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Effect of Mutation at the Lid Subdomain of Aspergillus Niger Lipase on Its Activity

Author: XueLongYin
Tutor: HuangJianZhong
School: Fujian Normal University
Course: Microbiology
Keywords: Aspergillus niger Lipase Lid domain Mutation Active
CLC: TQ925
Type: Master's thesis
Year: 2010
Downloads: 117
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Abstract


Aspergillus niger lipase (Aspergillus niger lipase, ANL) is widely used in industrial fields of oil processing , food additives and detergent additives , biological catalyst with good bio - security . Xinjiang crater lipase overproducing strain Aspergillus niger FJNU01 strains isolated from the papers preserved in our group as a starting material , clone lipase gene . Through Comparative Aspergillus niger lipase lid structure and its two sides hinge zone ferulic acid esterase of the amino acid residue sequence of Aspergillus niger ( A. niger Feruloyl esterase ANE ) differences in the amino acid residue sequence of the corresponding region , to determine the mutation point constructed a series of mutant lipase . By overlap extension PCR , site-directed mutagenesis of Aspergillus niger lipase lid structure on both sides of the hinge region of amino acid residues in the anl - S84G the anl - D99P , the anl - mutation K108E three , fat gene ; will form a lipase replace the entire lid of the α-helix structure of the amino acid residue sequence into the ferulic acid esterase to the amino acid residue sequence corresponding to the domain , to obtain the lipase mutant gene ANL- anelid ; the amino acid composition of the right and left sides of the hinge region in the lid structure of the lipase residue sequence of ferulic acid esterase to replace the corresponding region amino acid residue sequence obtained the lipase mutant gene ANL- lidLR . Lipase molecule After transformation mutant gene was inserted into pPIC9K plasmid to construct a series of recombinant expression vectors after the SAC I linearized ; recombinant expression vector introduced into Pichia pastoris GS115, screening recombinants . Recombinant transformants were induced by methanol , the fermentation supernatant collected by centrifugation , and mutant recombinant lipase enzyme activity was measured with alkaline titration . Experimental results show that : GS - pPIC9K- ANL- D99P and GS-pPIC9K-anl-K108E recombinant fermentation supernatant lipase activity were 21.37U/mL and 16.30U/mL , relative to wild -type lipase were 85.48 % and 65.20% , the GS-pPIC9K-anl-S84G, GS-pPIC9K-anl-anelid and GS- pPIC9K- anl - lidLR fermentation supernatant was not detected in lipase activity . The fermentation supernatant of recombinant protein was purified using Ni-NTA purified proteins by SDS-PAGE results showed that : ANL-S84G, ANL-D99P, ANL-K108E, ANL-anelid showed a single band , while anl- not purified lidLR .

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CLC: > Industrial Technology > Chemical Industry > Other chemical industries > Fermentation industry > Enzyme preparation ( enzyme )
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