Optimization, gene cloning, expression, and molecular docking insights for enhanced cellulase enzyme production by Bacillus amyloliquefaciens strain elh1.

Abstract

Background: In this study, we isolated a cellulase-producing bacterium, Bacillus amyloliquefaciens strain elh, from rice peel. We employed two optimization methods to enhance the yield of cellulase. Firstly, we utilized a one-variable-at-a-time (OVAT) approach to evaluate the impact of individual physical and chemical parameters. Subsequently, we employed response surface methodology (RSM) to investigate the interactions among these factors. We heterologously expressed the cellulase encoding gene using a cloning vectorin E. coli DH5α. Moreover, we conducted in silico molecular docking analysis to analyze the interaction between cellulase and carboxymethyl cellulose as a substrate.

Results: The bacterial isolate eh1 exhibited an initial cellulase activity of 0.141 ± 0.077 U/ml when cultured in a specific medium, namely Basic Liquid Media (BLM), with rice peel as a substrate. This strain was identified as Bacillus amyloliquefaciens strain elh1 through 16S rRNA sequencing, assigned the accession number OR920278 in GenBank. The optimal incubation time was found to be 72 h of fermentation. Urea was identified as the most suitable nitrogen source, and dextrose as the optimal sugar, resulting in a production increase to 5.04 ± 0.120 U/ml. The peak activity of cellulase reached 14.04 ± 0.42 U/ml utilizing statistical optimization using Response Surface Methodology (RSM). This process comprised an initial screening utilizing the Plackett-Burman design and further refinement employing the BOX -Behnken Design. The gene responsible for cellulase production, egl, was effectively cloned and expressed in E. coli DH5α. The transformed cells exhibited a cellulase activity of 22.3 ± 0.24 U/ml. The egl gene sequence was deposited in GenBank with the accession number PP194445. In silico molecular docking revealed that the two hydroxyl groups of carboxymethyl cellulose bind to the residues of Glu169 inside the binding pocket of the CMCase. This interaction forms two hydrogen bonds, with an affinity score of -5.71.

Conclusions: Optimization of cultural conditions significantly enhances the yield of cellulase enzyme when compared to unoptimized culturing conditions. Additionally, heterologous expression of egl gene showed that the recombinant form of the cellulase is active and that a valid expression system can contribute to a better yield of the enzyme.