【作者】 符丹丹；

【导师】 邬敏辰；

【作者基本信息】 江南大学 ， 发酵工程， 2005， 硕士

【摘要】 本研究论文以一株木聚糖酶产生菌宇佐美曲霉（Aspergillus usamii） E001为实验菌株,较系统地研究了发酵培养基组分和培养条件对该菌株固态发酵产木聚糖酶的影响。通过Plackett-Burman和Box-Behnken实验设计方法,确定了最佳的培养基组成为:麸皮3.4 g,玉米芯4.6 g,NH4NO₃ 1%,KH2PO4 0.3%,CaCl₂0.1%,MgSO₄ 0.15%,Tween-80 0.5% （其中百分数均相对于固体料,即麸皮+玉米芯）,液固比为1.2 : 1。最佳培养条件为:自然pH,28℃静置培养72 h;期间翻曲2³次。成熟麸曲酶活性最高达7442 IU/g干曲。木聚糖酶粗酶性质的研究表明:酶的最适作用温度50℃,低于50℃时较稳定;酶的最适作用pH 4.6,稳定范围为pH 5.0¹0.0。Ca²⁺对酶的活性略有激活作用,而Mn²⁺、Mg²⁺、Ba²⁺则具有强烈的抑制作用,其它金属离子也均有不同程度的抑制作用。固态发酵成熟麸曲经水浸提、硫酸铵盐析、Phenyl-Sepharose CL-4B疏水层析、Sephadex G-75凝胶过滤层析、DEAE Sepharose fast flow阴离子交换层析等分离纯化步骤,最终获得了PAGE、SDS-PAGE、PAGE酶谱及HPLC纯的两种木聚糖酶Xyn I和Xyn II。采用SDS-PAGE测得Xyn I、Xyn II的分子量分别为24.4 kDa和26.8 kDa,而用凝胶过滤层析测得两纯酶的分子量约为25.0 kDa,两种方法测定的分子量相近（25.0 kDa与24.4/26.8 kDa）,表明Xyn I和Xyn II均为单体酶（即酶蛋白由单亚基组成）;苯酚-硫酸法测得两种酶几乎都不含糖基;采用等电聚焦测得Xyn I和Xyn II的等电点分别为pI 5.0和pI 4.2;由氨基酸分析仪测定得知两种酶的氨基酸组成中酸性氨基酸含量较高;纯酶Xyn II的N末端15个氨基酸残基序列测定结果为:NH2-Ser-Ala-Gly-Ile-Asn-Tyr-Val-Gln-Asn-Tyr-Asn-Gly-Asn-Leu-Gly-。纯酶Xyn I和Xyn II的最适作用温度均为50℃,最适作用pH分别为5.0和4.5;Xyn I在45℃以下、pH 5⁹范围内稳定,而Xyn II则在55℃以下,pH 3⁹范围内稳定。测得Xyn I和Xyn II对桦木木聚糖的Km值分别为0.83 mg/mL和9.2 mg/mL;而对燕麦木聚糖的Km值分别为3.8 mg/mL和13.6 mg/mL。对酶水解产物的HPLC分析推知两种酶可能均为内切酶。更多还原

【Abstract】 The xylanase production was investigated with solid fermentation by Aspergillususamii E001. Multivariant statistical approaches were employed to evaluate theeffects of several variables (carbon and nitrogen source,initial pH and temperature)on xylanase production. Xylanase activity reached 7442 IU/g dry medium under theoptimized conditions at 28℃ for 72 h.Cross enzyme showed an optimum temperature and pH of 50℃ and pH 4.6,respectively. Xylanase was stable below 50℃ and within pH 5.0~10.0. Its activitywas increased by Ca2+ ion and strongly inhibited by Mn2+、Mg2+ and Ba2+ ions.Two xylanases Xyn I and Xyn II from solid-state fermentation culture werepurified by extracting with water, ammonium sulfate precipitation, and Phenyl-Sepharose CL-4B, Sephadex G-75 and DEAE-Sepharose fast flow column chromato-graphies. Two purified xylanases Xyn I and Xyn II were homogeneous on SDS-PAGE、PAGE、HPLC as well as zymogram on RBB-xylan, respectively. Themolecular weights of Xyn I and Xyn II were determined as 24.4 kDa and 26.8 kDarespectively on SDS-PAGE and as 25.0 kDa on gel filtration, indicating that they werea monomer. They didn’t contained carbohydrate by phenol-vitriol method. Theisoelectric points of Xyn I and Xyn II were estimated to be 5.0 and 4.2 by isoelectricfocusing. Analysis of amino acid composition showed that content of acid amino acidwas high. The sequence of 15 amino acids at N-terminus of Xyn II was NH2-Ser-Ala-Gly-Ile-Asn-Tyr-Val-Gln-Asn-Tyr-Asn-Gly-Asn-Leu-Gly-。The optimum temperatures of two xylanases were all 50℃. The pH values ofXyn I and Xyn II were pH 5.0 and pH 4.5 respectively. Xyn I was stable below 45℃and within pH 5~9;while Xyn II was stable below 55℃ and within pH 3~9. Kineticscoefficients Km of Xyn I and Xyn II were 0.83 mg/mL and 9.2 mg/mL with birchxylan as substrate;while were 3.8 mg/mL and 13.6 mg/mL with oat spelt xylan assubstrate respectively. Analysis of hydrolysis products by HPLC showed that twoenzymes may be endo-xylanase.更多还原