Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 85, Issue 1, Pages 117-126Publisher
SPRINGER
DOI: 10.1007/s00253-009-2067-8
Keywords
Disulfide bond; Rhizomucor miehei; Thermostability; Lipase; Yeast surface display
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Funding
- National Natural Science Foundation of China [U0773001]
- Ministry of Science and Technology of the People's Republic of China [B1070230]
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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 60A degrees C for 5 h and increased synthetic activity in organic solvent at 60A 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.
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