Enantioselective Biosynthesis of L-Phenyllactic Acid From Phenylpyruvic Acid In Vitro by L-Lactate Dehydrogenase Coupling With Glucose Dehydrogenase.

As a valuable versatile building block, L-phenyllactic acid (L-PLA) has numerous applications in the fields of agriculture, pharmaceuticals, and biodegradable plastics. However, both normally chemically synthesized and naturally occurring PLA are racemic, and the production titer of L-PLA is not satisfactory. To improve L-PLA production and reduce the high cost of NADH, an in vitro coenzyme regeneration system of NADH was achieved using the glucose dehydrogenase variant LsGDHD255C and introduced into the L-PLA production process. Here an NADH-dependent L-lactate dehydrogenase-encoding variant gene (L-Lcldh1Q88A/I229A) was expressed in Pichia pastoris GS115. The specific activity of L-LcLDH1Q88A/I229A (Pp) was as high as 447.6 U/mg at the optimum temperature and pH of 40°C and 5.0, which was 38.26-fold higher than that of wild-type L-LcLDH1 (Pp). The catalytic efficiency (k cat/K m) of L-LcLDH1Q88A/I229A (Pp) was 94.3 mM-1 s-1, which was 67.4- and 25.5-fold higher than that of L-LcLDH1(Pp) and L-LcLDH1Q88A/I229A (Ec) expressed in Escherichia coli, respectively. Optimum reactions of L-PLA production by dual-enzyme catalysis were at 40°C and pH 5.0 with 10.0 U/ml L-LcLDH1Q88A/I229A (Pp) and 4.0 U/ml LsGDHD255C. Using 0.1 mM NAD+, 400 mM (65.66 g/L) phenylpyruvic acid was completely hydrolyzed by fed-batch process within 6 h, affording L-PLA with 90.0% yield and over 99.9% ee p. This work would be a promising technical strategy for the preparation of L-PLA at an industrial scale.

[Expression of a Lactobacillus casei L-lactate dehydrogenase mutant in Pichia pastoris for asymmetric reduction of phenylpyruvate].

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.

Expression of a Lactobacillus casei L-lactate dehydrogenase mutant in Pichia pastoris for asymmetric reduction of phenylpyruvate / 生物工程学报

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.