恶臭假单胞菌腈水合酶βAla97影响催化己二腈的区域选择性  

βAla97 of nitrile hydratase from Pseudomonas putida affects regioselectivity towards adiponitrile

作　　者：张君[1] 崔文璟[1] 刘义[1] 崔幼恬[1] 周哲敏[1] 

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《生物加工过程》2015年第3期41-46,共6页Chinese Journal of Bioprocess Engineering

基　　金：国家自然科学基金(31070711)

摘　　要：对恶臭假单胞菌(Pseudomonas putida)腈水合酶(Pp NHase)催化己二腈进行研究,发现Pp NHase只能催化己二腈中1个氰基生成单酰胺产物5-氰基戊酰胺,具有区域选择性;睾丸酮丛毛单胞菌(Comamonas testosteroni)腈水合酶(Ct NHase)能催化己二腈直接生成己二酰二胺,不具有催化区域选择性。为了阐明这一区域选择性差异的机制,对Ct NHase和Pp NHaseβ亚基的氨基酸序列进行比对,结果显示同源性为94.1%,Ct NHase存在βAla97缺失。基于此分析,构建了Pp NHaseβ97位Ala的缺失突变体(ΔAla97)。利用高效液相色谱检测ΔAla97催化己二腈的产物,发现ΔAla97能将底物完全催化为己二酰二胺,表明βAla97是影响催化己二腈由5-氰基戊酰胺向己二酰二胺转化的关键氨基酸。Nitrile hydratase （NHase） from Pseudomonas putida （PpNHase） to catalyzes adiponitrile （ a kind of dinitriles） only to 5-cyanopentanamide （ monoamide）, indicating that only one of the cyano group was catalyzed to amide whereas the others not. This finding demonstrates that PpNHase exhibits catalyzing regioselectivity. On the other hand, NHase derived from Comamonas testosterone （CtNHase） catalyzes adiponitrile directely to adipamide （ diamide ）, without catalyzing regioselectivity towards adiponitrile. In order to illustrate the mechanism of regioselectivity of PpNHase towards adiponitrile, alignment of protein sequence of β subunit between PpNHase and CtNHase was compared. Though they shows high homology （94. 1%）, the β subunit of PpNHase has an additional 97 alanine compared to that of the CtNHase. Therefore, a mutant PpNHase with deletion of Ala97 （AAla97） was constructed. High- performance liquid chromatography （HPLC） analysis showed that the mutant AAla97 converted adiponitrile to adipamide completely, indicating that Ala97 locating at the β-subunit is the pivotal aminoacid influencing conversion of 5-cyanopentanamide to adipamide. Those results will be helpful to elucidate the regioselective mechanism.

关 键 词：腈水合酶 己二腈 生物催化 区域选择性 

分 类 号：Q814[生物学—生物工程] Q78