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›› 2007, Vol. 7 ›› Issue (5): 1004-1008.

• 生化工程专栏 • 上一篇    下一篇

烯丙基化合物对面包酵母立体选择性的影响机理

李彦,倪宏亮,姚善泾   

  1. 浙江大学 化学工程与生物工程学系
  • 出版日期:2007-10-20 发布日期:2007-10-20

Mechanism of Effects of Allyl Compounds on Asymmetric Reduction of Baker¢s Yeast

LI Yan,NI Hong-liang,YAO Shan-jing   

  1. Department of Chemical and Biochemical Engineering, Zhejiang University
  • Online:2007-10-20 Published:2007-10-20

摘要: 添加烯丙基化合物可以控制酵母催化不对称还原反应的立体选择性,以4-氯-3-羰基丁酸乙酯(COBE)为模型底物,用丙烯酰胺和烯丙基醇对酵母进行预处理,产物(S)-4-氯-3-羟基丁酸乙酯(S-CHBE)的对映体过量值(e.e.)可以增加至94%;而用烯丙基溴预处理,则可以得到R型产物. 通过DNS法和API ZYM试验条证明烯丙基类化合物对细胞及胞内酶系存在毒害作用,且作用大小取决于烯丙基所带基团. 预处理后的酵母细胞经PBS缓冲液多次洗涤后,其活性可以逐渐得到恢复. 随着底物浓度的增加,经烯丙基醇预处理的酵母细胞,其S型产物的e.e.值维持在90%左右,而经烯丙基溴处理后,反应得到的R型产物的e.e.值则从90%下降到45%左右. 说明烯丙基类化合物竞争性地吸附于酶的活性区域,且其所带基团决定了被吸附酶的类型. 以抑制剂类似物处理酵母细胞,其反应结果说明酵母体内催化不对称还原反应的两类酶存在着空间位阻效应.

关键词: 不对称还原, 酶抑制剂, 酶抑制剂, 立体选择性, 催化机理

Abstract: In the process of asymmetric reduction of b-ketoesters by baker¢s yeast, the enantioselectivity of yeast cells could be controlled by allyl compounds added in the reaction system. 4-Chrolo-3-oxobutanoate (COBE) was used as the model substrate. After yeast cells were pretreated with acrylamide and allyl alcohol, the enantiomeric excess (e.e.) of S-CHBE increased to 94%, while pretreated with allyl bromide, R-CHBE could be detected. The allyl compounds were proved to be toxic to the cells and intracellular enzymes by DNS and API ZYM system respectively. Furthermore, the toxicity was determined by the groups the allyl carried. Washed with PBS buffer for several times, the activity of pretreated yeasts could be recovered gradually. As the concentration of substrate increased, the e.e. of S-CHBE catalyzed by ally alcohol-pretreated yeast remained at 90%. On the other hand, the e.e. of R-CHBE catalyzed by allyl bromide-pretreated yeast dropped from 90% to 45%. These results suggested that allyl compounds acted as competitive enzyme inhibitors adsorbed on the active site of enzymes, and different groups of allyl compounds determined the types of subjected enzymes. The result in pretreatment of the yeast with inhibitor analogs showed that the enzymes had steric hindrance for both inhibitor and substrate.

Key words: asymmetric reduction, 4-chrolo-3-oxobutanoate (COBE, enzyme inhibitor, enantioselectivity, catalysis mechanism