Purification, gene cloning and characterization of an acidic β-1,4-glucanase from Phialophora sp. G5 with potential applications in the brewing and feed industries(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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An extracellular β-1,4-glucanase (CelG5, 〜55.0 kDa) was isolated from the culture filtrate of Phialophora sp. G5, and its encoding gene was cloned. The deduced amino acid sequence of CelG5 was at most 73.6% and 44.0%, respectively, identical with a hypothetical protein from Sordaria macrospora and an experimentally verified GH 7 endo-β-1,4-glucanase of Neurospora tetrasperma FGSC 2508. Native CelG5 had pH and temperature optima of pH 4.5-5.0 and 55-60℃. The enzyme showed some properties superior than most fungal β-1,4-glucanases, such as high activity over a wide pH range (exhibiting >50% of the maximum activity at pH 2.0-7.0), excellent stability in extreme acidic to alkaline conditions (pH 2.0-9.0), and strong resistance against pepsin and trypsin (retaining 89% and 94% activity, respectively). Recombinant CelG5 produced in Pichia pastoris had a molecular mass and a pH optimum similar to native CelG5, but with maximal activity at 65℃. Application tests showed that native CelG5 was stable under simulated gastric conditions (retaining >70% activity), and had capacity to decrease the viscosity of barley-bean feed (8.9% by 200 U CelG5) and mash (6.1% by 50 U CelG5) and increase the filtration rate of mash (18.4% by 50 U CelG5). These properties make CelG5 a good candidate for utilization in the animal feed and brewing industries.