PJB-2023-1560
Random mutagenesis of koji mold (Aspergillus oryzae) to enhance the catalytic efficiency and thermostability of glucoamylase
Anam Saqib and Muhammad Hamid Rashid
Abstract
Current report explains the effects of γ-ray mutagenesis on kinetics and thermodynamics of stability-function of Koji glucoamylase (GA). It is generally believed that stability and function are inversely proportional. Our results are novel as it reports about the simultaneous improvement in stability and function of glucoamylase through random mutagenesis. Aspergillus oryzae strain mutated by 60 kRad g-rays exposure was termed as M-60(5) and the mutant Koji showed enhanced (2.6 fold) production of GA. The pKa1 & pKa2 of active site residues (ASRs) evidenced about carboxyl and imidazole as ionizable groups. Thermostability of mutated GA increased ~2 fold at 55°C. The change in active site conformation due to γ-ray mediated random mutagenesis has improved kinetics of starch hydrolysis; constants for parent GA: Km (% w/v), Vmax /Km & Vmax (U mg-1 protein) for starch hydrolysis at 50°C and pH 5.0 were 0.022, 79.34 & 3606, whereas for mutant GA these were 0.065, 246 & 3785. The DHi of ASRs ionization confirmed that the conformation of mutant “M-60(5)” GA was altered due to mutagenesis. The mutation altered the conformation of the GA active site that contributed to improve the functional energy (ΔG*) from 99.35 kJ mol-1 for parent to 105.62 kJ mol-1 for mutant GA, resulted stabilization of the transition state (ΔG* is the heat content a protein molecule has to resist against thermal unfolding), which made it thermostable and highly efficient in starch hydrolysis
To Cite this article:
Saqib, A. and M.H. Rashid. 2023. Random mutagenesis of koji mold (Aspergillus oryzae) to enhance the catalytic efficiency and thermostability of glucoamylase. Pak. J. Bot., 55(5): DOI: http://dx.doi.org/10.30848/PJB2023-5(38)
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