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Improvement of Functions of Novel Enzymes in the Desulfurization Metaoblism of Petroleum by Protein Engineering and Molecular Genetics

蛋白质工程和分子遗传学改进石油脱硫代谢新型酶的功能

基本信息

批准号：
15580063
负责人：
IZUMI Yoshikazu
金额：
$ 2.43万
依托单位：
National University Corporation Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15580063/
关键词：
desulfurization dibenzothiophene Rhodococcus erythropolis desulfinase DszB DszC Bds Bfl BdsA desuofurization

项目摘要

It is difficult to remove polycyclic sulfur compounds in fossil fuels. Dibenzothiophene(DBT) is considered as a model polycyclic sulfur compound contained in fossil fuels. Some bacteria such as Rhodococcus erythropolis D-1 utilize DBT as a sole source of sulfur without breaking its carbon-carbon backbone through the sulfur-specific pathway. In this pathway, DBT is oxidized to DBT sulfone via DBT sulfoxide by DszC,DBT sulfone is converted to 2-hydroxybiphenyl 2-sulfinic acid (HBPSi) by DszA, and HBPSi is desulfurized to 2-hydroxybiphenyl by DszB. Flavin reductase is necessary for monooxygenase reactions by DszC and DszA.The research results of this study are summarized as follows.1.DszB of R.erythropolis KA2-5-1 and its C27S mutant complexed with biphenyl-2-sulfinic acid were crystallized and preliminary X-ray crystallographic analyses were conducted.2.The gene of thermostable falvin reductase (Bfl) from Bacillus sp.DSM411, which coupled with DszC, was overproduced in Escherichia coli, and the recombinant enzymes were purified and characterized.3.Following the above 1, the mutant Q65H of DszB showed higher thermostability and optimal temperature than the parent DszB enzyme.4.Three enzymes (BdsC,A,B) involved in the DBT desulfurization were purified from the moderately thermophilic bacterium, Bacillus subtilis WU-S2B and characterized.

化石燃料中多环硫化合物的去除较为困难。二苯并噻吩（DBT）被认为是化石燃料中含有的典型多环硫化合物。一些细菌，如红红红球菌D-1利用DBT作为硫的唯一来源，而不会通过硫特异性途径破坏其碳-碳主链。在该途径中，DBT被DszC通过DBT亚砜氧化为DBT砜，DBT砜被DszA转化为2-羟基联苯2-亚磺酸（HBPSi）， HBPSi被DszB脱硫为2-羟基联苯。黄素还原酶是DszC和DszA进行单加氧酶反应所必需的酶。本研究的研究成果总结如下：1。对红孢霉KA2-5-1的DszB及其C27S突变体与联苯-2-亚磺酸络合进行了结晶，并进行了初步的x射线晶体学分析。从Bacillus sp.DSM411中获得了与DszC偶联的热稳定性falvin reductase （Bfl）基因在大肠杆菌中过量产生，并对重组酶进行了纯化和鉴定。根据上述1，DszB突变体Q65H比亲本DszB酶表现出更高的热稳定性和最适温度。从中等嗜热菌枯草芽孢杆菌WU-S2B中纯化出三种参与DBT脱硫的酶（BdsC，A，B）并对其进行了表征。