Thermophilic lipase from Thermomyces lanuginosus: Gene cloning, expression and characterization

An extracellular lipase gene In? from thermophilic fungus Thermomyces lanuginosus HSAUP(03)80006 was cloned through RT-PCR and RACE amplification. Its coding sequence predicted a 292 residues protein with a 17 amino acids signal peptide. The deduced amino acids showed 78.4% similarity to another lipase Igy from T. lanuginosus while shared low similarity with other fungi lipases. Higher frequencies hydrophobic amino acids related to lipase thermal stability, such as Ala, Val, Leu and Gly were observed in this lipase (named LN). The sequence, -Gly-His-Ser-Leu-Gly-, known as a lipase-specific consensus sequence of mould, was also found in LN. High level expression for recombinant lipase was achieved in Pichia pastoris GS115 under the control of strong AOX1 promoter. It was purified to homogeneity through only one step DEAE-Sepharose anion exchange chromatography and got activity of 1328 U/ml. The molecular mass of one single band of this lipase was estimated to be 33 kDa by SOS-PAGE. The enzyme was stable at 60 degrees C and kept 65% enzyme activity after 30 min incubation at 70 degrees C. It kept half-activity after incubated for 40 min at 80 degrees C. The optimum pH for enzyme activity was 9.0 and the lipase was stable from pH 8.0 to 12.0. Lipase activity was enhanced by Ca2+ and inhibited by Fe2+, Zn2+, K+, and Ag+. The cell-free enzyme hydrolyzed and synthesized esters efficiently, and the synthetic efficiency even reached 81.5%. The physicochemical and catalytic properties of the lipase are extensively investigated for its potential industrial applications. (C) 2011 Elsevier B.V. All rights reserved.