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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
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 11
Quote: 0
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Abstract


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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