Maximum Enzyme Production Research Articles

Characterization of partially purified alkaline protease secreted by halophilic bacterium Citricoccus sp. isolated from agricultural soil of northern India

Abstract The present study reports the isolation of protease producing bacteria Bact2 from agricultural soil of Regional Centre of Soil Salinity Research Institute, Lucknow, India. It was identified as Citricoccus sp. (KC522120.1) by 16S rRNA gene sequencing. The optimum pH and temperature for maximum enzyme production was observed to be at 10 and 40 °C respectively. The enzyme protease was partially purified and recovery of enzyme was 77.16%. A 1.66 fold increase in enzyme activity was also observed in comparison to original. After purification, the partially purified enzyme (PPE) was characterized. The optimum pH and temperature of partially purified enzyme (PPE) activity was observed to be at 10 and 40 °C respectively. The activity of PPE was enhanced in presence of Ca2+ and Mg2+ while it was inhibited by Cu++ and Na+. The PPE was completely inhibited by Phenyl methyl sulphonyl fluoride (PMSF) indicating that it belongs to serine protease group. Finally, enzyme was found to be compatible with detergents and effective in removing stains of tea and ink indicating it's application in detergent industry. Besides, to our knowledge till date production of alkaline protease from Citricoccus sp. has been reported first time in the present manuscript.

Statistical optimization of xylanase and alkaline protease co-production by Bacillus spp using Box-Behnken Design under submerged fermentation using wheat bran as a substrate

Abstract This study explores the co-production of xylanase and alkaline protease from Bacillus licheniformis NRRL 14209 in single step using agricultural waste as a substrate. The aim of the present study was to screen the low cost and easily available agricultural lignocellulosic wastes as a substrate to substitute commercially available xylan. Five agricultural wastes were screened as a substrate and wheat bran was found to be most suitable for co-production of xylanase and alkaline protease. Optimization of various fermentation parameters affecting enzyme co-production by B. licheniformis was investigated. Maximum enzyme productivity of xylanase 4.31 U/mL and alkaline protease 3.66 U/mL was obtained at 24 h of incubation period, initial media pH 8.5 with 0.5% w/v wheat bran and 4% (v/v) inoculum concentration at 30 °C temperature. Box-Behnken Design was applied to obtain optimal concentrations of significant variables such as substrate concentration (0.25% w/v), inoculum concentration (4%v/v) and pH of 7.5. The maximum xylanase and alkaline protease production after Box-Behnken Design was 5.49 U/mL and 4.87 U/mL respectively, which are 1.27 and 1.33 fold higher than initial one factor at a time media optimization. Xylanase and alkaline protease isolated in this study can be useful in pulp pre-bleaching process to remove the hemicelluloses and in detergent industry respectively.

Hydrolytic Enzyme Production by Thermophilic Bacteria Isolated from Saudi Hot Springs.

Hydrolytic enzyme production by thermophilic bacteria isolated from hot springs in the southern region of Saudi Arabia was investigated. The physical and chemical properties of the hot springs prove to be an important environment for hydrolytic-enzyme-producing thermophilic bacteria. Eighty-four bacterial isolates were obtained from three hot springs: Al-Majardah, Al-Khubah and Al-Ardah. Screening of the isolates for enzyme production indicated that 78 isolates showed activity for one or more enzymes. Molecular identification and phylogenic analysis of selected promising isolates confirmed the identity of the isolates as Bacillus aerius, Bacillus licheniformis and Bacillus sonorensis, which have potential to produce the target enzymes α-amylase, protease and lipase, respectively. Optimization of hydrolytic enzyme production by bacterial strains was investigated using kitchen waste as a cheap carbon energy source. Maximum enzyme production was achieved after 72 hours of incubation at the beginning of the stationary phase of growth. Enzyme production was dependent on the initial pH value in the range of pH 7.5-8.5 and an optimal incubation temperature of between 55-60°C. Enzyme production increased gradually in proportion to the kitchen waste concentration; whereas maximum lipase production was gained at 5.0% (w/v) kitchen waste, 7.0% (w/v) of waste was optimal for both α-amylase and protease productivity. The results indicated that hot springs in Saudi Arabia are a substantial source of thermophilic bacteria producing industrially important enzymes using cheap and unexploited waste.

Poly(l-lactide)-Degrading Enzyme Production by Laceyella sacchari LP175 Under Solid State Fermentation Using Low Cost Agricultural Crops and Its Hydrolysis of Poly(l-lactide) Film

The aim of this study was to investigate the enhancement of poly(l-lactide) (PLLA)-degrading enzyme production by Laceyella sacchari LP175 under solid state fermentation (SSF) using low-cost agricultural crops as substrates and its hydrolysis of poly(l-lactide) film. Cassava chip, soybean meal and corncob were used to investigate the enzyme production using a statistical mixture design method. The effect of various inducers and SSF growth conditions parameters affecting PLLA-degrading enzyme production were also investigated. The fermentation of solid substrates was up-scale in a static tray bioreactor. The crude enzyme extracted from the fermented solid substrate was used to evaluate the biological degradation of PLLA film at 50 °C for 24 h. The results of substrate combination showed that 5 g of the substrate mixture, consisting of 4.3 g cassava chips and 0.7 g soybean meal, yielded 320 U/g dry solid. The addition of 0.1 g peptone to the mixture of solid materials increased enzyme production up to 456 U/g dry solid. The production of enzyme in a static tray bioreactor with optimized physical conditions yielded the maximum enzyme production, 472 U/g dry solid. The 20 mL reaction consisting of 8600 mg/L PLLA film was degraded by the crude enzyme extracted from the fermented solid substrate. Agricultural crops and wastes contain significant amounts of nutrients for microbial growth and products. This is the first reported of PLLA degrading enzyme production under SSF using low-cost substrates showing the possibility for application in large-scale biological recycling of bio-plastic as a future sustainable process.

Process modeling and optimization of high yielding L-methioninase from a newly isolated Trichoderma harzianum using response surface methodology and artificial neural network coupled genetic algorithm

Abstract A high yielding L-methioninase producing fungus was isolated from soil samples and was identified as Trichoderma harzianum. The enzyme was purified using chromatographic techniques and the purified enzyme had an apparent molecular mass of 48 kDa on SDS PAGE. The purified L-methioninase showed a specific activity of 74.4 U/mg, which is the highest among other L-methioninase, reported and can be a potential candidate as an anticancer agent. The media components such as lactose, L -methionine, KH2PO4, K2HPO4 and Zinc chloride was found to have a significant effect on enzyme production by classical optimization method. Response surface methodology and artificial neural network linked genetic algorithm was employed to develop an optimized medium for L-methioninase production. Maximum enzyme production obtained using RSM was 30.2 U/ml at medium composition of 12.5 g/l of lactose, 10 g/l of L -methionine, 2 g/l of KH2PO4, 4 g/l of K2HPO4 and 0.0125 g/l of zinc chloride. ANN model was found to be superior to RSM with a higher coefficient of determination (R2) of 0.995, lower RSME (0.306) and MSE (0.093). A higher enzyme production of 33.32 U/ml was achieved using ANN-GA optimized medium; 13.9 g/l of lactose, 11.37 g/l of L -methionine, 1.58 g/l of KH2PO4, 3.98 g/l of K2HPO4 and 0.01 g/l of zinc chloride, which is in agreement with the predicted value of 33.76 U/ml.

Characterization and optimization of extracellular L-Asparaginase production by selected Actinomycete strain isolated from an algerian wheat bran

L-Asparaginase is an enzyme that hydrolyses the amino acid L-Asparagine into aspartic acid and ammonia. As a medication, L-Asparaginase is used in chemotherapy to treat acute lymphoblastic leukaemia by depleting circulating Asparagine and depriving tumor cells. Interest in Actinomycetes as potential producers of antibiotics and enzymes encouraged us to investigate an isolated strain (CA01) from soft wheat bran.The Actinomycete strain was characterized based on its morphological and biochemical characteristics and selected due to a proved promising ability to produce L-Asparaginase optimized in both solid and liquid media cultures.The conditions of enzyme production were standardized according to a one-factor-at-a-time (OFAT) experimental design.To obtain optimal medium combination, a Box-Behnken Response Surface Methodology (RSM) has been adopted by choosing the most influential factors. The optimal conditions for the enzyme production were (g/l): L-Asparagine 10.7; Glucose 2.7; starch 7, in based medium containing (g/l): K2HPO4 0.5; MgSO4, 7H2O 0.1, corresponding to an optimal enzymatic activity of 8.03 IU/ml at 27.83°C. The maximum production of enzyme was reached on the sixth day of experiment. The ANOVA test (P value ˂ 0.05) and adjusted R2 values close to the experimental R2 show that the obtained model of the active L-Asparaginase of CA01 strain production is significant with the following linear terms: temperature, substrate concentration, Glucose concentration and there squared.

Purification, Characterization of Alkaline Cold Active Lipase from Acinetobacter radioresistens PR8 and Development of a New Zymography Method for Lipase Detection.

Bacterial lipases find so many industrial applications. Therefore, new source of lipase suitable for industrial conditions is always required. Lipase zymography methods use costly chromogenic substrates and indicator dyes and are few in numbers. The objectives of this work include lipase purification and its characterization from Acinetobacter radioresiens PR8 and development of new zymography method for lipase detection. The lipase was purified using conventional method and cation exchange chromatography and it was characterized biochemically and analytically. Based on these characterization new in-gel lipase zymography method was developed. In this present work, an alkalophilic lipase producing bacterium was isolated from soil; screened for extracellular lipase activity and identified to be Acinetobacter radioresistens PR8 (Genbank accession ID: MF073322). Enzyme production kinetics showed maximum production (4.16 U/ml at pH 9) of enzyme after 72 h. The lipase activity was found to be highest in olive oil (1% v/v; 8.1 U/ml). Low molecular weight (27 kDa) alkaline (pH 9) cold active (20 °C) lipase was purified from Acinetobacter radioresistens PR8. Lipase was characterized using PMF, FT-IR and its high conformational stability (Transition temperature: 122.3 °C) was attributed from its DSC spectrum. The importance of magnesium and sodium ions for enhancing lipase activity was obtained from flux balance analysis. Based on the lipase activating role of Mn2+ and Na+ ions, optimum temperature, pH with no chromogenic substrates and indicator dyes, a new in gel zymography method for lipase detection was developed.

Optimization of culture conditions and bench-scale production of anticancer enzyme L-asparaginase by submerged fermentation from Aspergillus terreus CCT 7693

L-Asparaginase amidohydrolase (EC 3.5.1.1) has received significant attention owing to its clinical use in acute lymphoblastic leukemia treatment and non-clinical applications in the food industry to reduce acrylamide (toxic compound) formation during the frying of starchy foods. In this study, a sequential optimization strategy was used to determine the best culture conditions for L-asparaginase production from filamentous fungus Aspergillus terreus CCT 7693 by submerged fermentation. The cultural conditions were studied using a 3-level, central composite design of response surface methodology, and biomass and enzyme production were optimized separately. The highest amount of biomass (22.0 g·L−1) was obtained with modified Czapek–Dox medium containing glucose (14 g·L−1), L-proline (10 g·L−1), and ammonium nitrate (2 g·L−1) fermented at 37.2 °C and pH 8.56; for maximum enzyme production (13.50 U·g−1), the best condition was modified Czapek–Dox medium containing glucose (2 g·L−1), L-proline (10 g·L−1), and inoculum concentration of 4.8 × 108 espore·mL−1 adjusted to pH 9.49 at 34.6 °C. The L-asparaginase production profile was studied in a 7 L bench-scale bioreactor and a final specific activity of 13.81 U·g−1 was achieved, which represents an increase of 200% in relation to the initial non-optimized conditions.

Bioprocess optimization for pectinase production using Aspergillus niger in a submerged cultivation system

BackgroundPectinase enzymes present a high priced category of microbial enzymes with many potential applications in various food and oil industries and an estimated market share of $ 41.4 billion by 2020.ResultsThe production medium was first optimized using a statistical optimization approach to increase pectinase production. A maximal enzyme concentration of 76.35 U/mL (a 2.8-fold increase compared with the initial medium) was produced in a medium composed of (g/L): pectin, 32.22; (NH4)2SO4, 4.33; K2HPO4, 1.36; MgSO4.5H2O, 0.05; KCl, 0.05; and FeSO4.5H2O, 0.10. The cultivations were then carried out in a 16-L stirred tank bioreactor in both batch and fed-batch modes to improve enzyme production, which is an important step for bioprocess industrialization. Controlling the pH at 5.5 during cultivation yielded a pectinase production of 109.63 U/mL, which was about 10% higher than the uncontrolled pH culture. Furthermore, fed-batch cultivation using sucrose as a feeding substrate with a rate of 2 g/L/h increased the enzyme production up to 450 U/mL after 126 h.ConclusionsStatistical medium optimization improved volumetric pectinase productivity by about 2.8 folds. Scaling-up the production process in 16-L semi-industrial stirred tank bioreactor under controlled pH further enhanced pectinase production by about 4-folds. Finally, bioreactor fed-batch cultivation using constant carbon source feeding increased maximal volumetric enzyme production by about 16.5-folds from the initial starting conditions.

Improved production of cellulase by Trichoderma reesei (MTCC 164) from coconut mesocarp-based lignocellulosic wastes under response surface-optimized condition.

Experimental investigations were carried out to develop economic production process of cellulase using coconut mesocarp as an inexpensive lignocellulosic inducer while replacing commercial cellulose. Cellulase production was initially investigated from commercial cellulose in different submerged conditions using Trichoderma reesei (MTCC 164). Maximum enzyme production was achieved 6.3g/l with activity level 37 FPU/ml in the condition where cellulose to water content ratio was maintained at 5:35 (W/V). To achieve similar maximum production of cellulase from coconut mesocarp, response surface methodology was implemented to optimize most influencing parameters. Most influencing nutritional parameters such as coconut mesocarp, glucose and peptone were optimized in the concentration ranges of 35g/l, 35g/l and 25g/l, respectively. Selecting optimized parameter values, fermentations were conducted inside the fermenter with 2L operating volume to ensure high concentration and activity profiles of enzyme. Enzyme concentration was achieved 7.20g/l after 96h of batch fermentation with specific activity levels of 42 FPU/ ml and CMCase 75U/ml. Enzyme concentration was further improved to 9.58g/l with activity levels of 54 FPU/ml and CMCase 93U/ml by adopting sequential feeding of coconut mesocarp in fed-batch fermentation mode. The presence of pure cellulase in the sample was confirmed by FTIR analysis.

Optimization of cellulose production by bacteria isolated from saw dust

Cellulase enzymes are of enormous value in various industries as well as in treatment of wastes particularly lignocellulosic wastes. This high degree of applications of cellulase necessitates inexhaustible search for more sources of these enzymes. The aim of this study was to optimize the production of cellulase by bacteria from saw dust. Bacteria were isolated from sawdust samples, characterized phenotypically and screened for cellulase production. The production of the enzyme was optimized using different carbon and nitrogen sources, temperature, pH and lignocellulosic wastes. Five cellulase-producing bacteria were isolated and identified as A72 and A74 (Bacillus spp.) B32, B53 and 2B53 (Pseudomonas spp.). Two percent (2%) of carboxymethyl cellulose and 1 % yeast extract gave the highest cellulase production for all the isolates except A72 in the case of yeast extract while pH 6.0 was preferred for maximum enzyme production for all except 2B53 which preferred pH 7. The highest sugar was produced by isolate 2B53 (1.38±0.54 mg/ml) in the presence of sawdust among the lignocellulosic wastes. This study showed that the cellulase produced by these isolates can be used to generate sugar for industrial uses from lignocellulosic wastes and plant origin.Keywords: Bacteria, Sawdust, Cellulase, Pseudomonas, Bacillus

Enhanced production of xylanase enzyme by Fusariummoniliforme using submerged fermentation

Hemicellulose degrading enzymes play an important role in bioconversion of agro and agro-industrial wastes. In this study, production of hemicellulase by six fungal isolates was determined under submerged culture using corn cobs xylan as a carbon source and enzyme inducer at different incubation periods. The results indicated that Alternariatenuisshowed the lowest enzyme productivity (156.95 ± 2.07U/l)while the highest enzyme production (2,594.44 ± 62.25U/l) was found by Fusariummoniliforme. One-factor-at-a-time (OFAT) revealed maximum enzyme productivity of 10,950.11 ± 98.45 U/l at; corn cobs xylan (6g/l), yeast extract (4g/l), inorganic salts (MgSO4, KH2PO4, CaCl2, FeSO4, and MnSO4), initial pH (5), initial inoculum size (4%), 150 rpm and temperature (30 ).

One-factor-at-a-time (OFAT) optimization of hemicellulases production from Fusarium moniliforme in submerged fermentation

ABSTRACTHemicellulose degrading enzymes play an important role in bioconversion of lignocellulosic and agro-industrial wastes. In this study, production of hemicellulase by six fungal isolates was determined under submerged culture using corn cobs xylan as a carbon source and enzyme inducer at different incubation periods. The results indicated that Alternaria tenuis showed the lowest enzyme productivity (156.95 ± 2.07U/l) while the highest enzyme production (2,594.44 ± 62.25 U/l) was obtained by Fusarium moniliforme. One-factor-at-a-time revealed maximum enzyme productivity of 10,950.11 ± 98.45 U/l at corn cobs xylan (6 g/l), yeast extract (4 g/l), inorganic salts (1.5 g/l KH2PO4, 1.0 g/l MgSO4.7H2O, 0.2 g/l CaCl2, 0.4 g/l FeSO4.7H2O, and 0.3 g/l MnSO4.5H2O), initial pH (5), initial inoculum size (4%), 150 rpm, and temperature (30°C) in a submerged fermentation process.

Adding value to the oil cake as a waste from oil processing industry: Production of lipase in solid state fermentation

Abstract Riccinus communis (castor) seed cake is a by-product generated after oil extraction from the seed. This deoiled seed cake contains a toxic protein ricin that restricts its application in livestock feed. In the present study, solid-state fermentation (SSF) was used to value addition in castor de-oiled cake (CDOC). It was used as a substrate for the production of lipase under SSF using different fungal species. Initially, seven fungal strains were grown on CDOC and out of seven, only four are able to grow on it. From these four strains, only two Aspergillus oryzae (NCIM 1212) and Aspergillus japonicas (MTCC no. 1975) were observed to produce lipase. The effect of different physical parameter viz., moisture content, pH and incubation time on lipase production were also determined. The maximum lipase yield was determined to be 25 U/gm. Maximum enzyme production was observed at 6th day of incubation. The optimum condition for the lipase production was pH 7.0, moisture content 100%. From these results, it can be concluded that CDOC could be a good and cheap substrate for lipase production through SSF.

Biodelignification of some agro-residues by Stenotrophomonas sp. CFB-09 and enhanced production of ligninolytic enzymes

Abstract Biological delignification is an environmental friendly pretreatment method for enhanced enzymatic hydrolysis of lignocellulosic biomass and fermentation processes. In an attempt to optimize delignification process which is crucial for effective biofuel production from agro-residues, this study investigated biodelignification of some agro-residues (coconut shell {CS}, coconut husk {CH}, palm kernel shell {PKS}, oil palm empty fruit bunch {OPEFB} and sawdust {SD}) by Stenotrophomonas sp. CFB-09 under submerged fermentation conditions. Initial lignin and residual lignin contents of each agro-residue were determined at the beginning and end of 216 h biodelignification period. Production of ligninolytic enzymes was monitored at 12 h intervals. Effects of nitrogen sources, temperature and pH on production of ligninolytic enzymes were determined over the biodegradation period. Remarkably, biodelignification of the agro-residues was in the range of 41–55%. Laccase had highest yield (31 U/mg) on CH, PKS and OPEFB. Lignin peroxidase production (381 U/mg) was maximum on OPEFB, and manganese peroxidase had highest yield (53 U/mg) on PKS. Surprisingly, there was appreciable production of ligninolytic enzymes at higher temperatures of biodegradation with 40–55% yield at 80 °C. Maximum enzyme production was achieved at 40 °C, pH 8.0 with ammonium nitrate as nitrogen source. Results provide deeper insights into biodelignification of agro-residues and demonstrate potential of Stenotrophomonas sp. CFB-09 for use in biodegradation of agro-residues, especially in pretreatment of lignocellulosic biomasses for production of biofuel and other value-added products.

Influence of medium composition and physical factors on enhanced production of endoglucanase by locally isolated fungal strain in solid state fermentation

Endoglucanase is one of the most important enzymes of the cellulase group. Endoglucanase are involved in the catalytic hydrolysis of cellulose and plays a pivotal role in different sectors like pharmaceutical, textile, detergent, and food processing as well as paper and pulp industry. With consumers getting more and more aware of environmental issues, industries find enzymes as a better option over other chemical catalysts. In the current research different thermophilic fungal strains were isolated from the different sources. Qualitative screening was carried out on the basis of cellulose hydrolysis zone. The quantitative screening was carried out employing solid state fermentation. The fungal culture, showing highest EG potential was selected identified and assigned the code Aspergillus fumigatus BBT2. Different fermentation media were evaluated and M 2 containing wheat bran gave maximum EG production. The maximal enzyme productivity was recorded in 72 hours, 40°C, pH 5, inoculum size 1.5ml, and moisture content (1:1). Glucose (1%) and peptone (1%) were optimized as best carbon and nitrogen sources, respectively.

Extracellular Pectinase from a Novel Bacterium Chryseobacterium indologenes Strain SD and Its Application in Fruit Juice Clarification.

Pectinase is one of the important enzymes of industrial sectors. Presently, most of the pectinases are of plant origin but there are only a few reports on bacterial pectinases. The aim of the present study was to isolate a novel and potential pectinase producing bacterium as well as optimization of its various parameters for maximum enzyme production. A total of forty bacterial isolates were isolated from vegetable dump waste soil using standard plate count methods. Primary screening was done by hydrolysis of pectin. Pectinase activity was determined by measuring the increase in reducing sugar formed by the enzymatic hydrolysis of pectin. Among the bacterial isolates, the isolate K6 exhibited higher pectinase activity in broth medium and was selected for further studies. The selected bacterial isolate K6 was identified as Chryseobacterium indologenes strain SD. The isolate was found to produce maximum pectinase at 37°C with pH 7.5 upon incubation for 72 hours, while cultured in production medium containing citrus pectin and yeast extract as C and N sources, respectively. During enzyme-substrate reaction phase, the enzyme exhibited its best activity at pH of 8.0 and temperature of 40°C using citrus pectin as substrate. The pectinase of the isolate showed potentiality on different types of fruit juice clarification.

Cellulase production by Aspergillus unguis in solid state fermentation

Lignocellulosic substrates are a good carbon source and provide rich growth media for a variety of microorganisms which produce industrially important enzymes. Cellulases are a group of hydrolytic enzymes such as filter paperase (FPase), carboxymethyl cellulase (CMCase) and β-glucosidase—responsible for release of sugars in the bioconversion of the lignocellulosic biomass into a variety of value-added products. This study examined cellulase production by a newly isolated Aspergillus unguis on individual lignocellulosic substrates in solid state fermentation (SSF). The maximum peak production of enzymes varied from one substrate to another, however, based on the next best solid support and local availability of groundnut fodder supported maximum enzyme yields compared with other solid supports used in this study. Groundnut fodder supported significant production of FPase (5.9 FPU/g of substrate), CMCase (1.1 U/g of substrate) and β-glucosidase activity (6.5 U/g of substrate) in SSF. Considerable secretion of protein (27.0 mg/g of substrate) on groundnut fodder was recorded. Constant increment of protein content in groundnut fodder due to cultivation of A. unguis is an interesting observation and it has implications for the improvement of nutritive value of groundnut fodder for cattle.

Enhancement of prodigiosin synthetase (PigC) production from recombinant Escherichia coli through optimization of induction strategy and media

ABSTRACTPigC is a synthetase that catalyzes the condensation of 4-methoxy-2,2′-bipyrrole-5-carboxyaldehyde and 2-methyl-3-amylpyrrole to produce prodigiosin, which has a wide variety of impressive biological properties. In this study, we optimized PigC production from engineered Escherichia coli BL21(DE3). Investigation of different induction strategies revealed that autoinduction was the most appropriate method for PigC expression. As a result, PigC activity was elevated to 75.7 U/mL, nearly 2.1-fold higher than that with induction by isopropy-β-d-thiogalactoside. To achieve maximum enzyme production, the automedium components were optimized. “Single-factor experiments” showed that PigC production was greatly affected by the concentrations of glucose, yeast extract, and lactose. The Box–Behnken design for response surface methodology was then used to determine the optimal concentrations of these three components. According to a statistical approach, the optimum values of the three most influential parameters were 0.73 g/L glucose, 13.17 g/L yeast extract, and 5.86 g/L lactose. In the optimized automedium, the best PigC activity was obtained at 179.3 U/mL, which was 2.4-fold higher than using the initial medium. This study maximized PigC production as a foundation for further study and future industrial application.

English

In the present study, the isolation, purification and partial characterization of thermostable serine alkaline protease produced from Bacillus thuringinsis SH-II-1A was reported. The culture was isolated from soil of slaughter house waste and identified further from ribosomal sequence. The crude enzyme was purified by ammonium sulfate precipitation, dialysis and Sephadex G-200 gel permeation chromatography up to 17.04 fold with recovery of 8.47%. Relative molecular weight (67 kDa) of purified enzyme was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Maximum production of enzyme and cell biomass was observed for 48 h of incubation period at 45°C. Strong activity of enzyme was observed at pH 10 to 11; also stability of up to 2 and 20 h incubation at the same pH range confirms alkaline protease. Optimum temperature recorded for protease activity was 45°C, and 100% thermal stability up to 350 min of incubation was recorded. Among different natural substrates tried, casein was found as ideal substrate. Enzyme activity was strongly enhanced by metal ions like Ca2+, Mg2+ and Mn2+ whereas, 100% enzyme activity was inhibited by phenylmethylsulphonyl fluoride (PMSF), and up to 92% inhibition by diisopropyl fluorophosphates (DFP) confirmed serine protease. Detergent compatibility of the enzyme was studied in the presence of 10 mM CaCl2 and 1 M glycine at 45°C. This indicates 80 to 100% stability for a period of 0.5 to 2.5 h incubation. Improved washing performance and removal of blood stains from the cotton cloth was observed when detergent Surf excel was used with enzyme. Overall, the observed properties of isolated protease conclude its commercial application in detergent and leather industries. Key words: Thermostable serine alkaline protease, Bacillus thuringinsis, purification, partial characterization.