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Construction and evaluation of a novel bifunctional phenylalanine–formate dehydrogenase fusion protein for bienzyme system with cofactor regeneration

  • Biocatalysis
  • Published:
Journal of Industrial Microbiology & Biotechnology

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.

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References

  1. An JM, Kim YK, Lim WJ, Hong SY, An CL, Shin EC, Cho KM, Choi BR, Kang JM, Lee SM (2005) Evaluation of a novel bifunctional xylanase–cellulase constructed by gene fusion. Enzyme Microb Technol 36:989–995

    Article  CAS  Google Scholar 

  2. Asano Y, Nakazawa A, Endo K (1987) Novel phenylalanine dehydrogenases from Sporosarcina ureae and Bacillus sphaericus. Purification and characterization. J Biol Chem 262:10346–10354

    CAS  PubMed  Google Scholar 

  3. Asano Y, Tanetani M (1998) Thermostable phenylalanine dehydrogenase from a mesophilic Microbacterium sp. strain DM 86-1. Arch Microbiol 169:220

    Article  CAS  PubMed  Google Scholar 

  4. Asano Y, Yamada A, Kato Y, Yamaguchi K, Hibino Y, Hirai K, Kondo K (1990) Enantioselective synthesis of (S)-amino acids by phenylalanine dehydrogenase from Bacillus sphaericus: use of natural and recombinant enzymes. J Org Chem 55:5567–5571

    Article  CAS  Google Scholar 

  5. Aÿ J, Götz F, Borriss R, Heinemann U (1998) Structure and function of the Bacillus hybrid enzyme GluXyn-1: native-like jellyroll fold preserved after insertion of autonomous globular domain. Proc Natl Acad Sci USA 95:6613–6618

    Article  PubMed  PubMed Central  Google Scholar 

  6. Bülow L, Mosbach K (1991) Multienzyme systems obtained by gene fusion. Trends Biotechnol 9:226–231

    Article  PubMed  Google Scholar 

  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  8. Crasto CJ, Feng JA (2000) LINKER: a program to generate linker sequences for fusion proteins. Protein Eng 13:309–312

    Article  CAS  PubMed  Google Scholar 

  9. Gilbert M, Bayer R, Cunningham AM, DeFrees S, Gao Y, Watson DC, Young NM, Wakarchuk WW (1998) The synthesis of sialylated oligosaccharides using a CMP-Neu5Ac synthetase/sialyltransferase fusion. Nat Biotechnol 16:769–772

    Article  CAS  PubMed  Google Scholar 

  10. Grifantini R, Galli G, Carpani G, Pratesi C, Frascotti G, Grandi G (1998) Efficient conversion of 5-substituted hydantoins to D-α-amino acids using recombinant Escherichia coli strains. Microbiology 144:947–954

    Article  CAS  PubMed  Google Scholar 

  11. Hölsch K, Weuster-Botz D (2010) Enantioselective reduction of prochiral ketones by engineered bifunctional fusion proteins. Biotechnol Appl Biochem 56:131–140

    Article  PubMed  Google Scholar 

  12. Hong SY, Lee JS, Cho KM, Math RK, Kim YH, Hong SJ, Cho YU, Kim H, Yun HD (2006) Assembling a novel bifunctional cellulase–xylanase from Thermotoga maritima by end-to-end fusion. Biotechnol Lett 28:1857–1862

    Article  CAS  PubMed  Google Scholar 

  13. Huang T, Warsinke A, Kuwana T, Scheller FW (1998) Determination of l-phenylalanine based on an NADH-detecting biosensor. Anal Chem 70:991–997

    Article  CAS  PubMed  Google Scholar 

  14. Hummel W, Schütte H, Schmidt E, Wandrey C, Kula M-R (1987) Isolation of l-phenylalanine dehydrogenase from Rhodococcus sp. M4 and its application for the production of l-phenylalanine. Appl Microbiol Biotechnol 26:409

    CAS  Google Scholar 

  15. Hummel W, Weiss N, Kula MR (1984) Isolation and characterization of a bacterium possessing l-phenylalanine dehydrogenase activity. Arch Microbiol 137:47–52

    Article  CAS  Google Scholar 

  16. Iturrate L, Sánchez-Moreno I, Oroz-Guinea I, Pérez-Gil J, García-Junceda E (2010) Preparation and characterization of a bifunctional aldolase/kinase enzyme: a more efficient biocatalyst for C–C bond formation. Chem A Eur J 16:4018–4030

    Article  CAS  Google Scholar 

  17. Kim GJ, Lee DE, Kim HS (2000) Construction and evaluation of a novel bifunctional N-Carbamylase–d-Hydantoinase fusion enzyme. Appl Environ Microbiol 66:2133–2138

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kula MR, Pohl M, Slusarczyk H (1997) sowie Verwendung dieser Formiatdeydrogenasen. J Bacteriol 179:P4480–P4485

    Google Scholar 

  19. Li H, Zhu D, Hyatt BA, Malik FM, Biehl ER, Hua L (2009) Cloning, protein sequence clarification, and substrate specificity of a leucine dehydrogenase from Bacillus sphaericus ATCC4525. Appl Biochem Biotech 158:343–351

    Article  CAS  Google Scholar 

  20. Li N, Jiang XN, Cai GP, Yang SF (1996) A novel bifunctional fusion enzyme catalyzing ethylene synthesis via 1-aminocyclopropane1-carboxylic acid. J Biol Chem 271:25738–25741

    Article  CAS  PubMed  Google Scholar 

  21. Liu W, Luo J, Zhuang X, Shen W, Zhang Y, Li S, Hu Y, Huang H (2014) Efficient preparation of enantiopure l-tert-leucine through immobilized penicillin G acylase catalyzed kinetic resolution in aqueous medium. Biochem Eng J 83:116–120

    Article  CAS  Google Scholar 

  22. Lu P, Feng MG (2008) Bifunctional enhancement of a β-glucanase–xylanase fusion enzyme by optimization of peptide linkers. Appl Microbiol Biotechnol 79:579–587

    Article  CAS  PubMed  Google Scholar 

  23. Lu Q (2005) Seamless cloning and gene fusion. Trends Biotechnol 23:199–207

    Article  CAS  PubMed  Google Scholar 

  24. Maeda Y, Ueda H, Kazami J, Kawano G, Suzuki E, Nagamune T (1997) Engineering of functional chimeric protein G–VargulaLuciferase. Anal Biochem 249:147–152

    Article  CAS  PubMed  Google Scholar 

  25. Mihara H, Muramatsu H, Kakutani R, Yasuda M, Ueda M, Kurihara T, Esaki N (2005) N-Methyl-l-amino acid dehydrogenase from Pseudomonas putida. FEBS J 272:1117–1123

    Article  CAS  PubMed  Google Scholar 

  26. Mohamadi HS, Omidinia E (2007) Purification of recombinant phenylalanine dehydrogenase by partitioning in aqueous two-phase systems. J Chromatogr B 854:273–278

    Article  CAS  Google Scholar 

  27. Nakamura K, Fujii T, Kato Y, Asano Y, Cooper AJ (1996) Quantitation ofL-amino acids by substrate recycling between an aminotransferase and a dehydrogenase: application to the determination of l-phenylalanine in human blood. Anal Biochem 234:19–22

    Article  CAS  PubMed  Google Scholar 

  28. Nampally M, Moerschbacher BM, Kolkenbrock S (2012) Fusion of a novel genetically engineered chitosan affinity protein and green fluorescent protein for specific detection of chitosan in vitro and in situ. Appl Environ Microbiol 78:3114–3119

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Nixon AE, Ostermeier M, Benkovic SJ (1998) Hybrid enzymes: manipulating enzyme design. Trends Biotechnol 16:258–264

    Article  CAS  PubMed  Google Scholar 

  30. Paradisi F, Collins S, Maguire AR, Engel PC (2007) Phenylalanine dehydrogenase mutants: efficient biocatalysts for synthesis of non-natural phenylalanine derivatives. J Biotechnol 128:408–411

    Article  CAS  PubMed  Google Scholar 

  31. Paradisi F, Moynihan E, Maguire AR, Engel PC (2004) Enantioselective synthesis of non-natural amino acids using phenylalanine dehydrogenases modified by site-directed mutagenesis. Org Biomol Chem 2:2684–2691

    Article  PubMed  Google Scholar 

  32. Rivero A, Allué JA, Grijalba A, Palacios M, Merlo SG (2000) Comparison of two different methods for measurement of phenylalanine in dried blood spots. Clin Chem Lab 38:773–776

    CAS  Google Scholar 

  33. Seah SYK, Britton KL, Rice DW, Asano Y, Engel PC (2002) Single amino acid substitution in Bacillus sphaericus phenylalanine dehydrogenase dramatically increases its discrimination between phenylalanine and tyrosine substrates. Biochemistry 41:11390–11397

    Article  CAS  PubMed  Google Scholar 

  34. Trujillo M, Duncan R, Santi D (1997) Construction of a homodimeric dihydrofolate reductase-thymidylate synthase bifunctional enzyme. Protein Eng 10:567–573

    Article  CAS  PubMed  Google Scholar 

  35. Wilson P, Anastasaki A, Owen MR, Kempe K, Haddleton DM, Mann SK, Johnston AP, Quinn JF, Whittaker MR, Hogg PJ (2015) Organic arsenicals as efficient and highly specific linkers for protein/peptide–polymer conjugation. J Am Chem Soc 137:12

  36. Yang B, Wu YJ, Zhu M, Fan SB, Lin J, Zhang K, Li S, Chi H, Li YX, Chen HF (2012) Identification of cross-linked peptides from complex samples. Nat Methods 9:904–906

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the State Key Program of National Natural Science Foundation of China (No. 21336009), the National Natural Science Foundation of China (No.41176111, No.41306124), the Fundamental Research Funds for the Central Universities (No.2013121029), the Foundation of South Oceanographic Research Center of China in Xiamen (No.: 14GYY011NF11), and the Public science and technology research funds projects of ocean (No.: 201505032-6).

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Authors and Affiliations

  1. Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China

    Wei Jiang & Bai-Shan Fang

  2. The Key Laboratory for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, 361005, China

    Wei Jiang & Bai-Shan Fang

  3. The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen, 361005, Fujian, China

    Bai-Shan Fang

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  1. Wei Jiang
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Correspondence to Bai-Shan Fang.

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Jiang, W., Fang, BS. Construction and evaluation of a novel bifunctional phenylalanine–formate dehydrogenase fusion protein for bienzyme system with cofactor regeneration. J Ind Microbiol Biotechnol 43, 577–584 (2016). https://doi.org/10.1007/s10295-016-1738-6

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  • DOI: https://doi.org/10.1007/s10295-016-1738-6

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