Effect Of Mutation At The Lid Subdomain Of Aspergillus Niger Lipase On Its Activity

Aspergillus niger lipase, as safe biocatalyst, was widely used in food and oil processing, detergent additive and other industrial fields. In this study, the corresponding gene of A. niger lipase FJNU01 which isolated from Xinjiang crater was cloned. We compare the amino acid residue of lid structure and the lid hinge region between A.niger lipase and A. niger feruloyl esterase, several sites of A. niger lipase were definition to design the lipase molecular with a permanently open lid conformation.Using overlap extension PCR, the amino acid residue of lid hinge region of Aspergillus niger lipase was mutanted, three mutant genes of anl-S84G, anl-D99P and anl-K108E were obtained; and the amino acid residues of a-helix structure of A.niger lipase were replaced by the corresponding amino acid residues of A. niger feruloyl esterase, then anl-anelid was obtained; the amino acid residus of both sides of the lid structure were replaced by the corresponding amino acid residues of A. niger feruloyl esterase, anl-lidLR was obtained.The lipase mutants were inserted into pPIC9K to construct series of recombinant expression vector, then introduced into Pichia pastoris GS115. After methanol induction, the mutant lipase activity was quantitatively determined by an alkali titration method, the fermentation supernatant activity of GS-pPIC9K-anl-D99P and GS-pPIC9K-anl-K108E were 21.37U/mL and 16.30U/mL respectively, and the relative activity were 85.48% and 65.20% respectively, while the ANL-S84G, ANL-anelid and ANL-lidLR lipase activity was not detected. After purification in a Ni-NTA agarose column, SDS-PAGE results of purified lipase mutants show that ANL-S84G, ANL-D99P, ANL-K108E and ANL-anelid exhibited a single band, but GS-pPIC9K-anl-lidLR was not purified.