Media optimization for the production of alkaline protease by Bacillus cereus PW3A using response surface methodology

Abstract

The present study reports production, partial purification, and media optimization for alkaline protease using Bacillus cereus PW3A. A profiling study for protease production indicates maximum enzyme activity (17.22 U/ml) was observed after 48 h of incubation. The studies also showed that the enzyme activity increased with the decrease in carbon content indicating the growth associated with nature protease production. Partial purification of protease was done using ammonium sulfate precipitation and dialysis. Further studies were conducted to assess significant media ingredients influencing protease production using the one-factor-at-a-time approach and Plackett-Burman design. Fructose and yeast extract were identified as the most significant variables. Response surface methodology was applied to optimize the factors for maximizing protease production. The results showed that the production increased from 17.22 U/ml to 47.43 U/ml indicating a three-fold augment in enzyme activity. Characterization of protease showed that the highest enzyme activity was shown at pH 8.0 and temperature 50°C; however, significant enzyme activity was retained till pH 10 and temperature 60°C. Using casein as substrate, the enzyme showed maximum activity V max 39 U/ml and K m 18 μM. The activity was enhanced by MgCl 2 and CuSO 4 and inhibited by HgCl 2 . Since the enzyme has both pH and temperature stability with greater substrate affinity, this protease finds many useful industrial applications.

Production Optimization, Partial Characterization, and Gluten-Digesting Ability of the Acidic Protease from Clavispora lusitaniae PC3

“…The predicted R 2 was 0.9194, indicating a close resemblance with R 2 and adjusted R 2 . This reveals the goodness of the model and the factors optimized [38,57].…”

“…Fermentation 2024, 10, x FOR PEER REVIEW 14 of 27 0.9194, indicating a close resemblance with R 2 and adjusted R 2 . This reveals the goodness of the model and the factors optimized [38,57]. Protease production by solid-state fermentation (SSF) was evaluated with a TP and BW mix as a substrate for Clavispora lusitaniae PC3.…”

“…Using a range of casein concentrations of 1, 2, 3, 4, 5, 10 and 15 mg/mL, the kinetic characteris-tics of the partially purified enzyme were determined under conventional test condi-tions. Tennalli et al [38] determined the kinetic rate constants Km and Vmax using the Lineweaver Burk plot.…”

“…The p-value of each factor was also evaluated. Factors with p-values less than 0.05 (p-value < 0.05) were considered significant factors for protease production [28,38] Protease production was influenced by factors with a significant positive or negative effect (p ≤ 0.05) (Table 3) The Fisher of the model F-value = 15 with p = 0.024 (≤0.05) is very significant, allowing us to conclude that the model was adequate and that the production of the protease was well explained by humidity, inoculum, and incubation period. This is also supported by the value of R 2 (coefficient of determination) of the model, at 0.9756 (a value > 0.75 indicates aptness of the model) [56].…”

“…The p-value and F-value in the ANOVA (Table S3) were determined, and the lesser the p-value was, the greater the importance of the corresponding coefficient was [35,38].…”