Hydroxy(phenyl)pyruvic acid reductase in Actaea racemosa L.: a putative enzyme in cimicifugic and fukinolic acid biosynthesis.

MAIN CONCLUSION: Hydroxy(phenyl)pyruvic acid reductase from Actaea racemosa catalyzes dual reactions in reducing 4-hydroxyphenylpyruvic acid as well as ß-hydroxypyruvic acid. It thus qualifies to be part of fukinolic and cimicifugic acid biosynthesis and also photorespiration. The accumulation of fukinolic acid and cimicifugic acids is mainly restricted to Actaea racemosa (Ranunculaceae) and other species of the genus Actaea/Cimicifuga. Cimicifugic and fukinolic acids are composed of a hydroxycinnamic acid part esterified with a benzyltartaric acid moiety. The biosynthesis of the latter is unclear. We isolated cDNA encoding a hydroxy(phenyl)pyruvic acid reductase (GenBank OR393286) from suspension-cultured material of A. racemosa (ArH(P)PR) and expressed it in E. coli for protein production. The heterologously synthesized enzyme had a mass of 36.51 kDa and catalyzed the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvic acid to 4-hydroxyphenyllactic acid or ß-hydroxypyruvic acid to glyceric acid, respectively. The optimal temperature was at 38 °C and the pH optimum at pH 7.5. NADPH is the preferred cosubstrate (Km 23 ± 4 µM). Several substrates are accepted by ArH(P)PR with ß-hydroxypyruvic acid (Km 0.26 ± 0.12 mM) followed by 4-hydroxyphenylpyruvic acid (Km 1.13 ± 0.12 mM) as the best ones. Thus, ArH(P)PR has properties of ß-hydroxypyruvic acid reductase (involved in photorespiration) as well as hydroxyphenylpyruvic acid reductase (possibly involved in benzyltartaric acid formation).

Directed Evolution of 4-Hydroxyphenylpyruvate Biosensors Based on a Dual Selection System.

Biosensors based on allosteric transcription factors have been widely used in synthetic biology. In this study, we utilized the Acinetobacter ADP1 transcription factor PobR to develop a biosensor activating the PpobA promoter when bound to its natural ligand, 4-hydroxybenzoic acid (4HB). To screen for PobR mutants responsive to 4-hydroxyphenylpyruvate(HPP), we developed a dual selection system in E. coli. The positive selection of this system was used to enrich PobR mutants that identified the required ligands. The following negative selection eliminated or weakened PobR mutants that still responded to 4HB. Directed evolution of the PobR library resulted in a variant where PobRW177R was 5.1 times more reactive to 4-hydroxyphenylpyruvate than PobRWT. Overall, we developed an efficient dual selection system for directed evolution of biosensors.

Poly(vinyl chloride)/Nanocarbon Composites for Advanced Potentiometric Membrane Sensor Design.

Polymer nanocomposites filled with carbon nanoparticles (CNPs) are a hot topic in materials science. This article discusses the current research on the use of these materials as interfacial electron transfer films for solid contact potentiometric membrane sensors (SC-PMSs). The results of a comparative study of plasticized poly (vinyl chloride) (pPVC) matrices modified with single-walled carbon nanotubes (SWCNTs), fullerenes-C60, and their hybrid ensemble (SWCNTs-C60) are reported. The morphological characteristics and electrical conductivity of the prepared nanostructured composite films are reported. It was found that the specific electrical conductivity of the pPVC/SWCNTs-C60 polymer film was higher than that of pPVC filled with individual nanocomponents. The effectiveness of this composite material as an electron transfer film in a new potentiometric membrane sensor for detecting phenylpyruvic acid (in anionic form) was demonstrated. Screening for this metabolic product of phenylalanine in body fluids is of significant diagnostic interest in phenylketonuria (dementia), viral hepatitis, and alcoholism. The developed sensor showed a stable and fast Nernstian response for phenylpyruvate ions in aqueous solutions over the wide linear concentration range of 5 × 10-7-1 × 10-3 M, with a detection limit of 10-7.2 M.

Enzymatic synthesis of halogen derivatives of L-phenylalanine and phenylpyruvic acid stereoselectively labeled with hydrogen isotopes in the side chain.

Halogenated, labeled with deuterium, tritium or doubly labeled with deuterium and tritium in the 3S position of the side chain isotopomers of L-phenylalanine and phenylpyruvic acid were synthesized. Isotopomers of halogenated L-phenylalanine were obtained by addition of ammonia from isotopically enriched buffer solution to the halogenated derivative of (E)-cinnamic acid catalyzed by phenylalanine ammonia lyase. Isotopomers of halogenated phenylpyruvic acid were obtained enzymatically by conversion of the appropriate isotopomer of halogenated L-phenylalanine in the presence of phenylalanine dehydrogenase. As a source of deuterium was used deuterated water, as a source of tritium was used a solution of highly diluted tritiated water. The labeling takes place in good yields and with high deuterium atom% abundance.

Competitive, multi-objective, and compartmented Flux Balance Analysis for addressing tissue-specific inborn errors of metabolism.

The inborn error of metabolism phenylketonuria (PKU, OMIM 261600) is most often due to inactivation of phenylalanine hydroxylase (PAH), which converts phenylalanine (Phe) into tyrosine (Tyr). The reduced PAH activity increases blood concentration of phenylalanine and urine levels of phenylpyruvate. Flux balance analysis (FBA) of a single-compartment model of PKU predicts that maximum growth rate should be reduced unless Tyr is supplemented. However, the PKU phenotype is lack of development of brain function specifically, and Phe reduction rather than Tyr supplementation cures the disease. Phe and Tyr cross the blood-brain barrier (BBB) through the aromatic amino acid transporter implying that the two transport reactions interact. However, FBA does not accommodate such competitive interactions. We here report on an extension to FBA that enables it to deal with such interactions. We built a three-compartment model, made the common transport across the BBB explicit, and included dopamine and serotonin synthesis as parts of the brain function to be delivered by FBA. With these ramifications, FBA of the genome-scale metabolic model extended to three compartments does explain that (i) the disease is brain specific, (ii) phenylpyruvate in urine is a biomarker, (iii) excess of blood-phenylalanine rather than shortage of blood-tyrosine causes brain pathology, and (iv) Phe deprivation is the better therapy. The new approach also suggests (v) explanations for differences in pathology between individuals with the same PAH inactivation, and (vi) interference of disease and therapy with the functioning of other neurotransmitters.

Underrepresented Impurities in 4-Hydroxyphenylpyruvate Affect the Catalytic Activity of Multiple Enzymes.

Macrophage migration inhibitory factor (MIF) is a key immunostimulatory protein with regulatory properties in several disorders, including inflammation and cancer. All the reported inhibitors that target the biological activities of MIF have been discovered by testing against its keto/enol tautomerase activity. While the natural substrate is still unknown, model MIF substrates are used for kinetic experiments. The most extensively used model substrate is 4-hydroxyphenyl pyruvate (4-HPP), a naturally occurring intermediate of tyrosine metabolism. Here, we examine the impact of 4-HPP impurities in the precise and reproducible determination of MIF kinetic data. To provide unbiased evaluation, we utilized 4-HPP powders from five different manufacturers. Biochemical and biophysical analyses showed that the enzymatic activity of MIF is highly influenced by underrepresented impurities found in 4-HPP. Besides providing inconsistent turnover results, the 4-HPP impurities also influence the accurate calculation of ISO-1's inhibition constant, an MIF inhibitor that is broadly used for in vitro and in vivo studies. The macromolecular NMR data show that 4-HPP samples from different manufacturers result in differential chemical shift perturbations of amino acids in MIF's active site. Our MIF-based conclusions were independently evaluated and confirmed by 4-hydroxyphenylpyruvate dioxygenase (HPPD) and D-dopachrome tautomerase (D-DT); two additional enzymes that utilize 4-HPP as a substrate. Collectively, these results explain inconsistencies in previously reported inhibition values, highlight the effect of impurities on the accurate determination of kinetic parameters, and serve as a tool for designing error-free in vitro and in vivo experiments.

Production of Salvianic Acid A from l-DOPA via Biocatalytic Cascade Reactions.

Salvianic acid A (SAA), as the main bioactive component of the traditional Chinese herb Salvia miltiorrhiza, has important application value in the treatment of cardiovascular diseases. In this study, a two-step bioprocess for the preparation of SAA from l-DOPA was developed. In the first step, l-DOPA was transformed to 3,4-dihydroxyphenylalanine (DHPPA) using engineered Escherichia coli cells expressing membrane-bound L-amino acid deaminase from Proteus vulgaris. After that, the unpurified DHPPA was directly converted into SAA by permeabilized recombinant E. coli cells co-expressing d-lactate dehydrogenase from Pediococcus acidilactici and formate dehydrogenase from Mycobacterium vaccae N10. Under optimized conditions, 48.3 mM of SAA could be prepared from 50 mM of l-DOPA, with a yield of 96.6%. Therefore, the bioprocess developed here was not only environmentally friendly, but also exhibited excellent production efficiency and, thus, is promising for industrial SAA production.

DHPPA, a major plasma alkylresorcinol metabolite reflecting whole-grain wheat and rye intake, and risk of metabolic syndrome: a case-control study.

PURPOSE: Whole-grain intake assessed through self-reported methods has been suggested to be inversely associated with the metabolic syndrome (MetS) risk in epidemiological studies. However, few studies have evaluated the association between whole-grain intake and MetS risk using objective biomarkers of whole-grain intake. The aim of this study was to examine the association between plasma 3-(3,5-Dihydroxyphenyl)-1-propanoic acid (DHPPA), a biomarker of whole-grain wheat and rye intake, and MetS risk in a Chinese population. METHODS: A case-control study of 667 MetS cases and 667 matched controls was conducted based on baseline data of the Tongji-Ezhou Cohort study. Plasma DHPPA concentrations were assessed by high-performance liquid chromatography-tandem mass spectrometry. The MetS was defined based on criteria set by the Joint Interim Statement. RESULTS: Plasma DHPPA was inversely associated with MetS risk. After adjustment for age, sex, body mass index, smoking status, alcohol drinking status, physical activity and education level, the odds ratios (ORs) for MetS across increasing quartiles of plasma DHPPA concentrations were 1 (referent), 0.86 (0.58-1.26), 0.77 (0.52-1.15), and 0.59 (0.39-0.89), respectively. In addition, the cubic spline analysis revealed a potential nonlinear association between plasma DHPPA and MetS, with a steep reduction in the risk at the lower range of plasma DHPPA concentration. CONCLUSION: Our study revealed that individuals with higher DHPPA concentrations in plasma had lower odds of MetS compared to those with lower DHPPA concentrations in plasma. Our findings provided further evidence to support health benefits of whole grain consumption.

Production of phenylpyruvic acid by engineered L-amino acid deaminase from Proteus mirabilis.

OBJECTIVES: This study aimed to develop an efficient enzymatic strategy for the industrial production of phenylpyruvate (PPA) from L-phenylpyruvic acid (L-Phe). RESULTS: L-amino acid deaminase from Proteus mirabilis was expressed in Escherichia coli BL21 (DE3) and modified to release product inhibition by employing conformational dynamics engineering. Based on structural analysis, two residues (E145/L341) were identified for reducing interactions between the product and enzyme and increasing flexibility of the protein, thereby facilitating the product release. The mutant M2E145A/E341A exhibited a 3.84-fold reduction in product inhibition and a 1.35-fold increase in catalytic efficiency in comparison to the wild type. Finally, 81.2 g/L PPA production with a conversion of 99.6% was obtained in a 5-L bioreactor. CONCLUSIONS: The engineered catalyst can significantly reduce product inhibition and facilitate the effective industrial synthesis of PPA.

Enzymatic conversion of dehydrocoelenterazine to coelenterazine using FMN-bound flavin reductase of NAD(P)H:FMN oxidoreductase.

Coelenterazine (CTZ) is known as luciferin (a substrate) for the luminescence reaction with luciferase (an enzyme) in marine organisms and is unstable in aqueous solutions. The dehydrogenated form of CTZ (dehydrocoelenterazine, dCTZ) is stable and thought to be a storage form of CTZ and a recycling intermediate from the condensation reaction of coelenteramine and 4-hydroxyphenylpyruvic acid to CTZ. In this study, the enzymatic conversion of dCTZ to CTZ was successfully achieved using NAD(P)H:FMN oxidoreductase from the bioluminescent bacterium Vibrio fischeri ATCC 7744 (FRase) in the presence of NADH (the FRase-NADH reaction). CTZ reduced from dCTZ in the FRase-NADH reaction was identified by HPLC and LC/ESI-TOF-MS analyses. Thus, dCTZ can be enzymatically converted to CTZ in vitro. Furthermore, the concentration of dCTZ could be determined by the luminescence activity using the CTZ-utilizing luciferases (Gaussia luciferase or Renilla luciferase) coupled with the FRase-NADH reaction.

Biosynthesis of phenylpyruvic acid from l-phenylalanine using chromosomally engineered Escherichia coli.

The efficiency of whole-cell biotransformation is often affected by the genetic instability of plasmid-based expression systems, which require selective pressure to maintain the stability of the plasmids. To circumvent this shortcoming, we constructed a chromosome engineering strain for the synthesis of phenylpyruvic acid (PPA) from l-phenylalanine. First, l-amino acid deaminase (pmLAAD) from Proteus myxofaciens was incorporated into Escherichia coli BL21 (DE3) chromosome and the copy numbers of pmLAAD were increased by chemically induced chromosomal evolution (CIChE). Fifty-nine copies of pmLAAD were obtained in E. coli BL8. The PPA titer of E. coli BL8 reached 2.22 g/L at 6 h. Furthermore, the deletion of lacI improved PPA production. In the absence of isopropyl-ß-d-thiogalactopyranoside, the resulting strain, E. coli BL8△recA△lacI, produced 2.65 g/L PPA at 6 h and yielded a 19.37% increase in PPA production compared to E. coli BL8△recA. Finally, the engineered E. coli BL8△recA△lacI strain achieved 19.14 g/L PPA at 24 h in a 5-L bioreactor.

Four-Step Pathway from Phenylpyruvate to Benzylamine, an Intermediate to the High-Energy Propellant CL-20.

Benzylamine is a commodity chemical used in the synthesis of motion-sickness treatments and anticonvulsants, in dyeing textiles, and as a precursor to the high-energy propellant CL-20. Because chemical production generates toxic waste streams, biosynthetic alternatives have been explored, recently resulting in a functional nine-step pathway from central metabolism (phenylalanine) in E. coli. We report a novel four-step pathway for benzylamine production, which generates the product from cellular phenylpyruvate using enzymes from different sources: a mandelate synthase (Amycolatopsis orientalis), a mandelate oxidase (Streptomyces coelicolor), a benzoylformate decarboxylase (Pseudomonas putida), and an aminotransferase (Salicibacter pomeroyi). This pathway produces benzylamine at 24 mg/L in 15 h (4.5% yield) in cultures of unoptimized cells supplemented with phenylpyruvate. Because the yield is low, supplementation with pathway intermediates is used to troubleshoot the design. This identifies conversion inefficiencies in the mandelate synthase-mediated synthesis of (S)-mandelic acid, and subsequent genome mining identifies a new mandelate synthase (Streptomyces sp. 1114.5) with improved yield. Supplementation experiments also reveal native redirection of ambient phenylpyruvate away from the pathway to phenylalanine. Overall, this work illustrates how retrosynthetic design can dramatically reduce the number of enzymes in a pathway, potentially reducing its draw on cellular resources. However, it also shows that such benefits can be abrogated by inefficiencies of individual conversions. Addressing these barriers can provide an alternative approach to green production of benzylamine, eliminating upstream dependence on chlorination chemistry.

Metabolomics Analysis of Litchi Leaves during Floral Induction Reveals Metabolic Improvement by Stem Girdling.

Prolonged exposure to cold temperatures often results in a relatively low flowering rate in litchi (Litchi chinensis Sonn.) trees with younger leaves. This study aimed to verify the impact of stem girdling on litchi flowering by identifying and characterizing the induced metabolic changes. After a 60 day exposure to cold treatment at 15 °C/10 °C (12 h/12 h), the flowering rate of the girdled trees was 100%, while that of the non-girdled trees was 20%, indicating that girdling improved litchi flowering at its turning stage. The metabolic profiles of litchi leaves with and without stem girdling during floral induction were compared and 505 metabolites potentially associated with litchi flowering were detected. Most metabolites were involved in the metabolism of starch and sucrose, fatty acid, and phenylpyruvic acid. The metabolic pathways concerned with the biosynthesis of epinephrine, sucrose, and d-maltose were induced in leaves after girdling treatment. The level of galactitol, phenylpyruvic acid, acetyl-CoA, linoleic acid, alpha-linolenic acid, and 13-HPOT biosynthesis remained stable in the leaves from girdled trees but changed drastically in the leaves from non-girdled trees. In addition, 379 metabolites concerning flowering rate were characterized. Metabolism pathways of starch and sucrose, galactose, and linoleic acid are of great significance to the flowering of litchi. Linoleic acid exhibited the most significant variations between girdled trees and non-girdled trees with fold changes of up to 13.62. These results contribute to understanding the biological mechanism of litchi floral induction and the metabolic changes after stem girdling.

Synthesis and Herbicidal Activity of Triketone-Aminopyridines as Potent p-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

Exploring novel p-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) inhibitors has become one of the most promising research directions in herbicide innovation. On the basis of our tremendous interest in exploiting more powerful HPPD inhibitors, we designed a family of benzyl-containing triketone-aminopyridines via a structure-based drug design (SBDD) strategy and then synthesized them. Among these prepared derivatives, the best active 3-hydroxy-2-(3,5,6-trichloro-4-((4-isopropylbenzyl)amino)picolinoyl)cyclohex-2-en-1-one (23, IC50 = 0.047 µM) exhibited a 5.8-fold enhancement in inhibiting Arabidopsis thaliana (At) HPPD activity over that of commercial mesotrione (IC50 = 0.273 µM). The predicted docking models and calculated energy contributions of the key residues for small molecules suggested that an additional π-π stacking interaction with Phe-392 and hydrophobic contacts with Met-335 and Pro-384 were detected in AtHPPD upon the binding of the best active compound 23 compared with that of the reference mesotrione. Such a molecular mechanism and the resulting binding affinities coincide with the proposed design scheme and experimental values. It is noteworthy that inhibitors 16 (3-hydroxy-2-(3,5,6-trichloro-4-((4-chlorobenzyl)amino)picolinoyl)cyclohex-2-en-1-one), 22 (3-hydroxy-2-(3,5,6-trichloro-4-((4-methylbenzyl)amino)picolinoyl)cyclohex-2-en-1-one), and 23 displayed excellent greenhouse herbicidal effects at 150 g of active ingredient (ai)/ha after postemergence treatment. Furthermore, compound 16 showed superior weed-controlling efficacy against Setaria viridis (S. viridis) versus that of the positive control mesotrione at multiple test dosages (120, 60, and 30 g ai/ha). These findings imply that compound 16, as a novel lead of HPPD inhibitors, possesses great potential for application in specifically combating the malignant weed S. viridis.

Protein Substitutes in PKU; Their Historical Evolution.

Protein substitutes developed for phenylketonuria (PKU) are a synthetic source of protein commonly based on L-amino acids. They are essential in the treatment of phenylketonuria (PKU) and other amino acid disorders, allowing the antagonistic amino acid to be removed but with the safe provision of all other amino acids necessary for maintaining normal physiological function. They were first formulated by a chemist and used experimentally on a 2-year-old girl with PKU and their nutritional formulations and design have improved over time. Since 2008, a bioactive macropeptide has been used as a base for protein substitutes in PKU, with potential benefits of improved bone and gut health, nitrogen retention, and blood phenylalanine control. In 2018, animal studies showed that physiomimic technology coating the amino acids with a polymer allows a slow release of amino acids with an improved physiological profile. History has shown that in PKU, the protein substitute's efficacy is determined by its nutritional profile, amino acid composition, dose, timing, distribution, and an adequate energy intake. Protein substitutes are often given little importance, yet their pharmacological actions and clinical benefit are pivotal when managing PKU.

Energy metabolism profile of the effects of amino acid treatment on hepatocytes: Phenylalanine and phenylpyruvate inhibit glycolysis of hepatocytes.

OBJECTIVES: Amino acids are not only the building blocks of proteins, but also can be metabolized to energy substances or function as signaling molecules. The aim of this study was to profile whether amino acid treatment (essential amino acids and alanine) affects the energy metabolism (glycolysis, mitochondrial respiration) of cultured hepatocytes. METHODS: AML12 hepatocytes were treated with 5 mM of each amino acid for 1 h and the energy metabolism was then measured by using an extracellular flux analyzer. RESULTS: The results showed that phenylalanine and lysine decreased the extracellular acidification rate (ECAR), an indirect indicator of glycolysis, whereas isoleucine and histidine increased the ECAR. Amino acids did not affect the oxygen consumption rate, an indirect indicator of mitochondrial respiration. The glycolysis stress test revealed that treatment of the hepatocytes with phenylalanine inhibited glycolysis when the concentration of the substrate for glycolysis was sufficient in cultured media. We also investigated the effect of metabolites derived from conversion of phenylalanine on glycolysis in hepatocytes and found that phenylpyruvate inhibited glycolysis, whereas tyrosine and phenylethylamine did not affect glycolysis. CONCLUSIONS: The findings from the present study complement basic knowledge of the effects of amino acid treatment on energy metabolism in cultured hepatocytes and indicate that phenylalanine and phenylpyruvate inhibit glycolysis.

Taxonomic notes on the antlion genus Distoleon Banks (Neuroptera: Myrmeleontidae) from Pakistan.

The species of the antlion genus Distoleon Banks, 1910 (Myrmeleontidae, Myrmeleontinae, Nemoleontini) from Pakistan are revised. Three species, Distoleon sambalpurensis Ghosh, 1984, Distoleon tappa (Walker, 1853), and Distoleon verendus (Walker, 1853), all known from northern Pakistan, are re-described. Myrmeleon vesanus Walker, 1853 syn. nov. is assigned as a junior synonym of Distoleon verendus (Walker, 1853). The female of Distoleon tappa is described for the first time. Discussions about the geographical distribution of all these species are provided in detail. A key to known species of Distoleon from Pakistan is provided.

Human Serum Phenylpyruvate Quantification Using Responsive 2D Photonic Crystal Hydrogels via Chemoselective Oxime Ligation: Progress toward Developing Phenylalanine-Sensing Elements.

There is a need to develop at-home phenylalanine (Phe) test kits, analogous to home glucose meters, for phenylketonuria patients who must measure their blood Phe levels frequently to adjust their diet. Unfortunately, such test kits are not available yet because of the lack of simple and inexpensive Phe-sensing elements. With the goal of developing a Phe-sensing element, we fabricated two-dimensional photonic crystal (2DPC) hydrogels that quantify human serum phenylpyruvate (PhPY), which is the product of the reaction between Phe and the enzyme phenylalanine dehydrogenase. The PhPY-sensing hydrogels have oxyamine recognition groups that link PhPY to the hydrogel polymer network via chemoselective oxime ligation. This structural modification induces the hydrogel to swell, which then increases interparticle spacings within the embedded 2DPC. The PhPY-induced particle spacing changes are measured from light diffraction and used to quantify the PhPY concentrations. The estimated limit of detection of PhPY in human serum for a detection time of 30 min is 19 µM, which is comparable to the minimum blood Phe concentrations of healthy people. Besides the potential application for developing Phe-sensing elements, this new hydrogel sensing approach via chemoselective oxime ligation is generalizable to the development of other chemical sensors working in complex biological environments.

Process properties of an l-amino acid oxidase from Hebeloma cylindrosporum for the synthesis of phenylpyruvic acid from l-phenylalanine.

The biocatalytic oxidation of amino acids represents an attractive approach towards the synthesis of α-keto acids, which are interest for various industrial applications. As l-amino acids are readily available from fermentation processes, these natural amino acids can serve as substrates in combination with an l-amino acid oxidase. Besides an aqueous phase as reaction medium, a further advantage of such a process is the utilization of air as oxidation agent. In this study, we studied the organic-synthetic properties of a literature-known recombinant l-amino acid oxidase from the fungus Hebeloma cylindrosporum with respect to its suitability to catalyze the formation of α-keto acids exemplified for the synthesis of phenylpyruvic acid starting from l-phenylalanine as a substrate. In our study the enzyme displayed a reasonable operational stability in the reaction system and as well as promising applicability data with respect to substrate and product inhibition. In a biotransformation, 20 mM of substrate were converted after 4 h reaction. The formation of undesired by-products was suppressed using a commercially available catalase enzyme.

[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.