Abstract
A (−) gamma-lactamase fromMicrobacterium hydrocarbonoxydans was purified to homogeneity by chromatography methods. SDS-PAGE showed the molecular weight of the enzyme was about 31 kDa. The purified enzyme had a specific activity of 61.3±2.5 U mg−1 for 2-azabicyclo [2.2.1] hept-5-en-3-one [(−) gamma-lactam]. The enantioselectivity factor (E) of the purified enzyme was 9.5±0.8 for unreacted (+) gamma-lactam. TheK m andV max value were 2.3±0.2 mM and 80.0±15.4 U mg−1 respectively. The highest activity was found at 30 °C and pH 8.0. ESIMS mass spectrometry analysis results and N-terminal sequence indicated the (−) gamma-lactamase might be a new enzyme.
References
Brabban A.D., Littlechild J., Wisdom R. (1996). Stereospecific gamma-lactamase activity in aPseudomonas fluorescens species. J. Ind. Microbiol., 16: 8–14.
Bradford M.M. (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.
Chen C.S., Wu S.H., Girdaukas G.S., Sih C.J. (1982). Quantitative analyses of biochemical kinetic resolution of enantiomers. J. Am. Chem. Soc., 104: 7294–7299.
Daluge S., Vince R. (1978). Synthesis of carbocyclic aminonucleosides. J. Org. Chem., 43: 2311–2320.
Fessner W.D., Jones J.B. (2001). Biocatalysis and biotransformation: from discovery to application. Curr. Opin. Chem. Biol., 5: 103–105.
Hauer B., Roberts S.M. (2004). Biocatalysis and biotransformation: probing the potential usefulness and the mechanisms of action of some novel biocatalysts. Curr. Opin. Chem. Biol., 8: 103–105.
Laemmli U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680–685.
Li H.Q., Su L., Yang L., Wang J.J., Zheng G.J. (2006). Study on the screening of lactamase and its fermentation conditions. Acta Microbiol. Sin., 46: 571–575.
Line K., Isupov M.N., Littlechild J.A. (2004). The Crystal Structure of a (−) gamma-lactamase from anAureobacterium species reveals a tetrahedral intermediate in the active site. J. Mol. Biol., 338: 519–532.
Mahmoudian M., Lowolon A., Jones M. (1999). A practical enzymatic procedure for resolution of N-substituted 2-azabicyclo [2.2.1] hept-5-en-3-ones. Tetrahedron-Asymmetr., 10: 1201–1206.
Nakano H., Iwasa K., Okayama Y. (1996). Lipase-catalyzed resolution of 2-azabicyclo [2.2.1] hept-5-en-3-ones. Tetrahedron-Asymmetr., 7: 2381–2386.
Schippers A., Bosecker K., Spröer C., Schumann P. (2005).Microbacterium oleivorans sp. nov. andMicrobacterium Hydrocarbonoxydans sp. nov., novel crude-oil-degrading Gram-positive bacteria. Int. J. Syst. Evol. Microbiol., 55: 655–660.
Schulze B., Wubbolts M.G. (1999). Biocatalysis for industrial production of fine chemicals. Curr. Opin. Biotechnol., 10: 609–615.
Shevchenko A., Wilm M., Vorm O., Mann M. (1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem., 68: 850–858.
Toogood H.S., Brown R.C., Line K., Keene P.A., Taylor S., McCague R., Littlechild J.A. (2004). The use of a thermostable signature amidase in the resolution of the bicyclic synthon (rac)-gamma-lactam. Tetrahedron, 60: 711–716.
Velazquez F., Olivo H.F. (2002). The application of chiral oxazolidinethiones and thiazolidine-thioned in asymmetric synthesis. Curr. Org. Chem., 6: 303–340.
Zheng G.J., Zhang G.J., Wang J.J. (2007).Microbacterium Hydrocarbonoxydans and its usage in the production of chiral intermediates. PATENT CN 200710118064,1 June 28.
Author information
Authors and Affiliations
Corresponding author
Additional information
These authors contributed equally to this work.
Rights and permissions
About this article
Cite this article
Wang, J., Guo, X., Zheng, G. et al. Purification and characterization of a novel (−) gamma-lactamase fromMicrobacterium hydrocarbonoxydans . Ann. Microbiol. 59, 345–348 (2009). https://doi.org/10.1007/BF03178337
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF03178337