The Protein Engineering of Hydrogenase and Its Application

氢化酶的蛋白质工程及其应用

基本信息

批准号：
62480028
负责人：
KARUBE Isao
金额：
$ 4.35万
依托单位：
Research Center for Advanced Science and Technoogy, University of Tokyo (1988)Tokyo Institute of Technology (1987)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-62480028/
关键词：
Escherichia coli Hydrogenase Hydrogen production Protein engineering hydA hydB 大腸菌

项目摘要

This research aimed the improvement of the enzyme character of hydrogenase by protein engineering. This enzyme dominates the hydrogen metabolism in anaerobic bacteria, therefore, the improvement of this enzyme will enhance the microbial hydrogen production efficiency.First, we investigated the roll and the products of both hydA and hydB. According to the gene product analyses, we assumed, 1)hydA product seems to control the expression level of hydrogenase in vivo, and 2)hydB product seems to be one of the subunit constructing hydrogenase.We then attempted the improvement of hydrogenase activity in vivo, by modifing hydA by site directed mutagenesis using synthetic DNA, synthesized by DNA synthesizer (Biosearch; Cyclone). The alternation of one amino acid, cysteine to serine, resulted in the improvement of hydrogenase activity (133% of wild strain). We assumed that the alternation of amino acid sequence in hydA might affect and change the expression level of hydrogenase.We have also found that hvdB mutant restored its hydrogenase activity by cultivating in a medium containing nickel ion. We also found that hydB mutant cultivated in media containing iron or vanadium ion restored only hydrogen uptake activity but not hydrogen evolution activity of hydrogenase. The cultivation in the presence such metals might result in incorporation of these metals in active center of hydrogenase instead of nickel, hence the alternation in its character might occur.In conclusion, we have been suceeded in the modification of hydrogenase by means of proteinengineering. These results will contribute to the research of microbial hydrogen production.

这项研究旨在通过蛋白质工程改善氢化酶的酶特征。这种酶在厌氧细菌中主导了氢代谢，因此，该酶的改善将提高微生物氢的生产效率。首先，我们研究了HYDA和HYDB的卷和产物。 According to the gene product analyses, we assumed, 1)hydA product seems to control the expression level of hydrogenase in vivo, and 2)hydB product seems to be one of the subunit constructing hydrogenase.We then attempted the improvement of hydrogenase activity in vivo, by modifing hydA by site directed mutagenesis using synthetic DNA, synthesized by DNA synthesizer (Biosearch; Cyclone).一种氨基酸（半胱氨酸与丝氨酸）的交替导致氢化酶活性的改善（野生应变的133％）。我们假设HYDA中氨基酸序列的交替可能会影响并改变氢化酶的表达水平。我们还发现，HVDB突变体通过在含有镍离子的培养基中培养来恢复其氢化酶活性。我们还发现，在含铁或钒离子的培养基中培养的HYDB突变体仅恢复氢的活性，而不是氢化氢化氢化氢的氢进化活性。这种金属在存在下的培养可能导致这些金属将这些金属掺入活性的氢化酶中心而不是镍中心，因此可能发生交替的交替。总而言之，我们通过蛋白质开发的方式对氢化酶进行了修饰。这些结果将有助于研究微生物氢的产生。