Purification and Kinetic Parameters Characterization of an Alkaline Protease Produced from Bacillus subtilis through Submerged Fermentation Technique (original) (raw)
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Stability and Activity Profile of Alkaline Protease, Produced from Bacillus Subtilis
2015
The present study gives an insight into the effect of different activators and inhibitors on the activity and stability of alkaline proteases produced by Bacillus subtilis IH-72. The alkaline protease was strongly activated both by bivalent and monovalent cations such as Mg 2+ , Mn 2+ , Na + and K + . The enzyme activity was considerably enhanced in the presence of fructose, galactose, glucose and mannitol. The enzyme was stabilized up to 10 days by immobilization on activated charcoal and was efficiently stabilized up to 2 months by lyophilization. The enzyme remained stable up to 19 days both at 4 o C and 30 o C in the presence of Mn 2+ . However, it exhibited significant stability up to 22 days at 4 o C and 30 o C in the presence of fructose, galactose and polyethylene glycol.
2012
In this study, we have shown the microbial alkaline protease production by using isolated Bacillus subtilis. Bacillus subtilis which produces an extracellular alkaline protease was isolated from meet surface purchased from local Egyptian markets. Maximum enzyme activity was achieved when the bacterium was grown on corn steep liquor (2.0 %, w/v) instead of soybean meal (2 %) followed by casein hydrolysate (2%, w/v) and 12 % cane sugar molasses as a carbon source at pH 10.0 and 37oC over 24 h incubation period (maximum enzyme production at 48 hr.). The enzyme has an optimum pH of around 10.0 and maintained its stability over a broad pH range between 5.0 to 12.0. Its optimum temperature is around 37 °C, and exhibited a stability of up to 50 oC.
Production, optimization and partial purification of protease fromBacillus subtilis
Journal of Taibah University for Science, 2015
Proteases have characteristics of biotechnological interest and have thus become important industrial enzymes. We report the production of thermostable protease and its characterization in Bacillus species, which is a thermotolerant bacterium; Bacillus subtilis is widely used for isolating protease enzyme. Gelatin was used as the substrate in nutrient agar medium for screening and showed the maximum zone of activity (22 mm) after overnight incubation and addition of the indicator. Under submerged fermentation conditions, a high level of protease production was found at 45 • C after 36 h at pH 10, with continuous agitation (180 rpm). The presence of galactose and peptone in the medium enhanced enzyme production by 0.5% when compared with other carbon and nitrogen sources. Thus, such additions can augment protease production and their application in various industries.
2012
The present study describes the production, purification and characterization of alkaline protease from mutant strain of Bacillus subtilis EMS-8. The enzyme was purified using ammonium sulphate precipitation which gave 2.64 fold purification with 81.5% yield at 70% saturation. The molecular weight of the enzyme was determined using SDS-PAGE and it was found to be 25 KDa. The optimum pH of enzyme activity was 8.5; however the enzyme remained stable up to pH 10 after 24 hrs of incubation. Similarly, the optimum temperature for enzyme activity was 40oC, whereas it remained stable up to 90oC with greatly reduced activity. Alkaline protease showed highest specificity towards casein. Among different inhibitors, Phenylmethylsulphonyl fluoride (PMSF) completely inhibited the enzyme activity indicating the serine nature of protease. Similarly, the protease activity was greatly reduced in the presence of MnCl2, whereas MgCl2 enhanced its activity. The shelf life of the protease was also deter...
Kuwait Journal of Science, 2021
Proteases have gained more commercial value to date due to multiple applications in different industrial sectors. Current research was aimed to use the cheaper agricultural waste for optimal protease production. Maximum level of protease production was achieved at 37 °C, incubation period of 24 h, pH 9.0, inoculum size 3%, 1.5 g sucrose as a carbon source and 30% moisture content by using solid-state fermentation. Among the various inorganic and organic nitrogen sources, ammonium nitrate and yeast extract tremendously increased the production of protease. Among metal ions and surfactants tested, Ca2+ and Tween 40 showed the optimal protease production. The purification of protease was carried out by ammonium salt precipitation followed by sephadex G-100 gel filtration chromatography. The purification resulted in 1.3 fold of purified protease with a specific activity of 51.5 and a total yield of 37.5 %. Molecular weight of purified protease was predicted upon SDS-PAGE with a single b...
Production, optimization and partial purification of protease fromBacillus subtilis
Journal of Taibah University for Science, 2015
Proteases have characteristics of biotechnological interest and have thus become important industrial enzymes. We report the production of thermostable protease and its characterization in Bacillus species, which is a thermotolerant bacterium; Bacillus subtilis is widely used for isolating protease enzyme. Gelatin was used as the substrate in nutrient agar medium for screening and showed the maximum zone of activity (22 mm) after overnight incubation and addition of the indicator. Under submerged fermentation conditions, a high level of protease production was found at 45 • C after 36 h at pH 10, with continuous agitation (180 rpm). The presence of galactose and peptone in the medium enhanced enzyme production by 0.5% when compared with other carbon and nitrogen sources. Thus, such additions can augment protease production and their application in various industries.
Journal of microbiology, biotechnology and food sciences
Alkaline proteases are the most important group of industrial enzymes that hydrolyse the peptide bond of proteins into small peptides. The industrial demand of alkaline protease predominantly from the microbial origin has been recently increased and enhances the research for alkaline protease with high stability at extreme industrial conditions. Thus, this study is aimed to characterize the alkaline protease from bacterial isolate, Bacillus subtilis VBC7 screened from dairy waste dumped soil. Extracellular alkaline protease production was carried out in alkaline broth by submerged fermentation. The production medium with 10% ground nut extract at pH 10 and 40 °C enhanced the alkaline protease activity (712 U/mL) than other wastes such as coconut pulp extract and sesame seed extract. This optimized media increased the bacterial growth rate and activity of alkaline protease compared to the unsupplemented basal medium. Further, alkaline protease was partially purified and assessed thei...
Production of alkaline protease by Bacillus subtilis using solid state fermentation
African Journal of Microbiology Research, 2013
The present study describes the optimization of nutritional and cultural parameters for the production of alkaline protease by Bacillus subtilis under solid state conditions. Among cultural conditions, incubation temperature, incubation period and moisture level of the substrate were optimized and it was found that maximum production of alkaline protease was observed at 37°C and moisture to substrate ratio of 1:1 after 48 h of incubation period. Among different nutritional parameters, the effects of different diluents, carbon and nitrogen sources on the enzyme production were studied. Maximum enzyme production (101.23 U/g) was observed when D 2 {(% w/v) CaCO 3 , 0.05; peptone, 0.1; glucose, 0.1 and yeast extract, 0.1} was used to moisten the substrate. The best carbon source for the production of alkaline protease by B. subtilis was found to be sucrose at a concentration of 1%. Similarly, nutrient broth (1.5%) and diammonium hydrogen phosphate (0.1%) were found to be best organic and inorganic nitrogen sources, respectively. It was also found that the maximum protease (126.8 U/g) was produced when 25% (v/w) inoculum was used to inoculate the fermentation flasks.
Proteases is family of enzymes and it has crucial role due to their physiological roles and very valuable commercial applications. Alkaline protease are produced by Bacillus species are particular importance because of their thermal stability and stability at different pH values. This study aimed to investigate the effect of physical and chemical factors in production of alkaline protease enzyme fermentation by members of the genus Bacillus. In this study, alkaline protease enzyme production were evaluated in submerged fermentation by Bacillus strains which were isolated from alkaline soils of Guilan province. Factors incubation were optimized such as time, pH, amount of inoculation and ammonium sulfate in alkaline protease enzyme production whit using response surface methodology (RSM) in culture. The maximum enzymatic activity was observed in incubation time of 36 hours, pH=9, inoculation amount of 15% (V) and ammonium sulfate 1.5% (W/V). Factors had significant effect on the production of alkaline protease enzyme such as pH and ammonium sulfate.