Study On Enzymatic Preparation Of D-alanine

D-alanine has antibacterial and analgesic effect,and it is an important organic chiral resource.It is widely used in medicine,food,pesticide,cosmetics and other fields.At present,the mainly industrial preparation method of D-alanine is enzymatic resolution.It requires the preparation of DL-alanine derivatives,which makes the reaction process complicated.The aim of this paper is to find a cheap and efficient biological enzymatic method to prepare D-alanine.Recombinant aspartate racemase and Recombinant D-amino acid aminotransferase were constructed through gene engineering technology by using E.coli and yeast expression system.The preparation of D-alanine by double enzyme method was investigated,and finally a new and efficient method to prepare D-alanine was obtained.The main research is as follows:1.Recombinant aspartate racemase(AspR)and D-amino acid transaminase(DaaT)with his tag were expressed in prokaryotic expression system,and the preparation of D-Ala by double enzyme cascade reaction was investigated.The optimum conditions for racemization catalyzed by AspR whole cell were pH 7.0,40?,100 g/L L-Asp.The optimum conditions for transamination catalyzed by the purified DaaT enzyme were 4 mmol/L PLP,pH 8.0,42?,n(D-Asp):n(PA)=10:1,25 g/L D-Asp.D-alanine was prepared by AspR whole cell and DaaT purified enzyme liquid.By catalyzing 25 mL 10%Asp,the yield of D-alanine finally reaches up to 12.1 g/L.2.Recombinant aspartate racemase and D-amino acid transaminase were expressed in yeast expression system,and the preparation of D-Ala by double enzyme coupled reaction was investigated.The fermentation of recombinant strainX-33/pPICZaA-DaaT and X-33/pPICZaA-AspR were induced by methanol for 120 h.The concentration of DaaT in fermentation supernatant reached 0.58mg/ml,and the concentration of AspR in concentrated fermentation supernatant was 0.12mg/ml.D-Ala was prepared by coupled reaction of concentrated X-33/pPICZaA-AspR fermentation supernatant and X-33/pPICZaA-DaaT fermentation supernatant,which catalyzed 1%L-Asp reaction for 20 h,and the yield of D-Ala was up to 82%.3.A new enzymatic preparation of D-Ala by cascade reaction has been developed.The fermentation conditions of the engineered strains BL21/pET-Duet-AspR and X-33/pPICZaA-DaaT were optimized respectively.The optimum fermentation conditions of the former were:30?,pH 8,add 25 mL fermented liquid to 250 mL Erlenmeyer flask,200 rpm,13h.The optimized fermentation conditions of the latter were:30?,pH 6,add 75 mL fermented liquid to 500 mL Erlenmeyer flask,250rpm,add methanol every 12h to end concentration 1.25%,144 h.Then,using the whole cell of BL21/pET-Duet-AspR and fermentation centrifugal supernatant of X-33/pPICZaA-DaaT,which were both obtained in the optimal fermentation conditions,to prepare D-alanine.The yield of D-alanine reached 14.47 g/L,and 6.7 g D-Ala was separated by cation exchange resin.The obtain rate of the alanine was 78%,the enantiomeric excess of alanine is 98%.