Expression, Directed Modification Of Lactobacillus Casei L-lactate Dehydrogenase And Its Application

L-Lactate dehydrogenase?L-LDH,EC 1.1.1.27?is a crucial biocatalyst which,in theory,is able to convert substrate into the single enantiomer of product with a maximum yield of100%.Optically pure L-phenyllactic acid?L-PLA?,a highly value-added organic acid and preservative existed in nature,can be produced from the asymmetric reduction of phenylpyruvic acid?PPA?mediated by L-LDH.As a promising compound,L-PLA has been widely applied in food,feed,pharmaceutical and material fields.To date,a variety of L-LDHs that display the reduction activity towards PPA have been discovered.However,the industrial application of L-LDHs still remains a challenge,due to their inherent drawbacks,such as low catalytic activity,unsatisfactory stereoselectivity and poor stability.In this work,to obtain an L-LDH possessing high activity and stereoselectivity towards PPA,three L-LDH-encoding genes derived from Lactobacillus casei CICIM B1192 were successfully expressed in Escherichia coli BL21?DE3?.Subsequently,using PPA as the substrate,L-Lc LDH1 having the highest activity among these three recombinant L-LDHs was selected to be the object of study.Then,in order to enhance its catalytic activity further,the directed modification was carried out followed by preliminary research of its application.The main results are as follows:Using L.casei total DNA as the template,three L-LDH-encoding genes?Lcldh1,Lcldh2and Lcldh3?were amplified by PCR technique.Then,the genes were ligated to the expression vector pET-22b?+?and induced into E.coli BL21?DE3?to construct three corresponding transformants?E.coli/Lcldh1,/Lcldh2 and/Lcldh3?.The asymmetric reduction of 10 mM PPA catalyzed by E.coli/Lcldh1 yielded 6.9 mM L-PLA with more than 99%enantiomeric excess?ee_p?,which was higher than those by E.coli/Lcldh2 and/Lcldh3.The activities of L-LcLDH1,L-LcLDH2 and L-LcLDH3 were determined to be 21.9,18.4 and 16.9 U·mg^-1when using their cell free extracts,respectively.Therefore,L-LcLDH1 has high activity towards PPA.After subjected to the affinity column chromatography,the purified L-Lc LDH1 displayed a specific activity of 75.5 U·mg^-1.The enzymatic properties of L-Lc LDH1 were analyzed.As a result,its optimal temperature and p H were 40°C and 5.0,and it was stable at or below 40°C as well as in a pH range of 4.5–6.0.Its activity was significantly affected by 3 mM Ag⁺and 5mM Pb²⁺.The kinetic parameters,K_m and k_cat/K_m values of L-Lc LDH1 towards PPA were8.23 mM and 11.5 mM^-1·s^-1,respectively.Four recombinant vectors,pET-22b-Lcldh1^Q88R,-Lcldh1^D183N,-Lcldh1^I229A and-Lcldh1^T235G,were constructed by whole-plasmid PCR technique as designed theoretically,and expressed in E.coli BL21?DE3?,respectively.Using PPA as the substrate,two purified L-LcLDH1's mutants,L-LcLDH1^Q88R and L-LcLDH1^I229A,displayed the specific activities of451.5 U·mg^-1 and 512.4 U·mg^-1.Their k_cat/K_m values were 4.8-and 5.2-fold that of L-LcLDH1.Then,using recombinant vectors pET-22b-Lcldh1^Q88R and pET-22b-Lcldh1^I229A229A as the template,two double-site mutagenesis library,E.coli/Lcldh1^Q88R/I229X and E.coli/Lcldh1^I229A/Q88X,were constructed.Finally,E.coli/Lcldh1^Q88R/I229Q and E.coli/Lcldh1^I229A/Q88A having relatively high L-LDH activities towards PPA were screened.The specific activities of L-Lc LDH1^Q88R/I229Q and L-LcLDH1^I229A/Q88A were separately 19.9-and38.8-fold higher than that of L-Lc LDH1,while their k_cat/K_m values were 6.8-and 35.2-fold that of L-Lc LDH1.A total of 90 m M PPA can be completely reduced in 10 h by using E.coli/Lcldh1^I229A/Q88A229A/Q88A whole cells,affording>99%ee_p L-PLA with a yield greater than 95%.In the fed-batch biotransformation process,99.8 mM L-PLA was prepared from 120 mM PPA with a space-time yield of 1.4 g·L^-1·h^-1,in which the yield of L-PLA was 3.5 times higher than the yield of L-PLA by asymmetric reduction of 150 mM PPA in the traditional batch bioconversion mode.This study can lay the foundation for producing L-PLA via biocatalysis.