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Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from 40 to 70 degrees C, with optimum at 55 degrees C. It was stable for 2 h at temperatures below 60 degrees C and stable over a broad pH range of 4.0-9.0 for 24 h. The apparent K (m) and V (max) of Chi72 for colloidal chitin were 0.23 mg ml(-1) and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the recycling of chitin waste.

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In this study, we increased β-glucan production from brewer’s yeast, Saccharomyces carlsbergensis RU01, by using tannic acid. High-pressure freezing and transmission electron microscopy (HPF-TEM) revealed that the yeast cell wall obtained from yeast malt (YM) medium supplemented with 0.1% w/v tannic acid was thicker than that of yeast cultured in YM medium alone. The production of β-glucan from S. carlsbergensis RU01 was optimized in 3% w/v molasses and 0.1% w/v diammonium sulfate (MDS) medium supplemented with 0.1% w/v tannic acid. The results showed that MDS medium supplemented with 0.1% w/v tannic acid significantly increased the dry cell weight (DCW), and the β-glucan production was 0.28±0.01% w/v and 11.99±0.04% w/w. Tannic acid enhanced the β-glucan content by up to 42.23%. β-Glucan production in the stirred tank reactor (STR) was 1.4-fold higher than that in the shake flask (SF) culture. Analysis of the β-glucan composition by Fourier transform infrared (FTIR) spectroscopy showed that the β-glucan of S. carlsbergensis RU01 cultured in MDS medium supplemented with 0.1% w/v tannic acid had a higher proportion of polysaccharide than that of the control. In addition, β-glucans from brewer’s yeast can be used as prebiotic and functional foods for human health and in animal feed.

Bacillus licheniformis SK-1 naturally produces chitinase 72 (CHI72) with two truncation derivatives at the C-terminus, one with deletion of the chitin binding domain (ChBD), and the other with deletions of both fibronectin type III domain (FnIIID) and ChBD. We constructed deletions mutants of CHI72 with deletion of ChBD (CHI72ΔChBD) and deletions of both FnIIID and ChBD (CHI72ΔFnIIIDΔChBD), and studied their activity on soluble, amorphous and crystalline substrates. Interestingly, when equivalent amount of specific activity of each enzyme on soluble substrate was used, the product yield from CHI72-ΔChBD and CHI72ΔFnIIIDΔChBD on colloidal chitin was 2.5 and 1.6 fold higher than CHI72, respectively. In contrast, the product yield from CHI72ΔChBD and CHI72ΔFnIIID-ΔChBD on β-chitin reduced to 0.7 and 0.5 fold of CHI72, respectively. These results suggest that CHI72 can modulate its substrate specificities through truncations of the functional domains at the C-terminus, producing a mixture of enzymes with elevated efficiency of hydrolysis. [BMB reports 2011; 44(6): 375-380]