Recently there is huge demand for chirally pure D-lactate since stereocomplex PLA definitely requires PDLA using D-lactate as monomer. We investigated lactate fermentation metabolism in Lactobacillus coryniformis subsp. torquens KCTC 3535 for production of optically pure D-lactate. L. coryniformis was known to produce D-lactate as dominant fermentation product at cultivation temperature around 30 ?. However, considerable production of L-lactate was observed when fermentation temperature was over 40 ?. Because optically pure isomers are produced using chiral-specific D- or L-lactate dehydrogenase (D- or L-LDH), we assumed that the thermostability of L-LDHs are significantly higher than that of D-LDHs, which may be a reason why L-lactate was produced at higher cultivation temperature. To elucidate above assumption, firstly we synthesized two kinds of D-LDH gene and six kinds of L-LDH gene based on the genomic information of Lactobacillus coryniformis subsp. torquens KCTC 3535. All the D-LDHs and L-LDHs were cloned and expressed in Escherichia coli. Among the total 8 LDHs tested, Only three LDHs including D-LDH1, L-LDH3 and L-LDH1702 showed activity and T5060 values, which is the temperature at which 50% of initial enzymatic activity remains after 60 min heat treatment, of each LDHs were determined 38.8, 49.0 and 52.0 ?, respectively. In addition, through Western blotting analysis for LDHs extracted from L. coryniformis cells, D-LDH1 and L-LDH3 were found dominantly expressed lactate dehydrogenase, which imply that two LDHs play a major role in the synthesis of lactate from pyruvate. Thermally stable property of L-LDH3 compared with that of D-LDH1 can be a major reason why the enantiopurity of D-lactate was decreased from 99 % to 50 % at relatively high fermentation temperature over 40 ?. Elucidation of temperature effect on the quality of D-lactate will give a direction for metabolic engineer to develop efficient microbial strain for optically pure D-lactate.

Elucidation of cultivation temperature effect on the production of chirally pure D-lactate in <i>Lactobacillus coryniformis</i>

245th National Meeting (2013)

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Elucidation of cultivation temperature effect on the production of chirally pure D-lactate in Lactobacillus coryniformis