# Gama rays mediated improvement of catalytic efficiency and thermostability of glucoamylase by replacing active site leucine to isoleucene from super koji (Aspergillus oryzae)

## TL;DR

This study uses gamma rays to improve the efficiency and heat stability of a key food industry enzyme by altering its active site.

## Contribution

A novel point mutation (Leu203 → Ile) in glucoamylase from Aspergillus oryzae enhances catalytic efficiency and thermostability via gamma ray-induced mutation.

## Key findings

- Mutant glucoamylase M-60(5) showed 1.62-fold higher kcat and 4.75-fold higher kcat/Km compared to the parent enzyme.
- The mutant enzyme had a 1.92-fold longer half-life at 55°C and improved thermodynamic parameters for starch hydrolysis.
- The point mutation altered the enzyme's active site conformation, stabilizing the transition state and enhancing performance.

## Abstract

Glucoamylase is considered as an essential enzyme in food industry. However, lowere catalytic efficiency and weak thermostability confine its application in food industry. Therefore, the current study was aimed to improve catalytic efficiency and thermostability of glucoamylase by replacing active site leucine to isoleucene from Super Koji (Aspergillus oryzae) using gama rays mediated point mutation. High catalytic efficiency and thermostability of glucoamylase from mutant Aspergillus oryzae M-60(5) (screened from 51 mutants) was achieved due to a point mutation, i.e., Leu203 → lle in active site. The SDS-PAGE molecular mass of parent and mutant glucoamylase was 63.1 kDa, while mutant glucoamylase showed; productivity =  9.7 U ml‒1, kinetic constants kcat = 118 (1.62 fold), (kcat/Km) = 1899 (4.75 fold) and half-life at 55 °C for 45 min (1.92 fold). Thermodynamics parameters for starch hydrolysis of parent glucoamylase were; ΔH*= 47.755 kJ mol‒1 and ΔG*= 67.975 kJ mol‒1 while for mutant ΔH*= 44.263kJ mol‒1 and ΔG*= 66.514 kJ mol‒1. The ΔG* of irreversible thermostability for parent and mutant at 55 °C was 104.95 kJ mol‒1 and 101.52 kJ mol‒1respectively. The point mutation altered the conformation of the glucoamylase active site that contributed to improve the functional energy (ΔG*), resulted the stabilization of transition state which made it thermostable and highly efficient in starch hydrolysis.