Effect Of Mutation At The Lid Subdomain Of Penicillium Expansum Lipase On Its Activity

In order to bring facility for molecular designing and alterating Penicillium expansum lipase gene, we amplify the mature peptide of Penicillium expansum lipase, and insert into pPIC9K vector to construct recombinant expression plasmid. By aligning Penicillium expansum lipase with feruloyl esterase, we mutagenesis the sixty-sixth, eihty-first, ninty-fourth amino acids, the "lid", the right side of hinge region of Penicillium expansum lipase, so as to study their effect on lipase activity.Firstly, by means of the previous constructed recombinant expression plasmid template, we site-directed mutagenesis three amino acids in both sides of the hinge region of "lid" (T66G, T81P, K94E) by circuLar plasmids step PCR, and abtain three recombinant expression plasmids containing mutant amino acids. After expressed in Pichia pastoris GS115 strain, we purifiy the expression product by Ni-column, then measure their hydrolysis activity on 4-nitrophenyl decanoate laurate. The results show that:PELT66G recombinant lipase relative activity (5.09U/mg) is similar to the wild-type PEL lipase, while PELT81P and PELK94E recombinant lipase relative activity have been improved 2.95 times and 2.57 times, respectively up to 19.08966U/mg and 17.2391U/mg.Secondly, we exchange the "lid" domain by the way of introducing restriction sites then back mutation by circuLar plasmid step PCR and construct recombinant expression plasmid with pPIC9K vector. However, after expressed in Pichia pastoris GS115 strain, it does not show any lipase activity on olive oil-rhodamine B agar plate. While the purified lipase by Ni-column can be seen one strap about 28KDa by SDS-PAGE electrophoresis.At last we also replace the right hinge region of lipase "lid" by overlap extension PCR. As the "lid" mutant lipase, the concentrated fermentation broth of this recombinant lipase do not show any lipase activity.In a word, Penicillium expansum lipase "lid" domain play very important role in its activity, especially the mutations on "lid", the right hinge region, T81P, K94E, which will resuLt in the loss or whole-fold improvement of lipase activity.