Directed Evolution of a Hyperthermophilic Endoglucanase Cel12B from Thermotoga maritima

Hao Shi, Lili Wang, Xun Li, Liangliang Wang, Yu Zhang, Xiangqian Li, Fei Wang

Abstract


The cel12B gene was cloned, optimized through directed evolution using error-prone polymerase chain reaction, and then expressed in the Escherichia coli BL21 (DE3) host strain. Five mutants promoting the enzyme activities were selected. The specific activity of the best-evolved Cel16 (L20R, D37V, I108T) was improved approximately 3-fold compared to the parental enzyme. The residual enzyme activity of Cel16 retained 90% of the original when incubated at 90 °C for 2 h, which was similar to the thermostability of the wild type. In addition, the best mutant Cel16, which had two prominent mutant sites, L20R and I108T, was able to increase the cavity polarity because the side chains of arginine and threonine could form hydrogen bonds with the substrate, shrinking the enzyme cavity to some extent and therefore enhancing the enzyme activity.

Keywords


Endoglucanase; Error-prone PCR; Directed evolution; Thermotoga maritima

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