A higher β-glucosidase-producing strain was isolated and identified as Tolypocladium cylindrosporum syzx4 based on its morphology and internal transcribed spacer（ITS） rDNA gene sequence.The present study is to ferm...A higher β-glucosidase-producing strain was isolated and identified as Tolypocladium cylindrosporum syzx4 based on its morphology and internal transcribed spacer（ITS） rDNA gene sequence.The present study is to ferment,purify and characterize a β-glucosidase from T.cylindrosporum gams.The enzyme was purified to homogeneity by sulfate precipitataion,diethylaminoethyl cellulose anion exchange chromatography and Sephadex G-100 gel filtration with a 9.47-fold increase in specific activity and a recovery of 12.27%.The molecular weight（Mw） of the β-glucosidase was estimated to be 58600 by SDS-PAGE,which is much lower than that of the enzyme from other fungi.The β-glucosidase shows high activity over a wide range of temperature from 35℃ to 70℃ and the optimum pH is approximately 2.4.Then the kinetic parameters Km（0.85 mmol/L） and vmax[85.23 mmol/（L·s）] of the β-glucosidase were studied,respectively with high affinity p-nitrophenyl-β-D-glucopyranoside（p-NPG） as the substrate,inhibition constant Ki（39.5 mmol/L） with the tolerance of glucose.Although β-glucosidases have been purified and characterized from several other sources,T.cylindrosporum β-glucosidase with the unique optimal pH and higher tolerance of glucose distinguished from others makes the β-glucosidase a potential application in simultaneous saccharification and fermentation（SSF）.展开更多
基金Supported by the Important Agriculture Program of the Jilin Province Technology Department,China(No.20096013)the Jilin University Basic Science Research Fund,China(No.200903259)+1 种基金the Graduate Innovation Fund of Jilin University,China (No.20101043)the Project of Jilin Fuel Alcohol Company Ltd.,China
文摘A higher β-glucosidase-producing strain was isolated and identified as Tolypocladium cylindrosporum syzx4 based on its morphology and internal transcribed spacer（ITS） rDNA gene sequence.The present study is to ferment,purify and characterize a β-glucosidase from T.cylindrosporum gams.The enzyme was purified to homogeneity by sulfate precipitataion,diethylaminoethyl cellulose anion exchange chromatography and Sephadex G-100 gel filtration with a 9.47-fold increase in specific activity and a recovery of 12.27%.The molecular weight（Mw） of the β-glucosidase was estimated to be 58600 by SDS-PAGE,which is much lower than that of the enzyme from other fungi.The β-glucosidase shows high activity over a wide range of temperature from 35℃ to 70℃ and the optimum pH is approximately 2.4.Then the kinetic parameters Km（0.85 mmol/L） and vmax[85.23 mmol/（L·s）] of the β-glucosidase were studied,respectively with high affinity p-nitrophenyl-β-D-glucopyranoside（p-NPG） as the substrate,inhibition constant Ki（39.5 mmol/L） with the tolerance of glucose.Although β-glucosidases have been purified and characterized from several other sources,T.cylindrosporum β-glucosidase with the unique optimal pH and higher tolerance of glucose distinguished from others makes the β-glucosidase a potential application in simultaneous saccharification and fermentation（SSF）.
文摘目的以赤灵芝(Ganoderma lucidum)子实体为实验材料,优化赤灵芝粗多糖的提取工艺,探讨其抗氧化活性。方法通过单因素实验和响应面法优化赤灵芝粗多糖的提取条件,用热水浸提法提取多糖,Sevag法除蛋白,阴离子交换柱层析和凝胶柱层析纯化,通过紫外光谱、傅里叶红外光谱和原子力显微镜对其结构进行初步测定,通过检测1,1-二苯基-2-苦基肼(DPPH)自由基、2,2′-联氨-双(3-乙基苯并噻唑啉-6-磺酸)二胺盐(ABTS)自由基、羟自由基和超氧阴离子清除能力,评价赤灵芝多糖的抗氧化活性。结果赤灵芝粗多糖的最佳提取条件为提取温度92.4℃,提取时间5 h 5 min,液料比21:1,得率为(2.967±0.228)%。提取纯化获得的赤灵芝多糖符合多糖的结构特征,不含有核酸和蛋白质,原子力显微镜表明其大多数呈现链状构象。赤灵芝多糖可以显著清除DPPH自由基、ABTS自由基、羟自由基和超氧阴离子。结论赤灵芝多糖具有较好的抗氧化能力,本研究为赤灵芝的进一步开发应用提供依据。
