普鲁兰酶N467G突变体的酶学性质分析

Enzymatic characterization of the pullulanase mutant N467G

在淀粉制糖工业中,普鲁兰酶通常与糖化酶配合使用,其在酸性pH和较高温度下的催化活力是影响淀粉脱支效率的主要因素。该研究通过对长野芽胞杆菌(Bacillus naganoensis)普鲁兰酶蛋白质解折叠自由能的差值(ΔΔG)的计算预测和突变位点稳定性分析,选择突变位点并进行定点突变,获得突变体N467G。与野生型普鲁兰酶分子相比,突变体N467G的最适作用温度提高至60℃,最适作用pH降低至4.5;其在60℃条件下孵育2.5 h或在pH 3.5~5.5孵育1 h残留酶活力均保持在80%以上,稳定性明显提高。此突变体的获得为其后续高效表达与工业化应用奠定了重要基础。

Pullulanase is extensively used in preparation of glucose syrup from starch,combined with glucoamylase.However,its catalytic activity at low pH and high temperature is the main factor affecting its efficiency during starch debranching.In this study,the difference of the protein unfolding free energy(ΔΔG)of Bacillus naganoensis pullulanase(PulA)mutants was calculated and the stability of each mutation site was analyzed.A mutant,N467G,was obtained through site-directed mutagenesis of the Asn467 residue of PulA and its biochemical properties were examined.Compared to the wild-type PulA,the optimal temperature of mutant N467G was increased by 5℃to 60℃and the optimal pH was reduced by 0.5 pH value to 4.5.Its relative activity was over 80%after incubating the enzyme at 60℃for 2.5 h or at pH 4.0-5.0 for 1 h,which represented a significantly higher stability.The result lays a foundation for the subsequent high-efficiency protein production and industrial application of this mutant.