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Rational design of glycerol dehydratase:Swapping the genes encoding the subunitsof glycerol dehydratase to improve enzymatic properties

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Chinese Science Bulletin 2006年第24期51卷 2977-2985页

作者： QI Xianghui SUN Liang LUO Zhaofei WU Jiequn MENG Xiaolei TANG Yue WEI Yutuo HUANG Ribo Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization Guangxi University Nanning 530004 China Guangxi Academy of Sciences Nanning-530003 China Sinozyme Biotech. Co. Ltd. Nanning 530004 China

1,3-propanediol (1,3-PD) is an important material for chemical industry,and there has been always much interest in the production of 1,3-PD using all possible routes. The genes encoding glyc-erol dehydratase (GDHt) from Citrobacter freundii,Klebsiella pneumoniae and metagenome were cloned and expressed in E. coli. All glycerol dehy-dratases but the one from metagenome could be detected to show enzyme activities. In order to im-prove the enzymatic properties of GDHts,the genes encoding α and β-γ subunits were cloned,and the enzyme characteristics were evolved by rational de-sign based on their 3D structures which were con-structed by homology modeling. Six heteroenzymes were obtained by swapping the α subunit genes of these three different-source-derived GDHts. The pH,thermal stability and Vmax of some heteroenzymes were dramatically improved by 2―5 times compared with the wild one (GDHtKP). The GDHt cloned from metagenome,originally proved to be with no enzyme activity,was converted into active enzyme by swap-ping its subunits with other different GDHts. In addi-tion,the effect of subtle 3D structural changes on the properties of the enzyme was also observed.

关键词：甘油脱水酶基因交换技术异源同工酶设计异基因组基因克隆

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