Whole Genome Sequencing Analysis of Cellulose-degrading Bacterium DC11 Isolated from Silkworm Excrement and Characterization of Its Key Cellulase Gene ytoP: Running Head: Characterization of strain DC11 and its key gene ytoP

Abstract

In recent years, the microbial degradation of cellulose has emerged in addressing environmental pollution and acting as a substitute for scarce animal feeds. This study conducted a whole-genome sequencing analysis of a cellulose-degrading bacterium known as Bacillus subtilis DC11, previously isolated from the silkworm excrement. A critical cellulase gene was identified, namely ytoP. The ytoP gene was successfully cloned and expressed in E.coli using genetic engineering. ytoP is a segment of the endoglucanase gene in Bacillus subtilis DC11 and was found to be 1074 bp in size and encoded 357 amino acids. This study effectively constructed the cellulase expression vector and achieved successful expression of the ytoP gene from strain DC11 in E. coli BL21 (DE3). SDS-PAGE electrophoresis revealed that the protein had an approximate size range of 40–50 KDa and a concentration of around 3.675 mg/mL. An assay of enzyme activity demonstrated that the purified protein, with a concentration of approximately 100 μg/mL, exhibited a maximum activity of 12.980 U/mL. Through the integration of whole-genome sequencing and genetic engineering techniques, the critical cellulase gene ytoP from Bacillus subtilis DC11 has been successfully cloned and expressed, achieving highly efficient cellulase production. This study lays the foundation for large-scale applications of microbial cellulose degradation in the future.