| Phenyllactic acid?PLA?is a kind of novel natural biological preservative with wide application prospect in the food industry,which has the outstanding characteristics of high safety,stability and wide antibacterial spectrum.PLA can inhibit bacterial and fungal contamination in food so the shelf life can be extended.Lactate dehydrogenase?LDH?genes derived from Lactobacillus plantarum LY-78 were researched through bioinformatics analyses in this study.The engineering bacteria with ldh genes of biologically activity were recombined to highly expressed LDH in the recombinant engineering bacteria.LDH was isolated and purified and its enzyme activity was determined;the enzymatic properties of LDH were investigated by study on influencing factors such as temperature,p H and metal ions and so on.1.Bioinformatics analyses of lactate dehydrogenase genes.All the sequences of nucleotide and amino acid of five ldh genes have high conservation.The five lactate dehydrogenase proteins were non-transmembrane and non-secreting proteins of thermal stability and located in the cytoplasm.The L3-LDH may not have LDH activity due to lack of NAD+ binding domain and functional site.D1-LDH,D2-LDH,L1-LDH and L2-LDH were likely to have LDH activity because there were completely functional sites and domains and typical NAD+ binding site motif GXGXXG in them.Therefore ldh D1,ldh D2,ldh L1 and ldh L2 genes were chosen to do the following research.2.Construction of lactate dehydrogenase gene in recombinant engineering bacteria.ldh genes were obtained by PCR,four recombinant engineering bacteria with ldh genes were successfully constructed followed by clone and subclone and identification by double digestion and sequencing.The results of SDS-PAGE showed that ldh D1,ldh D2,ldh L1 and ldh L2 genes were highly expressed in recombinant engineering bacteria.The highly expressed LDH products were isolated and purified and the enzyme activity were identified by reversed phase high performance liquid chromatography.The results showed that D1-LDH,D2-LDH,L1-LDH and L2-LDH were successfully obtained,which can catalyzed phenylpyruvic acid to produce PLA.The molecular weights were about 37 k Da,35 k Da,34 k Da and 33 k Da,respectively,which were consistent with the theoretical values.3.Analyses of recombinant lactate dehydrogenase enzymatic properties.The specific activityof D1-LDH,D2-LDH,L1-LDH and L2-LDH to phenylpyruvic acid were 65.35 U/mg,0.4123 U/mg,38.80 U/mg and 0.1657 U/mg.The results showed that D1-LDH and L1-LDH had higher specific activity in four LDH of Lactobacillus plantarum LY-78;and further study showed that the optimum temperature for the interaction between D1-LDH and L1-LDH and phenylpyruvic acid was 30 ?and 40 ?,respectively;the both optimum p H was 6.0,L1-LDH had better thermal stability and p H stability than D1-LDH.Fe2+ and Cu2+ were inhibitors of D1-LDH and L1-LDH,while Mg2+ and Mn2+ were both activators.The kinetic parameters of D1-LDH and L1-LDH measured under the optimum conditions showed that the Km values were 2.86 m M and 3.77 m M,Vmax values were 1.272 ?mol/min and 1.954 ?mol/min,Kcat values were 102 s-1 and 219 s-1,Kcat/Km values were 36 m M-1s-1 and 58 m M-1s-1.The results indicated that D1-LDH had a stronger affinity to phenylpyruvic acid,which was more conducive to the synthesis of PLA.In summary,four LDH in Lactobacillus plantarum LY-78 had the ability to produce PLA by catalyzing Phenylpyruvic acid.But there was a significant difference in their activity,D1-LDH and L1-LDH had higher enzyme activity to Phenylpyruvic acid,indicating that these two enzymes played a prominent role in the biosynthesis of PLA in the Lactobacillus plantarum LY-78 pathway. |
PDF Full Text Request