Дисертації: "Enzymes"

1

Ekici, Özlem Doğan. "Design, synthesis, and evaluation of novel irreversible inhibitors for caspases." Available online, Georgia Institute of Technology, 2004:, 2003. http://etd.gatech.edu/theses/available/etd-04062004-164633/unrestricted/ekici%5Fozlem%5Fd%5F200312%5Fphd.pdf.

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2

Ekici, Ozlem Dogan. "Design, synthesis, and evaluation of novel irreversible inhibitors for caspases." Diss., Georgia Institute of Technology, 2003. http://hdl.handle.net/1853/5333.

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3

Müller, Roger. "Artificial enzymes: from catalytic antibodies toward de novo enzyme design /." Zürich : ETH, 2008. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17897.

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4

Ghadge, G. D. "Microbial enzymes." Thesis(Ph.D.), CSIR-National Chemical Laboratory, Pune, 1986. http://dspace.ncl.res.in:8080/xmlui/handle/20.500.12252/3251.

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5

Qian, Yuhui. "Study of Basic Wood Decay Mechanisms and Their Biotechnological Applications." Fogler Library, University of Maine, 2008. http://www.library.umaine.edu/theses/pdf/QianY2008.pdf.

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6

Zhao, Xueyan. "Nanoscale biocatalysts for bioelectrochemical applications." Akron, OH : University of Akron, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=akron1164149161.

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Thesis (M.S.)--University of Akron, Dept. of Chemical Engineering, 2006.
"December, 2006." Title from electronic thesis title page (viewed 06/27/2007) Advisor, Ping Wang; Committee members, Lu-Kwang Ju, Steven S. C. Chuang; Department Chair, Lu-Kwang Ju; Dean of the College, George K. Haritos; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.

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7

Moore, Robert Goodwin Douglas C. "Towards the understanding of complex biochemical systems the significance of global protein structure and thorough parametric analysis /." Auburn, Ala, 2009. http://hdl.handle.net/10415/1766.

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8

Epstein, Todd Matthew. "Structural and kinetic studies of two enzymes catalyzing phospholipase A2 activity." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file 2.39 Mb., 186 p, 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3200538.

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9

Kita, Keiko. "STUDIES ON NEW RESTRICTION ENZYMES AND MODIFICATION ENZYMES FROM BACTERIA." Kyoto University, 1989. http://hdl.handle.net/2433/78227.

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10

Ewing, Erin. "In vacuo glycation of enzymes: A novel approach for increasing enzyme stability." Thesis, University of Ottawa (Canada), 2006. http://hdl.handle.net/10393/27129.

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A novel approach for the thermostabilization of proteins was investigated. It is well established that proteins that are naturally highly glycosylated show an increased resistance to inactivation at high temperatures. However, non-enzymatic attachment of carbohydrate to proteins, otherwise known as protein glycation, under aqueous conditions is very difficult and has not been used as a general approach for increasing the thermostability of proteins. In the present study, advantage was taken of the in vacuo protein glycation procedure recently developed by Kaplan and his co-workers by which proteins can be extensively glycated in the absence of water and chemical reagents. In this procedure, reducing sugars react with the epsilon-amino groups of lysine side-chains to form a stable ketoamine derivative. The primary objective of the research presented in this thesis, was to determine the extent to which the thermostability of proteins can be increased by in vacuo glycation with reducing monosaccharides such as glucose. Another objective of this study was to investigate possible practical applications of this thermostabilization technology. (Abstract shortened by UMI.)

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11

Banaszczyk, Mariusz G. "Artificial hydrolytic enzymes." Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74289.

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The efficiencies of many rigidly held cis-aquahydroxotetraazacobalt(III) complexes in promoting the hydrolysis of phosphate esters (BDNPP, BNPP, NPP) have been compared. The phosphate diester bond in ((trpn)Co(OH)(BNPP)) $ sp{+}$ is hydrolyzed at about the same rate as BNPP bound to a real enzyme from Enterobacter aerogenes and about 10$ sp{10}$ times more rapidly than free BNPP. The dramatic increase in the activity of the Co(III) complex with change in the tetraamine ligand structure can be explained in terms of a detailed mechanism of the reaction.
Co(III) complexes, ((tren)Co(OH$ sb2)$(OH)) $ sp{2+}$ and ((trpn)Co(OH$ sb2)$(OH)) $ sp{2+}$ have been shown to be highly efficient in promoting the hydrolysis of phosphate di- and monoesters with poor leaving groups. ((trpn)Co(OH$ sb2)$(OH)) $ sp{2+}$ promoted hydrolysis of phosphate monoesters leads to a novel binuclear phosphato complex which is a model for the active site of Purple Acid Phosphatases.
For the first time it has been shown that hydrolysis of carboxylic esters catalyzed by ((trpn)Co(OH$ sb2)$(OH)) $ sp{2+}$ is truly catalytic. The hydrolytic activity of ((trpn)Co(OH$ sb2)$(OH)) $ sp{2+}$ has been linked to the fact that this complex chelates carboxylic acids as shown by $ sp{13}$C NMR spectroscopy and the crystal structure of ((trpn)Co($ eta sp2$-OCC(CH$ sb3) sb3) rbrack sp{2+}$. This complex is the first example of a cobalt(III) complex in which carboxylic acid is chelated.

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12

Harris, Rebecca Clare. "Polyketide synthase enzymes." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360886.

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13

Proctor, Mark Richard. "Carbohydrate modifying enzymes." Thesis, University of Newcastle Upon Tyne, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.416647.

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14

SILVA, JOSE A. A. da. "Aspectos de atividade biologica da giroxina (enzima trombina simile) isolada do veneno da cascavel brasileira, Crotalus durissus terrificus." reponame:Repositório Institucional do IPEN, 2004. http://repositorio.ipen.br:8080/xmlui/handle/123456789/11191.

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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

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15

Reichstädter, Marek. "Imobilizace vybraných glykanohydroláz." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2015. http://www.nusl.cz/ntk/nusl-217152.

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The theoretical part of this thesis deals with cellulolytic enzymes, their microbial producers, the possibilities of using such enzymes in the industry and how can be enzymes - not only cellulolytic - immobilized. Experimental part examines the preparations created by immobilizing various amounts of the commercially used cellulolytic complex Cellulast 1.5L onto various synthetic carriers made of polyethylene terephthalate - commercially used Sorsilen, PET carrier and glutaraldehyde-treated PET carrier. Enzyme activity of these preparations was determined by Somogyi - Nelson method by spectrophotometry. For the highest activity immobilized preparation was determined the temperature- and the pH-optimum. The difference in effects change between the free and immobilized enzyme by measuring viscosity decrease of the substrate depending on the degradation of glycosidic bonds was also studied.

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16

Snider, Catherine E. "Synthesis and biochemical evaluation of irreversible inhibitors of aromatase /." The Ohio State University, 1986. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487266362338344.

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17

Silveira, Alvito J. "Synthesis of 6-guanidinobenzoxazinones as potential inhibitors of trypsin-like enzymes." Thesis, Georgia Institute of Technology, 1989. http://hdl.handle.net/1853/26914.

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18

Sun, An-Qiang. "How Do Enzymes Wear Out? Effects of Posttranslational Modifications on Structure and Stability of Proteins; The Triosephosphate Isomerase Model." Thesis, University of North Texas, 1991. https://digital.library.unt.edu/ark:/67531/metadc798116/.

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Triosephosphate isomerase (EC 5.3.1.1., TPI) undergoes specific posttranslational modifications (deamidation and oxidation) which are believed to initiate protein turnover by destabilization of the dimer. The crystal structures, amino acid sequences, and aging related changes of TPI from various species have been independently characterized by several laboratories. TPI has thus become the prototype enzyme for examining the initial steps in protein turnover. The binding of substrate enhances the specific deamidation of the mammalian enzyme, and a general mechanism of 'molecular wear and tear' [Gracy, R. W., Yiiksel, K. 0., Chapman, M. L., and Dimitrijevich, S. D. (1990) in Isozymes-Structure, Function and Use in Biology and Medicine (Ogita, Z-I., and Markert, C. L., Eds) pp. 787-817, Wiley-Liss, New York] has been proposed to explain how enzymes may wear out.

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19

Haileselassie, Seble Sereke Berhan. "Production of enzyme-modified cheese and bioactive peptides by Lactobacillus and commercial enzymes." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/MQ50782.pdf.

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20

Lakay, Francisco Martin. "Fungal enzymes as animal feed additives." Thesis, Stellenbosch : Stellenbosch University, 2001. http://hdl.handle.net/10019.1/52280.

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Thesis (MSc)--Stellenbosch University, 2001.
ENGLISH ABSTRACT: The use of fungal enzymes as ruminant feed digestibility enhancers was investigated. Currently, ruminants may not digest 38 to 80 % of fibrous forages' content. A renewed interest in the potential of feed enzymes for ruminants was prompted by the high costs of livestock production, together with the availability of newer enzyme preparations. Direct application of enzyme preparations can improve in vitro dry matter (DM) and neutral detergent fibre (NDF) degradation, indicating that direct-fed fibrolytic enzymes may be effective in enhancing in vivo digestion of forages. Two commercial enzyme products, Fibrozyme and Celluclast, and fungal extracellular enzyme extracts from Aureobasidium pullulans, Trichoderma reesei, Aspergillus aculeatus, and Thermomyces lanuginosus were evaluated for enhancing in vitro feed digestibility. Fibrozyme addition to both wheat straw and lucerne hay did not improve their in vitro digestibilities, even after a two hour pre-incubation period. The four fungal enzyme extracts did not enhance wheat straw's digestibility, but marginal increases were evident for lucerne hay. Celluclast addition resulted in marginal increases in the digestibility of both oat hay and oat silage, with no enhanced effect on lucerne hay and NaOH-treated wheat straw. No relationship could be found between the level of enzyme activity and the degree of feed digestion in the in vitro assay. Enzyme hydrolysis with Celluclast, in the absence of rumen fluid, gave more conclusive results. All the feed samples tested showed a positive response to Celluclast addition, even the less digestible feeds, namely sugarcane bagasse and wheat straw. In vitro results show that the assays were unsuccessful, because almost all of the experiments conducted showed inconclusive results. Alternative feed evaluation assays, which include the in vivo, in sacco and in situ methods of analysis, as well as gas production measurement and in vitro analysis with the DAISyII system, should be evaluated. A more detailed study of feed digestibility should be motivated by determining which feeds are hydrolysable, their chemical composition, i.e. how accessible the feeds are, and also evaluation of feed mixtures. The enzyme supplements also need to be evaluated for optimum temperature and pH, as well as the compilation of enzyme cocktails.
AFRIKAANSE OPSOMMING: Die gebruik van swamensieme om die verteerbaarheid van herkouervoere te verhoog, is ondersoek. Tussen 38 en 80 % van veselagtige voere se inhoud is tans onverteerbaar. 'n Hernieude belangstelling in die potensiaal van voerensieme vir herkouers word deur die hoë koste van veeproduksie, asook die beskikbaarheid van nuwe ensiempreparate gedryf Direkte byvoeging van ensiempreparate kan die in vitro droëmateriaal (DM) en neutrale onoplosbare vesel (NOV) vertering verbeter, wat daarop dui dat fibrolitiese ensieme wat direk gevoer word, effektief mag wees tydens die in vivo vertering van voer. Twee kommersiële ensiemprodukte, Fibrozyme en Celluclast, en die vier ekstrasellulêre ensieme van vier swamme, naamlik Aureobasidium pullulans, Trichoderma reesei, Aspergillus aculeatus, en Thermomyces lanuginosus is vir hul vermoë om die in vitro verteerbaarheid van voere te verbeter getoets. Byvoeging van Fibrozyme by beide koringstrooi en lusernhooi het geen verbetering in hulonderskeie in vitro verteerbaarheid tot gevolg gehad nie, selfs nie eens na 'n twee uur vooraf inkubasieperiode nie. Koringstrooi se verteerbaarheid is nie verbeter deur die byvoeging van die vier swam-ensiempreparate nie, maar 'n minimale verbetering is wel waargeneem in die verteerbaarheid van lusernhooi. Byvoeging van Celluclast het 'n minimale verbetering in beide hawerhooi en hawerkuilvoer se verteerbaarheid tot gevolg gehad, maar geen effek op lusernhooi of NaOH-behandelde koringstrooi se verteerbaarheid nie. Geen verwantskap is tussen die vlak van ensiemaktiwiteit en die mate van vertering tydens die in vitro toets gevind nie. Ensiematiese afbraak met Celluclast, in die afwesigheid van rumenvloeistof, het meer konkrete resultate gelewer. Al die voermonsters het 'n positiewe respons op die byvoeging van Celluclast getoon, selfs ook die minder verteerbare voere, nl. suikerrietbagasse en koringstrooi. In die wyer konteks was die resulate van die in vitro verteringstoetse egter onbeduidend as gevolg van groot variasie in die metings. Alternatiewe voerontledingstoetse, wat moontlik beter resultate mag lewer, sluit in in vivo, in sacco en in situ analises, asook die meting van gasproduksie en in vitro analise met die DAISyII sisteem. 'n Meer uitgebreide studie van voerverteerbaarheid wat die bepaling van die afbraak van voere, hul chemiese samestelling, met ander woorde toeganklikheid van voere, en die ondersoek van voermengsels behels, behoort aandag te geniet. Die ensiemmengsels behoort ook ten opsigte van samestelling, optimum temperatuur en pH ondersoek teword.

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21

Karamohamed, Samer. "Pyrosequencing : enzymes and assays /." Stockholm : Department of Biotechnology, 1999. http://www.lib.kth.se/abs99/kara0923.pdf.

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22

Sule, Mohammed S. "Radiation inactivation of enzymes." Thesis, University of Salford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.261991.

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23

Naylor, Neil J. "Biotransformations involving hydrolytic enzymes." Thesis, University of Warwick, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263607.

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24

Banks, M. N. "Activators of lysosomal enzymes." Thesis, University of Manchester, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.376247.

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25

Saldanha, Sanjay Adrian. "Studies on pantothenate enzymes." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620554.

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26

Mackay, Lindsey Gillian. "Porphyrin-based synthetic enzymes." Thesis, University of Cambridge, 1993. https://www.repository.cam.ac.uk/handle/1810/272641.

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27

LEE, KANG-MIN. "Enzymes en milieux heterogenes." Université Louis Pasteur (Strasbourg) (1971-2008), 1989. http://www.theses.fr/1989STR13051.

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L'emploi des enzymes en synthese organique pose quelque probleme du fait de la faible solubilite des produits organiques dans l'eau. C'est pourquoi l'alcool dehydrogenase a ete cristallisee; les microemulsions offrent des proprietes favorables a l'activite enzymatique

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28

Wirth, Petra. "Enzymes en solvants organiques." Grenoble 2 : ANRT, 1988. http://catalogue.bnf.fr/ark:/12148/cb37619244x.

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29

Vyas, P. R. "Studies on chitinolytic enzymes." Thesis(Ph.D.), CSIR-National Chemical Laboratory, Pune, 1991. http://dspace.ncl.res.in:8080/xmlui/handle/20.500.12252/3012.

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30

Finnigan, William John Andrew. "The exploitation of thermophiles and their enzymes for the construction of multistep enzyme reactions from characterised enzyme parts." Thesis, University of Exeter, 2016. http://hdl.handle.net/10871/27323.

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Biocatalysis is a field rapidly expanding to meet a demand for green and sustainable chemical processes. As the use of enzymes for synthetic chemistry becomes more common, the construction of multistep enzyme reactions is likely to become more prominent providing excellent cost and productivity benefits. However, the design and optimisation of multistep reactions can be challenging. An enzyme toolbox of well-characterised enzyme parts is critical for the design of novel multistep reactions. Furthermore, while whole-cell biocatalysis offers an excellent platform for multistep reactions, we are limited to the use of mesophilic host organisms such as Escherichia coli. The development of a thermophilic host organism would offer a powerful tool allowing whole-cell biocatalysis at elevated temperatures. This study aimed to investigate the construction of a multistep enzyme reaction from well-characterised enzyme parts, consisting of an esterase, a carboxylic acid reductase and an alcohol dehydrogenase. A novel thermostable esterase Af-Est2 was characterised both biochemically and structurally. The enzyme shows exceptional stability making it attractive for industrial biocatalysis, and features what is likely a structural or regulatory CoA molecule tightly bound near the active site. Five carboxylic acid reductases (CARs) taken from across the known CAR family were thoroughly characterised. Kinetic analysis of these enzymes with various substrates shows they have a broad but similar substrate specificity and that electron rich acids are favoured. The characterisation of these CARs seeks to provide specifications for their use as a biocatalyst. The use of isolated enzymes was investigated as an alternative to whole-cell biocatalysis for the multistep reaction. Additional enzymes for the regeneration of cofactors and removal of by-products were included, resulting in a seven enzyme reaction. Using characterised enzyme parts, a mechanistic mathematical model was constructed to aid in the understanding and optimisation of the reaction, demonstrating the power of this approach. Thermus thermophilus was identified as a promising candidate for use as a thermophilic host organism for whole-cell biocatalysis. Synthetic biology parts including a BioBricks vector, custom ribosome binding sites and characterised promoters were developed for this purpose. The expression of enzymes to complete the multistep enzyme reaction in T. thermophilus was successful, but native T. thermophilus enzymes prevented the biotransformation from being completed. In summary, this work makes a number of contributions to the enzyme toolbox of well-characterised enzymes, and investigates their combination into a multistep enzyme reaction both in vitro and in vivo using a novel thermophilic host organism.

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31

Oudshoorn, Matthew Leslie. "Tests of predictions made by the Equilibrium Model for the effect of temperature on enzyme activity." The University of Waikato, 2008. http://hdl.handle.net/10289/2418.

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The Classical Model describing the effects of temperature on enzyme activity consists of two processes: the catalytic reaction defined by ΔG cat and irreversible inactivation defined by ΔG inact, this model however, does not account for the observed temperature- dependant behaviour of enzymes. The recent development of the Equilibrium Model is governed not only by ΔG cat and ΔG inact but also by two new intrinsic parameters ΔHeq and Teq, which describe the enthalpy and the temperature of the midpoint, respectively, of a active and reversibly inactive enzyme transition. Teq is central to the physiological adaptation of an enzyme to its environmental temperature and links the molecular, physiological and environmental aspects of life to temperature in a way that has not been previously possible. The Equilibrium Model is therefore a more complete and accurate description of the effects of temperature on enzymes, it has provided new tools for describing and investigating enzyme thermal adaptation and possibly new biotechnological tools. The effects of the incorporating in the new Model of the parameters Teq and ΔH eq yield major differences from the Classical Model, with simulated data calculated according to the Equilibrium Model fitting well to experimental data and showing an initial rate temperature optimum that is independent of assay duration. Simulated data simulated according to the Classical Model can not be fitted to experimental data. All enzymes so far studied (gt30) display behaviour predicted by the Equilibrium Model. The research described here has set out to: experimentally test observations made by Eisenthal et al., on the basis of enzyme reactor data simulated according to the Equilibrium Model; to test the Equilibrium Model using an unusual (rapidly renaturable) enzyme, RNAase; and to test the proposed molecular basis of the Equilibrium Model by examining the effect of a change at the enzymes active site. The experimental results gathered here on the effect of time and temperature on enzyme reactor output confirm the predictions made by Eisenthal et al. (2006) and indicate that the Equilibrium Model can be a useful aid in predicting reactor performance. The Equilibrium Model depends upon the acquisition of data on the variation of the Vmax of an enzyme with time and temperature, and the non-ideal behaviour of RNase A made it impossible to collect such data for this enzyme, as a result the Equilibrium Model could not be applied. The disulfide bond within the active site cleft of A.k 1 protease was cleaved as a probe of the mechanism of the Equilibrium Model, which is proposed to arise from molecular changes at the enzymes active site. Support for the proposed mechanism was gained through the comparison of experimentally determined temperature dependence of the native and reduced forms of the enzyme and application of this data to the Equilibrium Model.

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32

Garrett, Mark Denis. "A study on selectivity in microbial biotransformations of substituted arenes." Thesis, Queen's University Belfast, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.287620.

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33

Sha, Zhou. "A Chemical Approach to Detect and Characterize The Activities of Mitochondrial ATP-dependent Protease Lon and ClpXP." Case Western Reserve University School of Graduate Studies / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1595239191845497.

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34

Fisher, Oriana. "Subcloning, enzymatic characterization, and in silico docking of transglutaminase 2." Waltham, Mass. : Brandeis University, 2009. http://dcoll.brandeis.edu/handle/10192/23253.

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35

Vögeli, Bastian [Verfasser], and Tobias [Akademischer Betreuer] Erb. "Control of reactive intermediates in enzymes and enzyme complexes / Bastian Vögeli ; Betreuer: Tobias Erb." Marburg : Philipps-Universität Marburg, 2019. http://d-nb.info/1185068716/34.

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36

Jordaan, Justin. "Isolation and characterization of a novel thermostable and catalytically efficient laccase from Peniophora sp. strain UD4." Thesis, Rhodes University, 2005. http://eprints.ru.ac.za/211/.

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37

Tsekoa, Tsepo L. "Structure, enzymology and genetic engineering of Bacillus sp. RAPc8 nitrile hydratase." Thesis, University of the Western Cape, 2005. http://etd.uwc.ac.za/index.php?module=etd&amp.

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Microbial nitrile hydratases are important industrial enzymes that catalyse the conversion of nitriles to the corresponding amides. A thermostable, cobalt-type Bacillus sp. RAPc8 microbial nitrile hydratase was cloned and expressed in E.coli. In this study the primary aim was to determine the molecular structure of Bacillus sp. RAPc8 microbial nitrile hydratase.

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38

Nourizad, Nader. "Recombinant Enzymes in Pyrosequencing Technology." Doctoral thesis, KTH, Biotechnology, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3765.

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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.

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39

Basile, Lacy Jamel. "Cyanide-degrading enzymes for bioremediation." Texas A&M University, 2008. http://hdl.handle.net/1969.1/86035.

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Cyanide-containing waste is an increasingly prevalent problem in today's society. There are many applications that utilize cyanide, such as gold mining and electroplating, and these processes produce cyanide waste with varying conditions. Remediation of this waste is necessary to prevent contamination of soils and water. While there are a variety of processes being used, bioremediation is potentially a more cost effective alternative. A variety of fungal species are known to degrade cyanide through the action of cyanide hydratases, a specialized subset of nitrilases which hydrolyze cyanide to formamide. Here I report on previously unknown and uncharacterized nitrilases from Neurospora crassa, Gibberella zeae, and Aspergillus nidulans. Recombinant forms of four cyanide hydratases from N. crassa, A. nidulans, G. zeae, and Gloeocercospora sorghi were prepared after their genes were cloned with N-terminal hexahistidine purification tags, expressed in Escherichia coli and purified using immobilized metal affinity chromatography. These enzymes were compared according to their relative specific activity, pH activity profiles, thermal stability, and ability to degrade cyanide in the presence of high concentrations of copper and silver. Although all four were relatively similar, the N. crassa cyanide hydratase (CHT) has the greatest thermal stability and widest pH range where activity remained above 50%. N. crassa also demonstrated the highest rate of cyanide degradation in the presence of both metals tested. The CHT of A. nidulans and N. crassa have the highest reaction rate of the four fungal nitrilases evaluated in this work. These data help determine optimization conditions for the possible use of these enzymes in the bioremediation of cyanide-containing waste. Similar to known plant pathogenic fungi, in vivo expression of CHT in both N. crassa and A. nidulans were induced by growth in the presence of KCN (potassium cyanide).

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40

Ayixiamuguli, Nueraimaiti. "Lignin degradation using lignolytic enzymes." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/35262/.

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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.

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41

Hammond, Richard Andrew. "Inducible enzymes in equine endotoxaemia." Thesis, Queen Mary, University of London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312910.

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42

Hirst, Judy. "Electron transport in redox enzymes." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364043.

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43

Kaw, Semiko. "Endothelin converting and degrading enzymes." Thesis, Oxford Brookes University, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.261235.

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44

Muroni, Maurizio. "Rational design of artificial enzymes." Thesis, Cardiff University, 2011. http://orca.cf.ac.uk/55043/.

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Despite the endeavours of many decades, the design of artificial enzymes remains challenging. The work presented here investigates two known molecules as scaffolds for the design of artificial enzymes an 18 amino acids a helical peptide with two disulfide bridges - 'Apoxaldie' able to catalyse the decarboxylation of oxaloacetate, and the 86-amino acid colicin E9 immunity protein (Im9) with four a helices. Apoxaldie was modified such that the active site lysines were substituted by 2,4-diaminobutyric acid in order to increase the proximity of the enzyme active site to the chiral environment of the a helix. The designed peptide ('Apoxaldie-Dab') was synthesized with two different strategies and the correct formation of disulfide bonds was achieved. However, Apoxaldie-Dab did not show the expected activity for the decarboxylation of oxaloacetate. Circular dichroism studies showed a 30% loss of a helicity upon introduction of 2,4- diaminobutyric acid into Apoxaldie which can explain the decrease in activity. In the case of Im9, two series of mutants, constructed around histidine 10 and asparagine 78 respectively, were designed to introduce histidine-based active sites into hydrophobic clefts. Site directed mutagenesis, gene expression and variant protein purification were carried out for ten variant mutants together with the wild type. The secondary structure and thermal stability of each protein were studied and catalytic activities were examined by monitoring the hydrolysis of /-nitrophenol acetate via ultraviolet-visible spectroscopy. The Im9 variant Tm9-W74A/N78H' demonstrated three times more activity compared to Im9, indicating the modification of IM9 to possess a histidine based active site increased activity as hypothesized through its rational design. The initial work of applying directed evolution on Tm9-W74A/N78H' was accomplished by constructing a phage display library. A transition state analogue was synthesised to test screening the expressed library. This method can be further developed to assist the design of Im9-based artificial enzymes.

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45

Whitaker, Richard George. "The electrochemistry of redox enzymes." Thesis, University of Warwick, 1989. http://wrap.warwick.ac.uk/4235/.

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The work presented in this thesis is of two types. Firstly methods for the electrochemical immobilisation of redox enzymes in organic polymers are described. The electrochemical monitoring of the immobilised enzyme reaction by detection of one of the enzyme's products is discussed, and the results obtained for such a system under a variety of experimental conditions are presented. A good understanding of the way in which such a system operates' was obtained by using a specially developed kinetic model., This model is explained fully in the theory chapter of this thesis. A variety of organic polymers were used in the electrochemical immobilisation process, with varying degrees of success. The flexibility of this approach is demonstrated by the use of a variety of immobilisation matrices and also by the development of bienzyme and bilayer devices. The final experimental chapter presents work on the covalent modification of redox enzymes with a variety of, redox centres based. on ferrocene. Although attempts to electrochemically immobilise a modified enzyme were not successful, some interesting information about the kinetic behaviour and stability of a series Of modified enzymes was obtained. An indication of possible work forming an extension to this thesis is given in the final part of this thesis. The electrochemical immobilisation techniques and the procedure for covalently modifying, enzymes using electroactive, groups are relatively recent ideas. Much work remains to be done before a better understanding of these systems is gained.

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46

Souter, Nicola H. "Thermophilic enzymes from Thermus ruber." Thesis, Heriot-Watt University, 1993. http://hdl.handle.net/10399/1437.

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47

Francis, Stewart. "Immunochemical detection of industrial enzymes." Thesis, Heriot-Watt University, 1996. http://hdl.handle.net/10399/701.

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48

O'Donnell, Raymond William. "Chitinolytic enzymes of Candida albicans." Thesis, University of Aberdeen, 1991. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=158392.

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It has been envisaged that lytic enzymes may be crucial in determining the morphology and growth of fungi, and may therefore represent a target for antifungal agents. The chitinolytic system of Candida albicans was investigated using a range of 4-methylumbelliferyl glycosides as model substrates, and its potential as a target for antibiotics has been assessed. Maximum hydrolysis was obtained with substrates having monomer and tetramer chain lengths, being attributed to N-acetylglucosaminidase and endochitinase respectively. Activities were investigated in cell fractions, vacuoles, and also in whole cell preparations. The characteristics of both chitinase and N-acetylglucosaminidase were examined, including pH and temperature optima and Km values. Chitinase was semi-purified on Fast Protein Liquid chromatography system and activity could be located after native polyacrylamide gel electrophoresis. Analysis of chitinase activity during the growth of the yeast morphology of C.albicans revealed maximal activities during the logarithmic phase, suggesting a relationship of chitinase levels to active growth of the pathogen. It was found that the antibiotic allosamidin was a potent inhibitor of chitinase activity of C.albicans, but not of N-acetylglucosaminidase. Conversely, an analogue of N-acetylglucosamine was found to be a potent inhibitor of N-acetylglucosaminidase but not of chitinase. Treatment of yeast suspensions with allosamidin resulted in an increased chain length. No cell death, or discernible pattern of change in the radiolabelling was observed in the presence of either allosamidin or N-acetylglucosamine analogue. Similarly no consistent change in the optical density of cultures was observed in the presence of either inhibitor. Even in the presence of the membrane permeabilising agent amphotericin no effects were observed above those achieved with amphotericin alone. Comparative studies were carried out upon the chitinolytic activity of Kluyveromyces lactis toxin and bovine serum. The chitin synthetic system of Benjaminiella poitrasii was compared to that of C.albicans.

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49

Doherty, Sean. "Apoplastic proteins, enzymes and radicals." Thesis, Durham University, 2000. http://etheses.dur.ac.uk/4376/.

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The soluble and readily extractable part of the plant extracellular matrix has been termed, the apoplast and contains a wide range of components such as, complex carbohydrates, structural proteins, enzymes and radicals that are known to be responsive to stress and developmental pressures. This thesis describes the development of a technique for the selective enrichment of apoplastic components for a range of subsequent analyses. Using this technique a number of apoplastic proteins were N-terminally sequenced and revealed 2 cell wall related enzymes, an antifungal protein and 3 auxin-binding/germin-like proteins. This technique also provided a novel approach to the further study of auxin-binding proteins via the use of affinity chromatography at their putative site of action, the apoplast. Three potential auxin-binding protiens were identified. Many attempts were made to subject the material extracted from the apoplast to the highly resolving technique of 2-dimensional electrophoresis, and during the process two unusual 2D systems were developed. These systems could be run in a small format that permitted very rapid analysis and/or using an in-gel loading strategy to subject up to 500µg of protein to 2D separation therefore permitting N-terminal sequencing from single 2D gels. Unfortunately 2D separation of apoplastic proteins was never fully achieved within the time frame of this study due to the vast degree of heterogenity present in the sample material. It did however demonstrate the very complex nature of apoplastic components. A series of experiments revealed that the tobacco leaf apoplast contained compartment specific antioxidant enzymes, some of which share physical characteristics with similar enzymes from other species. The activity of these enzymes altered in response to stress and according to the developmental age of the tissue. The reduced activity of these enzymes directly correlated to the degree of oxidative modification of apoplastic proteins illustrating that these enzymes are important in the detoxification of apoplastic radicals. Follow on experiments following the apoplastic generation of the superoxide anion and nitric oxide from impact stressed potato tuber tissue showed that radicals play important roles in the responses of plant tissue to stress, and show the first involvement of nitric oxide in plants in response to abiotic stress.

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50

Ogilvie, Isla Marion. "Enzymes of mitochondrial #beta#-oxidation." Thesis, University of Newcastle Upon Tyne, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.384937.

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