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KMID : 1094720220270060921
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Biotechnology and Bioprocess Engineering
2022 Volume.27 No. 6 p.921 ~ p.929
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Characterization of Key Enzymes for D-lactic Acid Synthesis in Leuconostoc citreum KM20
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Jinxi Liu
Hongjie Piao
Changlei Liu
Bae Sang-Rak
Hushan Cui
Qing Jin
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Abstract
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Leuconostoc citreum is a heterofermentative lactic acid bacteria, which converts pyruvate to D-lactic acid using D-lactate dehydrogenase. For analyzing the characteristics of the key enzyme for D-lactic acid synthesis in Leuconostoc citreum KM20, the key gene of D-lactic acid synthesis was identified by genomics and transcriptomic analysis, which was cloned into the vector pET-15b and transformed into Escherichia coli BL21 (DE3). The D-lactate dehydrogenase was purified by Ni-NTA column affinity chromatography and its enzymatic property was studied. The results showed that LCK_00389 gene was the key gene among the seven genes related to lactic acid synthesis. The molecular mass of the enzyme encoded by LCK_00389 was 38.8 kDa; the specific activity was 272.71 U/mg; the optimum pH and temperature for pyruvate reduction were 8.0 and 20°C, those for lactate oxidation were 12.0 and 30°C, respectively; the Km and the kcat value of pyruvate were 0.24 ± 0.01 mmol/L and 165.99 ± 3.65 s?1, those of D-lactic acid were 33.27 ± 2.72 mmol/L and 14.67 ± 0.29 s?1; Ca2+, Cu2+ and Na+ promoted the activity of the enzyme, while Zn2+ and Sodium dodecyl sulfate (SDS) inhibited the activity of the enzyme; in addition to using pyruvate as substrate, it also had had strong catalytic activity for oxaloacetate, phenylpyruvate and 2-ketoglutarate.
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KEYWORD
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D-lactic acid, D-lactate dehydrogenase, Leuconostoc citreum, Lactobacillus, gene expression
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