近平滑假丝酵母NAD(H)依赖型次级醇脱氢酶的分离纯化及酶学性质  

Purification and Characterization of NAD(H) Dependent Second Alcohol Dehydrogenase from Candida parapsilosis

作　　者：羊明[1] 徐岩 穆晓清[1] 肖荣 

机构地区：[1]江南大学教育部工业生物技术重点实验室,江苏无锡214036 [2]新泽西路特格斯大学高级生物技术与药物中心,美国新泽西08854

出　　处：《应用与环境生物学报》2007年第1期121-125,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家973计划(No.2003CB716008);国家自然科学基金(No.20376031);长江学者和创新团队发展计划(2005)资助项目~~

摘　　要：从近平滑假丝酵母(Candida parapsilosis CCTCC 203011)中分离得到了NAD(H)依赖型的次级醇脱氢酶.粗酶经乙醇沉淀、Blue Sepharose Fast Flow亲和层析、DEAE Sepharose离子交换层析后在SDS-PAGE上显示为单一条带,其酶蛋白的亚基分子量(Mr)为37.5×103.该酶还原反应的最适pH为6.0,最适温度为45℃,是一个含Zn2+金属酶.该脱氢酶具有较高的底物的选择性,对2-酮具有较高的还原活力;同时,也能作用于醇类发生氧化反应,对次级醇的氧化活力最高.以β-羟基苯乙酮为底物,用纯化得到的酶催化反应后,还原产物为(R)-苯基乙二醇,因此该酶为(R)-专一性次级醇脱氢酶,是一种生产(R)-苯基乙二醇的有效的生物催化剂.经LC-MASS-MASS分析得到了酶蛋白中3个肽段的氨基酸序列,通过比对发现该酶与NCBI编号为BAA24528的次级醇脱氢酶具有较高的同源性.An NAD（H） dependent secondary alcohol dehydrogenase was separated from Candida parapsilosis CCTCC 203011. The enzyme gave a single band on SDS - PAGE, which was purified through ethanol precipitation, Blue Sepharose FF and DEAE Sepharose FF chromatography from cell-free extract. The molecular mass of the enzyme subunit was about 37.5 ×10^3. The optimum pH and temperature for reduction were 6.0 and 45 ℃, respectively. In addition, the alcohol dehydrogenase performed high substrate specificity towards 2-ketone for reduction and secondary alcohol for oxidation. For the asymmetric reduction of β- hydroxyaeetophenone, （R）-1-phenyl-1,2-ethanediol was produced by the purified enzyme, so the enzyme was an （R）-specific second alcohol dehydrogenase, which could be an effective bioeatalyst to produce （R）-1-phenyl-1,2-ethanediol. The amino acid sequences of three peptides from the purified enzyme were analyzed by LC-MASS-MASS, and the secondary alcohol dehydrogenase showed high identity to CpSADH reported. Fig 4, Tab 3, Ref 17 .

关 键 词：近平滑假丝酵母 次级醇脱氢酶 分离纯化 β-羟基苯乙酮 

分 类 号：Q814[生物学—生物工程]