Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Appl Microbiol Biotechnol. 2014 Sep;98(17):7491-9. doi: 10.1007/s00253-014-5710-y. Epub 2014 Apr 11.

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

Author information

  • 1Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, Nanjing Agriculture University, Nanjing, 210095, People's Republic of China.

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 K m and k cat 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 Mn(2+) and inhibited by Cu(2+), Zn(2+), Ni(2+), and Fe(2+). DamH was inhibited strongly by phenylmethylsulfonyl and SDS and weakly by ethylenediaminetetraacetic acid and dimethyl sulfoxide.

PMID:
24723294
[PubMed - in process]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Springer
    Loading ...
    Write to the Help Desk