期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 20, 期 7, 页码 -出版社
MDPI
DOI: 10.3390/ijms20071602
关键词
cellulase; endoglucanase; rational design; protein engineering; disulfide bonds; thermostability; cellulose biodegradation
资金
- Ministry of Science and High Education (MON) of Russia [RFMEFI61617X0081]
- German Federal Ministry of Education and Research (BMBF) of BioEconomoy International [FKZ: 031B0506]
Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15-21% increase in specific activity against carboxymethylcellulose and beta-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 degrees C for 2 h, they retained 52-58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 degrees C, the enzyme-engineered forms retained 15-22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.
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