CAJ | 학술논문

对恶臭假单胞菌（ Pseudomonas putida）腈水合酶（ PpNHase）催化己二腈进行研究，发现PpNHase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（ CtNHase）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CtNHase和PpNHase β亚基的氨基酸序列进行比对，结果显示同源性为94?1％，CtNHase存在βAla97缺失。基于此分析，构建了PpNHaseβ97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。
대악취가단포균（ Pseudomonas putida）정수합매（ PpNHase）최화기이정진행연구，발현PpNHase지능최화기이정중1개청기생성단선알산물5청기무선알，구유구역선택성；고환동총모단포균（ Comamonas testosteroni）정수합매（ CtNHase）능최화기이정직접생성기이선이알，불구유최화구역선택성。위료천명저일구역선택성차이적궤제，대CtNHase화PpNHase β아기적안기산서렬진행비대，결과현시동원성위94?1％，CtNHase존재βAla97결실。기우차분석，구건료PpNHaseβ97위Ala적결실돌변체（ΔAla97）。이용고효액상색보검측ΔAla97최화기이정적산물，발현ΔAla97능장저물완전최화위기이선이알，표명βAla97시영향최화기이정유5청기무선알향기이선이알전화적관건안기산。
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 (ΔAla97 ) was constructed?High?performance liquid chromatography ( HPLC ) analysis showed that the mutant ΔAla97 converted adiponitrile to adipamide completely,indicating that Ala97 locating at the β?subunit is the pivotal amino?acid influencing conversion of 5?cyanopentanamide to adipamide.Those results will be helpful to elucidate the regioselective mechanism.