A Neutral Thermostable <i>β</i>-1,4-Glucanase from <i>Humicola insolens</i> Y1 with Potential for Applications in Various Industries

<div><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>_m and <i>V</i>_max 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>