Academic literature on the topic 'Optimization of protease'

Thesis (MSc)--Stellenbosch University, 2012.<br>ENGLISH ABSTRACT: This study describes: a) Characterization of traditional methodologies and testing methods used to purify and quantify trypsin and α-chymotrypsin b) Re-engineering / development of a new method for purifying trypsin and α-chymotrypsin that delivered higher product yields and improved control exercised over the process by investigating: i. Extraction methods ii. Centrifugation iii. Ultrafiltration iv. Chymotrypsinogen and trypsin crystallization v. Column chromatography vi. Investigation into different raw material sources for pancreatic enzyme production c) Development of kinetic and ELISA testing methodologies for in-process QC analysis.<br>AFRIKAANSE OPSOMMING: Hierdie Studie beskryf: a) Karakterisering van die ou prosessering metodes en toets metodes wat gebruik word om Tripsien en Alpha-chimotripsien te suiwer en te kwantifiseer. b) Herontwerp / ontwikkeling van 'n nuwe metode vir die suiwering Tripsien en Chimotripsien wat „n hoër opbrengs lewer en meer kontrole oor die proses uit oefen deur ondersoek in te stel na: i. Ekstraksie- metodes ii. Sentrifugering iii. Ultrafiltrasie iv. Chymotripsienogeen - en tripsien kristallisasie v. Kolom chromatografie vi. Ondersoek na verskillende rou materiaal bronne vir die produksie van pankreas ensieme. c) Die ontwikkeling van kinetiese- en ELISA toets metodes vir die in-proses kwaliteitkontrole.

Proteases ácidas pertencem a um importante grupo de enzimas industriais produzidas por fungos filamentosos, com aplicações na indústria de alimentos, de couro, farmacêutica e de cosméticos. O objetivo principal deste trabalho foi avaliar a produção de proteases ácidas extracelulares de fungos filamentosos isolados do solo do cerrado do centro-oeste brasileiro. Inicialmente, foi realizada uma triagem para avaliar a capacidade de 17 linhagens de fungos quanto à produção de protease em meio de cultura contendo Agar-leite. O fungo Aspergillus foetidus foi selecionado como melhor produtor de protease ácida extracelular. Visando à otimização da produção de proteases pelo fungo selecionado, avaliou-se a influência de diversos fatores no cultivo (pH, temperatura, agitação e diferentes fontes de nitrogênio e carbono). Após essa etapa, um planejamento experimental estatístico foi realizado com as variáveis independentes temperatura, pH inicial do meio e fonte de carbono e nitrogênio. A produção máxima de protease foi encontrada (63,7 U/mL) nas condições: pH inicial do meio igual a 7,0 a 28 ºC, 150 rpm em peptona 2% (p/v). Os estudos em biorreator demonstraram produção de protease nas condições de agitação e aeração iguais à 300 rpm e 1,0 vvm, após 120 h de cultivo. Os ensaios com diferentes temperaturas para a estimativa dos parâmetros termodinâmicos demonstraram que a protease ácida produzida pelo fungo é altamente estável apresentando máxima atividade em pH 5,0 e temperatura ótima igual a 55ºC. E, finalmente, para a purificação da enzima foi realizada cromatografia de gel-filtração. A enzima apresentou massa molecular de 50,6 kDa, e a análise do zimograma confirmou a atividade proteolítica. Além disso, a protease purificada foi inibida pelo composto pepstatina, indicando uma característica de protease ácida. Esses resultados obtidos demonstram um fungo filamentoso produtor de uma nova protease ácida com potencial aplicação para indústria farmacêutica e de cosméticos.<br>The acid proteases belong to the most important group of industrial enzymes produced by filamentous fungi, with applications in the food, leather, pharmaceutical and cosmetics industries. This study aimed the evaluation of extracellular acid proteases production from filamentous fungi isolated from different samples of the midwestern Brazil cerrado. Initially, a screening was performed to assess the ability of the 17 strains of yeast for production of protease-agar medium containing milk culture. The Aspergillus foetidus was selected as the best producer. Aimed at optimizing the production of proteases by the selected fungus, first evaluated the influence of various factors on the cultivation (pH, temperatura, agitation and different sources of nitrogen and carbon). After this step, a statistical experimental design was carried out with the independent variables temperatura, initial pH of the medium and source of carbon and nitrogen. The best conditions for protease production were (63.7 U / mL): initial pH values greater than 7.0, at 28 °C, 150 rpm peptone 2% (w/v). Aiming future production of this protease in industrial scale, studies have shown better in bioreactor protease production under the conditions of agitation and aeration equal to 300 rpm and 1.0 vvm, after 120 h of cultive. The tests at different temperaturas to estimate the thermodynamic parameters showed that the acid protease produced by the fungus is highly stable with maximum activity at pH 5.0 and optimum temperatura of 55 °C. And finally, for the purification of the enzyme were performed gel-filtration chromatography. The enzyme had a molecular mass of 50.6 kDa, and the analysis of the zymogram showed a proteolytic band. Furthermore, the purified protease was inhibited by pepstatin compound, indicating a feature of acid protease. These results demonstrate a new filamentous fungus producing acid protease with potential application to pharmaceuticals and cosmetics.

Made available in DSpace on 2017-07-21T18:53:04Z (GMT). No. of bitstreams: 1 CRISTIANE BOURSCHEID.pdf: 1448163 bytes, checksum: 21f79c9c1335f39830bfef99ee3f5f19 (MD5) Previous issue date: 2015-03-11<br>The chicken slaughterhouse generate co-products during slaughter, for example, feathers, bones, blood and guts, usually intended for animal feed with low added value. In bone part of the meat remains bound even after deboning and is a good source of substrate for the enzymatic hydrolysis. The protein hydrolysates may be applied as a nutritional supplement in foods. Given this context, the objective was to optimize the enzymatic hydrolysis process for obtaining protein hydrolyzate coproduct of chicken bones (Gallus gallus domesticus) and the addition of the hydrolyzate as a protein supplement in burger. The co-product selected to carry out the work was thigh and drumstick bone from the chicken bones in a slaughterhouse. The study was conducted in two stages. In the first one a central composite design (CCRD), a total of 17 tests was adopted to evaluate the influence of temperature, enzyme: substrate ratio and time on the degree of hydrolysis. Following the statistical optimization was performed to obtain the best conditions of enzymatic hydrolysis of co-product of chicken bones. The co-product of chicken bones and the optimized protein hydrolyzate were characterized in terms of chemical composition, total amino acids and pattern. In the second step, the hydrolyzate was added as a protein supplement in poultry burger, where two formulations were prepared, Hamburger control (no addition of protein hydrolyzate) and hamburger with hydrolyzed (with addition of 8% protein hydrolyzate). The burgers were characterized in terms of physical-chemical, microbiological and sensory analysis. The optimum conditions for the enzymatic hydrolysis were temperature (T) of 50 ° C, enzyme: substrate ratio (E:S) from 4.96% to time (t) of 110.16 minutes under these conditions the degree of hydrolysis was 24, 0.22 ± 21%. The protein hydrolyzate has the potential to supplementation in food, it is good source of essential amino acids, meeting the recommendations established by FAO / WHO, except for leucine, phenylalanine, and valine, and had a higher concentration of protein fractions from 14.437 kDa and 3,496 kDa. The results obtained for microbiological analysis and physical-chemical analysis of the burger control and burger with hydrolyzed, are in accordance with the respective standards set by law. The burger with hydrolyzate showed 1.02% more protein than the burger control, giving a protein supplementation to the product developed. In the sensorial analysis for flavor attribute evaluators preferred the burger with protein hydrolyzate. The burger with protein hydrolyzate and the burger control achieved 84.2% and 81.8% of general acceptance, respectively. The intention to buy for the burger with added protein hydrolyzate was 76.4% and for the burger control was 67%. In this light, the protein hydrolyzate obtained from chicken bones of the co-product is an alternative to supplementation in foods, as well as add value to the co-product of chicken dessosa and increase the competitiveness of slaughterhouse.<br>Os frigoríficos de frango geram coprodutos durante o abate, por exemplo, penas, ossos, sangue e vísceras, geralmente, destinados para ração animal com baixo valor agregado. Uma parte de carne permanece aderida aos ossos, mesmo após a desossa, sendo uma boa fonte de substrato para a hidrólise enzimática. Os hidrolisados proteicos podem ser aplicados como suplemento nutricional em alimentos. Diante desse contexto, o objetivo do trabalho foi otimizar o processo de hidrólise enzimática para obtenção de hidrolisado proteico de coproduto da desossa de frango (Gallus gallus domesticus) e a adição do hidrolisado como suplemento proteico em hambúrguer. O coproduto selecionado para realização do trabalho foi osso de coxa e sobrecoxa proveniente da desossa de frango em frigorífico. O trabalho foi realizado em duas etapas. Na primeira um delineamento composto central rotacional (DCCR), totalizando 17 ensaios foi adotado para avaliar a influência da temperatura, relação enzima:substrato e tempo sobre o grau de hidrólise. Na sequência, foi realizada a otimização estatística para obter as melhores condições da hidrólise enzimática. O coproduto da desossa de frango e o hidrolisado proteico otimizado foram caracterizados em termos de composição centesimal, aminoácidos totais e perfil eletroforético. Na segunda etapa, o hidrolisado foi adicionado como suplemento proteico em hambúrguer de frango. Foram elaboradas duas formulações, hambúrguer controle (sem adição de hidrolisado proteico) e hambúrguer com hidrolisado (com adição de 8% de hidrolisado proteico). Os hambúrgueres foram caracterizados em termos de análises físico-químicas, análises microbiológicas e análise sensorial. As condições ótimas para a hidrólise enzimática foram temperatura (T) de 50ºC, relação enzima:substrato (E:S) de 4,96% e tempo (t) de 110,16 minutos, nessas condições o grau de hidrólise foi de 24,21% ±0,22. O hidrolisado proteico apresentou potencial para suplementação em alimentos, pois é boa fonte de aminoácidos essenciais, atendendo as recomendações estabelecidas pela FAO/WHO, exceto para leucina, fenilalanina e valina, bem como apresentou maior concentração de frações proteicas entre 14,437 kDa e 3,496 kDa. Os resultados obtidos para a análise microbiológica e análises físico-químicas do hambúrguer controle e do hambúrguer com hidrolisado, estão de acordo com os respectivos padrões estabelecidos pela legislação vigente. O hambúrguer com hidrolisado apresentou 6% mais proteína do que o hambúrguer controle, conferindo uma suplementação proteica ao produto desenvolvido. Em relação à análise sensorial para o atributo sabor, os avaliadores preferiram o hambúrguer com hidrolisado proteico. O hambúrguer com hidrolisado proteico e o hambúrguer controle obtiveram 84,2% e 81,8% de aceitação geral, respectivamente. A intenção de compra para o hambúrguer com adição de hidrolisado proteico foi de 76,4% e para o hambúrguer controle foi de 67%. Diante do exposto, o hidrolisado proteico obtido a partir de coproduto da desossa de frango é uma alternativa para a suplementação em alimentos, além de agregar valor ao coproduto da dessosa de frango e aumentar a competitividade dos frigoríficos.

Developing microbial systems capable of converting low cost lipids into value added products depends on the ability to acquire substrates from the growth media. Saccharomyces cerevisiae can acquire free fatty acids from the growth media and a portion of these lipids can be converted into new lipid products. However, they cannot acquire complex lipids from the growth media unless a nonspecific lipase is included. To circumvent lipase addition, we are genetically engineering S. cerevisiae to secrete a lipase into the growth media. We selected the LIP2 gene from Yarrowia lipolytica, which encodes a nonspecific lipase. Several modifications were made to the LIP2 gene to improve processing. Results identified strains secreting the most lipase. From these results, high producing strains were inserted into an oil inducible vector. Halo assays confirmed lipase secretion, while measuring the fatty acid composition confirmed triacylglycerol breakdown, and yeast uptake of the free fatty acids released.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Physics, May, 2020<br>Cataloged from student-submitted PDF version of thesis.<br>Includes bibliographical references (pages 315-348).<br>The quantitative composition of proteomes results from biophysical and biochemical selective pressures acting under system-level resource allocation constraints. The nature and strength of these evolutionary driving forces remain obscure. Through the development of analytical tools and precision measurement platforms spanning biological scales, we found evidence of optimization in bacterial gene expression programs. We compared protein synthesis rates across distant lineages and found tight conservation of in-pathway enzyme expression stoichiometry, suggesting generic selective pressures on expression setpoints. Beyond conservation, we used high-resolution transcriptomics to identify numerous examples of stoichiometry preserving cis-elements compensation in pathway operons. Genome-wide mapping of transcription termination sites also led to the discovery of a phylogenetically widespread mode of bacterial gene expression, 'runaway transcription', whereby RNA polymerases are functionally uncoupled from pioneering ribosomes on mRNAs. To delineate biophysical rationales underlying these pressures, we formulated a parsimonious ribosome allocation model capturing the trade-off between reaction flux and protein production cost. The model correctly predicts the expression hierarchy of key translation factors. We then directly measured the quantitative relationship between expression and fitness for specific translation factors in the Gram-positive species Bacillus subtilis. These precision measurements confirmed that endogenous expression maximizes growth rate. Idiosyncratic transcriptional changes in regulons were however observed away from endogenous expression. The resulting physiological burdens sharpened the fitness landscapes. Spurious system-level responses to targeted expression perturbations, called 'regulatory entrenchment', thus exacerbate the requirement for precisely set expression stoichiometry.<br>by Jean-Benoît Lalanne.<br>Ph. D.<br>Ph.D. Massachusetts Institute of Technology, Department of Physics

A demanda por antioxidantes naturais vem aumentando devido à toxicidade de alguns antioxidantes sintéticos. Estudos vêm identificando antioxidantes de origem natural, como a proteína da soja, que é capaz de contribuir na melhoria de propriedades funcionais e biológicas de alimentos. A hidrólise enzimática da proteína de soja aumenta sua atividade antioxidante, assim como a capacidade emulsificante e a capacidade de formação de espuma. O objetivo deste trabalho foi o estudo da hidrólise da proteína de soja através de uma protease produzida por Chryseobacterium sp., a verificação da capacidade antioxidante e aplicação do hidrolisado em diferentes tipos de carnes para evitar a oxidação lipídica. A eficácia da hidrólise foi determinada através da proteína solúvel utilizando o método de Folin enquanto que a atividade antioxidante foi avaliada pelos métodos referentes à captura do radical DPPH e ABTS. Os hidrolisados foram adicionados em carne de porco e peixe e foi verificada a inibição da oxidação lipídica. A influência de três parâmetros (temperatura, pH, relação enzima/substrato) na hidrólise foi estudada através um experimento fatorial 23 . Como respostas foram avaliadas a atividade antioxidante (DPPH e ABTS), atividade quelante de ferro, proteína solúvel, capacidade de formação de espuma e capacidade emulsificante. Observou-se um aumento na concentração de proteína solúvel em função do tempo, sendo que os hidrolisados foram capazes de inibir tanto o radical DPPH quanto o ABTS. Os hidrolisados inibiram em parte a oxidação lipídica em carne suína e peixe. Ainda foi possível concluir que dependendo da finalidade para que se deseja o hidrolisado, diferentes condições devem ser utilizadas. Os resultados demonstram uma potencial aplicação da protease microbiana para gerar hidrolisados antioxidantes da proteína de soja.<br>The demand for natural antioxidants has been increasing due to the toxicity of some synthetic antioxidants. Studies have identified naturally occurring antioxidants, such as soy protein, which can contribute to improve functional and biological properties of food. Enzymatic hydrolysis of soy protein increases its antioxidant activity, as well as emulsifying capacity and foaming capacity. The purpose of this work was to study the hydrolysis of soy protein, verifying the antioxidant capacity, application of the hydrolysate in different types of meat and optimization of hydrolysis. The efficiency of hydrolysis was determined by the soluble protein by the method of Folin while the antioxidant activity was evaluated by the methods related to the capture of the radical DPPH and ABTS. The hydrolysates were added to pork and fish and the extent of lipid oxidation was determined by TBARS. In optimizing of the hydrolysis three parameters were varied (T, pH, enzyme substrate), it was applied to a surface response methodology for conducting trials using a 23 factorial experiment. As answers were evaluated antioxidant activity (DPPH and ABTS), iron chelating activity, Lowry, foaming capacity and emulsifying capacity. There was an increase in soluble protein concentration versus time, and the hydrolysates were able to inhibit both the ABTS and the DPPH radical. The hydrolysates were able of inhibit lipid oxidation in pork and fish. Was still possible to conclude that depending on the finality that will be given to hydrolysates different treatment conditions should be used. The results demonstrate a significant potential for application of microbial protease to generate antioxidants of hydrolyzed soy protein.

Pseudomonas fragi ATCC 4973 was grown in trypticase soy broth (TSB), on a trypticase soy broth + 1.5% agar (TSA) surface, and in a defined citrate broth. The citrate broth contained glutamine as the sole nitrogen source, Pseudomonas fragi grown in TSB started proteinase production at 24 h, during the late logarithmic early stationary growth phase. Pseudomonas fragi grown on TSA surfaces initiated proteinase production at 4 h, 20 hours earlier than in liquid medium. Electron micrographs of P. fragi grown on TSA revealed extracellular vesicles ca. 20 nm in diameter "blebbing" off the surface of the cells. These vesicles were absent from the surface of P. fragi cells grown in TSB, although vesicles could be isolated from the culture supernatant. Isolated extracellular vesicles were ca. 20 nm in diameter and contained a proteinase similar to that found in the supernatant. Electrophoretic analysis showed the vesicles and outer cell membrane of P. fragi to share similarities in their composition. Use of the centroid search technique of Aishima and Nakai, showed the optimum cultural conditions for proteinase production by P. fragi, in defined citrate broth to be: incubation temperature, 12.5 C; incubation time, 38 h; initial pH, 6.8; organic nitrogen concentration, 314 mmole nitrogen/L (glutamine); a gas mixture containing 16.4% oxygen flowing over the medium (7.42 ppm dissolved oxygen). Oxygen was the major factor influencing proteinase production by P. fragi. A comparison of optimization techniques suitable for microbiological experiments showed that the centroid search technique of Aishima and Nakai, the modified super simplex of Nakai and Kaneko and the simplex technique of Morgan and Deming all required similar time and experiment numbers to obtain the optimum point.<br>Land and Food Systems, Faculty of<br>Graduate

In this study, the aim was to investigate the effects of pH on therapeutically important protein, recombinant human growth hormone (rhGH), production by Pichia pastoris considering the expression levels of regulatory genes. In this frame, firstly the host microorganism was selected between two different methanol utilization phenotypes of P. pastoris, Mut+ and MutS on media containing glycerol/methanol or sorbitol/methanol. The highest rhGH production, 120 g L-1, and hGH gene expression, 9.84x109 copies mg-1 CDW, were achieved in the medium containing 30 g L-1 sorbitol and 1% (v/v) methanol by P. pastoris hGH-Mut+ strain. Thereafter, effects of pH on rhGH production and stability were investigated in laboratory scale bioreactors. RhGH was more stable at pH 5.0. Throughout the production, it is seen that medium of pH decreased. Thereafter, effects of pH on rhGH were investigated in pH controlled pilot-scale bioreactor. In addition to rhGH concentration, AOX intracellular enzyme activity, extracellular proteases concentrations<br>expression levels of hGH, AOX, pep4, prb1 and prc1 genes were determined. The highest cell concentration was obtained as 53 g L-1 at pH 6.0 but hGH concentration was found as 24 mg L-1 at t=24 h. The highest rhGH concentration was obtained as 271 g L-1 with 42 g L-1 cell density at pH 5.0 in medium containing sorbitol at t=24 h. At this condition, the overall product and cell yield on total substrate were found as 2.08 mg g-1 and 0.15 g g-1. Furthermore, the highest expression levels of hGH and AOX were attained at pH 5.0. Moreover, by keeping pH at 5.0, expression levels of three types of vacuolar proteases were minimized.