Lipases are commonly applied enzymes in biocatalysis and organic chemistry. They catalyze numerous reactions, recognize many substrates and exhibit high regioselectivity and enantiospecificity, characteristics that contribute to their wide application. The temperature and solvent stability as well as broad substrate specificity of lipases have regarded as the most important characteristics for use in industry. In this study, we tried to improve such characteristics of lipase from Acinetobacter baylyi by random mutation.
Lipase gene of A. baylyi was isolated directly from the genomic DNA with the PCR-cloning method. The gene has an ORF of 972 bp encoding a protein of 323 amino acid residues. The three-dimensional structure of A. baylyi lipase was predicted based on homology modeling. The enzyme was a α/β globular protein comprising a central six-stranded β sheets connected by six α helices and showed 98% identity with Pseudomonas aeruginosa lipase. Analysis of the lipase gene sequence suggesting the involvement of the GHSHGS region as the active site and the catalytic triad (S120, D249 and H289) has been found at perfectly conserved positions. The gene sequence contained two potential calcium binding site (D249 and D291) and two Cys residues (-C223~C273-) involving in disulfide bridge formation. A short hydrophobic region (PIVLAHG) upstream of an H-G conserved dipeptide of the oxyanion loop of Acinetobacter lipases was also detected. Two amino acids (PM) near C223 are noted as the potential residues involving either the characteristic of solvent stability or thermostability.
A cognate lipase specific foldase chaperone has been found upstream of the lipase gene.