Purification and Characterization of Proteases from Bacillus amyloliquefaciens Isolated from Traditional Soybean Fermentation Starter (original) (raw)
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ScienceAsia
Eighty-two bacillus strains were isolated from Thai fermented soybeans (thua nao), of which thirty-nine were identified as Bacillus subtilis. Crude proteins from these B. subtilis strains were investigated for their proteolytic activity. When tested on skim milk agar, the crude proteins of B. subtilis strain 38 exhibited the highest proteolytic activity (a clear zone with an average area of 480 mm 2). Optimal pH and temperature of the proteases from B. subtilis strain 38 were found to be at 6.5 and 47 °C , respectively. The proteases were unstable and rapidly decreased in activity when heated at 60 °C. Various protease inhibitors were tried, only 1, 10-phenanthroline decreased the enzyme activity indicating the presence of metalloproteases. In addition, we attempted to produce fermented soybeans using the B. subtilis strain 38 as a starter culture.
Energy Reports, 2020
The objective of the study to determine the factors affecting the protease content generated during soybean fermentation by Bacillus subtilis. In this study, four factors were investigated during soybean fermentation: (1) pH, (2) fermentation temperature, (3) fermentation time and bacterial density, and (4) added glucose content on steamed soybean. The experimental results showed that at 33 • C temperature, 48 h fermentation time, bacterial density 10 4 CFU, pH 7, and supplementation of 1 g glucose into 50 g steamed soybean, Bacillus subtilis fermented soybean (another name is natto) had the highest protease activity. This suggests that the fermentation conditions in the study are consistent with the growth of Bacillus subtilis to produce proteases. Moreover, the optimization of fermentation conditions contributes to reducing the energy consumption to obtain the desired product, by increasing the yield in reaction. Therefore, this study is important at an industrial level, while contributing to the rational use of energy. c
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...
The aim of this experimental study was to isolate and partially purify protease enzyme from Bacillus cereus and Bacillus subtilis. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species in suitable nutrient plates. The partial purification was realized by applying, respectively, ammonium sulfate precipitation, dialysis, and DEAE-cellulose ion-exchange chromatography to the supernatant that was produced later. Optimum pH, optimum temperature, pH stability, and temperature stability were determined, as well as the effects of pH, temperature, substrate concentration, reaction time, and inhibitors and activators on enzyme activity. In addition, the molecular mass of the obtained enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of partially purified enzyme from B. subtilis was determined to be 84 U/mg. The final enzyme preparation was eight-fold more pure than the crude homogenate. The molecular mass of the partially purified enzyme was found to be 45 kDa by using SDS-PAGE. The protease enzyme that was partially purified from B. cereus was purified 1.2-fold after ammonium sulfate precipitation. The molecular mass of the partially purified enzyme was determined to be 37 kDa by using SDS-PAGE.
INFLUENCE OF CULTURE CONDITIONS ON PRODUCTION AND ACTIVITY OF PROTEASE FROM BACILLUS SUBTILIS BS1
Bacillus subtilis BS1 was used in the present study for the production of protease. For protease production optimum pH and temperature were found to be 11 and 50˚C, respectively. Soybean (197 PU/mg) and casein (168 PU/mg) proved as the best substrates for the production of enzyme. Maximum production of protease (126 and 121 PU/mg) was shown in 1.5 and 4.5% of Sodium chloride (NaCl) concentration, respectively. Maximum activity was observed at pH 9 at 90ºC, in crude extract, after 20 minutes of incubation. EDTA slightly enhanced proteolytic activity, whereas, Na, K, Ca, Li, Mg, Cu and Fe inhibited the activity of protease. Due to maximum production of protease in the presence of cheaper, low concentrations of substrate and stability at alkaline pH, high temperature and salt-tolerance, these characteristics makes the strain and its enzymes usefull in different industries.
Optimization of Protease Production by Bacillus mojavensis A21 on Chickpea and Faba Bean
Advances in Bioscience and Biotechnology, 2014
Response surface methodology (RSM) was employed to optimize the medium composition and culture conditions for the production of alkaline proteases by Bacillus mojavensis A21 on uncommon substrates: chickpea (CF) and faba bean (FF) flours. A significant positive influence of temperature, CF, FF, incubation time and inoculums size on the protease production was evaluated by Plackett Burman Design. Among these, CF was the most influential factor. The enhancement of protease to 9127 U/ml was achieved with the optimization procedure on the medium composed of (g/l): CF, 40; FF 30, NaCl 2.0; KH 2 PO 4 1; K 2 HPO 4 1; CaCl 2 , 0.1; MgSO 4 0.1. The cultures were conducted for 72 hours with an IS of 2%, at 30˚C, an agitation speed of 150 rpm and an initial pH of 8.0. More interestingly, the optimization was accomplished using two cheap and local fermentation substrates, CF and FF, which could result in a significant reduction in the cost of medium constituents. The maximum alkaline protease production was 9127 U/ml after 72 h of incubation and showed 5-fold increase in protease production over the initial level.
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.
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...