Open AccessBiological characterization of D‐lactate dehydrogenase responsible for high‐yield production of D‐phenyllactic acid in Sporolactobacillus inulinus
Author(s) -
Cheng YaYun,
Park Tae Hyeon,
Seong Hyunbin,
Kim TaeJip,
Han Nam Soo
Publication year - 2022
Publication title -
microbial biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.287
H-Index - 74
ISSN - 1751-7915
DOI - 10.1111/1751-7915.14125
Subject(s) - bioconversion , enzyme kinetics , escherichia coli , lactate dehydrogenase , dehydrogenase , chemistry , biochemistry , enzyme , food science , fermentation , active site , gene
PLA (3‐D‐phenyllactic acid) is an ideal antimicrobial and immune regulatory compound present in honey and fermented foods. Sporolactobacillus inulinus is regarded as a potent D‐PLA producer that reduces phenylpyruvate (PPA) with D‐lactate dehydrogenases. In this study, PLA was produced by whole‐cell bioconversion of S. inulinus ATCC 15538. Three genes encoding D‐lactate dehydrogenase ( d‐ldh 1 , d‐ldh 2 , and d‐ldh 3) were cloned and expressed in Escherichia coli BL21 (DE3), and their biochemical and structural properties were characterized. Consequently, a high concentration of pure D‐PLA (47 mM) was produced with a high conversion yield of 88%. Among the three enzymes, D‐LDH1 was responsible for the efficient conversion of PPA to PLA with kinetic parameters of Km (0.36 mM), k cat (481.10 s −1 ), and k cat /Km (1336.39 mM −1 s −1 ). In silico structural analysis and site‐directed mutagenesis revealed that the Ile307 in D‐LDH1 is a key residue for excellent PPA reduction with low steric hindrance at the substrate entrance. This study highlights that S. inulinus ATCC 15538 is an excellent PLA producer, equipped with a highly specific and efficient D‐LDH1 enzyme.