Non-conventional expression of recombinant chitinase A originated from Bacillus licheniformis DSM8785, in Saccharomyces cerevisiae INVSc1

Abstract

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.

Keywords

• chitinolytic enzymes
• molecular cloning
• dot blotting
• fluorescent assay
• glycosylation