Abstract
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.
Highlights
Phenyllactic acid (PLA), a natural organic acid with high value added, is considered a promising preservative widely used in food and feed due to its broad antimicrobial activity (Ning et al, 2017)
To improve the L-phenyllactic acid (L-PLA) production efficiency and reduce costs, we attempted the expression of L-LcLDH1Q88A/I229A in P. pastoris GS115
P. pastoris enables some posttranslational modifications of a recombinant protein, such as FIGURE. 3 | Optimization of bioconversion conditions of the recombinant L-LcLDH1Q88A/I229A (Pp). (A) The temperature optima were measured under the standard assay conditions, but temperatures ranged from 20 to 60°C. (B) To estimate the temperature stability, the recombinant L-LcLDH1Q88A/I229A (Pp) was incubated for 2 h from 20 to 65°C, respectively. (C) The pH optima were measured under the above-mentioned assay conditions, except for pH ranging from 3.5 to 7.0. (D) To estimate the pH stability, the recombinant L-LcLDH1Q88A/I229A (Pp) was incubated for 1 h in a different pH buffer at 4°C, and the relative activity was determined at the optimal temperature of 40°C and optimal pH of 5.0
Summary
Phenyllactic acid (PLA), a natural organic acid with high value added, is considered a promising preservative widely used in food and feed due to its broad antimicrobial activity (Ning et al, 2017). Few L-LDHs exhibited a high catalytic activity, catalytic efficiency (kcat/Km), and yield, especially towards the bulkier substrates such as phenylpyruvic acid (PPA), which made them unable to be utilized effectively—for instance, the activities of L-LpLDH from Lactobacillus plantarum and L-BsLDH from Geobacillus stearothermophilus were 28.11 and 7.39 U/mg wet cell, respectively (Aslan et al, 2016; Zhu et al, 2017). An NADH/ NAD+ regeneration system could be introduced into the recycle to increase the availability of NADH and improve the yield of L-PLA (Zhang et al, 2009). To break through these bottlenecks, one of the effective strategies was to excavate novel LDHs having a high activity and/or construct an efficient coenzyme NADH regeneration system
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