Abstract:The gene coding for a thermostable lipase LipTX (Teth514_0029) from thermophilic bacterium Thermoanaerobacter sp. strain X514 was cloned and the lipase LipTX was purified using affinity chromatography. The gene coding for lipase LipTX was amplified using polymerase chain reaction (PCR) method, and cloned into a pET15b vector, which was used to construct the recombinant plasmid pET15b-LipTX. The constructed pET15b-LipTX was transformed into host Escherichia coli BL21 (DE3) to express the recombinant enzyme via isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. The purified recombinant enzyme was obtained after heat treatment and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) result showed that the relative molecular mass of lipase LipTX was approximately 28 ku. Study of the enzymatic properties indicated that LipTX displayed optimal activity at 70 ℃ and pH 7.5. Substrate specificity and kinetics experiments showed that LipTX could hydrolyze p-nitrophenyl ester (pNP-ester) substrates with different acyl chain lengths (from C8 to C12), and the most suitable substrate was pNP-decanoate. Additionally, this enzyme could hydrolyze triacylglycerols with long acyl chains (from C4 to C16) and showed the strongest enzymatic activity with tributyrin as the substrate. Furthermore, the recombinant lipase LipTX was found to have strong resistance to non-polar organic solvents (methanol, ethanol, acetone, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexane, and chloroform) and denaturants (sodium dodecyl sulfate (SDS), Tween-20, and Triton X-100). These results demonstrate that the lipase LipTX has a broad application prospect for biocatalysis and organic synthesis.
