Skip to main content
SpringerLink
Log in

Resolution Mechanism and Characterization of an Ammonium Chloride-Tolerant, High-Thermostable, and Salt-Tolerant Phenylalanine Dehydrogenase from Bacillus halodurans

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

As phenylalanine dehydrogenase (PheDH) plays an important role in the synthesis of chiral drug intermediates and detection of phenylketonuria, it is significant to obtain a PheDH with specific and high activity. Here, a PheDH gene, pdh, encoding a novel BhPheDH with 61.0% similarity to the known PheDH from Microbacterium sp., was obtained. The BhPheDH showed optimal activity at 60 °C and pH 7.0, and it showed better stability in hot environment (40–70 °C) than the PheDH from Nocardia sp. And its activity and thermostability could be significantly increased by sodium salt. After incubation for 2 h in 3 M NaCl at 60 °C, the residual activity of the BhPheDH was found to be 1.8-fold higher than that of the control group (without NaCl). The BhPheDH could tolerate high concentration of ammonium chloride and its activity could be also enhanced by the high concentration of ammonium salts. These characteristics indicate that the BhPheDH possesses better thermostability, ammonium chloride tolerance, halophilic mechanism, and high salt activation. The mechanism of thermostability and high salt tolerance of the BhPheDH was analyzed by molecular dynamics simulation. These results provide useful information about the enzyme with high-temperature activity, thermostability, halophilic mechanism, tolerance to high concentration of ammonium chloride, higher salt activation and enantio-selectivity, and the application of molecular dynamics simulation in analyzing the mechanism of these distinctive characteristics.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Asano, Y., Nakazawa, A., & Endo, K. (1987). Novel phenylalanine dehydrogenases from Sporosarcina ureae and Bacillus sphaericus. Purification and characterization. The Journal of Biological Chemistry, 262(21), 10346–10354.

    CAS  PubMed  Google Scholar 

  2. Bommarius, B. R., Schürmann, M., & Bommarius, A. S. (2014). A novel chimeric amine dehydrogenase shows altered substrate specificity compared to its parent enzymes. Chemical Communications (Camb), 50(95), 14953–14955.

    Article  CAS  Google Scholar 

  3. 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. Applied Microbiology and Biotechnology, 26, 409.

    Article  CAS  Google Scholar 

  4. Seah, S. Y. K., Britton, K. L., Rice, D. W., Asano, Y., & Engel, P. C. (2002). Single amino acid substitution in Bacillus sphaericus phenylalanine dehydrogenase dramatically increases its discrimination between phenylalanine and tyrosine substrates. Biochemistry, 41(38), 11390–11397.

    Article  CAS  Google Scholar 

  5. Seah, S. Y. K., Britton, K. L., Rice, D. W., Asano, Y., & Engel, P. C. (2003). Kinetic analysis of phenylalanine dehydrogenase mutants designed for aliphatic amino acid dehydrogenase activity with guidance from homology-based modelling. European Journal of Biochemistry, 270(23), 4628–4634.

    Article  CAS  Google Scholar 

  6. Ye, L. J., Hui, H. T., Yang, Y., Adams, J. P., Snajdrova, R., & Li, Z. (2015). Engineering of amine dehydrogenase for asymmetric reductive amination of ketone by evolving Rhodococcus phenylalanine dehydrogenase. ACS Catalysis, 5(2), 1119–1122.

    Article  CAS  Google Scholar 

  7. Asano, Y., & Tanetani, M. (1998). Thermostable phenylalanine dehydrogenase from a mesophilic Microbacterium sp. strain DM 86-1. Archives of Microbiology, 169(3), 220–224.

    Article  CAS  Google Scholar 

  8. Hummel, W., Weiss, N., & Kula, M.-R. (1984). Isolation and characterization of a bacterium possessing L-phenylalanine dehydrogenase activity. Archives of Microbiology, 137(1), 47–52.

    Article  CAS  Google Scholar 

  9. Ohshima, T., Takada, H., Yoshimura, T., Esaki, N., & Soda, K. (1991). Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes. Journal of Bacteriology, 173(13), 3943–3948.

    Article  CAS  Google Scholar 

  10. Pasquo, A., Britton, K., Baker, P., Brearley, G., Hinton, R., Moir, A., Stillman, T., & Rice, D. (1998). Crystallization of NAD+-dependent phenylalanine dehydrogenase from Nocardia sp239. Acta Crystallographica Section D Biological Crystallography, 54(2), 269–272.

    Article  CAS  Google Scholar 

  11. Brunhuber, N. M., Banerjee, A., Jacobs Jr., W. R., & Blanchard, J. S. (1994). Cloning, sequencing, and expression of Rhodococcus L-phenylalanine dehydrogenase. Sequence comparisons to amino-acid dehydrogenases. The Journal of Biological Chemistry, 269(23), 16203–16211.

    CAS  PubMed  Google Scholar 

  12. Misono, H., Yonezawa, J., Nagata, S., & Nagasaki, S. (1989). Purification and characterization of a dimeric phenylalanine dehydrogenase from Rhodococcus maris K-18. Journal of Bacteriology, 171(1), 30–36.

    Article  CAS  Google Scholar 

  13. Asano, Y., Nakazawa, A., Endo, K., Hibino, Y., Ohmori, M., Numao, N., & Kondo, K. (1987). Phenylalanine dehydrogenase of Bacillus badius. Purification, characterization and gene cloning. European Journal of Biochemistry, 168(1), 153–159.

    Article  CAS  Google Scholar 

  14. Brunhuber, N. M., Thoden, J. B., Blanchard, J. S., & Vanhooke, J. L. (2000). Rhodococcus L-phenylalanine dehydrogenase: Kinetics, mechanism, and structural basis for catalytic specifity. Biochemistry, 39(31), 9174–9187.

    Article  CAS  Google Scholar 

  15. Chen, S., & Engel, P. C. (2009). Efficient screening for new amino acid dehydrogenase activity: directed evolution of Bacillus sphaericus phenylalanine dehydrogenase towards activity with an unsaturated non-natural amino acid. Journal of Biotechnology, 142(2), 127–134.

    Article  CAS  Google Scholar 

  16. Hanson, R. L., Howell, J. M., LaPorte, T. L., Donovan, M. J., Cazzulino, D. L., Zannella, V., Montana, M. A., Nanduri, V. B., Schwarz, S. R., & Eiring, R. F. (2000). Synthesis of allysine ethylene acetal using phenylalanine dehydrogenase from Thermoactinomyces intermedius. Enzyme and Microbial Technology, 26(5-6), 348–358.

    Article  CAS  Google Scholar 

  17. Mihara, H., Muramatsu, H., Kakutani, R., Yasuda, M., Ueda, M., Kurihara, T., & Esaki, N. (2005). N-Methyl-l-amino acid dehydrogenase from Pseudomonas putida. The FEBS Journal, 272(5), 1117–1123.

    Article  CAS  Google Scholar 

  18. Paradisi, F., Moynihan, E., Maguire, A. R., & Engel, P. C. (2004). Enantioselective synthesis of non-natural amino acids using phenylalanine dehydrogenases modified by site-directed mutagenesis. Organic & Biomolecular Chemistry, 2, 2684–2691.

    Article  Google Scholar 

  19. Huang, T., Warsinke, A., Kuwana, T., & Scheller, F. W. (1998). Determination of L-phenylalanine based on an NADH-detecting biosensor. Analytical Chemistry, 70(5), 991–997.

    Article  CAS  Google Scholar 

  20. Nakamura, K., Fujii, T., Kato, Y., Asano, Y., & Cooper, A. J. (1996). Quantitation ofL-amino acids by substrate recycling between an aminotransferase and a dehydrogenase: application to the determination ofL-phenylalanine in human blood. Analytical Biochemistry, 234(1), 19–22.

    Article  CAS  Google Scholar 

  21. Randell, E. W., & Lehotay, D. C. (1996). An automated enzymatic method on the Roche COBAS MIRA TM S for monitoring phenylalanine in dried blood spots of patients with phenylketonuria. Clinical Biochemistry, 29(2), 133–138.

    Article  CAS  Google Scholar 

  22. Rivero, A., Allué, J. A., Grijalba, A., Palacios, M., & Merlo, S. G. (2000). Comparison of two different methods for measurement of phenylalanine in dried blood spots. Clinical Chemistry and Laboratory Medicine, 38, 773–776.

    Article  CAS  Google Scholar 

  23. 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. The Journal of Organic Chemistry, 55(21), 5567–5571.

    Article  CAS  Google Scholar 

  24. Matthews, B. W. (1993). Structural and genetic analysis of protein stability. Annual Review of Biochemistry, 62(1), 139–160.

    Article  CAS  Google Scholar 

  25. Michels, P. C., & Clark, D. S. (1997). Pressure-enhanced activity and stability of a hyperthermophilic protease from a deep-sea methanogen. Applied and Environmental Microbiology, 63(10), 3985–3991.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Niehaus, F., Bertoldo, C., Kähler, M., & Antranikian, G. (1999). Extremophiles as a source of novel enzymes for industrial application. Applied Microbiology and Biotechnology, 51(6), 711–729.

    Article  CAS  Google Scholar 

  27. Vieille, C., & Zeikus, J. G. (1996). Thermozymes: identifying molecular determinants of protein structural and functional stability. Trends in Biotechnology, 14(6), 183–190.

    Article  CAS  Google Scholar 

  28. Innis, M. A., Myambo, K. B., Gelfand, D. H., & Brow, M. (1988). DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proceedings of the National Academy of Sciences, 85(24), 9436–9440.

    Article  CAS  Google Scholar 

  29. Petzelbauer, I., Kuhn, B., Splechtna, B., Kulbe, K. D., & Nidetzky, B. (2002). Development of an ultrahigh-temperature process for the enzymatic hydrolysis of lactose. IV. Immobilization of two thermostable β-glycosidases and optimization of a packed-bed reactor for lactose conversion. Biotechnology and Bioengineering, 77(6), 619–631.

    Article  CAS  Google Scholar 

  30. Oren, A. (2002). Diversity of halophilic microorganisms: environments, phylogeny, physiology, and applications. Journal of Industrial Microbiology & Biotechnology, 28(1), 56–63.

    Article  CAS  Google Scholar 

  31. Trincone, A. (2011). Marine biocatalysts: enzymatic features and applications. Marine Drugs, 9(4), 478–499.

    Article  CAS  Google Scholar 

  32. Setati, M. E. (2010). Diversity and industrial potential of hydrolaseproducing halophilic/halotolerant eubacteria. African Journal of Biotechnology, 9, 1555–1560.

    Article  CAS  Google Scholar 

  33. Jiang, W., Sun, D., Ren, H., Xu, C., Wang, Y., Zhang, Y., Wang, S., & Fang, B. (2016). Isolation, purification and characterization of a salt-active and organic-solvent-thermostable phenylalanine dehydrogenase from Bacillus nanhaiensis DSF-15A2. Journal of Molecular Catalysis B: Enzymatic, 133, 12–19.

    Article  CAS  Google Scholar 

  34. Warden, A. C., Williams, M., Peat, T. S., Seabrook, S. A., Newman, J., Dojchinov, G., & Haritos, V. S. (2015). Rational engineering of a mesohalophilic carbonic anhydrase to an extreme halotolerant biocatalyst. Nature Communications, 6(1), 10278.

    Article  CAS  Google Scholar 

  35. Beck, D. A., Jonsson, A. L., Schaeffer, R. D., Scott, K. A., Day, R., Toofanny, R. D., Alonso, D. O., & Daggett, V. (2008). Dynameomics: mass annotation of protein dynamics and unfolding in water by high-throughput atomistic molecular dynamics simulations. Protein Engineering, Design & Selection, 21(6), 353–368.

    Article  CAS  Google Scholar 

  36. Ouyang Ping Fang, X. H., Guo, A., & Yanfeng, L. (2005). Molecular simulation method and its application in molecular biology. China Journal of Bioinformation, 3, 33–36.

    Google Scholar 

  37. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2), 248–254.

    Article  CAS  Google Scholar 

  38. Arnold, K., Bordoli, L., Kopp, J., & Schwede, T. (2006). The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics, 22(2), 195–201.

    Article  CAS  Google Scholar 

  39. Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., Skeel, R. D., Kale, L., & Schulten, K. (2005). Scalable molecular dynamics with NAMD. Journal of Computational Chemistry, 26(16), 1781–1802.

    Article  CAS  Google Scholar 

  40. Ebel, C., Costenaro, L., Pascu, M., Faou, P., Kernel, B., Proust-De Martin, F., & Zaccai, G. (2002). Solvent interactions of halophilic malate dehydrogenase. Biochemistry, 41(44), 13234–13244.

    Article  CAS  Google Scholar 

  41. Darden, T., York, D., & Pedersen, L. (1993). Particle mesh Ewald: an N· log (N) method for Ewald sums in large systems. The Journal of Chemical Physics, 98(12), 10089–10092.

    Article  CAS  Google Scholar 

  42. Kaiser, J., Kinderman, S. S., van Esseveldt, B. C., van Delft, F. L., Schoemaker, H. E., Blaauw, R. H., & Rutjes, F. P. (2005). Synthetic applications of aliphatic unsaturated α-H-α-amino acids. Organic & Biomolecular Chemistry, 3(19), 3435–3467.

    Article  CAS  Google Scholar 

  43. Garofalo, A. W., Wone, D. W., Phuc, A., Audia, J. E., Bales, C. A., Dovey, H. F., Dressen, D. B., Folmer, B., Goldbach, E. G., & Guinn, A. C. (2002). A series of C-terminal amino alcohol dipeptide Aβ inhibitors. Bioorganic & Medicinal Chemistry Letters, 12(21), 3051–3053.

    Article  CAS  Google Scholar 

  44. Isaac, M., Slassi, A., Da Silva, K., Arora, J., MacLean, N., Hung, B., & McCallum, K. (2001). 5, 5-Diaryl-2-amino-4-pentenoates as novel, potent, and selective glycine transporter type-2 reuptake inhibitors. Bioorganic & Medicinal Chemistry Letters, 11(11), 1371–1373.

    Article  CAS  Google Scholar 

  45. Natchus, M. G., Bookland, R. G., Laufersweiler, M. J., Pikul, S., Almstead, N. G., De, B., Janusz, M. J., Hsieh, L. C., Gu, F., & Pokross, M. E. (2001). Development of new carboxylic acid-based MMP inhibitors derived from functionalized propargylglycines. Journal of Medicinal Chemistry, 44(7), 1060–1071.

    Article  CAS  Google Scholar 

  46. Paradisi, F., Collins, S., Maguire, A. R., & Engel, P. C. (2007). Phenylalanine dehydrogenase mutants: efficient biocatalysts for synthesis of non-natural phenylalanine derivatives. Journal of Biotechnology, 128(2), 408–411.

    Article  CAS  Google Scholar 

  47. Tadeo, X., López-Méndez, B., Trigueros, T., Laín, A., Castaño, D., & Millet, O. (2009). Structural basis for the aminoacid composition of proteins from halophilic archea. PLoS Biology, 7(12), e1000257.

    Article  Google Scholar 

  48. Costenaro, L., Zaccai, G., & Ebel, C. (2002). Link between protein-solvent and weak protein-protein interactions gives insight into halophilic adaptation. Biochemistry, 41(44), 13245–13252.

    Article  CAS  Google Scholar 

  49. Fukuchi, S., Yoshimune, K., Wakayama, M., Moriguchi, M., & Nishikawa, K. (2003). Unique amino acid composition of proteins in halophilic bacteria. Journal of Molecular Biology, 327(2), 347–357.

    Article  CAS  Google Scholar 

  50. Tardieu, A., Bonnete, F., Finet, S., & Vivares, D. (2002). Understanding salt or PEG induced attractive interactions to crystallize biological macromolecules. Acta Crystallographica, Section D: Biological Crystallography, 58(10), 1549–1553.

    Article  Google Scholar 

  51. Van Den Burg, B. (2003). Extremophiles as a source for novel enzymes. Current Opinion in Microbiology, 6(3), 213–218.

    Article  Google Scholar 

Download references

Funding

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 Foundation of South Oceanographic Research Center of China in Xiamen (No.: 14GYY011NF11), the Public science and technology research funds projects of ocean (No.: 201505032-6), and the high-level personnel activation fee of Huaqiao University (600005-Z17Y0072). 

Author information

Authors and Affiliations

  1. Fujian Provincial Key Laboratory of Biochemical Technology, Department of Bioengineering and Biotechnology, College of Chemical Engineering, Huaqiao University, Xiamen, 361021, Fujian, China

    Wei Jiang

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

    Ya-Li Wang & Bai-Shan Fang

  3. The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, 361005, China

    Ya-Li Wang & Bai-Shan Fang

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

    Bai-Shan Fang

Authors
  1. Wei Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ya-Li Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bai-Shan Fang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

WJ designed and performed the experiments and wrote the manuscript. YZW performed the experiments and revised the manuscript. BSF conceived the study, designed and supervised the experiments, and is a corresponding author. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Bai-Shan Fang.

Ethics declarations

Conflict of Interest

The authors declare that they have no competing interests.

Electronic supplementary material

ESM 1

(DOC 1.02MB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jiang, W., Wang, YL. & Fang, BS. Resolution Mechanism and Characterization of an Ammonium Chloride-Tolerant, High-Thermostable, and Salt-Tolerant Phenylalanine Dehydrogenase from Bacillus halodurans. Appl Biochem Biotechnol 186, 789–804 (2018). https://doi.org/10.1007/s12010-018-2767-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-018-2767-6

Keywords

Search

Navigation