Directed Control Of Enzymatic Stereoselectivity In Biocatalytic Asymmetric Catalysis

Nowadays Biocatalytic synthesis has many advantages such as high selectivity andpurity, mild reaction, environmentally friendly and draws a lot of attention all over theworld. Since the early new century, the technology to prepare chiral drugs andintermediates by enzymatic asymmetric catalysis has been booming, and several enzymaticchiral synthesis systems have been established. With directed control of enzymaticstereoselectivity in biocatalytic asymmetric catalysis, it is able to control the direction andextent of reaction in order to prepare target product with high optical purity or specialstructure. The study consists of researching catalytic sites selectivity by phospholipase Band enantioselectivity by oxidoreductase with their directed control methods. The mainresults were as follows:(1) The phospholipase production conditions of Candida albicans MYA-2876wereoptimized. The optimal cultivation parameters are30℃, pH6.5, the inoculum is8%, andthe culture time is72h. Under the optimized conditions, with30%saturation concentrationof (NH4)2SO4to get off impurity and80%saturation concentration of (NH4)2SO4to purifythe PLB, activity of phospholipase reached to the highest yield of201.36U/mg-protein.(2) The research on the enzymatic characterization of Pf-PLB showed that the optimalreaction temperature and pH were30°C and7.8respectively, and they make quite bigdifference on enzyme activity.0.1mM/L Mg2+and Ca2+both promote enzyme activity by23.5%and18.8%respectively, and best substrate of PLB is emulsified liquid ofphosphatidylcholine.(3) With research of effects on the PLB catalytic sites selectivity by directed controlmethods such as reaction condition (temperature, pH, metal ions), medium engineering(organic solvent, ionic liquid, two-phase system), enzyme chemical modification,immobilized enzyme. The main results were as follows: Ionic liquid make a big differenceon the phospholipase B catalytic sites selectivity, PLB Sn-1catalytic sites selectivity (CSS1)huge increased with [Omim][D-HL]（content2/3）, reaching nearly100%, and Sn-2catalytic sites selectivity (CSS2) bring up to the highest47.08%with [Emim]Cl; To two-phase system, the effects on phospholipase B catalytic sites selectivity were in generallevel, but a greater impact on the yield which was increased by more than3times comparedto most of the aqueous phase, in Hexane-water system(Volume ratio1:1) CSS1raise up to84.78%, and in2-heptano-water system (Volume ratio1:1) CSS2go up to the maximum43.77%.(4) After studying reaction between three enzymes(ADH-A3263, ADH-A7011andE-Ketoreductase) and eight substrates(2-octanone,2-heptanone, acetophenone,1-propiophenone,1-phenylethanol,1-phenyl-propanol,2-heptanol and2-octanol), it isfound that optimal temperature is30℃to35℃, ADH-A3263and ADH-A7011are fit foralkaline pH and E-Ketoreductase is suited to neutral pH, however pH change can notprevent the rapid inactivation of the enzyme.(5) To make sure stop buffer was4mol/L guanidine hydrochloride, we screened moreappropriate enzymatic substrates measured by optical rotation rate of reaction in differenttemperature and pH with automatic polarimeter: acetophenone,1-propiophenone,1-phenylethanol,1-phenyl-propanol.(6) With research of effects on the oxidoreductase enantioselectivity by directed controlmethods such as reaction condition (temperature, pH, metal ions), medium engineering(organic solvent, ionic liquid, two-phase system), enzyme chemical modification,immobilized enzyme, and coenzyme regeneration system. The main results were as follows:Organic solvents have a good effect on enantioselective of E-ketoreductase catalyzing1-propiophenone, S-type enantiomeric ratio (E) increased reaching maximum of30.885inisooctane(water content1%), and S-type E bring up to the maximum of3.207inn-Hexane(water content1%); Ionic liquid make a very significant effect on enantioselectiveof E-ketoreductase catalyzing1-propiophenone with a product conformational flipping,R-type E increased with [Emim][D-HL](content50%) reaching maximum of6.769, andS-type E huge bring up to the maximum of200or more with [Bmim][PF6](content50%);Two-phase system make a big difference on enantioselective of E-ketoreductase catalyzing1-propiophenone with a product conformational flipping and conversion rate increasingcompared to organic solvent systems. S-type E huge increased in isooctane–water system(Volume ratio1:1) reaching maximum of333.448and R-type E bring up to the maximum of7.890in ethyl octanoate-water system(Volume ratio1:1).