Mining And Characterization Study Of D-lactic Acid Synthesis Related Enzyme Genes In Leuconostoc Citreum

Lactic acid can be divided into D-lactic acid and L-lactic acid according to its optical activity.Due to the lack of specific metabolic enzymes of D-lactate in human body and the low renal clearance rate of D-lactate,D-lactate accumulation can lead to acidosis.Consumption of fermented foods such as kimchi and yogurt is one of the ways D-lactic acid builds up in the body.The production of D-and L-lactic acid in fermented food and the ratio of the production of D-and L-lactic acid depend on the types of fermenting microorganisms and fermentation conditions.As a food starter,Leuconostoc citreum is widely used in fermented foods such as kimchi.Studies have shown that Leuconostoc citreum only contains D-Lactate hehydrogenase（D-LDH）,which catalyzes the production of D-lactic acid from pyruvate,and its potential harm should be paid attention to.Although the genome of Leuconostoc citreum has been sequenced,the function of many genes has been slowly studied.In order to improve the application value of Leuconostoc citreum in practical production,it is necessary to excavate and study the key enzymes related to D-lactic acid production,so as to lay a foundation for improving the safety of Leuconostoc citreum by genetic engineering or knocking down D-LDH,and make it more suitable for food industry starter culture.In this study,genomic,transcriptomic and phylogenetic analyses were carried out to explore the coding genes of D-LDH.The D-LDH gene in KM20 was cloned and expressed,and the expression plasmid was constructed and transformed into Escherichia coli BL21（DE3）for overexpression.The recombinant D-LDH was purified by Ni-NTA column affinity chromatography,and its enzymatic characteristics were analyzed.The main findings are as follows:1.According to genomic analysis,7 genes encoding lactate dehydrogenase or related enzymes were obtained;phylogenetic analysis concluded that LCK₀0389 was evolutionarily conserved in Leuconostoc citreum KM20 and related lactic acid bacteria;KM20 was analyzed by full-length transcriptomic sequencing,LCK₀0389,LCK₀0222,and LCK₀0027 were inferred to be the genes encoding D-LDH through the KEGG metabolic pathway;by analyzing the gene expression levels,combined with the results of phylogenetic analysis,it was inferred that LCK₀0389 was the key D-LDH encoding gene.2.Using the Leuconostoc citreum KM20 genome as a template,clone the three D-LDH coding genes,construct an expression vector,and transform it into Escherichia coli BL21（DE3）to achieve overexpression.Through SDS-PAGE and WB experiments confirmed that the molecular mass of D-LDH-1 is about 38.8 kDa,the molecular mass of D-LDH-2 is about 38.5 kDa,and the molecular mass of D-LDH-3 is about 40.0 kDa.The purified three enzymes were detected by SDS-PAGE,and the result of electrophoresis was a single band,and the purification was successful.3.The characteristics of D-LDH in the purified Leuconostoc citreum KM20 were analyzed,indicating that pyruvate reduction was the main reaction of the three enzymes,and it was confirmed that D-LDH-1 was a key enzyme in the production of D-lactic acid by Leuconostoc citreum KM20.The specific activities of after purification was 279.00 U/mg.In the process of pyruvate reduction,the optimum pH values of the D-LDH-1 is 8,the optimum reaction temperature is 20℃,In the process of D-lactic acid oxidation,the optimum pH value of D-LDH-1 was 12 and the optimum temperature was 30℃.Ca2+,Cu2+and Na+can promote the enzymatic activities of D-LDH-1,Zn2+and SDS showed strong inhibitory effect on the enzymatic activities of D-LDH-1.The Km values for pyruvate and NADH of D-LDH-1 was 0.24 mM and 0.12 mM,respectively;The D-lactate and NAD+Km values for D-LDH-1 was 33.27 mM and 2.65 mM,respectively.4.The characteristics of purified D-LDH-2（LCK₀0222）were analyzed,and the results were as follows:the specific activity of purified D-LDH-2 was 156.99 U/mg.In the process of pyruvate reduction,the optimum pH values of the D-LDH-2 is 8,the optimum reaction temperature is 40℃.In the process of D-lactic acid oxidation,the optimum pH value of D-LDH-2 was 12 and the optimum temperature was 30℃.The Km values for pyruvate and NADH of D-LDH-2 was 6.11 mM and 0.32 mM,respectively;The D-lactate and NAD+Km values for D-LDH-2 was 44.30 mM and 4.32 mM,respectively.5.The characteristics of purified D-LDH-3（LCK₀0027）were analyzed,and the results were as follows:the specific activity of purified D-LDH-3 was 2.29 U/mg.In the process of pyruvate reduction,the optimum pH values of the D-LDH-3 is 8,the optimum reaction temperature is 40℃.The Km values for pyruvate and NADH of D-LDH-3 was 2.98 mM and 0.32 mM,respectively.This study provides theoretical support for the targeted knockout of D-lactate dehydrogenase gene or changing its activity to improve the safety of fermented food through the excavation and characterization study of D-lactate synthesis-related enzyme genes of Leuconostoc citreum,which is of great significance for the application of Leuconostoc citreum in the food industry.