Construction of Acinetobacter baylyi Producing the Organic Solvent-tolerant Lipase with a Broad Spectrum of Substrate Specificity

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.
Mutagenesis with UV irradiation was carried out in a UV chamber (40W) for 30 min by keeping the distance of the UV source fixed to 55 cm. Lipase secreted from the mutant of A. baylyi lose the thermophilic characteristic but still tolerate to many organic solvents especially hydrophobic solvents. Other characteristics are comparable with those from wildtype except the specificity of long-chain acyl substrate. The mutant lipase has optimum temperature at 20 ºC and highly stables at the temperature between 5 and 15 ºC suggesting cold active lipase, recent trend lipase for industrial applications. Amino acid substitution between D184 of wildtype and Q184 of mutant lipase is remarkable. This probably enhances losing of H-bond from the side chain of D184 to N54 and K55 backbone in β-sheet causing less stability of mutant active site. This is the reason why mutant lipase loses thermophilic characteristic. A hybrid bacterium, Geobacillus sp. was also constructed by genome shuffling between A. baylyi and thermophiles. The hybrid bacterium could produce lipase with higher specific to short-chain substrates and improved thermostability.

แหล่งข้อมูล:

• โครงการวิจัยชื่อ “Construction of Acinetobacter baylyi Producing The Organic Solvent-tolerant Lipase with a Broad Spectrum of Substrate Specificity” – ระยะเวลาโครงการ: August 2011 – July 2013