Physical and Chemical Characterization of Alkaline Protease from Bacillus Subtilis VBC7 Using Agro Waste as Substrate (original) (raw)
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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...
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.
Biological Sciences - PJSIR
In this study, proteases have been isolated from agricultural soil samples and then cultured by shake flask method. The growth of the Bacillus subtilis has been confirmed by microbiological test on the agar plate and skim milk agar in rough, raised and irregular colonies. The yield of the alkaline protease has been optimised by varying the main factors i.e., nitrogen source (peptone, yeast extract, beef extract, casein, ammonium carbonate and urea), carbon source (sucrose, fructose, mannose, lactose, glucose, maltose and starch), incubation period (12, 24, 36, 48, 72, 84 and 96 h), temperature (35, 40, 45, 50, 55, and 60 °C) and salts (potassium sulphate, magnesium sulphate, calcium sulphate and manganese sulphate). The results revealed that the maximum enzyme production was obtained using casein and minimum activity was obtained using urea as a nitrogen source. Similarly, other factors have shown significant effect on the activity of the enzyme.
Advances in Enzyme Research, 2013
Production of alkaline protease from Bacillus subtilis SHS-04 was investigated under different fermentation conditions involving low-cost substrates with the aim of optimizing yield of enzyme. Maximum enzyme production (1616.21 U/mL) was achieved using groundnut meal (0.75%) as nitrogen source and 0.5% glucose as carbon source at 48 h cultivation period, pH 9, 45˚C and 200 rpm. The yield was 348% increase over comparable control samples. The alkaline protease had optimum temperature of 60˚C and remarkably exhibited 80% relative activity at 70˚C. It was highly thermostable showing 98.7% residual activity at 60˚C after 60 minutes of incubation at pH 9.0 and was stable in the presence of organic solvents studied. These properties indicate the viability of the protease for biotechnological and industrial applications. The optimized yield of enzyme achieved in this study establishes groundnut meal as potential low-cost substrate for alkaline protease production by B. subtilis SHS-04.
Alkaline protease, 2018
Alkaline proteases have applications in numerous industries. In this study, we have isolated and screened proteolytic bacteria from poultry wastes mixed soil and identified two bacterial isolates as Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 based on 16S rDNA sequencing. Maximum level of protease production was achieved after 24 h of fermentation in a basal medium. The optimal temperature, initial pH of the media and agitation for alkaline protease production by these two isolates were 30 C, pH 9.0 and 120 rpm, respectively. The both bacterial isolates produced maximum level of protease with 3.0% organic municipal solid wastes (OMSW) as the sole source of carbon and nitrogen under previously optimized fermentation conditions. In comparison with the shake flask, protease production increased about 2.5-fold in the bioreactor with reduction in fermentation period. The partial purification of protease Production and partial characterization of dehairing alkaline protease from Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 by using organic municipal solid wastes. (http://creativecommons.org/licenses/by-nc-nd/4.0/). resulted in a final 45.67 and 34.86-fold purified protease with a specific activity of 8335.34 and 9918.91 U/mg protein and a typical yield of 9.75 and 9.41% from B. subtilis and E. indicum, respectively. The optimum temperature and pH of the partially purified protease from the both sources was 40 C and pH 9.0, respectively. Protease from the both isolates was stable at pH 7.0e12.0 and at temperatures up to 50 C. The effects of protease inhibitors indicated that the protease from B. subtilis might be serine and cysteine type and from E. indicum might be cysteine type. Mg 2þ , K þ and Ca 2þ stimulated but Zn 2þ , Hg 2þ , Co 2þ and Fe 3þ strongly inhibited the protease activity. The partially purified protease from B. subtilis substantially dehaired cow skin and decomposed gelatinous compound from X-ray film. Our study revealed that OMSW can be used as raw material for production of bacterial extracellular protease and alkaline protease from B. subtilis might be potential for industrial and biotechnological applications.
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.
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.
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.
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.