Applied Microbiology and Biotechnology

, Volume 99, Issue 7, pp 3069–3080 | Cite as

Characterization of a recombinant (+)-γ-lactamase from Microbacterium hydrocarbonoxydans which provides evidence that two enantiocomplementary γ-lactamases are in the strain

  • Jianjun Wang
  • Yaxin Zhu
  • Guogang Zhao
  • Junge Zhu
  • Sheng Wu
Biotechnologically relevant enzymes and proteins


A two-step method, i.e., the transfer acyl analysis and then the chiral HPLC analysis, was employed in the screening of the cosmid library of Microbacterium hydrocarbonoxydans genome. Two enantiocomplementary γ-lactamase clones were found. A 40-kb cosmid showed (−)-γ-lactamase activity, and the activity was from Mhg which was reported previously according to the results of PCR identifying experiment. The 37-kb (+)-γ-lactamase cosmid was further constructed into a pUC18 plasmid library and screened by the same two-step method. A plasmid clone harboring a 1.6-kb fragment showed (+)-γ-lactamase activity. A 555-bp ORF in the 1.6-kb fragment showed high (+)-γ-lactamase activity when it was expressed under the control of T7 promoter. The coding protein showed significant homology with bacterial isochorismatase. The (+)-γ-lactamase was characterized and compared with the (−)-γ-lactamase Mhg. This is another report that two enantiocomplementary γ-lactamases are present in the same strain.


Cosmid library Enantiocomplementary γ-Lactamase Isochorismatase 


  1. Allan R, Twitchin B (1980) Synthesis of analogues of GABA. IV. Three unsaturated derivatives of 3-aminocyclopentane-1-carboxylic acid. Aust J Chem 33(3):599–604. doi:10.1071/CH9800599 CrossRefGoogle Scholar
  2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410. doi:10.1006/jmbi.1990.9999S0022-2836(05)80360-2 CrossRefPubMedGoogle Scholar
  3. Bianchi DA, Moran-Ramallal R, Iqbal N, Rudroff F, Mihovilovic MD (2013) Enantiocomplementary access to carba-analogs of C-nucleoside derivatives by recombinant Baeyer-Villiger monooxygenases. Bioorg Med Chem Lett 23(9):2718–2720. doi:10.1016/j.bmcl.2013.02.085 CrossRefPubMedGoogle Scholar
  4. Brabban AD, Littlechild J, Wisdom R (1996) Stereospecific gamma-lactamase activity in a Pseudomonas fluorescens species. J Ind Microbiol 16(1):8–14. doi:10.1007/Bf01569915 CrossRefGoogle Scholar
  5. Caruthers J, Zucker F, Worthey E, Myler PJ, Buckner F, Van Voorhuis W, Mehlin C, Boni E, Feist T, Luft J, Gulde S, Lauricella A, Kaluzhniy O, Anderson L, Le Trong I, Holmes MA, Earnest T, Soltis M, Hodgson KO, Hol WG, Merritt EA (2005) Crystal structures and proposed structural/functional classification of three protozoan proteins from the isochorismatase superfamily. Protein Sci 14(11):2887–2894. doi:10.1110/ps.051783005 CrossRefPubMedCentralPubMedGoogle Scholar
  6. Cilia E, Fabbri A, Uriani M, Scialdone GG, Ammendola S (2005) The signature amidase from Sulfolobus solfataricus belongs to the CX3C subgroup of enzymes cleaving both amides and nitriles. Ser195 and Cys145 are predicted to be the active site nucleophiles. FEBS J 272(18):4716–4724. doi:10.1111/j.1742-4658.2005.04887.x CrossRefPubMedGoogle Scholar
  7. Fournand D, Pirat J-L, Bigey F, Arnaud A, Galzy P (1997) Spectrophotometric assay of aliphatic monohydroxamic acids and α-, β-, and γ-aminohydroxamic acids in aqueous medium. Anal Chim Acta 353(2–3):359–366. doi:10.1016/S0003-2670(97)87798-7 CrossRefGoogle Scholar
  8. Gonsalvez IS, Isupov MN, Littlechild JA (2001) Crystallization and preliminary X-ray analysis of a gamma-lactamase. Acta Crystallogr D Biol Crystallogr 57(Pt 2):284–286CrossRefPubMedGoogle Scholar
  9. Goral AM, Tkaczuk KL, Chruszcz M, Kagan O, Savchenko A, Minor W (2012) Crystal structure of a putative isochorismatase hydrolase from Oleispira antarctica. J Struct Funct Genom 13(1):27–36. doi:10.1007/s10969-012-9127-5 CrossRefGoogle Scholar
  10. Gubareva LV, Webster RG, Hayden FG (2001) Comparison of the activities of zanamivir, oseltamivir, and RWJ-270201 against clinical isolates of influenza virus and neuraminidase inhibitor-resistant variants. Antimicrob Agents Chemother 45(12):3403–3408. doi:10.1128/AAC.45.12.3403-3408.2001 CrossRefPubMedCentralPubMedGoogle Scholar
  11. Guo N, Zheng J, Tian J, Wu L, Zhou H (2013) Characterization and constitutive expression of an acidic mesophilic endo-1,4-beta-D-xylanohydrolase with high thermotolerance and catalytic efficiency in Pichia pastoris. World J Microbiol Biotechnol 29(11):2095–2103. doi:10.1007/s11274-013-1374-5 CrossRefPubMedGoogle Scholar
  12. Guterl JK, Andexer JN, Sehl T, von Langermann J, Frindi-Wosch I, Rosenkranz T, Fitter J, Gruber K, Kragl U, Eggert T, Pohl M (2009) Uneven twins: comparison of two enantiocomplementary hydroxynitrile lyases with alpha/beta-hydrolase fold. J Biotechnol 141(3–4):166–173. doi:10.1016/j.jbiotec.2009.03.010 CrossRefPubMedGoogle Scholar
  13. Hasegawa PA, Hasegawa P (2007) Pharmaceutical composition for treating, e.g. hypertension, includes anti-hypertensive agent from angiotensin II receptor antagonist or angiotensin converting enzyme inhibitor, and dipeptidyl peptidase-4 inhibitor. WO2007050485-A2Google Scholar
  14. Hogrefe HH, Cline J, Youngblood GL, Allen RM (2002) Creating randomized amino acid libraries with the QuikChange multi site-directed mutagenesis kit. Biotechniques 33(5):1158–1160, 1162, 1164–5PubMedGoogle Scholar
  15. Jagt JC, Van Leusen AM (1974) Diels-alder cycloadditions of sulfonyl cyanides with cyclopentadiene. Synthesis of 2-azabicyclo[2.2.1]hepta-2,5-dienes. J Org Chem 39(4):564–566. doi:10.1021/jo00918a033 CrossRefGoogle Scholar
  16. King CH, Meckler H, Herr RJ, Trova MP, Glick SD, Maisonneuve IM (2000) Synthesis of enantiomerically pure (+)- and (−)-18-methoxycoronaridine hydrochloride and their preliminary assessment as anti-addictive agents. Bioorg Med Chem Lett 10(5):473–476CrossRefPubMedGoogle Scholar
  17. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685CrossRefPubMedGoogle Scholar
  18. Li HQ, Su L, Yang L, Wang JJ, Zheng GJ (2006) Study on the screening of lactamase and its fermentation conditions. Wei Sheng Wu Xue Bao 46(4):571–575PubMedGoogle Scholar
  19. Li D, Huang F, Xia M, Jiang Y, Yang Y (2013) Molecular cloning and expression of a novel mesophilic alkaline protease from Bacillus sp. L010 in Escherichia coli. Wei Sheng Wu Xue Bao 53(11):1240–1250PubMedGoogle Scholar
  20. Line K, Isupov MN, Littlechild JA (2004) The crystal structure of a (−) gamma-lactamase from an Aureobacterium species reveals a tetrahedral intermediate in the active site. J Mol Biol 338(3):519–532. doi:10.1016/j.jmb.2004.03.001S0022283604002633 CrossRefPubMedGoogle Scholar
  21. Maruyama C, Hamano Y (2009) The biological function of the bacterial isochorismatase-like hydrolase SttH. Biosci Biotechnol Biochem 73(11):2494–2500CrossRefPubMedGoogle Scholar
  22. Merlani M, Barbakadze V, Amiranashvili L, Gogilashvili L, Yannakopoulou E, Papadopoulos K, Chankvetadze B (2010) Enantioselective synthesis and antioxidant activity of 3-(3,4-dihydroxyphenyl)-glyceric acid–basic monomeric moiety of a biologically active polyether from Symphytum asperum and S. caucasicum. Chirality 22(8):717–725. doi:10.1002/chir.20823 PubMedGoogle Scholar
  23. Meyer H (1957) The ninhydrin reaction and its analytical applications. Biochem J 67(2):333–340PubMedCentralPubMedGoogle Scholar
  24. Morohoshi T, Wang WZ, Someya N, Ikeda T (2011) Genome sequence of Microbacterium testaceum StLB037, an N-Acylhomoserine lactone-degrading bacterium isolated from potato leaves. J Bacteriol 193(8):2072–2073. doi:10.1128/Jb.00180-11 CrossRefPubMedCentralPubMedGoogle Scholar
  25. Muftic MK (1964) A new phenol-hypochlorite reaction for ammonia. Nature 201:622–623CrossRefPubMedGoogle Scholar
  26. Mugford PF, Wagner UG, Jiang Y, Faber K, Kazlauskas RJ (2008) Enantiocomplementary enzymes: classification, molecular basis for their enantiopreference, and prospects for mirror-image biotransformations. Angew Chem Int Ed Engl 47(46):8782–8793. doi:10.1002/anie.200705159 CrossRefPubMedGoogle Scholar
  27. Murzin AG, Brenner SE, Hubbard T, Chothia C (1995) SCOP: a structural classification of proteins database for the investigation of sequences and structures. J Mol Biol 247(4):536–540. doi:10.1006/jmbi.1995.0159S0022283685701593 PubMedGoogle Scholar
  28. Nastopoulos V, Vallone B, Politi L, Scotto D’Abusco A, Scandurra R, Tsernoglou D (2001) Crystallization and X-ray diffraction measurements of a thermophilic archaeal recombinant amidase from Sulfolobus solfataricus MT4. Acta Crystallogr D Biol Crystallogr 57(Pt 7):1036–1037CrossRefPubMedGoogle Scholar
  29. Ni Y, Song L, Qian X, Sun Z (2013) Proteomic analysis of Pseudomonas putida reveals an organic solvent tolerance-related gene mmsB. PLoS One 8(2):e55858. doi:10.1371/journal.pone.0055858 CrossRefPubMedCentralPubMedGoogle Scholar
  30. Parsons JF, Calabrese K, Eisenstein E, Ladner JE (2003) Structure and mechanism of Pseudomonas aeruginosa PhzD, an isochorismatase from the phenazine biosynthetic pathway. Biochemistry 42(19):5684–5693. doi:10.1021/bi027385d CrossRefPubMedGoogle Scholar
  31. Qin X, Wang J, Zheng G (2010) Enantioselective resolution of gamma-lactam by a whole cell of Microbacterium hydrocarbonoxydans (L29-9) immobilized in polymer of PVA-alginate-boric acid. Appl Biochem Biotechnol 162(8):2345–2354. doi:10.1007/s12010-010-9007-z CrossRefPubMedGoogle Scholar
  32. Reese MG (2001) Application of a time-delay neural network to promoter annotation in the Drosophila melanogaster genome. Comput Chem 26(1):51–56CrossRefPubMedGoogle Scholar
  33. Romao MJ, Turk D, Gomis-Ruth FX, Huber R, Schumacher G, Mollering H, Russmann L (1992) Crystal structure analysis, refinement and enzymatic reaction mechanism of N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. at 2.0 A resolution. J Mol Biol 226(4):1111–1130CrossRefPubMedGoogle Scholar
  34. Rusnak F, Liu J, Quinn N, Berchtold GA, Walsh CT (1990) Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization, and substrate specificity of isochorismatase. Biochemistry 29(6):1425–1435CrossRefPubMedGoogle Scholar
  35. Simon RC, Zepeck F, Kroutil W (2013) Chemoenzymatic synthesis of all four diastereomers of 2,6-disubstituted piperidines through stereoselective monoamination of 1,5-diketones. Chemistry 19(8):2859–2865. doi:10.1002/chem.201202793 CrossRefPubMedGoogle Scholar
  36. Singh R, Vince R (2012) 2-Azabicyclo[2.2.1]hept-5-en-3-one: chemical profile of a versatile synthetic building block and its impact on the development of therapeutics. Chem Rev 112(8):4642–4686. doi:10.1021/cr2004822 CrossRefPubMedGoogle Scholar
  37. Suzuki T, Usui T, Oka M, Kataoka T (1998) Synthesis and muscarinic activity of a series of quinolines and naphthalenes with a 1-azabicyclo[3.3.0]octane moiety. Chem Pharm Bull (Tokyo) 46(8):1265–1273CrossRefGoogle Scholar
  38. Tanaka K, Kato M, Toda F (2001) Optical resolution of 2-azabicyclo[2.2.1]hept-5-en-3-one by inclusion complexation with brucine. Heterocycles 54(1):405–410CrossRefGoogle Scholar
  39. Taylor JD (2010) COPD and the response of the lung to tobacco smoke exposure. Pulm Pharmacol Ther 23(5):376–383. doi:10.1016/j.pupt.2010.04.003 CrossRefPubMedGoogle Scholar
  40. Taylor SJC, Sutherland AG, Lee C, Wisdom R, Thomas S, Roberts SM, Evans C (1990) Chemoenzymatic synthesis of (−)-carbovir utilizing a whole cell catalysed resolution of 2-azabicyclo[2.2.1]hept-5-en-3-one. J Chem Soc Chem Commun 0(16):1120–1121CrossRefGoogle Scholar
  41. Taylor SJC, Mccague R, Wisdom R, Lee C, Dickson K, Ruecroft G, Obrien F, Littlechild J, Bevan J, Roberts SM, Evans CT (1993) Development of the biocatalytic resolution of 2-azabicyclo[2.2.1]hept-5-en-3-one as an entry to single-enantiomer carbocyclic nucleosides. Tetrahedron-Asymmetry 4(6):1117–1128. doi:10.1016/S0957-4166(00)80218-9 CrossRefGoogle Scholar
  42. Taylor SJC, Brown RC, Keene PA, Taylor IN (1999) Novel screening methods—the key to cloning commercially successful biocatalysts. Bioorg Med Chem 7(10):2163–2168. doi:10.1016/S0968-0896(99)00146-7 CrossRefPubMedGoogle Scholar
  43. Toogood HS, Brown RC, Line K, Keene PA, Taylor SJC, McCague R, Littlechild JA (2004) The use of a thermostable signature amidase in the resolution of the bicyclic synthon (rac)-gamma-lactam. Tetrahedron 60(3):711–716. doi:10.1016/j.tet.2003.11.064 CrossRefGoogle Scholar
  44. Torres LL, Schlie, Schmidt M, Silva-Martin N, Hermoso JA, Berenguer J, Bornscheuer UT, Hidalgo A (2012) Promiscuous enantioselective (−)-[gamma]-lactamase activity in the Pseudomonas fluorescens esterase I. Org Biomol Chem 10(17):3388–3392CrossRefPubMedGoogle Scholar
  45. van Leeuwen JG, Wijma HJ, Floor RJ, van der Laan JM, Janssen DB (2012) Directed evolution strategies for enantiocomplementary haloalkane dehalogenases: from chemical waste to enantiopure building blocks. Chembiochem 13(1):137–148. doi:10.1002/cbic.201100579 CrossRefPubMedGoogle Scholar
  46. Wang JJ, Guo XY, Zheng GJ, Wen C (2009) Purification and characterization of a novel (−) gamma-lactamase from Microbacterium hydrocarbonoxydans. Ann Microbiol 59(2):345–348CrossRefGoogle Scholar
  47. Wang JJ, Zhang X, Min C, Wu S, Zheng GJ (2011) Single-step purification and immobilization of gamma-lactamase and on-column transformation of 2-azabicyclo [2.2.1] hept-5-en-3-one. Process Biochem 46(1):81–87. doi:10.1016/j.procbio.2010.07.018 CrossRefGoogle Scholar
  48. Wang J, Zhao G, Zhang Z, Liang Q, Min C, Wu S (2014) Autodisplay of an archaeal gamma-lactamase on the cell surface of Escherichia coli using Xcc_Est as an anchoring scaffold and its application for preparation of the enantiopure antiviral drug intermediate (−) vince lactam. Appl Microbiol Biotechnol 98(16):6991–7001. doi:10.1007/s00253-014-5704-9 CrossRefPubMedGoogle Scholar
  49. Wen YD, Remmel RP, Pham PT, Vince R, Zimmerman CL (1995) Comparative brain exposure to (−)-carbovir after (−)-carbovir or (−)-6-aminocarbovir intravenous infusion in rats. Pharm Res 12(6):911–915CrossRefPubMedGoogle Scholar
  50. Wisniewski T, Bayne E, Flanagan J, Shao Q, Wnek R, Matheravidathu S, Fischer P, Forrest MJ, Peterson L, Song X, Yang L, Demartino JA, Struthers M (2010) Assessment of chemokine receptor function on monocytes in whole blood: in vitro and ex vivo evaluations of a CCR2 antagonist. J Immunol Methods 352(1–2):101–110. doi:10.1016/j.jim.2009.10.010 CrossRefPubMedGoogle Scholar
  51. Wu Q, Soni P, Reetz MT (2013) Laboratory evolution of enantiocomplementary Candida antarctica lipase B mutants with broad substrate scope. J Am Chem Soc 135(5):1872–1881. doi:10.1021/ja310455t CrossRefPubMedGoogle Scholar
  52. Yang M, Gao Q, Wu S, Wang J, Zheng G (2012) Characterization of a recombinant (−)gamma-lactamase from Microbacterium hydrocarbonoxydans. Biotechnol Lett 34(2):275–279. doi:10.1007/s10529-011-0758-6 CrossRefPubMedGoogle Scholar
  53. Zajc A, Romao MJ, Turk B, Huber R (1996) Crystallographic and fluorescence studies of ligand binding to N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. J Mol Biol 263(2):269–283CrossRefPubMedGoogle Scholar
  54. Zheng RC, Zheng YG, Shen YC (2007) A screening system for active and enantioselective amidase based on its acyl transfer activity. Appl Microbiol Biotechnol 74(1):256–262. doi:10.1007/s00253-006-0642-9 CrossRefPubMedGoogle Scholar
  55. Zhu S, Gong C, Song D, Gao S, Zheng G (2012) Discovery of a novel (+)-gamma-lactamase from Bradyrhizobium japonicum USDA 6 by rational genome mining. Appl Environ Microbiol 78(20):7492–7495. doi:10.1128/AEM.01398-12 CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Jianjun Wang
    • 1
  • Yaxin Zhu
    • 1
  • Guogang Zhao
    • 2
  • Junge Zhu
    • 1
  • Sheng Wu
    • 1
  1. 1.State Key Laboratory of Microbial Resources, Institute of MicrobiologyChinese Academy of SciencesBeijingPeople’s Republic of China
  2. 2.Department of Internal Medicine, College of MedicineUniversity of KentuckyLexingtonUSA

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