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Applied Microbiology and Biotechnology

, Volume 101, Issue 5, pp 1953–1964 | Cite as

Identification and characterization of a thermostable and cobalt-dependent amidase from Burkholderia phytofirmans ZJB-15079 for efficient synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid

  • Zhe-Ming Wu
  • Ren-Chao Zheng
  • Xiao-Ling Tang
  • Yu-Guo ZhengEmail author
Biotechnologically relevant enzymes and proteins
  • 321 Downloads

Abstract

Enantiomerically pure 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acids are important chiral building blocks for a series of pharmaceuticals. Here, a bacteria strain with 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide-degrading ability was screened and identified as Burkholderia phytofirmans ZJB-15079, from which a novel amidase (Bp-Ami) was cloned and demonstrated to be capable of kinetic resolution of rac-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide to optically pure (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid. Phylogenetic analysis revealed that Bp-Ami was closely located to the acetamidase/formamidase (FmdA_AmdA) family, and it shared high homology with acetamidases. Bp-Ami was found to be the first cobalt-dependent FmdA_AmdA family amidase. The enzyme activity was significantly increased by 37.7-fold in the presence of 1 mM Co2+, with a specific activity of 753.5 U/mg, K m value of 24.73 mM, and k cat /K m value of 22.47 mM−1 s−1. As an enzyme from mesophile, Bp-Ami exhibited extreme thermostability with a half-life of 47.93 h at 80 °C, which was even superior to other reported amidases from thermophiles. The whole cell catalysis of 200 g/L 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide by Escherichia coli harboring Bp-Ami (5 g/L) resulted in 44 % yield and an enantiomeric excess (ee p) of 95 % within 10 min (E = 86). The high substrate tolerance, high specific activity, and extreme thermostability demonstrated the great potential of Bp-Ami for efficient biocatalytic synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid.

Keywords

Amidase Biochemical characterization Enantioselective resolution (R)-3,3,3-Trifluoro-2-hydroxy-2-methylpropionic acid 

Notes

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (No. 21202150, No. 21602199), Natural Science Foundation of Zhejiang Province (Y4080334, LY13B060004), and National High Technology Research and Development Program of China (No. 2012AA022201B).

Compliance with ethical standards

Conflict of interest

The author declares that he has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2016_7921_MOESM1_ESM.pdf (559 kb)
ESM 1 (PDF 559 kb)

References

  1. Aicher TD, Anderson RC, Gao J, Shetty SS, Coppola GM, Stanton JL, Knorr DC, Sperbeck DM, Brand LJ, Vinluan CC (2000) Secondary amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase. J Med Chem 43(2):236–249CrossRefPubMedGoogle Scholar
  2. Borges CL, Parente JA, Barbosa MS, Santana JM, Báo SN, De Sousa MV, de Almeida Soares CM (2009) Detection of a homotetrameric structure and protein–protein interactions of Paracoccidioides brasiliensis formamidase lead to new functional insights. FEMS Yeast Res 10(1):104–113CrossRefGoogle Scholar
  3. Borges CL, Pereira M, Felipe MS, de Faria FP, Gomez FJ, Deepe GS, Soares CM (2005) The antigenic and catalytically active formamidase of Paracoccidioides brasiliensis: protein characterization, cDNA and gene cloning, heterologous expression and functional analysis of the recombinant protein. Microbes Infect 7(1):66–77CrossRefPubMedGoogle Scholar
  4. 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(1–2):248–254CrossRefPubMedGoogle Scholar
  5. Braun M (2008) Process for the production of fluorine containing α-hydroxy carboxylic acids. EP1881077 A1Google Scholar
  6. Carlos N, Farnaud S, Renée T, Clemente A, Brown PR (2002) Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity. Biochem J 365(3):731–738CrossRefGoogle Scholar
  7. Chen J, Zheng RC, Zheng YG, Shen YC (2009) Microbial transformation of nitriles to nigh-value acids or amides. Adv Biochem Eng Biotechnol 113:33–77PubMedGoogle Scholar
  8. Chung C, Niemela SL, Miller RH (1989) One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci 86(7):2172–2175CrossRefPubMedPubMedCentralGoogle Scholar
  9. Díaz-Sáez L, Srikannathasan V, Zoltner M, Hunter WN (2014) Structures of bacterial kynurenine formamidase reveal a crowded binuclear zinc catalytic site primed to generate a potent nucleophile. Biochem J 462(3):581–589CrossRefPubMedPubMedCentralGoogle Scholar
  10. Egorova K, Trauthwein H, Verseck S, Antranikian G (2004) Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila. Appl Microbiol Biotechnol 65(1):38–45CrossRefPubMedGoogle Scholar
  11. Fraser JA, Davis MA, Hynes MJ (2001) The formamidase gene of Aspergillus nidulans: regulation by nitrogen metabolite repression and transcriptional interference by an overlapping upstream gene. Genetics 157(1):119–131PubMedPubMedCentralGoogle Scholar
  12. Friedrich CG, Mitrenga G (1981) Utilization of aliphatic amides and formation of two different amidases by Alcaligenes eutrophus. Microbiology 125(2):367–374CrossRefGoogle Scholar
  13. Fu L, Li X, Xiao X, Xu J (2014) Purification and characterization of a thermostable aliphatic amidase from the hyperthermophilic archaeon Pyrococcus yayanosii CH1. Extremophiles 18(2):429–440CrossRefPubMedGoogle Scholar
  14. Fuhshuku K-i, Watanabe S, Nishii T, Ishii A, Asano Y (2014) Efficient preparation of both enantiomers of 3,3,3-trifluoro-2-hydroxy-2-methylpropanoic acid catalyzed by Shinella sp. R-6 and Arthrobacter sp. S-2. J Mol Catal B Enzym 102:115–119CrossRefGoogle Scholar
  15. Fuhshuku K, Watanabe S, Nishii T, Ishii A, Asano Y (2015) A novel S-enantioselective amidase acting on 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide from Arthrobacter sp. S-2. Biosci Biotechnol Biochem 79(10):1587–1596CrossRefPubMedGoogle Scholar
  16. Gao M, Wang DX, Zheng QY, Huang ZT, Wang MX (2007) Remarkable electronic and steric effects in the nitrile biotransformations for the preparation of enantiopure functionalized carboxylic acids and amides: implication for an unsaturated carbon-carbon bond binding domain of the amidase. J Org Chem 72(16):6060–6066CrossRefPubMedGoogle Scholar
  17. Hermes H, Sonke T, Peters P, Van Balken J, Kamphuis J, Dijkhuizen L, Meijer E (1993) Purification and characterization of an L-aminopeptidase from Pseudomonas putida ATCC 12633. Appl Environ Microbiol 59(12):4330–4334PubMedPubMedCentralGoogle Scholar
  18. Hynes M (1975) Amide utilization in Aspergillus nidulans: evidence for a third amidase enzyme. Microbiology 91(1):99–109Google Scholar
  19. Komeda H, Asano Y (2000) Gene cloning, nucleotide sequencing, and purification and characterization of the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3. Eur J Biochem 267(7):2028–2035CrossRefPubMedGoogle Scholar
  20. Konigsberger K, Prasad K, Repič O (1999) The synthesis of (R)-and (S)-α-trifluoromethyl-α-hydroxycarboxylic acids via enzymatic resolutions. Tetrahedron Asymmetry 10(4):679–687CrossRefGoogle Scholar
  21. Labahn J, Neumann S, Büldt G, Kula MR, Granzin J (2002) An alternative mechanism for amidase signature enzymes. J Mol Biol 322(5):1053–1064CrossRefPubMedGoogle Scholar
  22. Ma DY, Wang DX, Pan J, Huang ZT, Wang MX (2008) Nitrile biotransformations for the synthesis of highly enantioenriched β-hydroxy and β-amino acid and amide derivatives: a general and simple but powerful and efficient benzyl protection strategy to increase enantioselectivity of the amidase. J Org Chem 73(11):4087–4091CrossRefPubMedGoogle Scholar
  23. Mahenthiralingam E, Draper P, Davis EO, Colston MJ (1993) Cloning and sequencing of the gene which encodes the highly inducible acetamidase of Mycobacterium smegmatis. Microbiology 139(3):575–583Google Scholar
  24. Makhongela H, Glowacka A, Agarkar V, Sewell B, Weber B, Cameron R, Cowan D, Burton S (2007) A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity. Appl Microbiol Biotechnol 75(4):801–811CrossRefPubMedGoogle Scholar
  25. Menzel K, Machrouhi F, Bodenstein M, Alorati A, Cowden C, Gibson AW, Bishop B, Ikemoto N, Nelson TD, Kress MH (2009) Process development of a potent bradykinin 1 antagonist. Org Process Res Dev 13(3):519–524CrossRefGoogle Scholar
  26. Nampoothiri KM, Roopesh K, Chacko S, Pandey A (2005) Comparative study of amidase production by free and immobilized Escherichia coli cells. Appl Biochem Biotechnol 120(2):97–108CrossRefGoogle Scholar
  27. Nawaz MS, Franklin W, Cerniglia CE (1993) Degradation of acrylamide by immobilized cells of a Pseudomonas sp and Xanthomonas maltophilia. Can J Microbiol 39(2):207–212CrossRefPubMedGoogle Scholar
  28. Nel AJM, Tuffin IM, Sewell BT, Cowan DA (2011) Unique aliphatic amidase from a psychrotrophic and haloalkaliphilic Nesterenkonia isolate. Appl Environ Microbiol 77(11):3696–3702CrossRefPubMedPubMedCentralGoogle Scholar
  29. Nguyen DD, Pandian R, Kim D, Ha SC, Yoon H-J, Kim KS, Yun KH, Kim J-H, Kim KK (2014) Structural and kinetic bases for the metal preference of the M18 aminopeptidase from Pseudomonas aeruginosa. Biochem Biophys Res Commun 447(1):101–107CrossRefPubMedGoogle Scholar
  30. Nie J, Guo H-C, Cahard D, Ma J-A (2010) Asymmetric construction of stereogenic carbon centers featuring a trifluoromethyl group from prochiral trifluoromethylated substrates. Chem Rev 111(2):455–529CrossRefPubMedGoogle Scholar
  31. Nielsen JW, Poulsen NR, Johnsson A, Winther JR, Stipp S, Willemoës M (2012) Metal-ion dependent catalytic properties of Sulfolobus solfataricus class ii α-mannosidase. Biochemistry 51(40):8039–8046CrossRefPubMedGoogle Scholar
  32. Nojiri M, Taoka N, Yasohara Y (2014) Characterization of an enantioselective amidase from Cupriavidus sp. KNK-J915 (FERM BP-10739) useful for enzymatic resolution of racemic 3-piperidinecarboxamide. J Mol Catal B Enzym 109:136–142CrossRefGoogle Scholar
  33. Ohnmacht CJ, Russell K, Empfield JR, Frank CA, Gibson KH, Mayhugh DR, McLaren FM, Shapiro HS, Brown FJ, Trainor DA (1996) N-aryl-3,3,3-trifluoro-2-hydroxy-2-methylpropanamides: KATP potassium channel openers. Modifications on the western region. J Med Chem 39(23):4592–4601CrossRefPubMedGoogle Scholar
  34. Parker BM, Taylor IN, Woodley JM, Ward JM, Dalby PA (2011) Directed evolution of a thermostable l-aminoacylase biocatalyst. J Biotechnol 155(4):396–405CrossRefPubMedGoogle Scholar
  35. Parker JS, Bower JF, Murray PM, Patel B, Talavera P (2008) Kepner-Tregoe decision analysis as a tool to aid route selection. Part 3. Application to a back-up series of compounds in the PDK project. Org Process Res Dev 12(6):1060–1077CrossRefGoogle Scholar
  36. Pei X, Wang Q, Li C, Yin X, Chen R, Xie T (2013) Addition of Co2+ to culture medium decides the functional expression of a recombinant nitrile hydratase in Escherichia coli. Biotechnol Lett 35(9):1419–1424CrossRefPubMedGoogle Scholar
  37. Qian M, Huang Q, Wu G, Lai L, Tang Y, Pei J, Kusunoki M (2012) Crystal structure analysis of a recombinant predicted acetamidase/formamidase from the thermophile Thermoanaerobacter tengcongensis. Protein J 31(2):166–174CrossRefPubMedGoogle Scholar
  38. Ruan LT, Zheng RC, Zheng YG (2016) A novel amidase from Brevibacterium epidermidis ZJB-07021: gene cloning, refolding and application in butyrylhydroxamic acid synthesis. J Ind Microbiol Biotechnol 43(8):1071–1083CrossRefPubMedGoogle Scholar
  39. Sessitsch A, Coenye T, Sturz A, Vandamme P, Barka EA, Salles J, Van Elsas J, Faure D, Reiter B, Glick B (2005) Burkholderia phytofirmans sp. nov., a novel plant-associated bacterium with plant-beneficial properties. Int J Syst Evol Microbiol 55(3):1187–1192CrossRefPubMedGoogle Scholar
  40. Sharma M, Sharma NN, Bhalla TC (2009) Amidases: versatile enzymes in nature. Rev Environ Sci Bio 8(4):343–366CrossRefGoogle Scholar
  41. Shaw NM, Naughton A, Robins K, Tinschert A, Schmid E, Hischier M-L, Venetz V, Werlen J, Zimmermann T, Brieden W (2002) Selection, purification, characterisation, and cloning of a novel heat-stable stereo-specific amidase from Klebsiella oxytoca, and its application in the synthesis of enantiomerically pure (R)-and (S)-3, 3, 3-trifluoro-2-hydroxy-2-methylpropionic acids and (S)-3, 3, 3-trifluoro-2-hydroxy-2-methylpropionamide. Org Process Res Dev 6(4):497–504CrossRefGoogle Scholar
  42. Shaw NM, Naughton AB (2004) The substrate specificity of the heat-stable stereospecific amidase from Klebsiella oxytoca. Tetrahedron 60(3):747–752CrossRefGoogle Scholar
  43. Shen W, Chen H, Jia K, Ni J, Yan X, Li S (2012) Cloning and characterization of a novel amidase from Paracoccus sp. M-1, showing aryl acylamidase and acyl transferase activities. Appl Microbiol Biotechnol 94(4):1007–1018CrossRefPubMedGoogle Scholar
  44. Skouloubris S, Labigne A, De Reuse H (2001) The AmiE aliphatic amidase and AmiF formamidase of Helicobacter pylori: natural evolution of two enzyme paralogues. Mol Microbiol 40(3):596–609CrossRefPubMedGoogle Scholar
  45. Sonke T, Ernste S, Tandler RF, Kaptein B, Peeters WP, van Assema FB, Wubbolts MG, Schoemaker HE (2005) L-selective amidase with extremely broad substrate specificity from Ochrobactrum anthropi NCIMB 40321. Appl Environ Microbiol 71(12):7961–7973CrossRefPubMedPubMedCentralGoogle Scholar
  46. Soriano-Maldonado P, Martínez-Gómez AI, Andújar-Sánchez M, Neira JL, Clemente-Jiménez JM, Las Heras-Vázquez FJ, Rodríguez-Vico F, Martínez-Rodríguez S (2011) Biochemical and mutational studies of the Bacillus cereus CECT 5050 T formamidase support the existence of a CEEK tetrad in several members of the nitrilase superfamily. Appl Environ Microbiol 77(16):5761–5769CrossRefPubMedPubMedCentralGoogle Scholar
  47. Wang YS, Cheng F, Zheng RC, Wang YJ, Zheng YG (2011) Characterization of an enantioselective amidase with potential application to asymmetric hydrolysis of (R, S)-2, 2-dimethylcyclopropane carboxamide. World J Microbiol Biotechnol 27(12):2885–2892CrossRefGoogle Scholar
  48. Wang YS, Zheng RC, Xu JM, Liu ZQ, Cheng F, Feng ZH, Liu LL, Zheng YG, Shen YC (2010) Enantioselective hydrolysis of (R)-2, 2-dimethylcyclopropane carboxamide by immobilized cells of an R-amidase-producing bacterium, Delftia tsuruhatensis CCTCC M 205114, on an alginate capsule carrier. J Ind Microbiol Biotechnol 37(5):503–510CrossRefPubMedGoogle Scholar
  49. Wu ZM, Zheng RC, Ding X, Jin JQ, Zheng YG (2016a) Enzymatic production of key intermediate of gabapentin by recombinant amidase from Pantoea sp. with high ratio of substrate to biocatalyst. Process Biochem 51(5):607–613CrossRefGoogle Scholar
  50. Wu ZM, Zheng RC, Zheng YG (2016b) Exploitation and characterization of three versatile amidase super family members from Delftia tsuruhatensis ZJB-05174. Enzym Microb Technol 86:93–102CrossRefGoogle Scholar
  51. Wyborn NR, Mills J, Williams SG, Jones CW (1996) Molecular characterisation of formamidase from Methylophilus methylotrophus. Eur J Biochem 240(2):314–322CrossRefPubMedGoogle Scholar
  52. Wyborn NR, Scherr DJ, Jones CW (1994) Purification, properties and heterologous expression of formamidase from Methylophilus methylotrophus. Microbiology 140(1):191–195CrossRefGoogle Scholar
  53. Yang ZY, Ni Y, Lu ZY, Liao XR, Zheng YG, Sun ZH (2011) Industrial production of S-2, 2-dimethylcyclopropanecarboxamide with a novel recombinant R-amidase from Delftia tsuruhatensis. Process Biochem 46(1):182–187CrossRefGoogle Scholar
  54. Zhang J, Yin J-G, Hang B-J, Cai S, He J, Zhou S-G, Li S-P (2012) Cloning of a novel arylamidase gene from Paracoccus sp. strain FLN-7 that hydrolyzes amide pesticides. Appl Environ Microbiol 78(14):4848–4855CrossRefPubMedPubMedCentralGoogle Scholar
  55. Zheng RC, Zheng YG, Shen YC (2007) A simple method to determine concentration of enantiomers in enzyme-catalyzed kinetic resolution. Biotechnol Lett 29(7):1087–1091CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Zhe-Ming Wu
    • 1
    • 2
  • Ren-Chao Zheng
    • 1
    • 2
  • Xiao-Ling Tang
    • 1
    • 2
  • Yu-Guo Zheng
    • 1
    • 2
    Email author
  1. 1.Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and BioengineeringZhejiang University of TechnologyHangzhouPeople’s Republic of China
  2. 2.Engineering Research Center of Bioconversion and Biopurification of Ministry of EducationZhejiang University of TechnologyHangzhouPeople’s Republic of China

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