ABSTRACT
The nucleotide sequence and N-, C-terminal amino acid sequences of alpha,beta-subunit of glutaryl 7-ACA acylase C130 from Pseudomonas sp. 130 were determined. The alignment of the acylase C130 with the other acylases shows that it has high homology with the acylases from Pseudomonas sp. GK16 and C427, but low homology with the others. There is large difference in the N-terminal of alpha-subunit, while the N-terminal of beta-subunit has significant conservation.
Subject(s)
Amino Acid Sequence , Base Sequence , DNA, Bacterial , Genes, Bacterial , Molecular Sequence Data , Penicillin Amidase , Genetics , Pseudomonas , Genetics , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
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.