Enhancing thermostability of a Rhizomucor miehei lipase by engineering a disulfide bond and displaying on the yeast cell surface.
To increase the thermostability of Rhizomucor miehei lipase, the software Disulfide by Design was used to engineer a novel disulfide bond between residues 96 and 106, and the corresponding double cysteine mutants were constructed. The R. miehei lipase mutant could be expressed by Pichia pastoris in a free secreted form or could be displayed on the cell surface. The new disulfide bond spontaneously formed in the mutant R. miehei lipase. Thermostability was examined by measuring of hydrolysis activity using 4-nitrophenyl caprylate as a substrate. The engineered disulfide bond contributed to thermostability in the free form of the R. miehei lipase variant. The variant displayed on the yeast cell surface had significantly increased residual hydrolytic activity in aqueous solution after incubation at 60 degrees C for 5 h and increased synthetic activity in organic solvent at 60 degrees C. These results indicated that yeast surface display might improve the stability of R. miehei lipase, as well as amplifying the thermostability through the engineered disulfide bond.
Han
Zhen-lin
ZL
Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, People's Republic of China.
Han
Shuang-yan
SY
Zheng
Sui-ping
SP
Lin
Ying
Y
D002210:Caprylates / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004795:Enzyme Stability; D005818:Genetic Engineering / Q000379:methods; D006358:Hot Temperature; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020103:Rhizomucor / Q000201:enzymology* / Q000235:genetics; D013997:Time Factors
D002210:Caprylates; D004220:Disulfides; C034106:4-nitrophenyloctanoate; D008049:Lipase