Cloning And Heterologous Expression Of Lactate Dehydrogenase Gene From Lactobacillus Plantarum

Lactate dehydrogenase(EC1.1.1.27;LDH)is important glycolytic enzyme that catalyse the conversion of pyruvate to lactate reversibly.The enzyme is widely present in animals,bacteria and also in plants.In this research, the lactate dehydrogenase gene (ldh) was cloned from Lactobacillus plantarum. The ldh gene can be expressed in E.coli BL21(DE3) and P. pastoris X33. The results were as follows:(1) The ldh was cloned by PCR from L. plantarum. The gene size is 963 bp, 99% identity with GenBank AY513726.1. The amino acids sequence of LDH has 100% identity with the aboved strains.(2) The expression vector, pET-ldh, was constructed and transformed to E.coli BL21 (DE3). The induced protein molecular weight was 39 kD screened by SDS-PAGE gel when induced by 1.0 mmol/L IPTG. The results of enzyme activity analysis showed that the activity of LDH were 12.3±0.087 U/mg;(3) The optimized condition was built for increasing the solubale LDH and to overcome inclusion protein produced in E. coli. The results showed that more soluble LDH was induced at 16℃, 150 r/min after the addition of 10 mmol/L lactose at OD600=0.8;(4) Some properties of LDH were also studied. The results revealed that the LDH has the maximum activity at 45℃, pH 6.5;(5) The ldh gene was cloned into pPICZA vector to construct pPICZ-ldh and transformed to P. pastoris X33 by lithium chloride transformation method. The ldh gene expression was induced in the eukaryotic cell and the LDH activity was 4.83±0.07 U/mg;(6) The ldh gene was cloned into pGAPZαA vector to construct pGAP-ldh and transformed to P. pastoris X33 by electro transformation method. The LDH could be secreted into the medium when the transformed Pichia was cultivated in YPD medium after 96 h. The LDH activity was 5.94±0.07 U/mg.