Cloning Identification And Application Of D-lactate Dehydrogenases From Lactobacillus Fermentum

Phenyllactic acid?PLA?is a natural,small-molecule organic acid widely found in fermented foods,such as:honey and yogurt,sourdough,pickles,and cheese.Because of its high safety,good stability,broad spectrum of inhibition,and good solubility,it is considered to be a new type of natural biological preservative.In the food,medicine,cosmetics and other aspects have a broad application prospects.In this paper,the cloning and expression of D-lactate dehydrogenase?D-LDH?in Lactobacillus fermentum and its enzymatic properties were studied,and the co-expression strain of D-lactate dehydrogenase and glucose dehydrogenase were constructed to achieve coenzyme regeneration by whole cells.The conditions for the production of D-PLA were preliminary investigated.The main results are showed as follows:?1?A strain with strong D-phenyl lactic acid production,Lactobacillus fermentum JN248 was screened from 20 strains of lactic acid bacteria deposited in our laboratory.?2?Through genome-wide analysis,four D-lactate dehydrogenase genes from Lactobacillus fermentum JN248 were successfully cloned.They were:lf-d-ldh0171?972 bp?,lf-d-ldh0031?993 bp?,lf-d-ldh0653?999 bp?and lf-d-ldh0940?1179 bp?.Connected with pCold II plasmid to construct the corresponding recombinant expression vector and successfully transferred into BL21?DE3?. The enzyme activity assay revealed that all four recombinant proteins had D-LDHs activity and the molecular weights of the proteins were approximately 34.48 kDa,35.99 kDa,36.74 kDa,and 43.08kDa,respectively.The phenylpyruvate-reducing ability of a recombinant protein?LF-D-LDH0653?was obtained by verifying and comparing the enzyme activity.?3?Expression conditions of LF-D-LDH0653 was optimized.It was found that when OD₆₀₀reached 0.4,adding IPTG to reach a final concentration of 0.3 mmol·L^-1 was the best induction condition.The LF-D-LDH0653 was purified by nickel column and desalting column,and the enzymatic properties were studied:the optimum temperature was 50?,and the optimum pH was8.0.Higher activity can still be maintained at higher temperatures and weakly alkaline conditions.Pyruvic acid and phenylpyruvate have the strongest catalytic effects.Using phenylpyruvate as substrate K_m,k_cat and k_cat/K_m were 1.68 mmol·L^-1,122.66 s^-1,and 73.01 L·mmol^-1·s^-1,respectively.?4?The gdh gene?786 bp?from Bacillus subtilis ATCC 13952 was successfully cloned,corresponding to a molecular weight of 28.16 kDa.E.coli BL21?DE3?/pCold II-ldh-gdh was constructed and coenzyme regeneration was achieved.The conditions of whole cell synthesis of D-PLA were further optimized:20 mmol·L^-11 disodium hydrogen phosphate-citric acid buffer?pH8.0?,84 mmol·L^-1 glucose,1.5 g·L^-1 whole cell catalyst,60 mmol·L^-1 PPA,45?,200 r·min^-1 reaction 3 h,the conversion rate of 94.7%.