Li Jianfang

Mutation breeding of Aspergillus niger strain LW-1 for high-yield β-mannanase production

A parent strain of Aspergillus niger LW-1 producing β-mannanase, preserved in our laboratory, was isolated. A strain, N-9, was screened out and further treated with vacuum microwave irradiation and ethyl methane sulphonate (EMS). A mutant strain, E-30, producing a high and stable yield of β-mannanase was obtained through screening by solid-state cultivation on the basic fermentation medium and several generations of bevel subculture. Its enzyme activity (36 675 U/g) was increased by 2.15 times compared to that of A. niger LW-1 (17 048 U/g). The production of high-yield β-mannanase by E-30 remained stable when maintained at 4°C for 2 months.

定点饱和突变提高 Lactobacillus casei L-乳酸脱氢酶对苯丙酮酸的催化效率

背景光学纯L-苯乳酸是一种天然防腐剂，也是一种高附加值的手性分子，在食品、制药和材料等领域有广阔的应用前景。本实验室已发现来源于Lactobacillus casei CICIM B1192的NADH依赖型L-乳酸脱氢酶(L-LcLDH)可不对称还原苯丙酮酸制备L-苯乳酸，但其活性较低。为提高L-LcLDH催化苯丙酮酸的催化效率，构建了一个单突变体L-LcLDHQ88R，其催化效率kcat/Km是L-LcLDH的4.9倍。 目的为进一步提高L-LcLDHQ88R催化苯丙酮酸的催化效率，采用饱和突变技术将位于L-LcLDHQ88R底物结合口袋附近的氨基酸残基Ile229随机替换为其他氨基酸，以获得活性更高的优良突变体。 方法以重组表达质粒pET-22b-LcldhQ88R为模板，采用全质粒PCR技术对L-LcLDHQ88R基因(LcldhQ88R)中编码Ile229的密码子实施饱和突变，构建突变转化子文库。以催化苯丙酮酸的活性为指标，从文库中筛选出优良的突变转化子。 结果突变转化子(Escherichia coli/LcldhQ88R/I229Q)表达出一种由Arg和Gln分别替换了Gln88和Ile229的双突变体L-LcLDHQ88R/I229Q。重组表达产物L-LcLDHQ88R/I229Q的酶学性质分析表明：L-LcLDHQ88R/I229Q的比活性是L-LcLDH的18.5倍，是L-LcLDHQ88R的2.3倍；其催化效率分别为后两者的6.8倍和1.4倍。L-LcLDH突变前后的温度和pH特性改变不大。根据分子对接结果推测出，双突变Q88R/I229Q导致L-LcLDH的底物结合口袋的入口变大和构型的变化可能对其催化活性的提高发挥了重要作用。 结论双突变Q88R/I229Q显著提高了L-LcLDH的活性和催化效率，使得L-LcLDHQ88R/I229Q在不对称还原苯丙酮酸制备L-苯乳酸中成为有潜力的工具酶。