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Reducing 3,4‐dihydroxyphenylpyruvic acid to d ‐3,4‐dihydroxyphenyllactic acid via a coenzyme nonspecific d ‐lactate dehydrogenase from Lactobacillus reuteri
Author(s) -
Wang Y.H.,
Bai Y.J.,
Fan T.P.,
Zheng X.H.,
Cai Y.J.
Publication year - 2018
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.14077
Subject(s) - lactobacillus reuteri , biochemistry , lactate dehydrogenase , enzyme , chemistry , lactic acid , dehydrogenase , lactobacillus , bacteria , biology , fermentation , genetics
Aims The purpose of this work was to find an efficient enzyme to synthesize d ‐3,4‐dihydroxyphenyllactic acid ( d ‐DSS). Methods and Results Nineteen lactic acid bacteria strains were screened for production of d ‐DSS using 3,4‐dihydroxyphenylpyruvic acid (DPA) as a substrate. Lactobacillus reuteri JN516 exhibited the highest d ‐DSS yield. A nonspecific coenzyme, d ‐lactate dehydrogenase ( d ‐LDH82319), from L. reuteri JN516 with high DPA reducing activity was identified. This enzyme reduced DPA to form d ‐DSS with excellent optical purity (enantioselectivity >99%). Its molecular weight was 35 kDa based on SDS‐PAGE migration. The Michaelis–Menten constant ( K m ), turnover number ( k cat ), and catalytic efficiency ( k cat / K m ) of d ‐LDH82319 for DPA were 0·09 mmol l −1 , 2·17 s −1 and 24·07 (mmol l −1 ) −1 s −1 , respectively, with NADH as the coenzyme. The ( K m ), ( k cat ) and ( k cat / K m ) of d ‐LDH82319 for DPA were 0·10 mmol l −1 , 0·13 s −1 and 1·30 (mmol l −1 ) −1 s −1 , respectively, with NADPH as the coenzyme. The optimum temperature and pH of d ‐LDH82319 were 25°C and pH 8 respectively. Additionally, d ‐LDH82319 had a broad substrate range for alpha‐keto acids, among which the activity of reducing pyruvate was the strongest; therefore, it belongs to the group of d ‐lactate dehydrogenases. d ‐LDH82319 and glucose dehydrogenase (GDH) were coexpressed to produce d ‐DSS from DPA. Conclusions d ‐LDH82319 from L. reuteri JN516 with high DPA reducing activity has the characteristics of a nonspecific coenzyme. Significance and Impact of the Study d ‐LDH82319 is the first reported coenzyme nonspecific d ‐lactate dehydrogenase with DPA‐reducing activity. The coexpression system provided an effective method to produce d ‐DSS.