Effect of K202A Mutation in the Thermostability of Penicillum expansum Lipase
- VernacularTitle:K202A突变对扩展青霉脂肪酶热稳定性的影响
- Author:
You-Tu ZOU
;
Yi-Zhen WU
;
Wen-Fang SHI
;
Lin LIN
;
- Publication Type:Journal Article
- Keywords:
Penicillum expansum Lipase Site-directed mutagenesis Thermostability
- From:
China Biotechnology
2006;0(12):-
- CountryChina
- Language:Chinese
-
Abstract:
Lipase gene from Penicillium expansum(lip07) was cloned and over-expressed in Pichia pastoris.a random mutant named ep8,which contained a single amino acid substitution,was obtained by using the lip07 as an error-prone PCR template in previous study.ep8 shows higher thermostability than that of lip07,To further improve the thermostability of the lipase,the Lys of wild-type(lip07) and mutant(ep8) in 202 were substituted by Ala using the Overlap extension PCR technique respectively.The mutant genes(lip07-K202A and ep8-K202A) were subcloned into pAO815,and then transformed into the Pichia pastoris GS115 for extracelluar expression,respectively.15% SDS-PAGE analysis indicated that the molecular mass of PEL-ep8-K202A and PEL-lip07-K202A are both about 28kDa,which is same with the wild-type lipase.The Tm of PEL-ep8-K202A is 41.66℃,2.63℃ higher than that of the wild-type(39.03℃) and 1.21℃ higher than the random mutant(PEL-ep8:40.45℃);the Tm of single mutant(PEL-lip07-K202A) is 37.08℃,2℃ lower than that of the wild-type lipase.
