Strain improvement by mutation for enhanced production of starch‐saccharifying glucoamylase from Bacillus licheniformis

Abstract

A natural isolate, Bacillus licheniformis KIBGE-IB3, was subjected to mutational analysis for the enhanced production of glucoamylase. Randomly generated mutants were initially screened for enzyme production and after secondary screening B. licheniformis KIBGE-IB3M67 demonstrated maximum glucoamylase production with improved starch saccharifying capability. Enzyme activity from KIBGE-IB3M67 reached up to 25 kU/mg by strain improvement, which was nearly twofold increase in enzyme production as compared to the parent strain. SEM revealed that raw potato starch is more profoundly hydrolyzed when treated with mutant glucoamylase. Successful mutation also reduced the fermentation time from 48 to 24 h for glucoamylase production, optimum fermentation pH and temperature slightly shifted from 7.0 (wild) to 7.5 (mutant), from 30 to 508C (wild), and from 20 to 558C (mutant), respectively. Reduction in fermentation time and enhanced starch saccharification ability makes this mutant (KIBGE-IB3M67) an attractive candidate for the production of glucoamylase at commercial level.

Purification and Characterization of a Thermostable Starch‐Saccharifying Alpha‐1,4‐Glucan‐Glucohydrolase Produced by Bacillus licheniformis

“…In the current study, glucoamylase was purified to homogeneity produced from both strains and was used to study the kinetic properties of this biocatalyst. The wild‐type was previously subjected to mutational analysis and the data were presented earlier …”

“…Bacillus licheniformis KIBGE‐IB3 [GenBank: GU216260] (wild‐type) and its hyper‐productive mutant, B. licheniformis KIBGE‐IB3M67, which are capable of producing starch hydrolyzing glucoamylase was previously isolated indigenously . In our previous work, the production of starch hydrolyzing glucoamylase from the wild‐type stain was improved through mutation.…”

“…Glucoamylase activity was determined after production and after every step of purification. Detailed information regarding enzyme assay and enzyme activity conditions have been described previously . One unit of glucoamylase is expressed as kilo Units per mg of protein (kU mg –1 ) and is defined as “the amount of glucoamylase that catalyzes the conversion of 1 µ m of substrate (starch) per minute under standard assay conditions.”…”

“…Substrate specificity of the purified enzyme was tested using starch from different origins. Enzyme activity was performed as described earlier under standard assay conditions …”

“…Previously, Bacillus licheniformis KIBGE‐IB3 was mutated and a hyper‐productive mutant was selected for further characterization and purification . Current study is an effort to purify and characterize bacterial α‐1,4‐glucan‐glucohydrolase (glucoamylase), which is produced by B. licheniformis KIBGE‐IB3 and its mutant strain known as B. licheniformis KIBGE‐IB3M67 by keeping the commercial aspects in view.…”