Cloning, Expression, Purification and Product Characterization of a Novel Chitinase from Bacillus Aryabhattai

Abstract

Chitin, an abundant polysaccharide in the world, is a primary byproduct of the seafood industry. Efficiently converting chitin into valuable products is crucial. Chitinase transforms chitin into chitin oligomers, which holds significant industrial potential. However, chitin's crystalline and insoluble nature makes the conversion process challenging. This study isolated a novel marine bacterium, Bacillus aryabhattai, from the Arabian Sea. Bacterial growth in different crystalline chitin substrates like chitin powder, flakes, and colloidal chitin confirmed the chitinase presence in bacterium could act upon insoluble crystalline chitin and produce chitin oligomers. The domain architecture analysis of the chitinase confirmed the presence of two N-terminal LysM domains, which helps chitinase act on crystalline chitin. Statistical optimization of media and process parameters revealed glycerol, yeast extract, magnesium chloride, and manganese sulphate as significant media components and 1% colloidal chitin. The optimum process parameters such as pH 7, temperature 40℃, inoculum size 12.5% (v/v), and inoculum age 20 hours enhanced the specific chitinase activity to ±115.2 U⸳ mg-1, ±63.02 U⸳ mg-1 and ±146.1 U⸳ mg-1 against chitin powder, flakes and colloidal chitin respectively, which is five to six times higher than basal level activity. Also, TLC and LC-MS analysis confirmed that chitin oligomers (monomer to hexamer) were produced from insoluble chitin powder and flakes by spent media of Bacillus aryabhattai. A recombinant chitinase from the marine bacteria Bacillus aryabhattai (BaChiA) was developed in the future. The chitinase gene was cloned into the pET 23a plasmid and transformed into E. coli Rosetta pLysS. Chitinase characterization against colloidal chitin revealed optimum parameters such as Tris buffer at pH 8, temperature 55℃, with 400 mM sodium chloride. Enzyme kinetics studies for colloidal chitin substrates revealed Vmax of 112.3 μmole⸳ min-1 and Km of 2.5 mg⸳ mL-1. The specific chitinase activity against chitin powder and flakes reached 875 U⸳ mg-1 and 625 U⸳ mg-1, respectively. The chitinase showed antifungal activity against Candida albicans, Fusarium solani, and Penicillium chrysogenum growth with the zone of inhibition of 14 mm and 25 mm diameter for wildtype and recombinant BaChiA, respectively. Thin Layer Chromatography (TLC) and LC-MS confirmed the production of chitin trimer, tetramer, pentamer, and hexamer from chitin powder and flakes using recombinant chitinase.