Marija Atanaskovic1*, Ivana Moric1, Milos Rokic1, Lidija Senerovic1
1Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11042 Belgrade 152, Serbia
matanaskovic [at] imgge.bg.ac.rs
Abstract
Biofilms are ubiquitous in nature, and the food industry is vulnerable to the risks posed by biofilm formation. Not only do they interfere with the food production process, but they also pose a public health threat. However, complete elimination of biofilms on food and food contact surfaces cannot be achieved by conventional methods (cleaning and disinfection) alone. New biofilm control strategies must be developed to prevent its formation and/or persistence. Novel approaches may be based on enzymes that depolymerize components of the biofilm matrix, making bacterial cells accessible to antimicrobial agents.
Environmental microorganisms are an inexhaustible source of new enzymes. In Salmonella Enteritidis and Escherichia coli, known foodborne pathogens, cellulose is an important component of the biofilm matrix, so our isolates from untapped environments were tested for cellulolytic activity. Of the more than 70 isolates examined, isolate BG28 was selected as the most promising. Its genome was sequenced, annotated, and it was identified as Gram-positive Microbacterium sp. Genome mining revealed the presence of four complete genes for different β-glucosidases, one of three enzyme types of cellulase complexes. To select the best candidate for heterologous expression DeepTMHMM, ProtParam, and SoluProt were used to predict the presence/absence of signal peptide and transmembrane domains, instability index, aliphatic index, hydrophilicity, and soluble expression in E. coli. Based on the prediction results, the gene annotated as β-glucosidase B was selected for recombinant expression. In addition, I-TASSER was used to model the tertiary structure of the selected enzyme.
The β-glucosidase B was recombinantly expressed, purified, and tested for its anti-biofilm activity. It was active and showed a 50% inhibitory effect on S. Enteritidis and E. coli biofilm formation at a concentration of 100 µg/ml. To further evaluate this in silico approach in the preselection of candidate enzymes for recombinant expression and purification, we will use it to identify other enzymes of the cellulase complex.
Keywords: β-glucosidase, bioinformatics tools, enzyme, biofilm
Acknowledgement: This study has been funded by the Ministry of Education, Science and Technological Development, Serbia (grant number 451-03-68/2022-14/200042).
