The PT/S-Box of Modular Cellulase AcCel12B Plays a Key Role in the Hydrolysis of Insoluble Cellulose

Yuwei Li,Weiwei Han,Limei Wang,Zuoming Zhang,Gui Gao,Hao Tong,Mingwei Bu,Junling Wang

doi:10.3390/catal8030123

Abstract

Cellulases play key roles in the degradation of lignocellulosic materials. The function and mechanism of the catalytic domain (CD) and carbohydrate-binding module (CBM) of cellulases were earlier revealed by analysis and characterization of protein structure. However, understanding of the catalytic mechanism of the entire enzyme, and the analysis of the catalytic model, were inadequate. Therefore, the linker chain between CD and CBM has been extensively studied to bridge this gap. Cellulase AcCel12B and three mutants with different linker lengths (with no or 1–3 PT/S-box units) were successfully constructed and purified. Results showed that the activity of cellulases on Avicel and regenerated amorphous cellulose (RAC) increased with the number of PT/S-box units. Furthermore, the desorption of AcCel12B and its mutants from RAC and Avicel were significantly different. The energy of desorption of wild-type and mutant AcCel12B from cellulose decreased with the number of PT/S-box units. Thus, AcCel12B containing more PT/S-box units was more easily desorbed and had more opportunity to hydrolyze cellulose than other samples. The number of PT/S-box units in endocellulase affected the desorption of the enzyme, which is possibly responsible for the differences in the activity of wild-type and mutant AcCel12B on Avicel and RAC.

Highlights

Cellulose, the most abundant natural homopolymer of D-glucose units linked by β-1,4-glycosidic bonds, differs from other polysaccharides in terms of solubility, structural rigidity, and resistance to biological disintegration [1]
Our study proposes a PT/S-box unit of AcCel12B and clearly confirms that the number of PT/S-box units in endocellulases affects the desorption of the enzyme and that the differences in the activity of wild-type and mutant AcCel12B on Avicel and regenerated amorphous cellulose (RAC) is due to differences in desorption
We cloned, expressed, and purified wild-type AcCel12B and three mutants with different linker lengths and observed that the activity of cellulases on insoluble cellulose increased with the number

Summary

Introduction

The most abundant natural homopolymer of D-glucose units linked by β-1,4-glycosidic bonds, differs from other polysaccharides in terms of solubility, structural rigidity, and resistance to biological disintegration [1]. Cellulases can hydrolyze β-1,4-glycosidic bonds of cellulose to produce oligomeric cellulose or cellooligosaccharides. Most cellulases are typical modular enzymes that consist of one or two catalytic domains (CD), one or several carbohydrate-binding modules (CBM), and other modules of unknown function, which are separated by a distinct linker region (LR) [4]. The CBM adsorbed on the cellulose surface targets the CDs to the insoluble substrate and increases the local concentration of cellulases. The linker performs various functions, such as providing cold adaptation to cellulase of an Antarctic bacterium [4], separating the CD and CBM by a necessary distance, facilitating the dynamic adsorption of CBM on the substrate [6], acting as a molecular spring between two functional modules [6], and affecting

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Conclusion