Optimization of protease production in indigenous Bacillus species isolated from soil samples in Lagos, Nigeria using response surface methodology

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Abstract Proteases catalyse the hydrolysis of peptide bonds in proteins and offer a huge potential for application in industries, including detergent, dairy, leather, baking, pharmaceutical and beverage industries. In this study, indigenous Bacillus species were isolated from soil samples collected from abattoir, refuse and non-refuse sites in Lagos, Nigeria and optimized for protease production. The isolates were purified on Bacillus agar and screened for protease production on casein agar. Three strains showing high potential for protease production were identified as Bacillus cereus ABBA1, Bacillus subtilis RD7 and Bacillus subtilis NRD9 via amplification and analysis of 16S rRNA genes. Protease optimization was done Insilco using Box-Behnken Design (BBD) by response surface methodology (RSM) with Design-Expert software and then validated experimentally. Factors optimized include temperature, pH, carbon and nitrogen source and inoculum density. Statistical analysis was done using ANOVA. The results obtained from the Insilco experimental model revealed high protease activity of 159.43 U/ml, 141.28 U/ml and 138.17 U/ml while experimental validation generated a high protease activity of 200.56 U/ml, 176.00 U/ml and 163.76 U/ml for strains ABBA1, RD7 and NRD9, respectively in optimized medium. This corresponds to 33.54-, 42.21- and 36.64- fold increase in protease production compared to the unoptimized protease production medium. The optimum conditions for extracellular protease production obtained from quadratic model of RSM were 40 °C, pH 8.5, 2.5% (v/v) inoculum density, 1.5 g/L maltose and 2.0 g/L beef extract powder. The model prediction agreed with the experimental data (R2 = 0.98) and was statistically significant (p ≤ 0.05). This results further confirms the need to optimize the production parameters to achieve maximum yield and economical use of available resources during production of industrially important enzymes.