Applied Microbiology and Biotechnology

, Volume 98, Issue 17, pp 7491–7499

Purification of an amide hydrolase DamH from Delftia sp. T3-6 and its gene cloning, expression, and biochemical characterization

  • Fei Wang
  • Ying Hou
  • Jie Zhou
  • Zhoukun Li
  • Yan Huang
  • Zhongli Cui
Biotechnologically relevant enzymes and proteins

DOI: 10.1007/s00253-014-5710-y

Cite this article as:
Wang, F., Hou, Y., Zhou, J. et al. Appl Microbiol Biotechnol (2014) 98: 7491. doi:10.1007/s00253-014-5710-y

Abstract

A highly active amide hydrolase (DamH) was purified from Delftia sp. T3-6 using ammonium sulfate precipitation, diethylaminoethyl anion exchange, hydrophobic interaction chromatography, and Sephadex G-200 gel filtration. The molecular mass of the purified enzyme was estimated to be 32 kDa by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. The sequence of the N-terminal 15 amino acid residues was determined to be Gly-Thr-Ser-Pro-Gln-Ser-Asp-Phe-Leu-Arg-Ala-Leu-Phe-Gln-Ser. Based on the N-terminal sequence and results of peptide mass fingerprints, the gene (damH) was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). DamH was a bifunctional hydrolase showing activity to amide and ester bonds. The specific activities of recombinant DamH were 5,036 U/mg for 2′-methyl-6′-ethyl-2- chloroacetanilide (CMEPA) (amide hydrolase function) and 612 U/mg for 4-nitrophenyl acetate (esterase function). The optimum substrate of DamH was CMEPA, with Km and kcat values of 0.197 mM and 2,804.32 s−1, respectively. DamH could also hydrolyze esters such as 4-nitrophenyl acetate, glycerol tributyrate, and caprolactone. The optimal pH and temperature for recombinant DamH were 6.5 and 35 °C, respectively; the enzyme was activated by Mn2+ and inhibited by Cu2+, Zn2+, Ni2+, and Fe2+. DamH was inhibited strongly by phenylmethylsulfonyl and SDS and weakly by ethylenediaminetetraacetic acid and dimethyl sulfoxide.

Keywords

Delftia sp. T3-6Amide hydrolasePurifiedCloningCharacterization

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Fei Wang
    • 1
    • 2
  • Ying Hou
    • 3
  • Jie Zhou
    • 1
  • Zhoukun Li
    • 1
  • Yan Huang
    • 1
  • Zhongli Cui
    • 1
  1. 1.Key Laboratory of Agricultural Environmental Microbiology, Ministry of AgricultureNanjing Agriculture UniversityNanjingPeople’s Republic of China
  2. 2.College of Bioscience and BioengineeringJiangxi Agriculture UniversityNanchangPeople’s Republic of China
  3. 3.College of Food and BioengineeringHenan University of Science and TechnologyLuoyangPeople’s Republic of China