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 dia crylate (BDA) to 4-hydroxybutyl acrylate (4HBA) was found in Brevibacterium lines IFO 12171, and purified to homogeneity. The purified enzyme was acti ve toward a variety of diesters of ethylene glycol, 1,4-butanediol, and 1,6 -hexanediol. The K-m and k(cat) of the enzyme for BDA(.) were 3.04 mM and 2 03,000 s(-1), respectively. The reaction with the purified enzyme gave 98 m M 4HBA from 100 mM BDA for 60 min. The enzyme gene was cloned from the chromosomal DNA of the bacterium. The o pen 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 t he 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 th e gene product (a fusion protein with 6 amino acid residues from beta-galac tosidase) showed the same catalytic properties as the enzyme from the paren t strain.