Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Directed Evolution of Lipase from Penicillium Expansum FS1884 by Error Prone PCR

Author ChenMing
Tutor ShiBiHong
School Fujian Normal University
Course Biochemistry and Molecular Biology
Keywords lipase error-prone PCR directed evolution enzyme characteristics
Type Master's thesis
Year 2011
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Lipase (PEL) from Penicillium expansum FS1884 has been widly used in industry. To improve the enzyme’s applicability in industries, two continuous rounds of error-prone PCR was introduced to PEL for directed evolution, which contribute to a library of approximately 106 mutated clones in E. coli DH5a. The recombinants plasmid (pPIC3.5K-ep-PEL)harboring the mutated lipase genes were transformed to Pichia pastoris GS115,and screened by YPOM medium plate and olive oil plate, six mutants (GS-ep25-PEL, GS-ep10-PEL, GS-ep33-PEL, GS-ep43-PEL, GS-ep51-PEL and GS-ep81-PEL)were obtained for further enzyme characteristics study.The results demonstrated that, no changes were found in optimum reaction temperature of GS-ep25-PEL, GS-ep33-PEL, GS-ep43-PEL, GS-ep51-PEL and GS-ep81-PEL compared to wild type enzyme of GS-PEL, while GS-ep10-PEL showed the optimum temperature of 35℃,5℃lower than 40℃of GS-PEL. The enzyme activity of GS-ep25-PEL, GS-ep10-PEL, GS-ep33-PEL, GS-ep43-PEL, GS-ep51-PEL and GS-ep81-PEL at optimum reaction temperature were 1912.5U/mL,2401.25U/mL, 1561.67U/mL,895.44 U/mL,1448.4 U/mL and 1540.3 U/mL, respectively, whichvalent 130%,168.56%,106.2%,60.9%,98.5%and 104.7%to that of GS-PEL respectively. The melt temperature (Tm) of GS-ep43-PEL and GS-ep81-PEL were decreased 3.6℃and 1.5℃,respectively, compared with that of GS-PEL. And the optimum reaction pH of GS-ep25-PEL and GS-ep10-PEL were changed to 11.0.Sequence analysis revealed that the size of the mutated lipase gene in all mutants was 858bp, with the same size as that of wild type lipase; and one single amino acid substitution in GS-ep25-PEL (K253M), four amino acid substitutions in GS-ep10-PEL (I104T/A188T/G192S/K253M), three amino acid substitutions in GS-ep33-PEL (A18T/K147M/D150E) and double substitutions in GS-ep43-PEL (K63R/R128G).

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