RESUMO
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.
Assuntos
L-Lactato Desidrogenase , Genética , Lacticaseibacillus casei , Genética , Ácidos Fenilpirúvicos , Metabolismo , Pichia , Genética , Proteínas Recombinantes , Genética , MetabolismoRESUMO
Salvia miltiorrhiza is a medicinal plant widely used in the treatment of cardiovascular and cerebrovascular diseases. Hydrophilic phenolic acids, including rosmarinic acid (RA) and lithospermic acid B (LAB), are its primary medicinal ingredients. However, the biosynthetic pathway of RA and LAB in S. miltiorrhiza is still poorly understood. In the present study, we accomplished the isolation and characterization of a novel S. miltiorrhiza Hydroxyphenylpyruvate reductase (HPPR) gene, SmHPPR, which plays an important role in the biosynthesis of RA. SmHPPR contained a putative catalytic domain and a NAD(P)H-binding motif. The recombinant SmHPPR enzyme exhibited high HPPR activity, converting 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), and exhibited the highest affinity for substrate 4-hydroxyphenylpyruvate. SmHPPR expression could be induced by various treatments, including SA, GA, MeJA and Ag, and the changes in SmHPPR activity were correlated well with hydrophilic phenolic acid accumulation. SmHPPR was localized in cytoplasm, most likely close to the cytosolic NADPH-dependent hydroxypyruvate reductase active in photorespiration. In addition, the transgenic S. miltiorrhiza hairy roots overexpressing SmHPPR exhibited up to 10-fold increases in the products of hydrophilic phenolic acid pathway. In conclusion, our findings provide a new insight into the synthesis of active pharmaceutical compounds at molecular level.
Assuntos
Sequência de Aminoácidos , Benzofuranos , Vias Biossintéticas , Genética , Cinamatos , Depsídeos , Regulação da Expressão Gênica de Plantas , Genética , Oxirredutases , Genética , Fenilpropionatos , Metabolismo , Ácidos Fenilpirúvicos , Metabolismo , Filogenia , Proteínas de Plantas , Genética , Metabolismo , Raízes de Plantas , Química , Genética , Metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes , Salvia miltiorrhiza , Química , Genética , Metabolismo , Alinhamento de SequênciaRESUMO
Volta metric behavior of mesotrione using silver/amalgam electrode we examined in this paper. Mesotrione belongs to the triketone group of herbicides. It is used against weeds in corn and operates by inhibition of 4-hydroxyphenyl piruvate in plants. There is little information about mesotrione as its presence in the market is short. For its wider application in the future, it is important to know details of its chemical characteristics and redox processes, including biogeochemical transformation and migration after application to agricultural land, which could contribute to its more efficient and safe application. The technique of cyclic voltammeter with conventional three-electrode cell and electrochemical workstation was used for the investigation of electrochemical behavior of mesotrione. Cyclic voltamograms of mesotrione were recorded at pH values 6, 8, 10 and 12. Several peaks were obtained at certain values of the potential and they were attributed to the reduction of two carbonyl groups [in position 1 and 3 of cyclohexane ring], the third carbonyl group and nitro group. The maximum current value was at pH 12
Assuntos
Amálgama Dentário , Eletrodos , Ácidos FenilpirúvicosRESUMO
Phenylketonuria is an autosomal recessive disorder caused by a deficiency of phenylalanine hydroxylase. Transthyretin has been implicated as an indicator of nutritional status in phenylketonuria patients. In this study, we report that phenylalanine and its metabolite, phenylpyruvic acid, affect MAPK, changing transthyretin expression in a cell- and tissue-specific manner. Treatment of HepG2 cells with phenylalanine or phenylpyruvic acid decreased transcription of the TTR gene and decreased the transcriptional activity of the TTR promoter site, which was partly mediated through HNF4alpha. Decreased levels of p38 MAPK were detected in the liver of phenylketonuria-affected mice compared with wild-type mice. In contrast, treatment with phenylalanine increased transthyretin expression and induced ERK1/2 activation in PC-12 cells; ERK1/2 activation was also elevated in the brainstem of phenylketonuria-affected mice. These findings may explain between-tissue differences in gene expression, including Ttr gene expression, in the phenylketonuria mouse model.