红平红球菌LSSE8-1中辅因子再生系统的构建  

Construction of an Efficient Cofactor-regeneration System in Rhodococcus erythropolis LSSE8-1

作　　者：李海波[1,2] 邢建民[1] 熊小超[1,2] 刘会洲[1] 

机构地区：[1]中国科学院过程工程研究所生化工程国家重点实验室,绿色过程与工程重点实验室,北京100190 [2]中国科学院研究生院,北京100049

出　　处：《过程工程学报》2008年第4期784-788,共5页The Chinese Journal of Process Engineering

基　　金：国家重点基础研究发展规划(973)基金资助项目(编号:2006CB202507);国家高技术研究发展计划(863)基金资助项目(编号:2006AA02Z209)

摘　　要：采用PCR方法从红球菌Rhodococcus sp.RHA1基因组中克隆了编码甲酸脱氢酶的基因(fdh),将该基因片段插入大肠杆菌-红球菌穿梭质粒pBS306,构建了甲酸脱氢酶表达质粒pBS-PFG,转入专一性脱硫菌R.erythropolis LSSE8-1,得到重组菌LSSE8-1-FDH.2,3,5-三苯基氯化四氮唑(TTC)显色反应测得重组菌LSSE8-1-FDH的总脱氢酶活(OD496)为0.464,原始菌LSSE8-1的总脱氢酶活(OD496)为0.353,重组菌比原始菌酶活增加了31%.考察了不同浓度甲酸根对R.erythropolis LSSE8-1与重组菌LSSE8-1-FDH生长的影响,当甲酸根浓度为25mmol/L时重组菌LSSE8-1-FDH的生长最佳.油水相静息细胞脱硫实验表明,重组LSSE8-1-FDH菌比宿主菌的脱硫速率增加了12.5%,总脱硫率增加了约20%.The aim of this study was to develop a new biocatalyst with high NADH regeneration, which was helpful to increase desulfurizatin activity. Formate dehydrogenase gene （fdh） was amplified from Rhodococcus RHA1 and then introduced into an specific desulfurization bacterium, Rhodococcus erythropolis LSSE8-1, using an E. coli-R, erythropolis shuttle vector pBS306. TTC reaction showed that the dehydrogenase activity of LSSE8-1-FDH （OD496） was 0.464, the dehydrogenase activity of LSSE8-1 （OD496） was 0.353, and the dehydrogenase activity increased 31%. Recombinant strain showed a moderately faster growth rate in 25 mmol/L sodium formate solution. Desulfurization of DBT in the oil-aqueous two phases system by LSSE8-1 and LSSE8-1-FDH was conducted respectively. Compared with the orginal strain LSSE8-1, the recombinant strain LSSE8-1-FDH had an increase of 12.5% in desulfurizing activity, 4 h in desulfurizing time, and total desulfuriztion capacity increased 20%.

关 键 词：甲酸脱氢酶 微生物脱硫 红球菌 NADH 二苯并噻吩 

分 类 号：Q819[生物学—生物工程]