Journal articles: 'Optimization of protease'

1

Chourasia, P., B. Patel, M. M. Prakash, and S. Gaherwal. "Screening and Optimization of Extracellular Alkaline Protease Production from Bacillus Spp." Environment Conservation Journal 13, no. 3 (December 20, 2012): 49–52. http://dx.doi.org/10.36953/ecj.2012.130309.

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Protease enzyme catalyzes the hydrolysis of protein. Among the various proteases, bacterial proteases are most significant when compared with animal and fungal proteases. In the present study a protease producing bacteria were isolated from soil collected from Govt. Holkar Science College, Indore campus and identified as Bacillus spp. They were grown within a temperature range between 25°C & 45 °C and pH range of 6.0 to 11.0. The optimum condition for protease production obtained was 35 °C at pH 9. The best carbon and organic nitrogen sources for this bacterial strain were fructose and yeast extract, respectively, while the most effective inorganic nitrogen sources was urea. It is envisaged that the isolate can be a potential source of alkaline protease for use as additive in industrial applications like detergent industry.

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Patra, Madhumita Dandopath. "Rational Lead Optimization Based on the Modeled Structure of Cysteine Protease of Leishmania donovani." Asian Journal of Organic & Medicinal Chemistry 4, no. 4 (2019): 256–66. http://dx.doi.org/10.14233/ajomc.2019.ajomc-p239.

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In present study, the molecular modeling techniques were applied to generate a refined model of a cysteine protease of Leishmania donovani using the crystal structure of a homologous protease and used for lead optimization. The structures of a series of complexes of the protease with the designed inhibitors were predicted using a novel docking technique comprising of repeated cycles of molecular dynamics and energy minimization. Calculation of the free energies of binding of the model with the designed inhibitors suggested that three compounds can form stable complexes with dissociation constants in the nanomolar range (0.038-1.41 nM). Search in the human genome revealed that a number of proteases of the cathepsin family had high homology with the parasite protease with amino acid identity around 45 %. The X-ray structures of all these were available in the protein data bank. The structures of the complexes of the selected inhibitors with a few homologous human proteases of known 3-D structures were also predicted using the same technique of optimization. The electrostatic potentials around the binding sites of the proteases were highly negative, which served as a clue for the introduction of positively charged groups in the designed inhibitors for higher affinity. The comparison of interaction energies and hydrogen bonding patterns among these complexes and similar complexes with homologous human proteases allowed us to short-listed three molecules as effective antileishmanial cysteine protease inhibitors.

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Ferrall-Fairbanks, Meghan C., Chris A. Kieslich, and Manu O. Platt. "Reassessing enzyme kinetics: Considering protease-as-substrate interactions in proteolytic networks." Proceedings of the National Academy of Sciences 117, no. 6 (January 24, 2020): 3307–18. http://dx.doi.org/10.1073/pnas.1912207117.

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Enzymes are catalysts in biochemical reactions that, by definition, increase rates of reactions without being altered or destroyed. However, when that enzyme is a protease, a subclass of enzymes that hydrolyze other proteins, and that protease is in a multiprotease system, protease-as-substrate dynamics must be included, challenging assumptions of enzyme inertness, shifting kinetic predictions of that system. Protease-on-protease inactivating hydrolysis can alter predicted protease concentrations used to determine pharmaceutical dosing strategies. Cysteine cathepsins are proteases capable of cathepsin cannibalism, where one cathepsin hydrolyzes another with substrate present, and misunderstanding of these dynamics may cause miscalculations of multiple proteases working in one proteolytic network of interactions occurring in a defined compartment. Once rates for individual protease-on-protease binding and catalysis are determined, proteolytic network dynamics can be explored using computational models of cooperative/competitive degradation by multiple proteases in one system, while simultaneously incorporating substrate cleavage. During parameter optimization, it was revealed that additional distraction reactions, where inactivated proteases become competitive inhibitors to remaining, active proteases, occurred, introducing another network reaction node. Taken together, improved predictions of substrate degradation in a multiple protease network were achieved after including reaction terms of autodigestion, inactivation, cannibalism, and distraction, altering kinetic considerations from other enzymatic systems, since enzyme can be lost to proteolytic degradation. We compiled and encoded these dynamics into an online platform (https://plattlab.shinyapps.io/catKLS/) for individual users to test hypotheses of specific perturbations to multiple cathepsins, substrates, and inhibitors, and predict shifts in proteolytic network reactions and system dynamics.

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Mostafa, El-Sayed E., Moataza M. Saad, Hassan M. Awad, Mohsen H. Selim, and Helmy M. Hassan. "Optimization Conditions of Extracellular Proteases Production from a Newly IsolatedStreptomyces PseudogrisiolusNRC-15." E-Journal of Chemistry 9, no. 2 (2012): 949–61. http://dx.doi.org/10.1155/2012/168540.

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Microbial protease represents the most important industrial enzymes, which have an active role in biotechnological processes. The objective of this study was to isolate new strain ofStreptomycesthat produce proteolytic enzymes with novel properties and the development of the low-cost medium. An alkaline protease producer strain NRC-15 was isolated from Egyptian soil sample. The cultural, morphological, physiological characters and chemotaxonomic evidence strongly indicated that the NRC-15 strain represents a novel species of the genusStreptomyces, hence the nameStrptomyces pseudogrisiolusNRC-15. The culture conditions for higher protease production by NRC-15 were optimized with respect to carbon and nitrogen sources, metal ions, pH and temperature. Maximum protease production was obtained in the medium supplemented with 1% glucose, 1% yeast extract, 6% NaCl and 100 μmol/L of Tween 20, initial pH 9.0 at 50 °C for 96 h. The current results confirm that for this strain, a great ability to produce alkaline proteases, which supports the use of applications in industry.

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Alias, Norsyuhada, Mu’adz Ahmad Mazian, Abu Bakar Salleh, Mahiran Basri, and Raja Noor Zaliha Raja Abd Rahman. "Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12." Enzyme Research 2014 (June 30, 2014): 1–20. http://dx.doi.org/10.1155/2014/197938.

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Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE) strategy with an open reading frame (ORF) of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity) and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX) promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. The highest protease production (28.3 U/ml) was obtained from P. pastoris GS115 host (GpPro2) at 20°C after 72 hours of postinduction time with 0.5% (v/v) of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa.

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Usman, Abdilbar, Said Mohammed, and Jermen Mamo. "Production, Optimization, and Characterization of an Acid Protease from a Filamentous Fungus by Solid-State Fermentation." International Journal of Microbiology 2021 (April 29, 2021): 1–12. http://dx.doi.org/10.1155/2021/6685963.

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Acid proteases represent an important group of enzymes, extensively used in food and beverage industries. There is an increased demand for acid proteases adapting to the industrial extreme environment, especially lower pH. Thus, this necessitates the search for a better acid protease from fungi that best performs in industrial conditions. The fungal isolates were isolated from grape and dairy farm soil using potato dextrose agar and further screened for protease production based on the hydrolysis of clear zone on skim milk agar. The potential fungi were then subjected to secondary screening under solid-state fermentation (SSF). After the secondary screening, the potential fungus was identified to the genus level by the macroscopic and microscopic methods. The growth conditions and media composition for the potential fungus were further optimized under SSF. The crude enzyme produced by the potential isolate was characterized after partial purification by acetone and ammonium sulfate precipitation. A total of 9 fungal isolates showed protease production in primary and secondary screening; however, one potential isolate (Z1BL1) was selected for further study based on its protease activity. The isolate was identified to the genus Aspergillus based on their morphological features. The maximum acid protease from the isolate Z1BL1 was obtained using fermentation media containing wheat bran as a solid substrate, 1 mL of 3.2 × 106 inoculum size, 50% moisture content, and pH 4.5 upon 120-h incubation at 30°C. The acetone-precipitated enzyme exhibited the maximum activity at 50°C and pH 5 with stability at pH 4–6 and temperature 40–60°C. Thus, the acid protease produced from Aspergillus showed suitable enzyme characteristics required in the industry and could be a candidate for application in the food industry after further purification.

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Hashmi, Sidra, Sajid Iqbal, Iftikhar Ahmed, and Hussnain Ahmed Janjua. "Production, Optimization, and Partial Purification of Alkali-Thermotolerant Proteases from Newly Isolated Bacillus subtilis S1 and Bacillus amyloliquefaciens KSM12." Processes 10, no. 6 (May 25, 2022): 1050. http://dx.doi.org/10.3390/pr10061050.

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Proteases that can remain active under extreme conditions such as high temperature, pH, and salt concentration are widely applicable in the commercial sector. The majority of the proteases are rendered useless under harsh conditions in industries. Therefore, there is a need to search for new proteases that can tolerate and function in harsh conditions, thus improving their commercial value. In this study, 142 bacterial isolates were isolated from diverse alkaline soil habitats. The two highest protease-producing bacterial isolates were identified as Bacillus subtilis S1 and Bacillus amyloliquefaciens KSM12, respectively, based on 16S rRNA sequencing. Optimal protease production was detected at pH 8, 37 °C, 48 h, 5% (w/v) NaCl for Bacillus subtilis S1 (99.8 U/mL) and pH 9, 37 °C, 72 h, 10% (w/v) NaCl for Bacillus amyloliquefaciens KSM12 (94.6 U/mL). The molecular weight of these partially purified proteases was then assessed on SDS-PAGE (17 kDa for Bacillus subtilis S1 and 65 kDa for Bacillus amyloliquefaciens KSM12), respectively. The maximum protease activity for Bacillus subtilis S1 was detected at pH 8, 40 °C, and for Bacillus amyloliquefaciens KSM12 at pH 9, 60 °C. These results suggest that the proteases secreted by Bacillus subtilis S1 and Bacillus amyloliquefaciens KSM12 are suitable for industries working in a highly alkaline environment.

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CAI, KANGTAO, HUAYOU CHEN, XINYU HENG, LINGYU KANG, JUNMING WU, CHENXI LU, and XIAOYU LIANG. "Optimization of Small Peptide Feed from Milk Thistle Residue by Synergistic Fermentation of Multiple Strains and Proteases." Romanian Biotechnological Letters 26, no. 6 (December 30, 2021): 3102–9. http://dx.doi.org/10.25083/rbl/26.6/3102-3109.

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In order to improve the utilization rate of the milk thistle residue, this study used the synergistic fermentation of multiple strains and proteases to increase the small peptide content of the fermented feed produced by the milk thistle residue. Taking the small peptide content of the milk thistle residue fermented feed as an indicator, the optimal fermentation process was obtained by single-factor optimization experiments and the response surface methodology. The optimal fermentation process was as follows: fermentation time of 7 days, inoculum size of 15%, inoculation ratio of aerobic strains: anaerobic strains = 1: 2, solid-state fermentation water content of 66%, fermentation temperature of 36℃, and amount of protease was 0.25% acid protease+0.25% bromelain. Under the above process, the small peptide content of the fermented feed from milk thistle residue was greatly improved to 57.86%. These results inferred that the added proteases were beneficial to the growth of fermentative microorganisms, the secretion of protease and the increase of the small peptide content.

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Maheswari, P., S. Mahendran, and A. Kamilabanu. "Isolation and Optimization of Protease Producing Bacteria from Marine Sediment." International Journal of Trend in Scientific Research and Development Volume-2, Issue-3 (April 30, 2018): 122–32. http://dx.doi.org/10.31142/ijtsrd9674.

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10

Braaksma, Machtelt, Age K. Smilde, Mariët J. van der Werf, and Peter J. Punt. "The effect of environmental conditions on extracellular protease activity in controlled fermentations of Aspergillus niger." Microbiology 155, no. 10 (October 1, 2009): 3430–39. http://dx.doi.org/10.1099/mic.0.031062-0.

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Proteolytic degradation by host proteases is one of the key issues in the application of filamentous fungi for non-fungal protein production. In this study the influence of several environmental factors on the production of extracellular proteases of Aspergillus niger was investigated systematically in controlled batch cultures. Of all factors investigated in a series of initial screening experiments, culture pH and nitrogen concentration in particular strongly affected extracellular protease activities. For instance, at a culture pH of 4, protease activity was higher than at pH 5, and protease activity increased with increasing concentrations of ammonium as nitrogen source. Interestingly, an interdependence was observed for several of the factors studied. These possible interaction effects were investigated further using a full factorial experimental design. Amongst others, the results showed a clear interaction effect between nitrogen source and nitrogen concentration. Based on the observed interactions, the selection of environmental factors to reduce protease activity is not straightforward, as unexpected antagonistic or synergistic effects occur. Furthermore, not only were the effects of the process parameters on maximum protease activity investigated, but five other protease-related phenotypes were studied as well, such as maximum specific protease activity and maximum protease productivity. There were significant differences in the effect of the environmental parameters on the various protease-related phenotypes. For instance, pH significantly affected final levels of protease activity, but not protease productivity. The results obtained in this study are important for the optimization of A. niger for protein production.

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Mandragutti, Teja, Muni Kumar Dokka, Pavani Sanapala, Chandana Vineela Karrotu, and Sudhakar Godi. "Screening and optimization of candidate alkaline protease for dehairing potential from marine Bacillus paramycoides M2." Research Journal of Biotechnology 17, no. 2 (January 25, 2022): 78–89. http://dx.doi.org/10.25303/1702rjbt7889.

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Alkaline proteases are active from neutral to alkaline pH range and have extensive applications in detergent and leather industries. In the present research, bacteria isolated from marine water samples were screened for proteolytic activity. Among the isolates, M2 showed maximum proteolysis with a clear zone when cultured on skim milk agar plates at 37°C for 24 h. Molecular identification using 16S rRNA sequencing and phylogenetic analysis revealed that M2 has sequence identity (99.93%) to Bacillus paramycoides. SEM analysis was carried for determining the morphology of M2 and also for enzyme treated skin. FAME analysis using GCMS was performed for the determination of fatty acids in the strain. The selected isolate was inoculated into protease production medium under submerged fermentation conditions at 37ºC for 48 h with a constant agitation of 120 rpm. Protease activity was determined under varying conditions of pH, incubation temperature, carbon and nitrogen sources, metal ions and NaCl (1- 5%) using casein as substrate. The isolate M2 utilized molasses and peptone as carbon and nitrogen sources for better alkaline protease production at 40°C and pH 10 under optimal conditions. The dehairing experiments with M2 alkaline protease revealed dehairing efficacy of protease over chemical treatment. Hence, extracellular alkaline protease from M2 isolate could find potential application in leather processing industries and can be exploited commercially.

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Zafrida, Siska, Stalis Norma Ethica, Aditya Rahman Ernanto, and Wijanarka Wijanarka. "Optimization of Crude Protease Production from Bacillus thuringiensis HSFI-12 and Thrombolytic Activity Its Enzyme Dialysate." Trends in Sciences 19, no. 23 (November 10, 2022): 1952. http://dx.doi.org/10.48048/tis.2022.1952.

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Cardiovascular disease (CVD) is among the leading causes of death in the world caused by thrombosis. Thrombosis is the formation of excessive blood clots on the walls of blood vessels called thrombus. This could lead to fatal blockage of the heart muscle or brain. Thrombosis can be treated using enzyme-type drugs such as thrombolytic proteases. There have been many studies of bacteria producing thrombolytic enzymes isolated from fermented foodstuffs. However, commercial production of bacterial enzymes to fulfill the need of thrombolytic agents is still limited, causing high price of CVD therapy. Bacillus sp. HSFI-12 (Holothuria scabra Fermented Intestine-12) isolated from the fermented intestine of sea cucumber Holothuria scabra had been reported to have a competitive thrombolytic activity to the commercial Nattokinase. This study aimed to determine bacterial optimum incubation time based on enzyme activity after bacterium molecular identification was done. It also aimed to obtain the dialysate of bacterial crude protease and then determine the thrombolytic activity of the obtained dialysate. Thrombolytic activity was tested on 4 different types of blood (A, B, AB and O). Results of molecular identification showed that strain HSFI-12 shared similarity level of 99.80 %, with Bacillus thuringiensis. Activity of crude protease from the incubated cultures peaked at 48 h of incubation with activity of 191.5 U/mL The activity of concentrated protease after precipitation and dialysis process (dialysate) was 2-times higher by 355,7 U/mL. The percentage of lysis of blood clots produced by crude blood groups A, B, AB and O showed a range of values of 66.423 - 67.656 % while those that produce dialysate are 77.564 - 78.861 %. As conclusion, the best (optimized) incubation time to produce crude enzyme from B. thuringiensis HSFI-12 with the highest activity was 48 h. The process of concentrating the crude thrombolytic protease of B. thuringiensis HSFI-12 increases both activity and thrombolytic ability of the bacterial enzyme. HIGHLIGHTS The most optimum incubation time for protease production from Bacillus thuringiensis is 48 h Partial purification on bacterial crude protease through resulted in 86 % increased activity Partial purification on bacterial crude protease resulted in 17 % increased blood clot lysis ability Clot lysis ability of both crude and dialysate proteases are higher than that of Nattokinase (NK) GRAPHICAL ABSTRACT

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Chang, Kyeong-Ok, Yunjeong Kim, Scott Lovell, Athri Rathnayake, and William Groutas. "Antiviral Drug Discovery: Norovirus Proteases and Development of Inhibitors." Viruses 11, no. 2 (February 25, 2019): 197. http://dx.doi.org/10.3390/v11020197.

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Proteases are a major enzyme group playing important roles in a wide variety of biological processes in life forms ranging from viruses to mammalians. The aberrant activity of proteases can lead to various diseases; consequently, host proteases have been the focus of intense investigation as potential therapeutic targets. A wide range of viruses encode proteases which play an essential role in viral replication and, therefore, constitute attractive targets for the development of antiviral therapeutics. There are numerous examples of successful drug development targeting cellular and viral proteases, including antivirals against human immunodeficiency virus and hepatitis C virus. Most FDA-approved antiviral agents are peptidomimetics and macrocyclic compounds that interact with the active site of a targeted protease. Norovirus proteases are cysteine proteases that contain a chymotrypsin-like fold in their 3D structures. This review focuses on our group’s efforts related to the development of norovirus protease inhibitors as potential anti-norovirus therapeutics. These protease inhibitors are rationally designed transition-state inhibitors encompassing dipeptidyl, tripeptidyl and macrocyclic compounds. Highly effective inhibitors validated in X-ray co-crystallization, enzyme and cell-based assays, as well as an animal model, were generated by launching an optimization campaign utilizing the initial hit compounds. A prodrug approach was also explored to improve the pharmacokinetics (PK) of the identified inhibitors.

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Steuer, Christian, Karl H. Heinonen, Lars Kattner, and Christian D. Klein. "Optimization of Assay Conditions fo r Dengue Virus Protease: Effect of Various Polyols and Nonionic Detergents." Journal of Biomolecular Screening 14, no. 9 (September 2, 2009): 1102–8. http://dx.doi.org/10.1177/1087057109344115.

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The aim of this work was to perform a systematic study of the effect of nonionic detergents on the activity of the dengue virus NS2B-NS3 protease. To ensure a high activity of the protease, the assay procedures for the dengue virus and other flaviviral proteases published to date are performed in the presence of up to 35% glycerol, which does not represent the cellular physicochemical environment. In addition, the high viscosity of glycerol-containing solutions leads to various experimental problems in miniaturized assays. Using an internally quenched peptide substrate, the authors show that glycerol is not essential for enzymatic activity if certain nonionic detergents are added to the assay buffer. In addition, nonionic detergents may help to avoid false-positive screening results caused by “promiscuous” inhibitors. Other polyalcohols can substitute glycerol and have less effect on the viscosity of the assay buffer. The assay was used to screen a compound library and allowed the identification of small-molecular nonpeptidic inhibitors of dengue NS3 protease. Finally, the authors discuss the mode of action of nonionic detergents and the influence that they may have on the conformational properties of the NS2B-NS3 protease. ( Journal of Biomolecular Screening 2009:1102-1108)

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Shivasharanappa, Kirankumar, Jayashree V. Hanchinalmath, Y. Sai Sundeep, Debajit Borah, and V. S. S. L. Prasad Talluri. "Optimization and Production of Alkaline Proteases from Agro Byproducts Using a Novel Trichoderma viridiae Strain VPG 12, Isolated from Agro Soil." International Letters of Natural Sciences 14 (April 2014): 77–84. http://dx.doi.org/10.18052/www.scipress.com/ilns.14.77.

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In recent years, there has been a phenomenal increase in the use of alkaline proteases as industrial catalysts. The aim of this work was to isolate potent fungal strain from the agricultural field of Gulbarga region of India, for the production of alkaline protease by utilizing the agricultural by products viz, red and green gram and Bengal gram as substrate under submerged fermentation process. Optimization of fermentation process parameters such as substrate (Red gram husk, green gram husk and Bengal gram husk) utilization, utilization, temperature, pH and incubation period for alkaline protease production was carried out. The maximum production of alkaline protease by Trichoderma VPG 12 was found at pH 8, temperature 35 °C, incubated for 120 h. But the activity of the enzyme could also be seen in a wide range of pH (5-9) and temperature (20-40 °C). With all these properties, the strain can be considered for industrial grade production of alkaline protease.

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de Oliveira, Rodrigo Lira, Emiliana de Souza Claudino, Attilio Converti, and Tatiana Souza Porto. "Use of a Sequential Fermentation Method for the Production of Aspergillus tamarii URM4634 Protease and a Kinetic/Thermodynamic Study of the Enzyme." Catalysts 11, no. 8 (August 11, 2021): 963. http://dx.doi.org/10.3390/catal11080963.

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Microbial proteases are commonly produced by submerged (SmF) or solid-state fermentation (SSF), whose combination results in an unconventional method, called sequential fermentation (SF), which has already been used only to produce cellulolytic enzymes. In this context, the aim of the present study was the development of a novel SF method for protease production using wheat bran as a substrate. Moreover, the kinetic and thermodynamic parameters of azocasein hydrolysis were estimated, thus providing a greater understanding of the catalytic reaction. In SF, an approximately 9-fold increase in protease activity was observed compared to the conventional SmF method. Optimization of glucose concentration and medium volume by statistical means allowed us to achieve a maximum protease activity of 180.17 U mL−1. The obtained enzyme had an optimum pH and temperature of 7.0 and 50 °C, respectively. Kinetic and thermodynamic parameters highlighted that such a neutral protease is satisfactorily thermostable at 50 °C, a temperature commonly used in many applications in the food industry. The results obtained suggested not only that SF could be a promising alternative to produce proteases, but also that it could be adapted to produce several other enzymes.

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Parthasarathy, M., and J. Joel Gnanadoss. "Medium Formulation and its Optimization to Enhance Protease Production by Streptomyces sp. Isolated from Mangroves." Biosciences, Biotechnology Research Asia 15, no. 3 (September 1, 2018): 719–28. http://dx.doi.org/10.13005/bbra/2680.

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Streptomyces sp. LCJ12A was isolated from the soil and sediments of Pichavaram Mangrove Forest, Tamil Nadu, India. Production of protease from the Streptomyces sp. LCJ12A was carried out by using submerged fermentation. To enhance the protease production, the fermentation medium was formulated and optimized. Different carbon, nitrogen and inducer sources were used for the optimization. In that fructose, sodium nitrate and red gram husk showed greater quantity of protease production and their different concentrations were optimized in Protease production broth (PPB). Response surface methodology (RSM) was employed for the medium optimization at a low cost for the production of industrially important enzyme. The optimized values showed that fructose at 2.0 g/L enhances the yield of protease up to 120.08±2.2 U/mL, sodium nitrate at 2.0 g/L maximize the protease production up to 180.35±1.9 U/mL and red gram husk at 2.0 g/L yields 194.16±2.2 U/mL which was 1.6 times higher when compared to the unoptimized medium. Statistical optimization by using RSM predicted that 327.16 U/mL of protease enzyme can be produced. Through experimentation based on RSM, the protease yield reached up to 323.4 U/mL. When compared to unoptimized medium, the statistically optimized medium produced 3 times higher yield. As a result of the optimization studies, an increase in protease activity was reached compared to the unoptimized conditions and thus offers a new approach for industrial enzyme production.

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Self, Rachel A., Mark D. Harrison, Valentino S. Te’o, and Steve Van Sluyter. "Development of simple, scalable protease production from Botrytis cinerea." Applied Microbiology and Biotechnology 106, no. 5-6 (February 16, 2022): 2219–33. http://dx.doi.org/10.1007/s00253-022-11817-1.

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Abstract Heat haze-forming proteins are stable during winemaking and are typically removed via adsorption to bentonite. Proteolytic degradation is an alternative method to prevent wine-haze and offers the opportunity to reduce the environmental impacts and labor cost of the process. Herein, we describe the development of a production system for Botrytis cinerea proteases for the enzymatic degradation of heat haze-forming proteins. The effect of culture medium on the secretion of glucan by B. cinerea was investigated and methods to inactivate B. cinerea laccase in liquid culture medium were assessed. Protease production by B. cinerea was scaled up from 50 mL in shake flasks to 1 L in bioreactors, resulting in an increase in protease yield from 0.30 to 3.04 g L−1. Glucan secretion by B. cinerea was minimal in culture medium containing lactose as a carbon source and either lactic or sulfuric acid for pH control. B. cinerea laccases were inactivated by reducing the pH of culture supernatant to 1.5 for 1 h. B. cinerea proteases were concentrated and partially purified using ammonium sulfate precipitation. SWATH-MS identified aspartic acid protease BcAP8 amongst the precipitated proteins. These results demonstrate a simple, affordable, and scalable process to produce proteases from B. cinerea as a replacement for bentonite in winemaking. Key points • Isolates of B. cinerea that produce proteases with potential for reducing wine heat-haze forming proteins were identified. • Media and fermentation optimization increased protease yield tenfold and reduced glucan secretion. • Low pH treatment inactivated laccases but not proteases. Graphical abstract

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Shivasharanappa, Kirankumar, Jayashree V. Hanchinalmath, Y. Sai Sundeep, Debajit Borah, and V. S. S. L. Prasad Talluri. "Optimization and Production of Alkaline Proteases from Agro Byproducts Using a Novel <i>Trichoderma viridiae</i> Strain VPG 12, Isolated from Agro Soil." International Letters of Natural Sciences 14 (April 29, 2014): 77–84. http://dx.doi.org/10.56431/p-vif609.

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In recent years, there has been a phenomenal increase in the use of alkaline proteases as industrial catalysts. The aim of this work was to isolate potent fungal strain from the agricultural field of Gulbarga region of India, for the production of alkaline protease by utilizing the agricultural by products viz, red and green gram and Bengal gram as substrate under submerged fermentation process. Optimization of fermentation process parameters such as substrate (Red gram husk, green gram husk and Bengal gram husk) utilization, utilization, temperature, pH and incubation period for alkaline protease production was carried out. The maximum production of alkaline protease by Trichoderma VPG 12 was found at pH 8, temperature 35 °C, incubated for 120 h. But the activity of the enzyme could also be seen in a wide range of pH (5-9) and temperature (20-40 °C). With all these properties, the strain can be considered for industrial grade production of alkaline protease.

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Muthukrishnan, N., R. Ragunathan, and Jesteena Johney. "Statistical Optimization of Alkaline Protease Enzyme Produced by Bacillus subtilis MH266414 and its Application in Different Industries." International Journal of Current Microbiology and Applied Sciences 12, no. 5 (May 10, 2023): 125–35. http://dx.doi.org/10.20546/ijcmas.2023.1205.018.

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Proteases are an important class of enzymes with numerous industrial applications. Under submerged cultivation conditions, the Bacillus subtilis MH266414 generated alkaline protease. In order to maximise the factors such as pH, incubation period, and temperature, response surface methodology-based central composite design (CCD) was utilised. The quadratic model was shown to be reasonably adjusted with the experimental data by the CCD design. To assess the changes in the response surface and to understand the connection between the culture conditions and the enzyme yield, statistics-based contour and 3-D plots were created. In this investigation, a highest yield of protease activity of 63.0725 U/mL were obtained after optimization. The crude enzyme was purified using ammonium sulphate precipitation and DEAE Sephadex column chromatography. The fraction obtained from the column shows a specific activity of 3512U/mg-1. The isolated protease also gives better result of washing and dehairing results. This result implies that this purified enzyme from Bacillus subtilis MH266414 can be used as biotechnological tool for various industries.

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Wu, Jin-Chao, Jie Cheng, and Xiao-lai Shi. "Preparation of ACE Inhibitory Peptides fromMytilus coruscusHydrolysate Using Uniform Design." BioMed Research International 2013 (2013): 1–6. http://dx.doi.org/10.1155/2013/290120.

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The angiotensin-I-converting enzyme (ACE) inhibitory peptides from mussel,Mytilus coruscus, were investigated and the variable factors, protease concentration, hydrolysis time, pH, and temperature, were optimized using Uniform Design, a new statistical experimental method. The results proved that the hydrolysate of alkali proteases had high ACE-inhibitory activity, especially the alkali protease E1. Optimization by Uniform Design showed that the best hydrolysis conditions for preparation of ACE-inhibitory peptides fromMytilus coruscuswere protease concentration of 36.0 U/mL, hydrolysis time of 2.7 hours, pH 8.2, and Temperature at 59.5°C, respectively. The verification experiments under optimum conditions showed that the ACE-inhibitory activity (91.3%) were agreed closely with the predicted activity of 90.7%. The amino acid composition analysis ofMytilus coruscusACE-inhibitory peptides proved that it had high percent of lysine, leucine, glycine, aspartic acid, and glutamic acid.

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Hong, Tran Thi, Ton That Huu Dat, Nguyen Phuong Hoa, Tran Thi Kim Dung, Vu Thi Thu Huyen, Le Minh Bui, Nguyen Thi Kim Cuc, and Pham Viet Cuong. "Expression and characterization of a new serine protease inhibitory protein in Escherichia coli." Biomedical Research and Therapy 7, no. 2 (February 29, 2020): 3633–44. http://dx.doi.org/10.15419/bmrat.v7i2.590.

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Introduction: Proteases are enzymes that catalyze the hydrolysis of peptide bonds and play an important role in almost all biological processes. However, excessive protein proteolysis can be implicated in several diseases, such as cancer, as well as cardiovascular, inflammatory, neurodegenerative, bacterial, viral and parasitic diseases. In these cases, protease inhibitors can be used as one of versatile tools for regulating proteolytic activity of target proteases as well as therapeutic applications. In this study, we expressed and characterized a new serine protease inhibitory protein (PI-QT) from the metagenome of sponge-associated microorganisms in Escherichia coli. Methods: The gene PI-QT encoding for a new serine protease inhibitory protein was expressed in E. coli BL21(DE3). In addition, the expressed protein was purified and characterized. Results: Optimization of expression of the recombinant protein PI-QT in E. coli showed that suitable conditions for expression of the protein were pre-induction cell density (OD600) of 0.6 - 0.7, IPTG concentration of 1 mM and temperature of 25oC. The protease inhibitory protein was also purified and identified by mass spectrometry LC-MS/MS. The recombinant protein showed inhibitory activity against trypsin anda-chymotrypsin with activity values of 97526 U/mg and 41714 U/mg, respectively. Maximum activity of the protease inhibitory protein was obtained at pH 7 and temperature 20-35oC. The inhibitor was stable over pH 4-9 and up to temperature 50oC. Addition of Zn2+, Mg2+ and Ca2+ enhanced inhibitory activity, whereas other metal ions, surfactants and oxidants reduced inhibitory activity of the protease inhibitor. Conclusion: The recombinant protein PI-QT is a potential protease inhibitor for therapeutic applications.

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Rathakrishnan, P., P. Nagarajan, and Rajesh Kannan. "Response surface optimization of medium composition for protease production by Bacillus subtilis using cassava waste." Chemical Industry and Chemical Engineering Quarterly 17, no. 2 (2011): 215–22. http://dx.doi.org/10.2298/ciceq100927006r.

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Optimization of the growth condition for maximum growth rate and protease production was carried out using Bacillus subtilis. The optimization of protease production using agro industrial waste product such as cassava waste as substrate was performed with statistical methodology based on experimental designs. The screening of twelve nutrients for their influence on protease production was achieved using a Plackett-Burman design. MgSO4.7H2O, casein and glucose were selected based on their positive influence on protease production. The selected components were optimized using Response Surface Methodology (RSM). The optimum conditions are (% w/w): MgsO4.7H2O- 0.14, casein- 1.4 and glucose- 2.64. These conditions were validated experimentally which revealed an enhanced protease yield of 202.048 U/gds.

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Dorcas, Kusuma, and Pavan Kumar Pindi. "Optimization of Protease Production from Bacillus cereus." International Journal of Current Microbiology and Applied Sciences 5, no. 6 (June 10, 2016): 470–78. http://dx.doi.org/10.20546/ijcmas.2016.506.054.

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Sherrill, Ronald G., C. Webster Andrews, William J. Bock, Ronda G. Davis-Ward, Eric S. Furfine, Richard J. Hazen, Randy D. Rutkowske, Andrew Spaltenstein, and Lois L. Wright. "Optimization of pyrrolidinone based HIV protease inhibitors." Bioorganic & Medicinal Chemistry Letters 15, no. 1 (January 2005): 81–84. http://dx.doi.org/10.1016/j.bmcl.2004.10.029.

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Vasantha, Soumya Thrivikraman, and Abhilash Thankappan Subramanian. "Optimization of cultural conditions for the production of an extracellular protease by Pseudomonas species." International Current Pharmaceutical Journal 2, no. 1 (December 8, 2012): 1–6. http://dx.doi.org/10.3329/icpj.v2i1.12870.

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The aim of the study was to identify new sources of protease and a protease producing bacteria was isolated from slaughter house soil and identified as Pseudomonas species. The protease production efficiency of the organism was measured with different environmental and nutritional parameters. Optimization of the fermentation medium for maximum protease production was carried out. The culture condition like temperature, pH, incubation time, carbon and nitrogen sources were optimized. The optimum condition found for protease production was 30°C at pH 7 in the medium for 48 hour. Carbon source casein and nitrogen source beef extract stimulated the production of protease. Protease production was higher with immobilized cells. Pseudomonas species can be used profitably for the large scale production of protease to meet the present day demand of the industrial sector.DOI: http://dx.doi.org/10.3329/icpj.v2i1.12870 International Current Pharmaceutical Journal 2012, 2(1): 1-6

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Ainutajriani, Ainutajriani, Sri Darmawati, Dewi Seswita Zilda, Muhammad Ardi Afriansyah, Ragil Saptaningtyas, and Stalis Norma Ethica. "PRODUCTION OPTIMIZATION, PARTIAL PURIFICATION, AND THROMBOLYTIC ACTIVITY EVALUATION OF PROTEASE OF Bacillus cereus HSFI-10." BIOTROPIA 30, no. 2 (August 1, 2023): 147–57. http://dx.doi.org/10.11598/btb.2023.30.2.1765.

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Cardiovascular disease is the primary cause of mortality in the world due to the formation of blood clots or thrombi in blood vessels. Bacterial proteases commonly function as thrombus dissolver agents in the pharmaceutical industry. Bacterial isolate HSFI-10 (Holothuria scabra Fermented Intestine-10) previously isolated from Rusip fermented sea cucumber had demonstrated thrombolytic activity. This study aimed to produce crude protease of HSFI-10 strain at an optimized incubation time and determine the thrombolytic activity of crude and dialysate proteases on A, B, AB, and O blood types. Isolate HSFI-10 was first molecularly identified and found to be Bacillus cereus with a homology level of 99.80% with Bacillus cereus strain ST06. The optimum crude enzyme was obtained after 48-h incubation with an activity of 222.52 U/mL, which increased to 438.84 U/mL after ammonium sulfate precipitation and dialysis. Clot lysis activity of crude enzymes was measured based on the gravimetry method on blood in the ABO system, showing results that ranged from 68.99% to 69.76%, while the dialysate ranged from 81.16% to 82.52%. In conclusion, partial purification of bacterial protease could increase both its specific and thrombolytic activities on human blood in the ABO system, with only 1% activity variability between A, B, AB, and O blood types.

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Bezerra, Victor Hugo Souto, Samuel Leite Cardoso, Yris Fonseca-Bazzo, Dâmaris Silveira, Pérola Oliveira Magalhães, and Paula Monteiro Souza. "Protease Produced by Endophytic Fungi: A Systematic Review." Molecules 26, no. 22 (November 22, 2021): 7062. http://dx.doi.org/10.3390/molecules26227062.

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The purpose of this systematic review was to identify the available literature of production, purification, and characterization of proteases by endophytic fungi. There are few complete studies that entirely exhibit the production, characterization, and purification of proteases from endophytic fungi. This study followed the PRISMA, and the search was conducted on five databases: PubMed, PMC, Science Direct, Scopus Articles, and Web of Science up until 18 May 2021, with no time or language restrictions. The methodology of the selected studies was evaluated using GRADE. Protease production, optimization, purification, and characterization were the main evaluated outcomes. Of the 5540 initially gathered studies, 15 met the inclusion criteria after a two-step selection process. Only two studies optimized the protease production using statistical design and two reported enzyme purification and characterization. The genus Penicillium and Aspergillus were the most cited among the eleven different genera of endophytic fungi evaluated in the selected articles. Six studies proved the ability of some endophytic fungi to produce fibrinolytic proteases, demonstrating that endophytic fungi can be exploited for the further production of agents used in thrombolytic therapy. However, further characterization and physicochemical studies are required to evaluate the real potential of endophytic fungi as sources of industrial enzymes.

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Ratnaningrum, D., W. Kosasih, E. S. Endah, A. K. N. Lathifa, A. M. Diwan, V. Nida, V. Saraswaty, and C. Risdian. "Protease production by soil bacteria for green technology: Screening and optimization." IOP Conference Series: Earth and Environmental Science 1201, no. 1 (June 1, 2023): 012094. http://dx.doi.org/10.1088/1755-1315/1201/1/012094.

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Abstract Protease is a type of enzyme that hydrolyzes proteins into peptides and amino acids. The demand for protease for green technology in some industries like detergent, food, and leather is increasing nowadays. Some soil bacteria, especially the Bacillus strains, are known to have the ability to produce protease. Compared to other sources like plant and animal origin, microbial protease has more advantages as it can be produced at an industrial scale, short cultivation time, and are easy to harvest. This research aimed to screen the proteolytic bacteria from the bulk soil samples collected under mango trees and to study the optimum condition for protease production using the response surface methodology (RSM). Three bacterial strains (SH2CR, SH3CR, and SC4CR) were isolated and shown to have proteolytic activity. Based on 16S rRNA gene analysis, the strain SH2CR was close to Priestia megaterium NBRC 15308T (96.97% similarity), while the strains SH3CR and SC4CR were related to Bacillus zanthoxyli strain 1433T (100% similarity). One of them, SH2CR, was further studied using a fermenter at a one-liter production scale with the medium containing skim milk at 30°C. The best condition for protease production by SH2CR was achieved at 48 h incubation time, 300 rpm of agitation, and 1.25% skim milk.

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Bagdonas, Martynas, Kamilė Čerepenkaitė, Aurelija Mickevičiūtė, Rūta Kananavičiūtė, Birutė Grybaitė, Kazimieras Anusevičius, Audronė Rukšėnaitė, et al. "Screening, Synthesis and Biochemical Characterization of SARS-CoV-2 Protease Inhibitors." International Journal of Molecular Sciences 24, no. 17 (August 30, 2023): 13491. http://dx.doi.org/10.3390/ijms241713491.

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The severe acute respiratory syndrome-causing coronavirus 2 (SARS-CoV-2) papain-like protease (PLpro) and main protease (Mpro) play an important role in viral replication events and are important targets for anti-coronavirus drug discovery. In search of these protease inhibitors, we screened a library of 1300 compounds using a fluorescence thermal shift assay (FTSA) and identified 53 hits that thermally stabilized or destabilized PLpro. The hit compounds structurally belonged to two classes of small molecules: thiazole derivatives and symmetrical disulfide compounds. Compound dissociation constants (Kd) were determined using an enzymatic inhibition method. Seven aromatic disulfide compounds were identified as efficient PLpro inhibitors with Kd values in the micromolar range. Two disulfides displayed six-fold higher potency for PLpro (Kd = 0.5 µM) than for Mpro. The disulfide derivatives bound covalently to both proteases, as confirmed through mass spectrometry. The identified compounds can serve as lead compounds for further chemical optimization toward anti-COVID-19 drugs.

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Sana Ghorri, Ouided Benslama, Ouafa BENSERRADJ, and Ilhem Mihoubi. "Application of Plackett-Burman design for the optimization of protease production by Aspergillus niger." South Asian Journal of Experimental Biology 12, no. 4 (August 4, 2022): 515–21. http://dx.doi.org/10.38150/sajeb.12(4).p515-521.

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Proteolytic enzymes represent one of the largest groups of industrial enzymes. Fungal proteases have been used for the large-scale production of industrial enzymes. The optimization of enzyme production is a very important objective, where the aim is to further improve the production process. Therefore, the application of an experimental statistical method is essential. In this study, the production of the enzyme protease was carried out by fermentation of a mold (Aspergillus niger) using two media, one based on wheat bran and the other based on peas chick. The optimization of the synthesis of the enzyme was carried out using a statistical method of experimental planning based on the Plackett and Burman matrices. The variables used are the 3 production factors temperature, pH, and substrate concentration. The results were modeled according to multiple linear regression. The results obtained revealed that the strain A.niger gave an excellent activity reaching 740 IU for the wheat bran medium and 194.54 IU for the chickpea medium. The study of pH optima and incubation temperature has shown that the protease produced by A. niger has an optimum pH equal to 6 and an optimum temperature of 40°C for wheat bran medium and has an optimum pH equal to 5 and an optimum temperature of 30°C for chickpeas. The results obtained from the experimental designs of Plackett and Burman proved to be ideal for the selection of factors influencing protease production.

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Jiang, Hai Tao, Zhong Ping Qiu, Yang Liu, Zheng Jun Gong, and Yuan Yue Liu. "Optimization of Determination Conditions for Protease in Landfill." Advanced Materials Research 750-752 (August 2013): 1499–504. http://dx.doi.org/10.4028/www.scientific.net/amr.750-752.1499.

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Protease was a key hydrolase in nitrogen transformation in landfill. The activity could show transformation states of nitrogen, that had important significance to study the biotransformation of organic nitrogenous substances in landfill. This paper designed six factors and five levels (L25 (56)) orthogonal test to measure the activity of protease in landfill. The results showed that the optimal experimental conditions for protease in landfill were 16h of culture period, 30°C of culture temperature, 6mL 1% casein, 0.3mL toluol, 0.2mL 0.1N H2SO4 and 1.5mL 20% Na2SO4. The experimental results were 84.23% to 368.86% more compared with that by the method of traditional method. In the optimized tests, the Relatively Standard Deviation (RSD) was less than 3.37%, and average recovery rates was 97.15%. The result showed the designed optimal experiments could provide more accurate and stable experimental data.

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BalaKumaran, M. D., and R. Santhi. "Keratinolytic Protease Production by Bacillus Cereus Strain Ps03 under Submerged Fermentation: Optimization and Characterization." International Journal of Applied Sciences and Biotechnology 4, no. 3 (September 26, 2016): 397–401. http://dx.doi.org/10.3126/ijasbt.v4i3.15780.

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In the present study, chicken feather powder was screened for its application as the substrate for the production of keratinolytic protease by Bacillus subtilis strain PS03. Bacillus subtilis produced a high level of keratinolytic protease using chicken feather powder as substrate. With feather powder as substrate, physical factors such as incubation time, pH and temperature were optimized for increased keratinolytic protease production by Bacillus subtilis. The enzyme production was enhanced when using maltose as carbon source and yeast extract as nitrogen sources. SDS-PAGE analysis indicated the molecular weight of 46 kDa of the partially purified keratinolytic protease. The keratinolytic protease enzyme was stable over a pH range of 6 – 9 and temperature range of 35 - 50°C with maximum activity at pH 9 and 40°C. Based on the results, the use of feather powder as substrate for keratinolytic protease production is cost effective and is easy to scale up. Considering the availability and cost, chicken feather powder is considered as an ideal substrate for keratinolytic protease production in an industrial point of view. Int J Appl Sci Biotechnol, Vol 4(3): 397-401

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Sharma, Chhavi, Gad Elsayed Mohamed Salem, Neha Sharma, Prerna Gautam, and Rajni Singh. "Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1." Biomolecules 10, no. 1 (December 18, 2019): 3. http://dx.doi.org/10.3390/biom10010003.

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The present study demonstrates the production and thrombolytic potential of a novel thermostable thiol-dependent fibrinolytic protease by Bacillus cereus RSA1. Statistical optimization of different parameters was accomplished with Plackett–Burman design and validated further by central composite design with 30.75 U/mL protease production. Precipitation and chromatographic approaches resulted in 33.11% recovery with 2.32-fold purification. The molecular weight of fibrinolytic protease was 40 KDa and it exhibited a broad temperature and pH stability range of 20–80 °C and pH 5–10 with utmost activity at 50 °C and pH 8, respectively. The protease retained its fibrinolytic activity in organic solvents and enhanced the activity in solutions with divalent cations (Mn2+, Zn2+, and Cu2+). The enzyme kinetics revealed Km and Vmax values of 1.093 mg/mL and 52.39 µg/mL/min, respectively, indicating higher affinity of fibrinolytic activity towards fibrin. Also, complete inhibition of fibrinolytic activity with DFP and a 2-fold increase with DTT and β-mercaptoethanol indicates its thiol-dependent serine protease nature. MALDI–TOF analysis showed 56% amino acid sequence homology with Subtilisin NAT OS = Bacillus subtilis subsp. natto. The fibrinolysis activity was compared with a commercial thrombolytic agent for its therapeutic applicability, and fibrinolytic protease was found highly significant with absolute blood clot dissolution within 4 h in in vitro conditions. The isolated fibrinolytic protease of Bacillus cereus RSA1 is novel and different from other known fibrinolytic proteases with high stability and efficacy, which might have wide medicinal and industrial application as a thrombolytic agent and in blood stain removal, respectively.

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Maskey, Bunty, and Nabindra Kumar Shrestha. "Optimization of Crude Papaya (Carica papaya) Protease in Soft-Unripened Cheese Preparation." Journal of Food Science and Technology Nepal 12, no. 12 (December 19, 2020): 1–8. http://dx.doi.org/10.3126/jfstn.v12i12.30139.

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The use of plant protease instead of chymosin for producing cheese has become a trend which is aimed at lacto-vegetarian consumers and religion based ecological markets. In this context, the present investigation was carried out in order to utilize milk clotting enzyme from Papaya (Carica papaya). Numerical optimization study revealed that maximum milk clotting activity was achieved at pH 6.5, temperature 70℃ and enzyme concentration 1 g/1000 ml milk using papaya protease as coagulant. Protein, ash and calcium showed no significant (p>0.05) difference among the cheeses made using different coagulants. However, significantly (p<0.05) higher levels of moisture and ash, and lower levels of fat were observed in the cheese produced by papaya protease compared to that made using rennet. Papaya protease significantly enhanced the spreadability of cheese while the other sensory properties were similar to the control except aftertaste. The results revealed that the papaya latex as crude papaya protease may have potential application for the manufacture of soft-unripened cheese and further could be utilized as a milk coagulant in cheese making.

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Pranaw, Kumar, Surender Singh, Debjani Dutta, Surabhi Chaudhuri, Sudershan Ganguly, and Lata Nain. "Statistical Optimization of Media Components for Production of Fibrinolytic Alkaline Metalloproteases from Xenorhabdus indica KB-3." Biotechnology Research International 2014 (April 23, 2014): 1–11. http://dx.doi.org/10.1155/2014/293434.

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Xenorhabdus indica KB-3, a well-known protease producer, was isolated from its entomopathogenic nematode symbiont Steinernema thermophilum. Since medium constituents are critical to the protease production, the chemical components of the selected medium (soya casein digest broth) were optimized by rotatable central composite design (RCCD) using response surface methodology (RSM). The effects of all five chemical components (considered as independent variables), namely tryptone, soya peptone, dextrose, NaCl, and dipotassium phosphate, on protease production (dependent variable) were studied, and it was found that tryptone and dextrose had maximum influence on protease production. The protease production was increased significantly by 66.31% under optimal medium conditions (tryptone—5.71, soya peptone—4.9, dextrose—1.45, NaCl—6.08, and dipotassium phosphate—0.47 in g/L). To best of knowledge, there are no reports on optimization of medium component for protease production by X. indica KB-3 using RSM and their application in fibrinolysis. This study will be useful for industrial processes for production of protease enzyme from X. indica KB-3 for its application in the field of agriculture and medicine.

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Jingying, Chen, and Gu Yan. "Optimization of fermentation conditions for protease production from Bacillus subtilis." BIO Web of Conferences 59 (2023): 01006. http://dx.doi.org/10.1051/bioconf/20235901006.

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In the paper, the fermentation conditions and fermentation medium of bacillus subtilis protease production were optimized to improve the outcome of the protease produced from bacillus subtilis to provide premium raw materials for the subsequent preparation of chitin from crab shell. The optimal fermentation conditions are temperature at 39°C, pH value of 7.5, fermentation time of 60h, inoculum size of 5%, filling volume of 80/250mL, and rotational speed at 180r/min; the optimal medium component ratio is 1.25% for starch mass concentration, 1% for yeast paste mass concentration, and 0.3% for sodium dihydrogen phosphate mass concentration. The activity of protease produced from bacillus subtilis GC021 is 174.87U/mL under the optimal fermentation conditions and at the optimal medium component ratio.

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Puntambekar, Ashwini Nilesh, and Manjusha Sudhakar Dake. "ISOLATION, PURIFICATION, AND OPTIMIZATION OF THERMOPHILIC AND ALKALIPHILC PROTEASE ORIGINATING FROM HOT WATER SPRING BACTERIA." Asian Journal of Pharmaceutical and Clinical Research 10, no. 9 (September 1, 2017): 284. http://dx.doi.org/10.22159/ajpcr.2017.v10i9.19717.

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Objective: The main objective of this study is to investigate the industrial applications of a thermophillic alkaline protease from a hot water spring bacterial isolate “A” and to study its production, optimization, and purification.Methods: The alkaline protease was produced using shake flask studies maintaining a pH of 9.0 and a temperature of 50°C. Optimization studies of the enzyme were carried out using variable pH, temperature, organic carbon, and nitrogen sources followed by purification of the enzyme using DEAE-cellulose ion exchange chromatography technique. Stability of the enzyme was analyzed in the presence of organic solvents and surfactants. The efficiency of the enzyme in the removal of proteinaceous stains in the presence of strong detergents under extreme conditions was assessed. The fibrinolytic activity of the enzyme in dissolving the blood clot was confirmed.Results: The isolated alkaline protease was purified to homogeneity with a 16-fold increase. Media optimization studies revealed that 1% glucose and 1 % casein-induced the production of alkaline protease. The purified enzyme retained stability in the presence of ethanol, methanol, and acetone and surfactants such as 0.5% (w/v) sodium dodecyl sulfate (SDS) and 0.5% (v/v) Triton-X-100. The isolated alkaline protease successfully removed the proteinaceous stains and showed significant results in the dissolution of blood clot.Conclusion: The above experimental results confirm that the isolated enzyme has both thermophilic and alkaliphilic protease properties. Thereby the enzyme finds promising industrial applications even in extreme conditions.

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Lee, Sulhee, Dong-Hun Jang, Hyuk Jun Choi, and Young-Seo Park. "Optimization of Soymilk Fermentation by the Protease-producing Lactobacillus paracasei." Korean Journal of Food Science and Technology 45, no. 5 (October 31, 2013): 571–77. http://dx.doi.org/10.9721/kjfst.2013.45.5.571.

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Zhang, Hai Yue, Tian Tian, and Hua Chen. "Optimization of Enzymatic Hydrolysis for Protein from Black Bean by Response Surface Methodology." Advanced Materials Research 781-784 (September 2013): 875–79. http://dx.doi.org/10.4028/www.scientific.net/amr.781-784.875.

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Taking the degree of hydrolysis (DH) as the index, the optimal hydrolysis process for the protein from black bean was explored by response surface methodology (RSM) to prepare polypeptides. Four proteases were used to hydrolyze the black bean protein and determine the DH of black bean protein. The results indicated that the optimal reaction systems were as follows: The black bean protein solution was treated with the alkaline protease hydrolysis firstly and then the trypsin hydrolysis was used in order to improve the DH. The DH of protein was increased to 35.64% and the yield of peptides production was higher than using only alcalase.

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Liu, Hui, Xiaomiao Shi, Dongmei Guo, Zuowei Zhao, and Yimin. "Feature Selection Combined with Neural Network Structure Optimization for HIV-1 Protease Cleavage Site Prediction." BioMed Research International 2015 (2015): 1–11. http://dx.doi.org/10.1155/2015/263586.

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It is crucial to understand the specificity of HIV-1 protease for designing HIV-1 protease inhibitors. In this paper, a new feature selection method combined with neural network structure optimization is proposed to analyze the specificity of HIV-1 protease and find the important positions in an octapeptide that determined its cleavability. Two kinds of newly proposed features based on Amino Acid Index database plus traditional orthogonal encoding features are used in this paper, taking both physiochemical and sequence information into consideration. Results of feature selection prove thatp2,p1,p1′, andp2′are the most important positions. Two feature fusion methods are used in this paper: combination fusion and decision fusion aiming to get comprehensive feature representation and improve prediction performance. Decision fusion of subsets that getting after feature selection obtains excellent prediction performance, which proves feature selection combined with decision fusion is an effective and useful method for the task of HIV-1 protease cleavage site prediction. The results and analysis in this paper can provide useful instruction and help designing HIV-1 protease inhibitor in the future.

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PAGARRA, HALIFAH. "Isolation and Optimization of Endophytic Bacteria from Roots of Karst Area Ecosystems Producing Protease Enzymes." Journal of Research on the Lepidoptera 51, no. 2 (May 15, 2020): 431–39. http://dx.doi.org/10.36872/lepi/v51i2/301110.

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Singh, Rajnish Prakash, and Prabhat Nath Jha. "Characterization and Optimization of Alkaline Protease Production from Bacillus licheniformis HSW-16 Isolated from Sambhar Salt Lake." International Journal of Applied Sciences and Biotechnology 3, no. 2 (June 25, 2015): 347–51. http://dx.doi.org/10.3126/ijasbt.v3i2.12757.

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Halophilic microorganisms are recognized as potential source of secondary metabolites including enzymes and drugs with wide agricultural and industrial applications. In the present study protease producing halotolerant bacterium Bacillus licheniformis HSW-16 was isolated from hypersaline Sambhar lake, Rajasthan India. Protease production was performed by using azocasein as substrate. Confirmation of protease production was also done by amplification of alkaline protease gene and sequencing. The various nutritional factors such as carbon and nitrogen source and other physiological parameters like pH, temperature, incubation time and agitation speed were optimized for optimum protease production. The enzyme was active in pH range 7-10, temperature 25 °C-40 °C and salt concentration of 1.5M. The characteristics demonstrated by this isolate showed that it could be used as a potential source of enzyme.Int J Appl Sci Biotechnol, Vol 3(2): 347-351 DOI: http://dx.doi.org/10.3126/ijasbt.v3i2.12757

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Seung-Ki Kim, 김진우, Song-Yi Kim, Jae Min Jo, and Da Hae Gam. "Isolation of Protease Producing Bacillus from Korean Bean Paste and Optimization of Protease Production Conditions." Journal of Advanced Engineering and Technology 11, no. 3 (September 2018): 205–11. http://dx.doi.org/10.35272/jaet.2018.11.3.205.

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Liu, Hao, Chunhui Lv, Peng Liu, Lihui Wang, and Peng Wang. "Optimization of the Process of Compound Enzymatic Hydrolysis of Soluble Protease Preparation." E3S Web of Conferences 131 (2019): 01024. http://dx.doi.org/10.1051/e3sconf/201913101024.

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soluble protease preparations, including trypsin, papain and other preparations, are widely used in various fields, such as medicine, agriculture, industry and so on. Based on this, this paper takes the soluble protease as the main research object, and uses the experimental method of compound enzymolysis to observe and analyze the utilization of egg membrane protein, so as to improve the utilization rate of egg membrane protein.

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A. A. Al-Zahrani, Hind. "Optimization conditions of alkaline protease production by Streptomyces sp.H1 isolated from red sea coastal region in submerged culture." International Journal of Basic and Applied Sciences 7, no. 3 (August 23, 2018): 48. http://dx.doi.org/10.14419/ijbas.v7i3.14519.

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A potent alkaline protease producing strain characterized and identified as Streptomyces sp. H1 was isolated from soil around red sea shore. The enzyme was produced extracellulary in submerged culture revealed maximum level during early stationary phase. Alkaline protease showed the highest activity at incubation time, pH and inoculum size of 3 days, 9 and 8% respectively. Among different carbon sources beet molasses gave a maximum production followed by starch, sucrose and fructose. High yield of protease production was noticed with casein followed by peptone, yeast extract and ammonium sulphate. Furthermore, it was optimized with 7g/l NaCl resulted in higher level of protease. Optimization of the process parameters resulted in about 3.4 fold increase in the alkaline protease. Partial purification of the crude enzyme was achieved by fractional precipitation using ammonium sulfate at 50% saturation. Due to the maximum production of protease in the presence of cheaper substrate as beet molasses, stability at alkaline pH 9 and temperature up to 70 oC besides salt tolerance make the strain and its enzyme useful in different industrial applications.

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Kumari, Saravana, and Reshma R. "Effect of alkaline protease produced from fish waste as substrate by Bacillus clausii on destaining of blood stained fabric." Journal of Tropical Life Science 11, no. 1 (February 3, 2021): 59–66. http://dx.doi.org/10.11594/jtls.11.01.08.

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Alkaline protease or peptidases are the largest groups of enzymes in the biological industry with a variety of application in manufacturing units used in the process of substrate stabilization, dehairing, diagnosis, extraction, food production, destaining, etc., where the pH of the environmental conditions remain above neutral pH. Because of these wider applications of alkaline proteases in industries, their demand is increasing to compete with their chemical counterpart. An alkaline tolerant bacterial strain Bacillus clausii wasisolated from fish waste and used for mass production of alkaline protease using fish waste homogenate as media. Preliminary study on optimization of conditions for the mass production carried out. The optimum temperature for production ranges between 25°C and 35°C and pH determined as 9. The mass production of extracellular alkaline protease carried out using mobilized and immobilized cells of B. clausii at optimized condition using production media, the mixture of production media and fish waste homogenate and in nutrient broth. The recorded results showed that the maximum enzyme production obtained with immobilized cells in nutrient broth media and followed by fish waste homogenate media of 8900 U/ml and 8600 U/ml. Purification protease enzyme yielded 0.35 g/ml from the production media . Bloodstained cloth treated with immobilized enzyme removed the stain completely compared to treatment with non-immobilized enzyme and commercially used detergent. So, the current study suggests the usage of microbial alkaline protease in household detergents to replace the usage of synthetic detergents and save the environment from chemical pollutants.

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48

Kozlov, Igor, Peter Melnyk, Chanfeng Zhao, John Hachmann, Veronika Shevchenko, Anu Srinivasan, David Barker, and Michal Lebl. "A Method for Rapid Protease Substrate Evaluation and Optimization." Combinatorial Chemistry & High Throughput Screening 9, no. 6 (July 1, 2006): 481–87. http://dx.doi.org/10.2174/138620706777698535.

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49

Komiyama, Tomoko, Bryan VanderLugt, Martin Fugère, Robert Day, Randal J. Kaufman, and Robert S. Fuller. "Optimization of protease-inhibitor interactions by randomizing adventitious contacts." Proceedings of the National Academy of Sciences 100, no. 14 (June 27, 2003): 8205–10. http://dx.doi.org/10.1073/pnas.1032865100.

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Pant, Gaurav, Anil Prakash, J. V. P. Pavani, Sayantan Bera, G. V. N. S. Deviram, Ajay Kumar, Mitali Panchpuri, and Ravi Gyana Prasuna. "Production, optimization and partial purification of protease fromBacillus subtilis." Journal of Taibah University for Science 9, no. 1 (January 2015): 50–55. http://dx.doi.org/10.1016/j.jtusci.2014.04.010.

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Journal articles on the topic 'Optimization of protease'

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