Enhancing thermostability of β-amylase from Bacillus flexus CCTCC 2015368 based on in vitro molecular evolution

Abstract:

We used in vitro molecular evolution technology by error-prone PCR and high-throughput screening to improve thermostability of Bacillus flexus CCTCC 2015368 β-amylase. Mutant D476N with significant thermostability increase was selected by LB agar starch plate colorimetric assay and 96-well plate enzyme activity assay. The optimum pH was 6.5 for the mutant D476N, compared to 7.0 of the wild type. The optimal temperature was 55 ℃ for both mutant D476N and the wild type. The T50 value of the mutant D476N was 4 ℃ higher than that of the wild type. The half-life of mutant D476N at 55 ℃ was 35 min, 95% higher than that of the wild type. The Km of the mutant D476N was 97.98 μmol/L, 1.14 times of that of the wild type (85.86 μmol/L). The thermostability of the mutant D476N was slightly lower than that of the wild type. The three-dimensional structure of wild type and mutant D476N was simulated by SWISS-MODEL and analyzed by PyMol software. The mutated amino acid residue Asn476 was located on the loop of protein surface. The molecular free energy(ΔG) of D476N was calculated by MOE software was 106.0 kcal/mol, reduced by 10.3% compared to the wild enzyme. These results were consistent with the theory that the protein molecular free energy and thermostability were negatively correlated.

Key words:molecular evolution, error-prone PCR, β-amylase, thermostability