameters such as feeding substrate concentration and residence time. Constant hydrolysis profiles observed during the steady state of the reactor demonstrated its suitability. Regarding the flow of permeate, it remained constant along time, confirming an effective catalytic action of enzymes which avoided any fouling problem in the membrane. This PhD thesis presents for the first time a sustainable strategy for the saccharification of cellulose in membrane reactors, based on the combination of the technology of ionic liquids with enzyme technology, which allows the use of plant biomass for production of bioethanol.

Millions of years of evolution have resulted in an immense number of different proteins, which participate in virtually every process within cells and thus are of utmost importance for allknown forms of life. In addition, there are several examples of natural proteins which have found use in applications outside their natural environment, such as the use of enzymes infood industry and washing powders or the use of antibodies in diagnostic, bioseparation or therapeutic applications. To improve the performance of proteins in such applications, anumber of techniques, all collectively referred to as ‘protein engineering’, are performed in thelaboratory.Traditionally, methods involving ‘rational design’, where a few alterations are introduced atspecific protein locations to hopefully result in expected improvements have been applied.However, the use of more recent techniques involving a simultaneous construction of a large number of candidate variants (protein libraries) by various diversification principles, fromwhich rare clones showing enhanced properties can be isolated have contributed greatly to thefield of protein engineering.In the present thesis, different protein traits of biotechnological importance have beenaddressed for improvements by the use of such methods, in which there is a crucial need tomaintain a clonal link between the genotype and the phenotype to allow an identification of protein library members isolated by virtue of their functional properties. In all protein library investigations included in this thesis this coupling has been obtained by Escherichia coli bacterialcell-membrane compartmental confinement.In a first study, a combination of error prone PCR and gene-shuffling was applied to the Tobacco Etch Virus (TEV)-protease gene in order to produce collections from which genesencoding variants showing an enhanced soluble expression of the enzyme frequently used inbiotechnology to cleave fusion proteins were identified. Using Green Fluorescence Protein(GFP)-based cell fluorescence analysis, a clone with a five-fold increase in the yield of solubly produced protein was successfully isolated. In a second study, a novel and different GFPbased selection system, in addition also involving targeted in vivo protein degradation principles,was employed for investigations of the substrate sequence space of the same protease. In two additional studies, a selection system denoted Protein Fragment Complementation Assay(PCA), based on the affinity driven structural complementation of a genetically split β-lactamase enzyme was used to identify variants having desired target protein binding abilities,including both specificity and affinity. Using Darwinian principles concerning clonal growth advantages, affibody binding proteins showing sub-nanomolar dissociation constants to thehuman cytokine TNF-α were isolated. Taken together, these studies have shown that the bacterial format is very well suited for use in various aspects of protein library selection.
QC 20100729

Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose.

Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns.

Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.

High pressure (HP) processing has been emerging rapidly as a novel technique for non-thermal preservation of foods. Application of HP processing for shelf life extension of orange juice was the principal objective of the current research. To accomplish this objective, and to establish a scientific basis for HP processing of orange juice, a systemic approach was used which included the evaluation of: (a) HP inactivation kinetics of pectin methyl esterase (PME, the key enzyme in orange juice implicated with respect to quality changes), (b) destruction of spoilage microorganisms and changes in product quality, (c) HP process verification and finally, (d) storage studies on HP treated orange juice.
In preliminary studies, the effect of HP treatment on indigenous microorganisms, texture and color of selected fresh fruits and vegetables were evaluated. Results showed that HP had a significant effect on the destruction of microorganisms. Product texture and color were mildly affected, often resembling the appearance of mildly heat-treated products.
Pressure induced inactivation kinetics of pectin methyl esterase (PME) was investigated at pH 3.7 and 3.2 in freshly squeezed single strength (12.6°Brix) and concentrated (10--40° Brix) orange juice. Results showed a biphasic nature of pressure induced inactivation of PME in both juices. The first phase consisted of rapid change in inactivation of enzyme, designated as instantaneous pressure kill (IPK), due to pulse pressurization, followed by gradual inactivation of enzyme, characterized by a first order rate of inactivation during pressure hold-time.
Combination treatment involving pressure cycle, pressure level and pressure hold-time was then evaluated for inactivation of PME using a response surface methodology. Overall, pressure pulse had a lower effect on inactivation of PME compared to other factors.
Pressure destruction kinetics of Leuconostoc mesenteroides and Saccharomyces cerevisiae the spoilage organisms in orange juice, were then investigated. Pressure destruction kinetics followed the same dual effect behavior, as observed with PME inactivation. IPK effect increased with pressure cycles and was more pronounced with S. cerevisiae that Leu. mesenteroides.
Storage studies of HP treated single strength and concentrated orange juice were conducted at selected temperatures (4, 10 and 20°C). Results showed that treated juice was microbiologically stable from a few days to several weeks depending on type of juice, storage temperature and processing conditions. (Abstract shortened by UMI.)

R-phenylacetylcarbinol (PAC) is used as a precursor for production of ephedrine and pseudoephedrine, which are anti-asthmatics and nasal decongestants. PAC is produced from benzaldehyde and pyruvate mediated by pyruvate decarboxylase (PDC). A strain of Rhizopus javanicus was evaluated for its production of PDC. The morphology of R. javanicus was influenced by the degree of aeration/agitation. A relatively high specific PDC activity (328 U decarboxylase g-1 mycelium) was achieved when aeration/agitation were reduced significantly in the latter stages of cultivation. The stability of partially purified PDC and crude extract from R. javanicus were evaluated by examining the enzyme deactivation kinetic in various conditions. R. javanicus PDC was less stable than Candida utilis PDC currently used in our group. A kinetic model for the deactivation of partially purified PDC extracted from C. utilis by benzaldehyde (0?00 mM) in 2.5 M MOPS buffer has been developed. An initial lag period prior to deactivation was found to occur, with first order dependencies of PDC deactivation on exposure time and on benzaldehyde concentration. A mathematical model for the enzymatic biotransformation of PAC and its associated by-products has been developed using a schematic method devised by King and Altman (1956) for deriving the rate equations. The rate equations for substrates, product and by-products have been derived from the patterns for yeast PDC and combined with a deactivation model for PDC from C. utilis. Initial rate and biotransformation studies were applied to refine and validate a mathematical model for PAC production. The rate of PAC formation was directly proportional to the enzyme activity level up to 5.0 U carboligase ml-1. Michaelis-Menten kinetics were determined for the effect of pyruvate concentration on the reaction rate. The effect of benzaldehyde on the rate of PAC production followed the sigmoidal shape of the Monod-Wyman-Changeux (MWC) model. The biotransformation model, which also included a term for PDC inactivation by benzaldehyde, was used to determine the overall rate constants for the formation of PAC, acetaldehyde and acetoin. Implementation of digital pH control for PAC production in a well-stirred organic-aqueous two-phase biotransformation system with 20 mM MOPS and 2.5 M dipropylene glycol (DPG) in aqueous phase resulted in similar level of PAC production [1.01 M (151 g l-1) in an organic phase and 115 mM (17.2 g l-1) in an aqueous phase after 47 h] to the system with a more expensive 2.5 M MOPS buffer.

Bacterial L-asparaginases (E.C.3.5.1.1) are enzymes that catalyze the hydrolysis of L-asparagine to aspartic acid. For the past 30 years these enzymes have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. The presence of a low rate glutaminase activity however causes serious side-effects to patients in treatment, as glutamine depletion give rise to neurotoxicity, anaphylaxis, and other hypersensitivity reactions. The interest in the enzyme from Erwinia carotovora originates from the fact that it shows a decreased glutaminase activity, and therefore the enzyme is expected to exhibit fewer side effects when used in therapy.

The main focus of this thesis is the crystal structure determination of L-asparaginase from Erwinia carotovora in the presence of aspartic acid at 2.5 Å resolution. The structure was refined to an R/Rfree factor of 19.9/28.6 with good stereochemistry.

L-Asparaginases are homotetrameric enzymes with a known 222 symmetry and an identical fold. The Erwinia carotovora asparaginase consists of eight monomers of 330 amino acid residues each. In this case the enzyme is active as a dimer of tetramers. The two tetramers have an inner twofold non-crystallographic symmetry. Each monomer forms two identifiable domains a large N-domain and a small C-domain. The active sites are found at a topological switch-point between those domains.

Background: Insulin is an important hormone for glucose homeostasis. It is released from β-cells in the endocrine pancreas as a response to increased concentrations of plasma glucose. The major effect of insulin is the facilitation of cellular uptake and storage of glucose as glycogen. Insulinomas are tumours that produce excessive amounts of insulin resulting in hypoglycaemia. The condition has been observed in dogs and cats and is often malignant. One part of establishing the diagnosis is confirmation of elevated concentrations of insulin in a hypoglycaemic sample. Aim: The aim of this study was to evaluate if an equine-optimized insulin ELISA (Mercodia AB, Uppsala, Sweden) is useful for analysis of insulin in canine and feline serum samples when insulinoma is suspected. Material and methods: All samples were analysed with Equine insulin ELISA. Precision, linearity and effects of haemolysis were studied. The stability of insulin was evaluated after storage in 4°C, room temperature and after repeated freezing and thawing. A reference interval was constructed for both canine and feline samples. Results: Total precision expressed as CV was 4.4 – 18.9 %. The method was linear up to at least 100 mU/L for dogs and 15 mU/L for cats. Reference interval for cats was <11.6 mU/L, due to few healthy animals no reference interval for dogs could be established. Stability was acceptable for up to four days. No effects of haemolysis were detected. Conclusion: Mercodia Equine insulin ELISA is suitable for analysis of insulin in serum from dogs and cats when suspecting insulinoma.

Typ- 1 diabetes (T1D) är en autoimmun sjukdom med insulinbrist orsakad av nedbrytning av insulinproducerande betaceller i pankreas. Fyra olika antikroppar har identifierats som är riktade mot betacellsspecifika antigen; insulinautoantikroppar (IAA), glutamic acid decarboxylase antibodies (GADA), islet antigen2-antikroppar (IA-2A) och antikroppar riktade mot zinktransportören 8 (ZnT8A). I denna studie gjordes en jämförelse av metoderna 2screen islet cell autoantibody ELISA-kit (RSR, Cardiff, UK) och 3screen islet cell autoantibody ELISA- kit (RSR, Cardiff, UK), vars brunnar är coatade med GAD65/IA-2 antigen respektive GAD65/IA-2/ZnT8 antigen, för att undersöka ifall dessa båda kit ger jämförbar sensitivitet och specificitet i en adult population av nydebuterade patienter med T1D och friska vuxna blodgivare. RSR 2screen erhöll 1 % högre specificitet (98 %) jämfört med RSR 3screen (97 %) vid samma sensitivitet (92 %) och rekommenderas i första hand för screening av autoantikroppar i en population av vuxna patienter med ökad risk för T1D och friska vuxna blodgivare.
Type- 1 diabetes (T1D) is an autoimmune disease with insulin deficiency caused by degradation of insulin- producing betacells in pancreas. Four different autoantibodies that target beta- cell specific antigenes have been identified: insulinautoantibodies (IAA), glutamic acid decarboxylase antibodies (GADA), islet antigen2-antibodies (IA-2A) and antibodies against zinktransporter 8 (ZnT8A). In this study, a comparison between 2screen islet cell autoantibody ELISA-kit (RSR, Cardiff, UK) coated with GAD65/IA-2 and 3screen islet cell autoantibody ELISA- kit (RSR, Cardiff, UK) coated with GAD65/IA-2/ZnT8, was performed to investigate whether results from these two kits provide comparable sensitivity and specificity in an adult population of new onset patients with T1D and healthy adults. RSR 2screen obtained 1 % higher specificity (98 %) in comparison to RSR 3screen (97 %) on the same sensitivity (92 %) and is recommended primarily for screening of autoantibodies in a population of adult patients at increased risk for T1D and healthy adults blood donors.

Petit Manseng grapes harvested in 2011 and 2012 were fertilized with soil nitrogen at 0, and 30 kgN/ha, foliar nitrogen at 15kg/ha and foliar nitrogen plus sulfur at 15kg/ha and 5kg respectively. Point quadrat analysis demonstrated foliar nitrogen alone and nitrogen plus sulfur treatments increased percent gaps and lower leaf layer numbers. Berry juice samples differed in ammonia, arginine and yeast assimilable nitrogen concentration. Total glycosides were 25 percent higher in the foliar nitrogen treatment versus the control treatment. Electronic nose measurements on field clusters and laboratory berry analyses was different among treatments in volatile content. Harvest samples underwent acid or enzyme hydrolysis of precursor fractions. Solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC/MS) analysis identified 27 free aroma and flavor compounds and 52 bound compounds. Lactones and carboxylic acids were the major components of the free fractions while bound fractions had increased concentrations of alcohols, esters and terpenes compared to the free fraction. With nitrogen fertilization, acid and enzyme hydrolysis had reduced concentrations of some higher alcohols and carboxylic acids. Acid hydrolysis released more terpenes with nitrogen treatments versus enzymatic hydrolysis. Ester content was increased in both acid and enzyme hydrolysis fractions in vines receiving nitrogen treatments. For descriptive analysis, eight trained panelists described aroma, flavor, texture/mouthfeel and aftertaste attributes. Analysis of Variance (ANOVA) demonstrated that wines were a significant source of variation with 23 of the 24 attributes used. Wine principal component analysis (PCA) of aroma attributes explained 23.5% of the variation from PC1, while flavor-by-mouth and texture/mouthfeel attributes explained 26.3% of the variation due to PC1. The aim of this study was to develop descriptive terms for Petit Manseng and determine the influence of fruit nitrogen levels on the aroma and flavor profile of this cultivar.
Ph. D.

Pyrosequencing is a DNA sequencing method based on thedetection of released pyrophosphate (PPi) during DNA synthesis.In a cascade of enzymatic reactions, visible light isgenerated, which is proportional to the number of nucleotidesincorporated into the DNA template. When dNTP(s) areincorporated into the DNA template, inorganic PPi is released.The released PPi is converted to ATP by ATP sulfurylase, whichprovides the energy to luciferase to oxidize luciferin andgenerate light. The excess of dNTP(s) and the ATP produced areremoved by the nucleotide degrading enzyme apyrase.

The commercially available enzymes, isolated from nativesources, show batch-tobatch variations in activity and quality,which decrease the efficiency of the Pyrosequencing reaction.Therefore, the aim of the research presented in this thesis wasto develop methods to recombinantly produce the enzymes used inthe Pyrosequencing method. Production of the nucleotidedegrading enzyme apyrase by Pichia pastoris expression system,both in small-scale and in an optimized large-scale bioreactor,is described. ATP sulfurylase, the second enzyme in thePyrosequencing reaction, was produced inEscherichia coli. The protein was purified and utilizedin the Pyrosequencing method. Problems associated with enzymecontamination (NDP kinase) and batch-to-batch variations wereeliminated by the use of the recombinant ATP sulfurylase.

As a first step towards sequencing on chip-format,SSB-(single-strand DNA binding protein)-luciferase and KlenowDNA polymerase-luciferase fusion proteins were generated inorder to immobilize the luciferase onto the DNA template.

The application field for the Pyrosequencing technology wasexpanded by introduction of a new method for clone checking anda new method for template preparation prior the Pyrosequencingreaction.

Keywords:apyrase, Pyrosequencing technology, Z_basictag fusion, luciferase, ATP sulfurylase, dsDNAsequencing, clone checking, Klenow-luciferase, SSB-luciferase,Pichia pastoris, Echerichia coli.

An enzyme treatment process for the pre-tanning of sheepskins has been previously reported by the Leather and Shoe Research Association of New Zealand (LASRA) as an alternative to current industry operations. The newly developed process had marked benefits over conventional processing in terms of a lowered energy usage (73%), processing time (47%) as well as water use (49%), but had been developed as a “proof of principle’’. The objective of this work was to develop the process further to a stage ready for adoption by industry. A process ready for adoption by industry is one that is able to generate good quality products (good quality products are those that has solids removed to industry standards, without visible damages to grain surface, evenly dyed etc.), require minimal changes to plant or operation layout and reduce usage of resources (e.g. water and chemicals). Mass balancing was used to investigate potential modifications for the process based on the understanding developed from a detailed analysis of preliminary design trials. Results showed that a configuration utilising a 2 stage counter-current system for the washing stages and segregation and recycling of enzyme float prior to dilution in the neutralization stage was a significant improvement. Benefits over conventional processing include a reduction of residual TDS by 50% at the washing stages and 70% savings on water use overall. Benefits over the un-optimized LASRA process are reduction of solids in product after enzyme treatment and neutralization stages by 30%, additional water savings of 21%, as well as 10% savings of enzyme usage. The optimized (new) LASRA process uses existing equipment and requires no additional outlay of capital. The process is now developed to a point where it should be trialled at industrial scale.

Currently, the transplantation of islets of Langerhans is a viable means to maintain control of blood sugar levels and reduce the risk of hypoglycemia in defined populations with brittle type I diabetes mellitus or those requiring pancreatectomy. However, the process of islet isolation is highly variable and not all isolations result in islet numbers or quality suitable for transplantation. This thesis aimed to improve transplantation success through optimization and standardization of the isolation process and to identify pretransplant variables associated with early islet engraftment. A previously disregarded enzyme activity, tryptic-like activity (TLA), has been identified to influence pancreas digestion efficiency and islet isolation success in both the preclinical and clinical situations. For human pancreases, islet isolation success rates improved from 0% in the lowest TLA group to over 50% in the highest TLA groups without affecting islet quality. These findings should help standardize evaluation of enzymes for clinical islet isolation. A closed, automated, pump-made gradient system was compared to the open, manual method for islet separation. No differences were observed in expected gradient volumes, islet yields or total purities between the two methods. The pump-made gradient system successfully removed manual influences on density gradient production while fulfilling regulatory requirements for closed system processing. Islet quantification was evaluated with computer-assisted digital imaging analysis (DIA) and a semi-closed assessment system. By using the DIA system method, which measures islet purity and pellet volume instead of manual counting methods, variation in islet counts and purity reduced by almost half. By using a transplant outcome measurement of C-peptide adjusted by blood glucose and creatinine, we identified four pretransplant factors that affect early transplant outcome. Of the four factors, one was related to the organ transport time, one to function of the islets, and two to the transplanted tissue volume. When these four factors were put into a predictive model, it accounted for about 40% of the transplant outcome. The work contained in this thesis identifies and optimizes a number of critical elements related to islet isolation and transplantation protocols.

This thesis describes the rational design of a novel enzyme, a thiolester hydrolase, derived from human glutathione transferase (GST) A1-1 by the introduction of a single histidine residue. The first section of the thesis describes the design and the determination of the reaction mechanism. The design was based on the crystal structure of human GST A1-1 complexed with S-benzylglutathione. The resulting enzyme, A216H, catalyzed the hydrolysis of the non-natural substrate GSB, a thiolester of glutathione and benzoic acid. The reaction followed saturation kinetics with a kcat of 0.00078 min-1 and KM of 5 μM. The rate constant ratio, (kcat/KM)/kuncat, was found to be more than 107 M-1. The introduction of a single His residue in position 216 opened up a novel reaction pathway in human GST A1-1 and is a nice example of catalytic promiscuity. The substrate requirements were investigated and A216H was found to be selective since only two out of 18 GS-thiolesters tested were substrates for A216H. The reaction mechanism of the A216H-catalyzed hydrolysis of GSB was determined and found to proceed via an acyl intermediate at Y9. The hydrolysis was catalyzed by H216 that acts as a general base and the deacylation was found to be the rate-determining step. The Y9-intermediate could be selectively trapped by oxygen nucleophiles and primary alcohols, in particular 1-propanol and trifluoroethanol, were the most efficient. In addition, saturation kinetics was obtained in the acyl transfer reaction with 1-propanol indicating the presence of a second binding site in A216H. The second section of this thesis describes the site-specific covalent modification of human GST A1-1. The addition of GSB to the wild-type protein results in a site-specific benzoylation of only one tyrosine residue, Y9, out of ten present in the protein (one out of totally 51 nucleophiles). The reaction was tested with five GST classes (Alpha, Mu, Pi, Theta and Omega) and found to be specific for the Alpha class isoenzymes. The covalent modification reaction was further refined to target a single lysine residue, K216, providing a more stable linkage in the form of an amide bond. The reaction was found to be versatile and approximately 50% of the GS-thiolesters tested acylated K216, including a fluorophore.

On the day of the public defence the status of article II was: Submitted and article IV was: In press.

The processing of lambskins plays an important role in the New Zealand meat industry. The use of enzyme dewooling offers considerable advantages over the conventional depilation method which generates unpleasant working conditions and poses product quality risks when not properly handled. Prior to this work it was unclear from the literature why the practice of enzymatic depilation had not generally been adopted by industry. The aim of this work was to determine the problems associated with enzymatic depilation and provide a mechanistic understanding of the dewooling and damaging processes of enzyme depilation to provide underpinning knowledge for the design of a successful enzymatic depilation system. It was found that variability in depilation between different regions of the skin resulted in either over exposure of the skin to the enzyme regent and subsequent damage or underexposure of the skin to the enzyme reagent and incomplete depilation. Two approaches were taken in the work: Firstly an attempt was made for the first time to understand the variability in enzymatic depilation so that the variability observed in enzymatic depilation could potentially be reduced, thereby allowing a complete depilation process with no overexposure. Secondly an investigation was made for the first time to understand the cause of damage to skins during the process of enzymatic depilation so that the enzyme depilation process could potentially be modified to avoid damage. Experimental work characterising the time course of depilation and damage development was carried out and compared with the variation of physical properties across the skin. Correlations between depilation and physical properties such as thickness, grease content and follicle density were found. Reduction in the variability of these properties would likely improve the evenness of depilation but would not reduce it enough to eliminate damage due to over exposure. A range of techniques including: immunohistology, 2-dimensional electrophoresis, matrix assisted laser desorption ionisation, and atomic force microscopy were used to probe the structural and biochemical mechanism of enzyme depilation and damage. In this way it was found that the removal of minor collagen components were the likely cause of damage observed. In particular the removal of collagen VI was associated with a disruption of the smooth mesh of fine collagen fibres observed at the surface of the leather. The key requirement identified for a successful enzyme depilation system was the use of a broad spectrum protease which has no activity against collagen VI. The means to select a protease with these attributes was also developed by adopting a micro depilation assay incorporating immunohistology. This knowledge will enable the future development of non damaging enzyme depilatory reagents that could revolutionise the industry.

Diabetes has, in the past three decades, surged immensely. Because of this, new insulin analogues are constantly in the making.  In clinical studies, the presence of antidrug antibodies can prove a challenge when measuring insulin. In order to overcome the interference from antidrug antibody complexes on the total insulin measurement in human serum, several pre-treatment methods on insulin and polyclonal antibodies spiked samples were tried using ELISA analysis. Several different methods were tried, acid dissociation using a glycine buffer with and without ethanol in different concentrations, high ionic strength dissociation using MgCl2, Polyethylene glycol (PEG) and filtration. The best results were found when using the acid dissociation technique. Using glycine promising results were achieved, especially when 20 % ethanol was added to the acid mixture. Pre-treatment using PEG, MgCl2 and filtration was unsuccessful with the methods used. The main goal was reached through the use of glycine with the addition of 20% ethanol for acid dissociation. The proposed method still leaves significant room for optimisation and needs further verification on real patient samples. However, it is a good step in the direction of a global methodology using ELISA to overcome antidrug antibody interference for total insulin measurement in human serum.

Lignin is the only plant biomass that contains aromatic groups in its structure and can provide a wide range of low molecular weight aromatic chemicals if its depolymerisation can be achieved successfully. Currently, lignin is mainly produced as a waste by-product by the paper and pulp industry and biorefineries. Therefore, the transformation of the phenolic-rich lignin into value added aromatic platform chemicals can be regarded of primary concern to improve the economic profitability of biorefining. Moreover, being a renewable resource, the consumption of fossil fuels will be reduced if lignin can be utilised efficiently. Between chemical degradation and enzymatic degradation, the latter could be a more sustainable method to break down lignin due to its enhanced substrate specificity and ability to preserve the aromatic ring structure compared with chemical processing. Therefore, laccase from Trametes versicolor (LTV), lignin peroxidase (LiP) and manganese peroxidase (MnP) were studied to determine the scope to depolymerise both water-soluble and insoluble lignins nder mild reaction conditions. The enzymatic activity and stability of all three enzymes was investigated and optimum assay conditions were achieved. LTV was found to be the most stable enzyme as it maintained 55 % of its activity at least for the first 6 h at 30 °C whereas LiP was deactivated after 2 h at 25 °C, and MnP was deactivated after 1 h at 28 °C. However, LTV stability decreased at higher temperatures during the oxidation of 2,2’-azino-bis (3- ethylbenthiazoline-6-sulphonic acid (ABTS)). One of the non-phenolic lignin model compounds, veratryl alcohol, was oxidised by LTV in the presence of ABTS, thus confirming the published data. The enzymatic degradation of Organosolv lignin (OSL) by LTV resulted in the formation of 2,6-dimethoxy-1,4-benzoquinone (DBQ). The OSL degradation by LTV was not improved by ethanol addition as a co-solvent although ethanol could stabilise LTV at 40 % (v/v). LTV catalysed the degradation of Kraft lignin although it indicated little effect on lignosulphonates. Lastly, the effect of varying the concentrations of 92 ionic liquids (ILs) and their equivalent metal salts on LTV activity was investigated to find a suitable co-solvent to improve the poor mass transfer in OSL degradation. The study showed that 62 ILs were laccase compatible at an IL concentration of 6 % (w/v) and more than 50 % laccase activity was retained in 18 ionic liquids up to 10 % (w/v), and 80 % (v/v) of dioctyl sulfosuccinate quaternary ammonium salt, [N4,4,4,4][AOT]. However, there was a progressive loss of activity when the concentrations of the ILs increased. Further study on the enzymatic degradation of ILs-pre-treated OSL is currently ongoing in our research group so that the decomposition of water-insoluble lignin will be understood more comprehensively.

L'objectif de ce travail a été d'étudier l'immobilisation d'enzymes sur des matériaux membranaires en utilisant la technique de dépôt multicouches de polyélectrolytes. Pour cela, nous avons utilisé deux enzymes de taille et de charge différentes, la trypsine et l'uréase, afin de mettre en évidence le rôle de la localisation des enzymes dans le matériau (modification de surface ou dans les pores). La stabilité au cours du temps du stockage a été étudié et a montré que l'immobilisation des enzymes dans des couches de polyélectrolytes améliore leur stabilité de façon très significative. Deux techniques de séparation membranaire, l'ultrafiltration et la diffusion, ont été utilisées afin d'appréhender les mécanismes intervenant lors du transport des solutés. Ces résultats ont montré l'importance du temps de résidence à proximité de l'enzyme par la compétition entre la réaction et la filtration. Une étude des variations structurales de la trypsine a été faite par spectroscopie de fluorescence et anisotropie de fluorescence
The objective of this work is to study the enzyme immobilization on membrane materials by using the technique of deposit of polyelectrolyte multilayers. For that purpose, we used two enzymes with different size and charge, trypsin and urease, to study the role of the location of enzymes in the material (surface modification or into the pores). The storage stability was studied and showed that the immobilization of enzymes into the layers of polyelectrolytes improves their stability in a very significant way. Two techniques of membrane separation, the ultrafiltration and the diffusion, were used to understand the mechanisms occurring during the transport of solutes. These results showed the importance of time of residence near the enzyme by the competition between the reaction and the filtration. The structural variation of the trypsin was investigated by fluorescence spectroscopy and fluorescence anisotropy

Enzyme technology is widely regarded as an exciting new technology possessing great opportunities for commercial interests and is one of a small group of key technologies singled out by the Science Research Councils during the 1960's as worthy of special support. In this thesis I outline the basic characteristics of this technology analysing the nature of the Government's policy towards it. The approach I have chosen requires an in depth analysis of the innovation process for enzymes which forms the basis for a model. This model is then used to focus on aspects of the UK science policy towards innovation in enzyme technology, assessing its impacts, and appraising the usefulness of this approach for future policy initiatives.

As doenças raras são epidemiologicamente caracterizadas por ocorrerem entre 0,65-1:1.000 indivíduos. Houve importante incremento no surgimento de tratamentos específicos para muitas destas condições a partir da publicação de legislações específicas em diferentes países (sendo o pioneiro em 1983 nos EUA o chamado 'Orphan Drug Act'). Dentre as doenças que dispõem atualmente de tratamento específico está a Doença de Fabry, uma doença genética classificada erro inato do metabolismo do grupo das doenças Lísossômica se caracterizada pelo acúmulo de glicoesfíngol ipídíos no endotélio vascular, o que ocasiona problemas renais, cardíovasculares e neurológicos. Os problemas renais e cardíovasculares são os responsáveis pela maior morbi-mortalídade entre os portadores da doença. Esta ocorre em homens e mulheres, com manifestações heterogêneas entre indivíduos e diferentes gêneros. O tratamento específ ico da doença é através da chamada Terapia de Reposição Enzimática (TRE) havendo duas opções existentes: a agalsidase alfa e a agalsidase beta. Não há, porém, consenso sobre os resultados da TRE sobre desfechos clínicos relevantes. O alto custo associado a esta terapêutica (cerca de 200 mil dólares/ano/por paciente adulto) é o dificulta o acesso dos pacientes à mesma. Uma das formas se racionalizar a incorporação de tecnologias novas na área de saúde, que se caracteriza empreender uma avaliação objetiva de benefícios e custos associados é a chamada Avaliação de Tecnologias de Saúde. Este conjunto de métodos é cada vez mais empregado para auxiliar ou nortear decisões na área de saúde. Existem, no entanto , dificuldades associadas ao seu emprego no campo das doenças raras. Nossa proposta consiste em tendo como modelo a Doença de Fabry, obter um panorama da situação no Brasil a o acesso a tratamentos de alto custo para doenças raras, e, analisar a aplicabilidade dos princípios da avaliação de tecnologias de saúde no campo das doenças raras, tentando por fim colaborar no aprimoramento do processo de acessibilidade a estes tratamentos. OBJETIVOS Objetivo geral : Avaliar o acesso no Brasil a tratamentos de doenças raras e a aplicabilidade da avaliação de tecnolog ias em saúde no contexto específico destas. Objetivos específicos: 1) Caracterizar as políticas existentes no Brasil em relação ao acesso de medicamentos para doenças raras e avaliar as formas empregadas para este acesso. 2) Colaborar no aprimoramento do processo de incorporação destes tratamentos (de al to custo) no segmento das doenças raras, através da aplicação de principias de 'aval iação de tecnologias em saúde', usando como exemplo a Doença de Fabry. 3) Identificar e propor possíveis estratégias que possam colaborar para uma acessibilidade adequada e justa a tratamentos cl inicamente efetivos no campo das doenças raras. MÉTODOS 1) Foi realizada uma revisão da literatura caracterizando as politicas brasileiras na área de medicamentos e aval iando as formas de acesso utilizadas pelos pacientes; 2) Uma segunda revisão, de caráter sistemático, sobre os efeitos da TRE na DF, foca d a nos efe itos sobre a nefropatia e a cardiopatia (sistemas cujo comprometimento em pacientes com DF causa maior morbi-mortalidade) da doença foi feita. Baseado nos dados obtidos elaborou-se um modelo probabilístico (markoviano) para avaliar o efeito da TRE sobre a nefropatia da DF seguido de aval iação do custo-efetividade. A partir de uma análise destes resultados e da li teratura, foram identificados os aspectos que mais influenciariam a acessibilidade ao tratamento, seguida da elaboração de propostas para auxiliar a incorporação e a acessibil idade a estes tratamentos. RESULTADOS 1) A política de assistência farmacêutica brasileira atual é baseada em elencos de medicamentos divididos em atenção básica e do 'componente especiali zado'. Não existe uma política especifica direcionada aos tratamentos (de alto custo) dos portadores de doenças raras. 2) A revisão empreendida mostrou que dentre os dois sistemas aval iados de forma particular (pela morbi-mortalidade associada) havia mais dados quer sobre história natural , quer sobre efeitos da TRE em relação à nefropatia, tendo sido esta escolh ida então como foco da modelagem . O modelo construido foi capaz de identificar um subgrupo de pacientes com DF que quando tratado com TRE tem redução significativa (diferença absoluta de 10%) na probabilidade de ter progressão da nefropatia (evoluir a estágio dialítico). A despeito dos benefícios observados a análise mostrou ser esta opção não custo-efetiva no contexto existente (custo é superior ao limite preconizado pela OMS de s 3 vezes o PIB per capíta do Brasil). 3) A acessibilidade é ainda um obstáculo ao uso destes medicamentos. O custo é o limitante central da acessibilidade aos tratamentos específicos destas patologias. Atuar em aspectos associados a este e às pol íticas vigentes seriam formas de tentar mudar este cenário. CONCLUSÕES 1) Não há em nosso país políticas que sistematizem o processo de incorporação de tratamentos (de alto custo) para doenças raras. O acesso a estes é disperso, no caso da DF não existe disponibil idade da terapia de reposição enzimática via SUS. 2) A revisão sobre o efeito da TRE na nefropatia mostrou resultados muito heterogêneos. Foi possivel, no entanto, elaborar um modelo avaliando TRE na nefropatia da DM, o qual é o primeiro conhecido utilizando este desfecho especifico e elaborado dentro contexto brasileiro. A despeito de identificado um subgrupo de pacientes que poderia beneficiar-se significativamente com o tratamento especifico a estratégia não foi custo-efetiva para este desfecho. Os custos associados foram o limitante central. Novas opções em termos de efetivação da incorporação destas tecnologias devem ser consideradas.
BACKGROUND. According to the WHO definition rare diseases occur between 0.65-1/1,000 individuals. The U.S. 'Orphan Drug Act' in 1983', the first legislation with incentives for the development of therapies for rare diseases had an impact on the development of treatments for these diseases. Fabry Disease (FD) is a rare genetic disease characterized by accumulation of glycosphingolipids in vascular endothelium leading to systemic dysfunction (renal, cardiovascular, and neurologic disease). This disease has specific treatment available, with two options of recombinant enzymes (alfa or betagalsidase) for enzyme replacement therapy (ERT). There are few controlled trials evaluating their effects, indicating some improvements in neuropathic pain, in heart abnormalities and in globotriaosylceramide (GL-3) levels. Nevertheless most of the clinical benefits of ERT are still unclear. Another aspect is the high cost associated with this treatment which makes it not easily accessible. OBJECTIVES: General Objective: Evaluate access to rare disease treatments in Brazil and the applicability of health technology assessment (HTA) in the context of these diseases. Specific objectives: 1) Evaluate the Brazilian governmental policies for rare diseases treatment and how the access to the treatments is actually done. 2) Evaluate the applicability of health technology assessment (HTA) in the context of rare diseases, using the example of Fabry disease. 3) Identify and propose strategies that could contribute to a fair access to clinically effective treatments for of rare diseases. METHODS: An extensive literature review about the Brazilian policies in the area was performed. In the sequence a systematic review about the subject ERT and FD was conducted. After this, a model estimating the likelihood of nephropathy progression with or without ERT was built, followed by a cost-effectiveness analysis. Another literature review focused in the identification of obstacles to accessibility and possible strategies to overcome them was conducted. RESULTS: 1) There is no specific policy in Brazil regarding high cost drugs for the treatment of rare diseases. 2) ERT appears to slow the progression of nephropathy in the Fonly proteinuria subgroup '. However the cost associated is very high making this option not cost-effective. 3) The accessibility to innovative treatments for rare diseases is not adequate and the high cost of these therapies is a major obstacle to change this scenario. CONCLUSION: There are no policies in Brazil to systematize the access to the specific (high cost) treatments for rare diseases. The model evaluating TRE in FD nephropathy was able to identify benefits for a subgroup of patients. It was the first known model using this specific outcome and built focusing the Brazilian context. However the ERT strategy was not cost-effective for this outcome. The costs associated with these therapies are very high and an important limiting factor to the access. the central. New options should be considered to offer adequate access to the (effective) therapies.

Färgning av polyester (PET) sker i temperaturer omkring 130℃ och som en följd av den höga värmen migrerar oligomerer ut ur PET-fibern. Dessa oligomerer skapar därefter problem i form av vita fällningar och avlagringar, som leder till en reducering av maskinernas effektivitet, samt försämring av materialets utseende. En vanligt förekommande metod för att begränsa problemet är att tillsätta hjälpkemikalier i färgbadet. Novozymes A/S hävdar att genomförda studier med PET-garn och enzymet cutinase NS59038 har visat en minskning av det vita damm som kan ses på garnet i samband med migration av oligomerer. Eftersom enorma mängder PET produceras globalt varje år skulle endast en liten minskning av mängden kemikalier innebära en stor skillnad. Kemikalier som ersätts med andra kemikalier kan ifrågasättas om det är en bra metod eller om det bestrider syftet. Ur ett hållbarhetsperspektiv kan enzymer ses som ett bättre alternativ eftersom de enligt Jajpura (2017) är biologiskt hållbara, formar sällan någon biprodukt och dess reaktion kräver oftast mildare förhållanden. Hjälpkemikalien Sera Con P-NSI används av Ludvig Svensson AB som färgar garn av den flamskyddade polyesterfibern Trevira CS. Syftet med projektet har därmed varit att undersöka om enzymet cutinase NS59038 är ett alternativ till hjälpkemikalien Sera Con P-NSI, för att reducera de problem som uppstår med oligomerer i färgningsprocessen med Trevira CS. Garnets egenskaper har jämförts genom visuell analys med mikroskop, viktförändring, reflektionsspektrofotometer och dragprovning. Metoder som använts i syfte att detektera oligomerer har varit FTIR, UV-vis spektrofotometer och svart svavelfilterpapper. Utifrån genomförda analyser av garnets vikt, styrka, färgupptagning och visuell bedömning har det inte kunnat konstateras att cutinase NS59038 skulle vara ett alternativ till Sera Con P-NSI. Resultat från analysmetoderna visade skillnader i medelvärde, men inget samband mellan val av färgrecept och garnets egenskap har detekterats. De olika färgningsförsöken visade inte någon förekomst av fällningar/avlagringar som med använda testmetoder har kunnat bekräftas som oligomerer. Resultaten bedömdes därmed inte som tillräckliga för att konstatera om cutinase NS59038 påverkar garnets kvalitet i jämförelse med hjälpkemikalien Sera Con P-NSI.
In the dyeing process of polyester (PET) the temperature goes up to around 130℃ and as a result oligomers migrate out of the PET fiber. These oligomers create problems as white precipitates that deposits on the material and the inside of machines. This leads to a reduction in machine efficiency, as well as a change of material appearance. One common method for limiting the problem is to add help chemicals in conjunction with the dyeing process. According to results from previous studies, Novozymes A/S claims that the enzyme cutinase NS59038 has reduced the white dust on the yarn of polyester. Based on the enormous quantity of PET that are produced every year, even a small reduction of the chemicals that are used would mean a huge difference. Chemicals that replace other chemicals can be questioned whether or not it can be seen as an alternative or if it disputes the purpose. From a sustainable point of view enzymes could according to Jajpura (2017) be seen as a better alternative because they are biodegradable, their reaction seldom form any byproduct and often requires less energy. The help chemical Sera Con P-NSI is used by Ludvig Svensson AB and is added to the dye bath in the dyeing process of the specific polyester Trevira CS (a flame retardant polyester fiber). The purpose of the project has thus been to investigate whether the enzyme cutinase NS59038 is an alternative to the help chemical Sera Con P-NSI, to reduce the problems with oligomers in the dyeing process of Trevira CS. The properties of the yarn have been compared by visual analysis with microscope, weight change, measurement of the color change with spectrophotometer and tensile strength. Methods used to detect oligomers have been FTIR, UV-vis spectrophotometer and black sulfur filter paper. Based on the analysis of the weight of the yarn, strength, color uptake and visual assessment, it can not be established that cutinase NS59038 would be an alternative to Sera Con P-NSI. Results from the analysis methods show differences in the mean, but no connection between the choice of dye recipe and the properties of the yarn can be detected. None of the precipitates that was predicted to arise on the surface of the yarn was ever detected. The results are therefore not considered sufficient to determine whether cutinase NS59038 affects the quality of the yarn compared to the help chemical Sera Con P-NSI.

Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, September 2018.
Page 399 blank. Cataloged from PDF version of thesis.
Includes bibliographical references (pages 210-229).
RNA plays important and diverse roles in biology, yet molecular tools to measure and manipulate RNA are limited. Recently, the bacterial adaptive immune system, CRISPR, has revolutionized our ability to manipulate DNA, but no known RNA-targeting versions exist. To discover parallel bacterial RNA-targeting systems that could be used for transcriptome engineering, we developed a computational pipeline to mine for novel Class 2 CRISPR systems across more than 25,000 bacterial genomes. Among the many novel CRISPR systems, we found a programmable RNA-targeting CRISPR system, CRISPR-Cas 13, that could provide immunity to E. coli against the ssRNA MS2 phage and biochemically characterized the enzyme. We adapted CRISPR-Casl3 for modulating the transcriptome in mammalian and plant cells by heterologously expressing Casl 3 and engineering the enzyme to precisely knockdown, bind, and edit RNA. Cas 13 knockdown was as efficient as RNA interference, but much more specific, across many transcripts tested. RNA editing with Cas 13 was also highly efficient, with up to 90% base editing rates, and as low as 20 off-targets with engineered specificity versions. Lastly, we combined Cas13 with isothermal amplification to develop a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with single-molecule sensitivity and singlebase mismatch specificity. We used this Casl3a-based molecular detection platform, termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), to specifically detect pathogenic bacteria, genotype human DNA, and identify cell-free tumor DNA mutations. Our results establish CRISPR-Cas13 as a flexible platform for RNA targeting with wide applications in RNA biology, diagnostics, and therapeutics.
by Omar O. Abudayyeh.
Ph. D. in Medical Engineering and Medical Physics

New approaches to the application of membranes for the production of potable water are constantly being sought after in anticipation of future demands for increasingly rigorous water quality standards and reduced environmental impact. A major limitation, however, is membrane fouling, which manifests itself as a continual reduction in flux over time and thus restricts the practical implementation to restore flux. Mechanical and chemical methods have been implemented to restore flux to ultrafiltration systems, but these either result in a break in the process operation or lead to membrane damage or additional pollution problems. This project was aimed to develop a 'defouling on demand' stategy for cleaning membranes used during brown water ultrafiltration. The process involves the use of activatable peroxidase enzymes, which were immobilised onto flat sheet polysulphone membranes. Following flux decline which reaches a critical level with the build-up of the foulant layer, the immobilised enzyme layer was activated by the addition of a chemical activator solution, in this case hydrogen peroxidase and manganous sulphate. Manganese peroxidase was found to be the most effective enzyme at alleviating fouling by degrading the foulant layer formed on the membrane surface and hence restored flux to the ultrafiltration system. A 93% flux improvement was observed when manganese peroxidase was activated when 800uM manganous sulphate, 100mM hydrogen peroxide were added in the presence of a manganese chelator, lactate. The concept and the potential benefits this system holds will be discussed in further detail.