Efficient Preparation Of L-phenyllactic Acid By Coupling L-lactate Dehydrogenase With Glucose 1-dehydrogenase

L-phenyllactic acid?L-PLA?is a highly value-added natural organic acid with broad-spectrum and effective inhibitory activity against a variety of food-borne pathogens and putrefactive bacterias.And it is easily absorbed by human metabolism,so it can replace preservatives to use in food and feed.As a synthetic precursor of many drugs and materials,L-PLA has also attracted much attention for the important role of medicine and materials.The present research reports on biosynthesis of L-PLA showed low yield and production efficiency,which limited the large-scale production of L-PLA.In this study,a Lactobacillus casei L-lactate dehydrogenase mutant,L-LcLDH1^Q88A/I229A coupled with glucose1-dehydrogenase to construct coenzyme regeneration systems either in vivo or in vitro.Then the double-enzyme system in vitro was used in asymmetric reduction of phenylpyruvate?PPA?to L-PLA,laying the foundation for the industrial production of L-PLA.L-LcLDH1^Q88A/I229A and Thermoplasma aciddophilium glucose 1-dehydrogenase SyGDH were used to construct a coenzyme regeneration system either in vivo or in vitro.The recombinant plasmid pET-22b-Lcldh1^Q88A/I229A was transformed into E.coli/pET-28a-Sygdh to obtain a co-expressing recombinant E.coli/pET-22b-Lcldh1^Q88A/I229A/pET-28a-Sygdh,which was used for asymmetric reduction of PPA.Compared with E.coli/pET-22b-Lcldh1^Q88A/I229A,the yield of L-PLA was improved.Under the induction conditions of temperature 15?,0.6 mM IPTG,12 h of induction time for the co-expressing recombinant E.coli,the enzyme activities of L-LcLDH1^Q88A/I229A and SyGDH were 158.7U·g^-1?wet cells?and 44.6 U·g^-1?wet cells?,respectively,which were increased by 17.1%and39.5%compared with before optimization.E.coli/pET-22b-Lcldh1^Q88A/I229A and E.coli/pET-28a-Sygdh were induced by IPTG,and these E.coli cells were broken to obtain crude enzymes.Then crude enzymes were purified with Ni-NTA column to obtain pure enzymes.The crude and pure enzymes of L-LcLDH1^Q88A/I229A and SyGDH were coupled respectively to construct two coenzyme regeneration systems in vitro.Asymmetric reduction of 10 mM PPA by the co-expressing recombinant E.coli cells,crude-enzyme system,and pure-enzyme system,the yields of the L-PLA were 86.9%,91.3%,and 99.6%,respectively.However,there was no by-products only by the pure enzyme system.Therefore,the pure enzyme system is most suitable for asymmetric reduction of PPA to L-PLA.In order to solve the problem of high cost of pure enzymes,L-LcLDH1^Q88A/I229A88A/I229A was expressed in Pichia pastoris GS115 and crude enzyme of SyGDH expressed by E.coli was heat-treated.The result of SDS-PAGE analysis showed that the apparent molecular weight of L-LcLDH1^Q88A/I229A?Pp?was 36.8 kDa.The fermentation broth had few heteroproteins,and the specific activity was 270.5 U·mg^-1.After purification,the kinetic parameters were determined.V_max and catalytic efficiency k_cat/K_m of L-LcLDH1^Q88A/I229A?Pp?were 627.0U·mg^-1 and 94.3 mM^-1·s^-1,which were 26.1 and 25.5 times these of E.coli recombinase L-LcLDH1^Q88A/I229A?Ec?,respectively.SyGDH has good thermal stability with 93.1%of residual enzyme activity after heated at 50?for 1 h.SyGDH in heat treatment was coupled with reLcLDH1^Q88A/I229A to catalyze 10 mM PPA with a yield of 99.6%L-PLA,without any by-products.The asymmetric reduction of PPA?100 mM?was performed at 40°C and pH 5.0in an optimal biocatalytic system,containing 10 U·mL^-1 reLcLDH1^Q88A/I229A,1 U·mL^-1SyGDH,2 mM NAD⁺and 120 mM D-glucose,producing L-PLA with 99.2%yield and a total turnover number?TTN?of 49.6 for NADH recycling.To achieve efficient recycling of NAD⁺,a Lysinibacillus sphaericus G10 glucose1-dehydrogenase mutant LsGDH^D255C,with good affinity of NAD⁺,heat-and acid-resistant,was selected to replace SyGDH for the coenzyme regeneration in vitro.When the NAD⁺concentration was 0.1 mM,it catalyzed 100 mM PPA with a yield of 99.5%L-PLA and the TTN of 995 in 2 h.Then,the amount of LsGDH^D255C was optimized.When the amount of LsGDH^D255C was 4 U·m L^-1,the reaction time could be shortened to 30 min.Finally,the new coenzyme regeneration in vitro was used to produce L-PLA by feeding PPA three times,accumulating 359.8 mM L-PLA with an enantiomeric excess value?ee_p?more than 99%.In the meanwhile,the space time yield was 10.0 g·L^-1·h^-1 and the average conversion rate was269.3 g·g^-1·L^-1.The sample was isolated and purified,getting L-PLA with the yield of 70.6%.