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A halotolerant bacterium capable of chitinase production was previously isolated from saline soils of Gavkhooni marsh, Iran. The strain was identified as Bacillus pumilus strain SG2. This strain secretes two chitinases into the medium in response to the presence of chitin in the medium. The two chitinases, ChiS and ChiL, were active on both polymeric as well as oligosaccharide substrates. In this study, these two extracellular chitinases were purified from the supernatant of the Bacillus culture by ammonium sulfate precipitation. The optimum pH and temperature for both of these chitinases were pH 6 and 37°C, respectively. According to the conserved domain analysis, ChiS and ChiL were categorized in family 18 of the glycosylhydrolases. Three essential conserved amino acid residues in in ChiL were found within the active site. Antifungal activities of the two purified chitinases were assessed on Rhizoctonia solani, Verticillium sp., Nigrospora sp., Stemphyllium botryosum and Bipolaris sp. demonstrating inhibition of all the tested strains. However, these chitinases did not inhibit cell wall formation of the non-chitin-containing fungi (oomycetes) Phytophthora citricola and Phytophthora capsici. This is the first investigation demonstrating antifungal activity of chitinases purified from B. pumilus SG2.

A splice form of prochymosin lacking exon‐6 was subcloned and expressed. Expression was optimized by changing various parameters. The maximum amount of protein expressed by this method was 44.61% of total cellular protein, compared to the 45.4% predicted by the Qualitek‐4 software. The expressed protein was then purified using ion exchange chromatography. Activation of the recombinant prochymosin was carried out by changing different parameters. Optimal conditions for activation involved reduction of pH to 5 and 4 h of incubation with the acidic solution, followed by neutralization and 2 h of incubation under neutralized conditions. The activity of the subsequent enzyme sample was 26 units/mL.

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