| Chiral amino alcohol is a category of compound with optical activity applied in the area of asymmetric catalysis,chiral drug and chemical industry.Although the chemical synthesis technology of chiral amino alcohol is relatively mature,but it is facing serious drawbacks such as environmental pollution and harsh reaction conditions.Compared with the chemical synthesis,biosynthesis responds to the requirement of sustainable development.In the field of biosynthesis,it is easier to convert cheap substrate into value-added complex compound by multi-enzyme cascade reaction than single enzyme reaction.It is of great significance for multi-enzyme cascade reaction to make full use of the enzyme and expand the application of enzyme.In this paper,a new multi-enzyme cascade had been constructed to transform the racemic styrene oxide to(R)-phenylglycinol.The cascade reaction included three modules:hydrolysis,oxidation and transamination.Initially,styrene oxide was hydrolysized to phenylglycol by the epoxide hydrolase VrEH2 mutant.Then,glycerol dehydrogenase EaGDH was chosen to oxidize phenylglycol,coupling with the alanine dehydrogenase AlaDH to regenerate coenzyme NAD+.Finally,transamination of 2-hydroxyacetophenone was carried out by a transaminase mutant ω-TAy150F/V153A with L-alanine as the amino donor.The pyruvate,byproduct of transamination,is converted to alanine by the AlaDH cosuming NADH and ammonia.The resulting NAD+ was reused for the oxidation step.The internal coenzyme cycle was built by AlaDH between oxidation and transamination without extra addition of coenzymes(NAD+and NADH)and cosubstrate.The reaction conditions of single module including buffer and pH were investigated.The result showed reaction conditions of module could be compatible at the pH range of 7.5-8.5.The cascade system successfully obtained(R)-phenylglycinol from racemic styrene oxide in the glycine/NaOH buffer(pH 8.0)and the yield of target product(R)-phenylglycinol was 40.5%,the intermediate phenylglycol content was 59.1%.It was speculated that the equilibrium of the cascade reaction leaded to the accumulation of the intermediate phenylglycol by the reverse reaction from(R)-phenylglycinol to phenylglycol.In order to reduce the accumulation of intermediate,a novel reactor coupled with product adsorption in situ was designed for promoting cascade reaction yield toward(R)-phenylglycinol.Cation exchange resin was selected for the adsorption of product(R)-phenylglycinol to push forward the reaction equilibrium.The yield of target product increased from 40.5%to 81.9%,the content of intermediate reduced to 16.5%.This study indicated(R)-phenylglycinol could be achieved from styrene oxide by the multi-enzyme cascade and the reactor coupled with product adsorption in situ could improve the yield of target product. |
