초록
<P>We cloned a new glycoside hydrolase family 6 gene, <I>Hicel6C</I>, from the thermophilic fungus <I>Humicola insolens</I> Y1 and expressed it in <I>Pichia pastoris</I>. Using barley <I>β</I>-glucan as a substrate, recombinant HiCel6C protein exhibited neutral pH (6.5) and high temperature (70°C) optima. Distinct from most reported acidic fungal endo-<I>β</I>-1,4-glucanases, HiCel6C was alkali-tolerant, retaining greater than 98.0, 61.2, and 27.6% of peak activity at pH 8.0, 9.0, and 10.0, respectively, and exhibited good stability over a wide pH range (pH 5.0−11.0) and at temperatures up to 60°C. The <I>K</I><SUB>m</SUB> and <I>V</I><SUB>max</SUB> values of HiCel6C for barley <I>β</I>-glucan were 1.29 mg/mL and 752 μmol/min·mg, respectively. HiCel6C was strictly specific for the <I>β</I>-1,4-glucoside linkage exhibiting activity toward barley <I>β</I>-glucan, lichenan, and carboxy methylcellulose sodium salt (CMC-Na), but not toward laminarin (1,3-<I>β</I>-glucan). HiCel6C cleaved the internal glycosidic linkages of cellooligosaccharides randomly and thus represents an endo-cleaving enzyme. The predominant product of polysaccharide hydrolysis by HiCel6C was cellobiose, suggesting that it functions by an endo-processive mechanism. The favorable properties of HiCel6C make it a good candidate for basic research and for applications in the textile and brewing industries.</P>