Dissertation > Excellent graduate degree dissertation topics show

Effect of Mutation at the Lid Subdomain of Penicillium Expansum Lipase on Its Activity

Author: LinRuiFeng
Tutor: HuangJianZhong
School: Fujian Normal University
Course: Fermentation Engineering
Keywords: Lipase from Penicillium expansum The circular plasmid step PCR Overlap extension PCR Cover
CLC: Q55
Type: Master's thesis
Year: 2010
Downloads: 20
Quote: 0
Read: Download Dissertation

Abstract

In order to facilitate the molecular design and renovation, the paper will be extended Lipase from Penicillium mature peptide coding gene was inserted into the pPIC9K expression vector the recombinant expression the plasmid pPIC9K-pel, and lipase gene 5 'end of Penicillium expansum lipase gene Raw plus the coding sequence of (His) 6 tag. Extended by comparison Penicillium lipase and the amino acid sequence of the ferulic acid esterase from Aspergillus niger, select Penicillium expansum lipase \\In front of the recombinant expression plasmid pPIC9K-pel used as template the circular plasmid step PCR site-directed mutagenesis method the three Penicillium expansum lipase \) to obtain the recombinant plasmid containing the mutant coding sequence of amino acid residues, followed by: pPIC9K-PEL-T66G pPIC9K-PEL-T81P pPIC9K-PEL-K94E. The recombinant expression plasmids were introduced into Pichia pastoris GS115 auxotrophic strains induced by methanol. Expression product after Ni-NTA affinity purification, the determination of the hydrolysis of 4 - nitrobenzene decyl laurate specific activity. Experimental results show that: the pELT66G recombinant lipase specific activity of the wild-type PEL lipase similar recombinant Lipase for pELT81P and pELK94E 5.09U/mg; specific activity than the wild-type lipase were increased by 2.95 times and 2.57 times, respectively, to reach 19.1U/mg and 17.2 U / mg. Secondly, the introduction of restriction sites and then circular plasmid Reply The mutation method replacement Lipase from Penicillium expansum \the pPIC9K-pel (-lid): The recombinant plasmid imported into Pichia pastoris GS115 auxotrophic strain induced by methanol. The expressed product was purified by Ni-column-purified after SDS-PAGE electrophoresis to detect expression of the 28 kDa to a treaty strip. The expressed product was olive oil Rhodamine B flat qualitative detection of enzyme activity. The Finally overlap extension PCR replacement Lipase from Penicillium expansum \The recombinant plasmids were introduced into Pichia pastoris GS115 auxotrophic strain induced by methanol. The expressed product was olive oil Rhodamine B flat qualitative detection of enzyme activity. The experimental results show that: Lipase from Penicillium expansum \The mutations that cause lipase activity is completely lost or times improve.

Effect of Mutation at the Lid Subdomain of Penicillium Expansum Lipase on Its Activity

Abstract

Related Dissertations