Abstract
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 β-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52–58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °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.
Highlights
Cellulases are highly attractive enzymes for various industrial applications, such as lignocellulosic biomass conversion, cotton and paper manufacturing, juice extraction, and use as detergent enzymes and animal feed additives [1,2,3]
disulfide bond (DSB) engineering was performed as an emerging approach to increase the thermostability of endoglucanase II (EGLII) (Cel5A) from P. verruculosum
Protein engineering based on the DSB design was performed to improve the thermostability of EGLII from P. verruculosum
Summary
Cellulases are highly attractive enzymes for various industrial applications, such as lignocellulosic biomass conversion, cotton and paper manufacturing, juice extraction, and use as detergent enzymes and animal feed additives [1,2,3]. Cellulases are classified into three main groups according to their activities, including endo-1-4-β-glucanases (or endoglucanases), exo-cellobiohydrolases, and β-glucosidases. Endoglucanases have drawn much attention from researchers as an essential component of cellulase multienzyme cocktails for biomass degradation or as individual biocatalysts in some abovementioned applications [4,5,6,7]. The practical applications of endoglucanases are often limited because of the loss of enzymatic activity at industrially required temperatures. The enzyme thermal stabilization is an essential requirement for its efficient use in biotechnology [10]. Protein engineering holds a potential to develop thermostable enzymes as biocatalysts
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