法尔皮有孢汉生酵母(Hanseniaspora valbyensis)醇乙酰基转移酶的分离纯化及特性

Purification and Properties of Alcohol Acetyltransferase from Hanseniaspora valbyensis

乙酸苯乙酯是苹果酒里的重要香气物质之一,是在醇乙酰基转移酶(AATase)的催化下由乙酰辅酶A和苯乙醇缩合而成。从法尔皮有孢汉生酵母(Hanseniasporavalbyensis)中收集细胞膜片段,然后经过DEAESepharose、SephadexG-75、OctylSepharose三步层析分离得到纯酶。纯化后的酶经SDS-PAGE电泳,得到一条明显的条带,分子量在37kD左右。此酶的最适反应条件是在30℃、pH7.0,在10℃以下酶比较稳定,pH稳定在7.0～8.0。重金属离子Hg2+、Zn2+、Pb2+对酶活有明显的抑制,EDTA、Mn2+对酶活没有太大的影响,Mg2+能激活此酶。此酶也能催化其它重要乙酸酯的合成,但对乙酸苯乙酯的合成具有特异性。

Phenethyl acetate, one of the major characteristic aroma compounds in apple wine, was synthesized by the condensation of acetyl-CoA and phenethyl alcohol under the catalysis of alcohol acetyltransferase （EC 2.3.1.84）.The enzyme was dissolved from the cell membrane fraction of Hanseniaspora valbyensis, by 1% Triton and then purified by three steps of chromatographic separations： DEAE Sepharose, Sephadex G-75 and Octyl Sepharose. The purified enzyme had a single band on a SDS polyacrylamide gel, and its molecular mass was estimated to be about 37kD. The optimal temperature and pH for the purified AATase activity were 30℃ and pH7.0 respectively. It was stable between pH7.0 and pH8.0, but unstable at temperatures above 10℃. The activity was strongly inhibited by heavy metal ions such as Hg^2＋, Zn^2＋, Pb^2＋, but slightly stimulated by Mg^2＋ and slightly inhibited by EDTA and Mn^2＋. The enzyme catalyzes the estedfication of other alcohol with acetyl-COA to form acetate ester, but shows specificity to form phenethyl acetate.