Molecular characterization of transesterification activity of novel lipase family I.1.

Lipase's thermostability and organic solvent tolerance are two crucial properties that enable it to function as a biocatalyst. The present study examined the characteristics of two recombinant thermostable lipases (Lk2, Lk3) based on transesterification activity. Conversion of C12-C18 methyl ester with paranitrophenol was investigated in various organic solvent. Both lipases exhibited activity on difference carbon chain length (C12 - C18, C18:1, C18:2) of substrates. The activity of Lk2 was higher in each of substrate compared with that of Lk3. Experimental findings showed that the best substrates for Lk2 and Lk3 are C18:1 and C18:2 respectively, in agreement with the computational analysis. The activity of both enzymes prefers on nonpolar solvent. On nonpolar solvent the enzymes are able to keep its native folding shown by the value of radius gyration, solvent-enzyme interaction and orientation of triad catalytic residues. Lk3 appeared to be more thermostable, with maximum activity at 55°C. The presence of Fe3+ increased the activity of Lk2 and Lk3. However, the activity of both enzymes were dramatically decreased by the present of Ca2+ despite of the enzymes belong to family I.1 lipase known as calcium dependent enzyme. Molecular analysis on His loop of Lk2 and Lk3 on the present of Ca2+ showed that there were shifting on the orientation of catalytic triad residues. All the data suggest that Lk2 and Lk3 are novel lipase on the family I.1 and both lipase available as a biocatalyst candidate.

Effect of mutation at oxyanion hole residu (H110F) on activity of Lk4 lipase.

Mutant of lipase at oxyanion hole (H110 F) was constructed. The gene was highly expressed in Eschericia coli BL21 (DE3) and the recombinant protein was purified using Ni-NTA affinity chromatography. The activity of mutant enzyme was significantly increased compared to that the wild type. Further comparison showed that both of the enzymes exhibited same optimum pH and temperature, and showed highest lipolytic activity on pNP-decanoate (C10). The wild type appeared lost of activity on C14 and C16 substrates meanwhile the mutant still showed activity up to 20 %. In the presence of non polar organic solvent such as n-hexane, the wild type became inactive enzyme meanwhile the mutant still remained 50 % of its activity. The results suggested that mutation at oxyanion hole (H110 F) caused enzyme-substrate interaction change resulting on elevation of activity, better activity toward longer carbon chain substrate and improving the activity in the present of non polar organic solvent.

Cloning, heterologous expression, and characterization of a novel thioesterase from natural sample.

A novel thioesterse gene was successfully cloned and sequenced directly from natural sample of Domas Hot Spring, West Java, Indonesia. Homological analysis of the sequence showed that the gene appeared high homology to thioesterase genes with the highest to a putative thioesterase gene from uncultured Acidilobus sp. JCHS at 66% identity. However, phylogenetic analysis showed that the protein was separated from the branch with other known thioesterases. The size of the gene is around 500 base pairs, lied into 2 kb DNA fragment from a random PCR amplicon. The gene was overexpressed in Escherichia coli, a dominant band appeared at 17 kDa in SDS-PAGE with expression level at around 32% of total proteins. The activity of the purified protein using acetyl-CoA as substrate showed that the protein exhibited thioesterase activity. Furthermore, the enzyme also showed esterase activity on p-nitrophenyl ester as substrate. Detail characterization of esterolytic activity showed that the enzyme preferred p-nitrophenyl decanoate as substrate. The optimum activity of the enzyme was at 80 °C and pH 8. Activity of the enzyme was maintained after incubation at 80 °C up to 24 h. In addition, the enzyme was favorable on polar organic solvents. All the data obtained suggested that the enzyme is a novel alkaline thermostable thioesterase.