A new carboxylesterase from Brevibacterium linens IFO 12171 responsible for the conversion of 1,4-butanediol diacrylate to 4-hydroxybutyl acrylate: purification, characterization, gene cloning, and gene expression in Escherichia coli.
A carboxylesterase that is responsible for conversion of 1,4-butanediol diacrylate (BDA) to 4-hydroxybutyl acrylate (4HBA) was found in Brevibacterium lines IFO 12171, and purified to homogeneity. The purified enzyme was active toward a variety of diesters of ethylene glycol, 1,4-butanediol, and 1,6-hexanediol. The K(m) and kcat of the enzyme for BDA were 3.04 mM and 203,000 s-1, respectively. The reaction with the purified enzyme gave 98 mM 4HBA from 100 mM BDA for 60 min. The enzyme gene was cloned from the chromosomal DNA of the bacterium. The open reading frame encoding the enzyme was 1176 bp long, corresponding to a protein of 393 amino acid residues (molecular mass = 42,569 Da). The deduced amino acid sequence contained the tetra peptide motif sequence, STTK, and the serine residue was confirmed to be the catalytic center of BDA esterase by site-directed mutagenesis for several amino acid residues. The gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product (a fusion protein with 6 amino acid residues from beta-galactosidase) showed the same catalytic properties as the enzyme from the parent strain.
Sakai
Y
Y
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
Ishikawa
J
J
Fukasaka
S
S
Yurimoto
H
H
Mitsui
R
R
Yanase
H
H
Kato
N
N
D000179:Acrylates / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001951:Brevibacterium / Q000201:enzymology*; D002072:Butylene Glycols / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity
D000179:Acrylates; D002072:Butylene Glycols; C022288:butanediol diacrylate; C120172:4-hydroxybutyl acrylate; D002265:Carboxylic Ester Hydrolases