Expression of a Lactobacillus casei L-lactate dehydrogenase mutant in Pichia pastoris for asymmetric reduction of phenylpyruvate.
- Author:
Ting ZHANG
1
;
Jianfang LI
1
;
Die HU
1
;
Chuang LI
2
;
Bochun HU
2
;
Minchen WU
3
Author Information
- Publication Type:Journal Article
- Keywords: L-lactate dehydrogenase; L-phenyllactic acid; Pichia pastoris; coenzyme regeneration system; glucose 1-dehydrogenase
- MeSH: L-Lactate Dehydrogenase; genetics; Lactobacillus casei; enzymology; genetics; Phenylpyruvic Acids; metabolism; Pichia; genetics; Recombinant Proteins; genetics; metabolism
- From: Chinese Journal of Biotechnology 2020;36(5):959-968
- CountryChina
- Language:Chinese
- Abstract: To improve the productivity of L-phenyllactic acid (L-PLA), L-LcLDH1(Q88A/I229A), a Lactobacillus casei L-lactate dehydrogenase mutant, was successfully expressed in Pichia pastoris GS115. An NADH regeneration system in vitro was then constructed by coupling the recombinant (re) LcLDH1(Q88A/I229A) with a glucose 1-dehydrogenase for the asymmetric reduction of phenylpyruvate (PPA) to L-PLA. SDS-PAGE analysis showed that the apparent molecular weight of reLcLDH1(Q88A/I229A) was 36.8 kDa. And its specific activity was 270.5 U/mg, 42.9-fold higher than that of LcLDH1 (6.3 U/mg). The asymmetric reduction of PPA (100 mmol/L) was performed at 40 °C and pH 5.0 in an optimal biocatalytic system, containing 10 U/mL reLcLDH1(Q88A/I229A), 1 U/mL SyGDH, 2 mmol/L NAD⁺ and 120 mmol/L D-glucose, producing L-PLA with 99.8% yield and over 99% enantiomeric excess (ee). In addition, the space-time yield (STY) and average turnover frequency (aTOF) were as high as 9.5 g/(L·h) and 257.0 g/(g·h), respectively. The high productivity of reLcLDH1(Q88A/I229A) in the asymmetric reduction of PPA makes it a promising biocatalyst in the preparation of L-PLA.
