Response‐surface methodology for the production and the purification of a new H₂O₂‐tolerant alkaline protease from Bacillus invictae AH1 strain

Abstract

This work deals with the optimization of the culture conditions of Bacillus invictae AH1 in order to increase the production level of the proteolytic activity. Response‐surface methodology (RSM) was applied for the most significant fermentation parameters (concentration of wheat bran and K2HPO4/KH2PO4) that were earlier identified by Plackett–Burman Design from seven possible factors. A central composite design was used and the quadratic regression model of producing active protease was built. A maximum protease activity was reached and validated experimentally, using a maximum wheat bran concentration (50 g/L) with increased K2HPO4/KH2PO4 concentration (2.275 g/L). Protease production obtained experimentally coincident with the predicted value and the model was proven to be adequate. Interestingly, the use of RSM increased the protease production by four times (7,000 U/mL) using a low‐cost substrate and a culture time of 40 hr, as compared to the standard culture conditions. In the second part of this study, a H2O2‐tolerant alkaline protease produced from B. invictae AH1 with a molecular mass of about 41 kDa, noted P3, was purified by successive steps of ultrafiltration, gel filtration and ion exchange chromatography. The K m and Vmax values of the purified protease using casein, as substrate, were about 4 mg/mL and 27 μM/min, respectively. The highest enzyme activity was found at pH 9.0 and a temperature of 60°C. In addition, the enzyme showed a quasi‐total stability against H2O2 (5% for 1 hr) and against most of the tested solid and liquid detergents, suggesting its eventual use in bio‐detergent formulations.

Isolation and characterization of a new cold-active protease from psychrotrophic bacteria of Western Himalayan glacial soil

“…Moreover, in comparison to previous reports suggesting proteases from Bacilli had low K m between 0.57 and 4.0 mg/ml at higher temperatures (45–60 °C) 41 , 55 – 57 , the Apr-BO1 had a low K m (1.0 mg/ml) and V max (10.0 µmol/ml/min) at low temperature (20°), which indicates that it has high substrate affinity and efficient catalytic activity. The sequence of Apr-BO1 and other similar proteases showed low similarity after comparing with the database available at UniProt ( https://www.uniprot.org/blast/ ).…”

“…Inhibition observed in Apr-BO1 enzyme for the present study was not as sturdy as reported for various serine alkaline proteases from Bacillus pumilus AI 38 , Bacillus sp. ZJ1502 39 , Bacillus safensis S406 40 and Bacillus invictae AHI 41 , adds to its novelty.…”

PARTIAL PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR METALLOPEPTIDASE PRODUCED BY Bacillus amyloliquefaciens FE-K1

“…As shown in Table 1, the presence of PMSF, which is the inhibitor of serine peptidases, caused a decrease in the peptidase activity. It was determined that serine peptidases are generally stable at pH 8.0 (Mothe & Sultanpuram 2016, Hammami et al 2020). It was observed that the stability of the enzyme decreased in alkaline conditions, especially for more extended incubation periods (Fig.…”

Optimization of Alkaline Protease Production by Solibacillus silvestris, Isolated from Gir Region of Gujarat

“…The enzyme used in the detergent had to be stable in a wide range of temperature and had to be active in the presence of various surfactants, bleaching agents, and various other additives. Currently, a large proportion of commercially available protease is derived from Bacillus strains and most of them were isolated from coastal regions [7][8][9][10][11]. From the study of the previous literature, it can be seen that due to the longest coastal region of Gujarat the possibility of getting the isolate with very good alkaline protease production is high [12][13][14][15][16].…”

“…carbon source, nitrogen source, etc.) and therefore optimization of the factors is essential to improve the protease yield [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. A number of statistical experimental designs with response surface methodology (RSM) have been employed for optimizing enzyme production from microorganisms [21].…”