[Cloning, sequencing and high expression in Escherichia coli of D-hydantoinase gene from Burkholderia pickettii]

Sheng Wu Gong Cheng Xue Bao. 2002 Jan;18(2):149-54.
[Article in Chinese]

Abstract

A strain, MMR003, used for D-p-HPG production in industry was classified as Burkholderia pickettii by morphological observation and biochemical characterization. The gene encoding the D-hydantoinase enzyme was cloned, sequenced and expressed in Escherichia coli. The nucleotide sequence of the 5.0 kb insert of subclone pXZ-total was determined. One open reading frame of 1374 bp was found and predicted to encode a polypeptide consisting of 458 amino acids in size of 50 kD. The amino acid sequence alignment of D-hydantoinase from Burkholderia pickettii shows the 85% homologous with the corresponding enzyme from Agrobacterium radiobacter NRRL B11291. The D-hydantoinase gene (dha) harboured in the plasmid pXZPH2 in E. coli BL21(DE3) was highly expressed by IPTG induction. The D-hydantoinase activity for D, L-p-hydroxyphenylhydantion is 0.66 u/mL broth, which is 2-fold increase compared to the parent strain Burkholderia pickettii.

MeSH terms

  • Amidohydrolases / genetics*
  • Amidohydrolases / metabolism
  • Amino Acid Sequence
  • Animals
  • Burkholderia / classification
  • Burkholderia / enzymology*
  • Burkholderia / genetics
  • Cloning, Molecular
  • DNA Probes
  • Escherichia coli
  • Gene Expression*
  • Molecular Sequence Data
  • Recombination, Genetic
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid

Substances

  • DNA Probes
  • Amidohydrolases
  • dihydropyrimidinase