Research On Mechanism Of Effects Of Pretreatment Of Baker's Yeast On Asymmetric Reduction

Because of its high efficiency and high enantioselectivity, the biocatalysis has become one of the most important chiral synthesis methods. Baker's yeast can catalyse most asymmetric reduction of β-ketoeasters. The optical active products are important intermediate of synthesizing chiral drugs and other chiral chemicals. But the low enantioselectivity was a main obstacle in large scale production.In the process of asymmetric reduction of β-ketoesters by Baker's yeast, the enantioselectivity of yeast cells can be controlled by allyl compounds added in the reaction system. 4-chrolo-3-oxobutyrate (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 was increased to 94%, while pretreated with allyl bromide, R-CHBE can be detected. But the treat on yeast cells using allyl compounds will cause a yield drop.The allyl compounds were proved to be toxic to the cells and intracellular enzymes by assaying GLC consumption, ATP concentration, the activities of intracellular hydrolases and mitochondrial complexes respectively. And the toxicity was determined by the group binding to the allyl.Washed with PBS buffer for several times, pretreated yeasts can restore activity gradually. With the concentration of substrate increasing, the enantiomeric excess of S-CHBE catalyzed with ally alcohol-pretreated yeast can be kept at 90%, while the enantiomeric excess of R-CHBE catalyzed with ally bromide-pretreated yeast was dropped from 90% to 45%. These results suggested that allyl compounds adsorbed to the active area of enzymes and were considered as competitive enzyme inhibitors and two kinds of enzyme were adsorbed due to different groups connecting to the allyl. Using inhibitor analogs to pretreat the yeast showed that the enzymes have steric hindrance for inhibitor and substrate.Heat can influence the enantioselectivity of yeast cells as an eyzyme inhibitor. As the heat excited temperature rising, the yield of product drops and the e.e. of S-CHBE raises. When treated under 50℃, the e.e. of S-CHBE reaches a maximum. But when the temperature was over 55℃, the yield drops sharply.Heat treatment on yeast cells will cause the descending of the capability of GLC consumption and the activity of intracellular hydrolases. It proves the heat treatmentwill inhibit the activities of cells and most of the intracellular enzymes.Dehydrogenases in the respiration chain of yeast cells have different activities under different heat excite temperatures. And the trend is the same as the change of enantioselectivity of baker's yeast after heat treatment. It shows a possibility that the dehydrogenases in the respiration chain of yeast cells may be one of the enzyme which can catalyse COBE asymmetric reducing to S-CHBE. When treating yeast cells with relative high temperature, the activities of S-enzymes rise and R-enzymes are inhibited. So it reduces the yield of product and makes the e.e. of S-CHBE higher.