Biochemical Characterization of a Novel Thermostable β-1,3-1,4-Glucanase (Lichenase) from Paecilomyces thermophila

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• 作者：Shaoqing Yang ; Yan Qiaojuan ; Zhengqiang Jiang ; Guangsen Fan ; Lan Wang
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2008
• 出版时间：July 9, 2008
• 年：2008
• 卷：56
• 期：13
• 页码：5345 - 5351
• 全文大小：2099K
• 年卷期：v.56,no.13(July 9, 2008)
• ISSN：1520-5118

文摘

The purification and characterization of a novel extracellular β-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila J18 were studied. The strain produced the maximum level of extracellular β-glucanase (135.6 U mL⁻¹) when grown in a medium containing corncob (5%, w/v) at 50 °C for 4 days. The crude enzyme solution was purified by 122.5-fold with an apparent homogeneity and a recovery yield of 8.9%. The purified enzyme showed as a single protein band on SDS-PAGE with a molecular mass of 38.6 kDa. The molecular masses were 34.6 kDa and 31692.9 Da when detected by gel filtration and mass spectrometry, respectively, suggesting that it is a monomeric protein. The enzyme was a glycoprotein with a carbohydrate content of 19.0% (w/w). Its N-terminal sequence of 10 amino acid residues was determined as H₂N−A(?)GYVSNIVVN. The purified enzyme was optimally active at pH 7.0 and 70 °C. It was stable within pH range 4.0−10.0 and up to 65 °C, respectively. Substrate specificity studies revealed that the enzyme is a true β-1,3-1,4-D-glucanase. The K_m values determined for barley β-D-glucan and lichenan were 2.46 and 1.82 mg mL⁻¹, respectively. The enzyme hydrolyzed barley β-D-glucan and lichenan to yield bisaccharide, trisaccharide, and tetrasaccharide as the main products. Circular dichroism studies indicated that the protein contains 28% α-helix, 24% β-sheet, and 48% random coil. Circular dichroism spectroscopy is also used to investigate the thermostability of the purified enzyme. This is the first report on the purification and characterization of a β-1,3-1,4-glucanase from Paecilomyces sp. These properties make the enzyme highly suitable for industrial applications.