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Aims:  Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain. Methods and Results:  The keratinase gene with and without leader sequence from the chromosomal DNA of Bacillus licheniformis MKU3 was amplified by PCR and cloned into pET30b and transferred into Escherichia coli BL21. The ker gene without leader sequence only expressed in E. coli and the recombinant strain produced an intracellular keratinase activity of 74·3 U ml−1. The ker gene was further subcloned into E. coli‐Bacillus shuttle vector, pWH1520. Bacillus megaterium ATCC 14945 carrying the recombinant plasmid pWHK3 expressed the ker gene placed under xylA promoter and produced an extracellular keratinase activity of 95 U ml−1. Response surface methodology (RSM) was employed to optimize the fermentation condition and to improve the level of keratinase production by the recombinant strain. A maximum keratinolytic activity of 166·2 U ml−1 (specific activity, 33·25 U mg−1) was obtained in 18 h of the fermentation carried out with an initial inoculum of 0·4 OD600 nm and xylose concentration of 0·75% w/v. Conclusions:  Bacillus licheniformis keratinase was cloned and successfully expressed using T7 promoter in E. coli and xylose inducible expression system in B. megaterium. Response surface methodology was employed to optimize the process parameters, which resulted in a three‐fold higher level of keratinase production by the recombinant B. megaterium (pWHK3) than the wild type strain B. licheniformis MKU3. Significance and Impact of the Study:  This study suggests that B. megaterium is a suitable host for the expression of cloned genes from heterologous origin. Optimization of fermentation conditions improved the keratinase production by B. megaterium (pWHK3) and suggested that this recombinant strain could be used for the production of keratinase.

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Objective: Aim of the present investigation was to optimize the acid protease production from Aspergillus spp. through statistical method in solid state fermentation and to study the inhibitory enzyme kinetics.Methods: To fulfill above mentioned aim, seven solid substrates were screened though using PBD (Plackett-Burman Design) and concentrations of three significant were determined by using one of the Response surface methodologies (RSM), Box-Behnken design (BBD). Inhibitory enzymatic effects were carried by using previously developed models.Results: From PBD, wheat bran, soybean meal, and dried potato peel (DPP) were screened as major influencing nutritional factors for enzyme production. Better optimal values were determined by BBD as wheat bran: 8.841 g, soybean meal: 4.557 g, and DPP: 0.661 g with predicted protease activity as 817.83 U/g (±44.047 U/g). Linear, interactive, and quadratic effects of aforesaid substrates on enzyme activity were formulated by quadratic model through multiple regression model (R2Adj:Adjusted R square = 94.78%; R2Pre:Predicted R square = 98.13%). Partial substrate inhibition to crude acid protease activity was notified with casein concentration higher than 0.4 mmol and inhibitory constant, KN, was computed with previous developed mathematical models. Ratio of reaction rate constants, k4/k2, was found to be 0.233 that had confirmed partial casein inhibition to enzyme velocity. Improved activity and kinetics of caseinolysis make amicable for industrial applications.Conclusion: Quick optimization was performed with statistical methodology over conventional approach. Inhibitory enzyme kinetic studies were important for industrial applications of acid protease.