Production and characterisation of protease enzyme produced by a novel moderate thermophilic bacterium (EP1001) isolated from an alkaline hot spring, Zimbabwe (original) (raw)
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Optimization of Alkaline Protease production from bacteria isolated from soil
Protease producing bacteria were isolated from soil and were identified as Pseudomonas fluorescens, Bacillus subtilus, E.coli and Serratia marscens. Optimization of the fermentation medium for maximum protease production was carried out. The culture conditions like pH, temperature, carbon sources and nitrogen sources were optimized. The Optimum conditions for protease production were found to be 37 o C at pH 10 with Glucose as Carbon Source and Peptone as Nitrogen Source. Soycake and Calcium chloride stimulates the production of protease with 1ml of inoculums size for 48 hrs of incubation period with less concentration of EDTA. Among all studied bacterial isolates, the highest enzyme activity was observed in B. Subtilis. Sonia Sethi et al J. Microbiol. Biotech. Res., 2012, 2 (6):858-865 ______________________________________________________________________________ 859 Available online at www.scholarsresearchlibrary.com
A Review on Microbial Alkaline Protease: An Essential Tool for Various Industrial Approaches
Industrial Biotechnology
Proteolytic enzymes are present in all living organisms and help in cell growth and differentiation. Proteases are the hydrolytic enzymes that act as biocatalysts for the cleavage of proteins into smaller peptides and amino acids. Microorganisms have turned out to be a competent and inexpensive source of alkaline protease enzymes that can produce a continuous and consistent supply of desired product. Alkaline proteases have extensive application in various industrial sectors especially in detergent and leather industries. However, their application in food has not been much exploited. This review summarizes all the reports of applications of alkaline protease in different sectors with a main view on food applications. The effect of various physiochemical parameters on alkaline protease is discussed. Different sources of isolation and optimum pH and temperature of alkaline protease producing bacterial and fungal species are also reported.
An overview on fermentation, downstream processing and properties of microbial alkaline proteases
Applied Microbiology and …, 2002
Microbial alkaline proteases dominate the worldwide enzyme market, accounting for a two-thirds share of the detergent industry. Although protease production is an inherent property of all organisms, only those microbes that produce a substantial amount of extracellular protease have been exploited commercially. Of these, strains of Bacillus sp. dominate the industrial sector. To develop an efficient enzyme-based process for the industry, prior knowledge of various fermentation parameters, purification strategies and properties of the biocatalyst is of utmost importance. Besides these, the method of measurement of proteolytic potential, the selection of the substrate and the assay protocol depends upon the ultimate industrial application. A large array of assay protocols are available in the literature; however, with the predominance of molecular approaches for the generation of better biocatalysts, the search for newer substrates and assay protocols that can be conducted at micro/nano-scale are becoming important. Fermentation of proteases is regulated by varying the C/N ratio and can be scaled-up using fed-batch, continuous or chemostat approaches by prolonging the stationary phase of the culture. The conventional purification strategy employed, involving e.g., concentration, chromatographic steps, or aqueous two-phase systems, depends on the properties of the protease in question. Alkaline proteases useful for detergent applications are mostly active in the pH range 8-12 and at temperatures between 50 and 70C, with a few exceptions of extreme pH optima up to pH 13 and activity at temperatures up to 80-90C. Alkaline proteases mostly have their isoelectric points near to their pH optimum in the range of 8-11. Several industrially important proteases have been subjected to crystallization to extensively study their molecular homology and threedimensional structures.
Malaysian Journal of Microbiology, 2012
Aims: The research was done to study the conditions enhancing catalytic activities of alkaline proteases from Vibro sp., Lactobacillus brevis, Zymomonas sp., Athrobacter sp., Corynebacterium sp. and Bacillus subtilis. Methodology and Results: The proteolytic enzymes were purified in 2-step procedures involving ammonium sulphate precipitation and sephadex G-150 gel permeation chromatography. The upper and lower limits for the specific activities of proteases from the selected microorganisms were estimated at 20.63 and 47.51 units/mg protein with Zymomonas protease having the highest specific activity towards casein as its substrate and purification fold of 3.46, while that of Lactobacillus brevis protease was 8.06. The native molecular weights of these active proteins ranged from 30.4 to 45.7 kDa with Athrobacter sp. protease having the highest weight for its subunits. The proteolytic enzymes had optimum pH range of 8 to 10 and temperature range of 50 to 62 ºC accounting for the percentage relative activity range of 75 to 94% and 71 to 84 % respectively. The activities of Lactobacillus brevis and Bacillus subtilis proteases were maximum at pH 9 and 10 respectively. Lactobacillus brevis protease activity was maximum at temperature of 62 ºC, while beyond this value, a general thermal instability of these active proteins was observed. At above 70 ºC, the catalytic activities of Corynebacterium sp., Vibrio sp., Zymomonas sp. and Arthrobacter sp. proteases were progressively reduced over a period of 120 min of incubation, while Bacillus subtlis and Lactobacillus brevis proteases were relatively stable. Effect of metal ions was investigated on the catalytic activity of protease from the microorganisms. Lactobacillus brevis, Zymomonas sp., Arthrobacter sp., Corynebacterium sp. and Bacillus subtilis protease activities were strongly activated by metal ions such as Ca +2 and Mg +2. Enzyme activities were inhibited strongly by Cu 2+ and Hg 2+ but were not inhibited by ethylene diamine tetra acetic acid (EDTA), while a slight inhibition was observed with K + , Na + and Fe 2+. Conclusion, significance and impact of study: The outcome of this present study indicated useful physico-chemical properties of proteolytic enzymes that could be of biotechnological use in enhancing enzyme catalytic efficiency.
Bacillus strains isolated from the salteren pond (Kakinada) were screened and identified for high alkaline protease activity. The isolates which were positive on skim milk agar (1%) were selected as protease producing strains. Of the ten bacterial isolates screened, isolate S-8 was observed as a potential haloalkaline protease producer and it was identified as Bacillus cereus strain S8 (MTCC NO: 11901) by 16S rRNA gene sequencing, phylogenetic tree analysis and by different biochemical tests. Protease production was enhanced by optimizing the culture conditions. The nutritional factors such as carbon and nitrogen sources, NaCl and also physical parameters like temperature, incubation time, pH, inoculum size were optimized for the maximum yield of protease. Studies on the effect of different carbon and nitrogen sources revealed that maximum protease production was obtained in the medium supplemented with Molasses,1%(w/v); Potassium nitrate, 0.75%(w/v); salt solution-5%(v/v) {MgSo 4. 7H 2 O, 0.5%(w/v); KH 2 PO 4, 0.5%(w/v)}; FeSO 4. 7H 2 O, 0.01%(w/v) and CaCO 3, 0.5% respectively. Thus, with selected carbon and nitrogen sources along with 1 % NaCl and 2% inoculum the maximum protease production (205.0 U/ml) was obtained in the period of 72 h incubation at pH-12.0 under 160 rpm when compared to the initial enzyme production (165.0 U/ml). The crude enzyme extract of this strain was also characterized with respect to temperature, pH, incubation period and different concentrations of casein which was used as enzyme substrate. This study shows that the enzyme has wide range of pH stability from 8 to 11 with optimum activity at pH-10.0. It is thermostable with optimum activity at 70°C (392U/ml) with 1h incubation of enzyme with 1% casein as its substrate. From the above investigations it was concluded that the protease production by these microorganisms at wide temperatures and pH ranges could be explored for varied industrial applications.
Study of Protease Producing Bacteria and their Enzymatic Activity at Different Parameters
International Research Journal of Engineering & Technology, 2019
Bacterial alkaline proteases are among the important hydrolytic enzyme and have been used extensively since the advent of enzymology. Bacterial extracellular alkaline proteases are of great importance due to its wide spectrum applications in detergent industries, bioremediation, food industries, and leather processing and bio-film degradradation. The Optimization of the time of incubation, the effect of temperature, the effect of pH, the effect of different carbon source and effect of different nitrogen source and they were found to be 120hrs, 12, 170Unit/ml for galactose, and 70 Unit/ml for ammonium sulfate respectively for the production of protease and also optimize the parameters on enzyme activity they are temperature, pH, 1% casein substrate, and the different volume of the crude enzymes and the results was 370C,11, 3.5ml, 60 Unit/ml for 2 ml of crude enzymes respectively.