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Enhanced production of alkaline protease enzyme by mutated Aspergillus oryzae developed by random mutagenesis
Abstract
Thermostability is an important attribute and a prerequisite for the enzymes used in industrial processes. Aspergillus oyzae has the potential to produce proteases for industrial applications. In this study, A. oryzae mutants were used to identify hyper-producers of thermostable proteases. Initially, 22 mutants were selected on the basis of their growth performance, cultured on 1% skimmed milk media plates and Clearing Indexes (CI). Five mutants with higher ‘CI’ were selected for submerged fermentation employing 1% casein as substrate and following growth conditions were used: pH 6.5, temperature 30°C and shaking at 150 rpm for 84 hr. Irreversible thermostability was determined at 65°C using pseudo-first order plots. Mutant strain M-120(3) showed highest production and thermostability of protease compared to control and other mutants with half-life (t½) of 125 min. Effect of temperature and substrate concentration on growth kinetics of Mutant M-120(3) were determined showing optimum temperature of 32°C with a specific growth rate (µ) of 0.037h-1 and specific rate of product formation (qp) of 0.31 (U g-1h-1). Kinetics and thermodynamic parameters confirmed that mutant strain M-120(3) required less activation energy (Ea) to produce protease enzyme (Eap = 65.22 kJ mol-1) and cell mass (Eax = 29.074 kJ mol-1) than control strain. It had low maximum specific rate of cell mass formation (µmax) 0.043 kJ mol-1 and higher maximum specific rate of product formation (qp) 1.07 kJ mol-1. These parameters confirmed that Mutant M-120(3) had great potential for commercial scale production of thermostable proteases for industrial application
