Non-conventional expression of recombinant chitinase A originating from Bacillus licheniformis DSM8785, in Saccharomyces cerevisiae INVSc1: Scientific paper

Chitinases are glycosyl hydrolases, that cleave the β-1,4 linkage between N-acetyl glucosamines present in chitin chains. Chitin is the second most abundant polysaccharide on Earth after cellulose, and it is produced in the exoskeleton of crustaceans and insects, and in some parts of the cell walls of fungi. Enzymatic development and the extraction of superior derivatives from chitin wastes – such as chitooligosaccharides with vast importance in the medi­cal and biofuels industry – lead to the necessity of creating chitinases using dif­ferent strains of organisms. In this paper, the chiA gene from the Bacillus lich­eniformis DSM8785 encoding chitinase A (ChiA) with C-terminal hexahis­tid­ine tag was cloned and expressed in the extracellular expression system pYES2 from Saccharomyces cerevisiae INVSc1 as a hyperglycosylated enzyme. The production of recombinant ChiA was successfully confirmed by dot blotting, using anti-His antibodies. The optimal time of expression was identified to be 24 h when galactose was added only at the beginning of fermentation, the chit­in­ase activity starting to decrease after this threshold. Nevertheless, in another experiment, when galactose was added every 24 h for 72 h, the expression con­tinued for the entire period. The purified enzyme was detected, using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), as a het­ero­geneous diffuse band between 80 and 180 kDa. The molecular mass of the same ChiA enzyme expressed in Pichia pastoris KM71H and Escherichia coli BL21 (DE3) was compared using SDS-PAGE with ChiA expressed in S. cere­visiae INVSc1. The activity of ChiA was determined using the fluorogenic substrate, 4-methylumbelliferyl β-d-N,N,N-triacetylchitotrioside (4MUTC). Using a bioinformatics simulation, the number of the glycolsylation sites of the ChiA gene sequence and the proximity of these sites to the alpha factor sequ­ence were hypothesized to be a possible reason for which ChiA enzyme was internally expressed