title: Construction and evaluation of a novel bifunctional phenylalanine-formate dehydrogenase fusion protein for bienzyme system with cofactor regeneration.
abstract: Phenylalanine dehydrogenase (PheDH) plays an important role in enzymatic synthesis of L-phenylalanine for aspartame (sweetener) and detection of phenylketonuria (PKU), suggesting that it is important to obtain a PheDH with excellent characteristics. Gene fusion of PheDH and formate dehydrogenase (FDH) was constructed to form bifunctional multi-enzymes for bioconversion of L-phenylalanine coupled with coenzyme regeneration. Comparing with the PheDH monomer from Microbacterium sp., the bifunctional PheDH-FDH showed noteworthy stability under weakly acidic and alkaline conditions (pH 6.5-9.0). The bifunctional enzyme can produce 153.9 mM L-phenylalanine with remarkable performance of enantiomers choice by enzymatic conversion with high molecular conversion rate (99.87 %) in catalyzing phenylpyruvic acid to L-phenylalanine being 1.50-fold higher than that of the separate expression system. The results indicated the potential application of the PheDH and PheDH-FDH with coenzyme regeneration for phenylpyruvic acid analysis and L-phenylalanine biosynthesis in medical diagnosis and pharmaceutical field.
authors.affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.|The Key Laboratory for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, 361005, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China. fbs@xmu.edu.cn.|The Key Laboratory for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, 361005, China. fbs@xmu.edu.cn.|The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen, 361005, Fujian, China. fbs@xmu.edu.cn.
authors.forename: Bai-Shan
authors.initials: BS
authors.lastname: Fang
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology; D002175:Candida / Q000201:enzymology; D003067:Coenzymes / Q000096:biosynthesis* / Q000378:metabolism; D004795:Enzyme Stability; D005560:Formate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005561:Formates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010649:Phenylalanine / Q000096:biosynthesis / Q000378:metabolism; D010667:Phenylpyruvic Acids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013237:Stereoisomerism; D013696:Temperature
chemical_list: D003067:Coenzymes; D005561:Formates; D010667:Phenylpyruvic Acids; D011993:Recombinant Fusion Proteins; C030544:formic acid; D010649:Phenylalanine; D005560:Formate Dehydrogenases; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase; C031606:phenylpyruvic acid
keywords: Bifunctional enzyme; Biocatalyst; Biosynthesis; Cofactor regeneration; Formate dehydrogenase; Phenylalanine dehydrogenase
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