诱变选育D-泛解酸内酯水解酶高产菌及发酵条件优化

Mutagenesis breeding of D-lactonohydrolase high-producing bacteria and optimization of fermentation conditions

该文以实验室筛选得到的一株具有D-泛解酸内酯水解酶活力的镰孢霉菌(酶活力为1.42 U/mL)为出发菌株,先后利用常温室压等离子(atmospheric room temperature plasma,ARTP)和紫外-氯化锂(UV-LiCl)技术,对其进行了4轮递进诱变选育,以筛选得到高活性D-泛解酸内酯水解酶菌株。实验得到ARTP诱变的最佳处理时间为80 s,UV-LiCl诱变的最佳处理时间为80 s(6 g/L LiCl)。通过变色圈大小初筛和摇瓶复筛,最终筛选得到一株酶活力达3.46 U/mL的菌株4-80-6,酶活力较出发菌株提高了143.66%,并且通过8次传代培养,该菌株遗传性较为稳定。以20%的底物浓度催化反应时,突变株4-80-6水解率由原始菌11.6%提高到17.1%,光学纯度由93.5%提高到98.3%。对突变株4-80-6菌株进行发酵条件优化,得到最佳产酶条件为:培养温度25℃,培养基初始pH 8.5,接种量10.5%,培养时间48 h,该条件下酶活力为4.33 U/mL,比优化前提高了25.14%。研究结果为DL-泛解酸内酯酶法拆分的工业应用提供了一种有效策略。

A Fusarium strain with D-pantothenic acid lactone hydrolase activity was selected as the starting strain.A strain with high activity of D-lactonohydrolase was screened by four rounds of progressive mutagenesis with atmospheric room temperature plasma(ARTP)and UV-LiCl technology.The optimum treatment time of ARTP and UV-LiCl mutation was 80 s and the concentration of LiCl was 0.6%).A strain of 4-80-6 with an enzyme activity of 3.46 U/mL was finally screened through preliminary screening of color changing circle and secondary screening of shaking flask.The enzyme activity was 143.66%higher than that of the original strain.After eight generations,the heritability of the strain was relatively stable.When the substrate concentration was 20%,the hydrolysis rate of mutant strain of 4-80-6 increased from 11.6%to 17.1%,and the optical purity increased from 93.5%to 98.3%.The fermentation conditions of mutant strain of 4-80-6 were optimized.The optimum conditions for enzyme production were as follows:the culture temperature was 25℃,the initial pH of culture medium was 8.5,the inoculation amount was 10.5%and the culture time was 48 h.Under these conditions,the enzyme activity was 4.33 U/mL,which was 25.14%higher than that before optimization.The results provide an effective strategy for the industrial application of DL panthenolide enzymatic resolution.