【摘要】 从芽孢杆菌属短芽孢杆菌种分泌的胞外几丁质酶经过硫酸铵盐析、苯基琼脂糖疏水相互作用层析和DEAE阴离子交换层析第一次被提纯。和其他来源的几丁质酶相比 ,该纯酶的独特之处在于它是一个由两个完全相同的亚基以疏水相互作用聚合而成的二聚体。常温下经过巯基乙醇处理后 ,该酶在 1 0 %SDS聚丙烯酰胺电泳显示的分子量为 85kD。沸水处理 3min或用 50℃的 8mol/L尿素溶液处理 1 0min后 ,该酶在 1 0 %SDS聚丙烯酰胺电泳显示的分子量为 4 8kD。该酶的亚基解聚后 ,活性丧失。该酶的等电点为 5.5,水解几丁质的最适 pH为 8.0 ,最适温度为 6 0℃。该酶的酸碱稳定范围是 pH 6 .0到1 0 .0。Ag+和巯基乙醇能抑制酶活性。该纯酶的N端 1 0个氨基酸残基依次是AVSNSKIIGY ,表明该酶是一个尚未发现的新几丁质酶。该纯酶水解几丁质的特征研究表明该酶属一种内切几丁质酶 ,不具备外切几丁质酶的活力。和其他几丁质酶相比 ,该酶具有高度的热稳定性和蛋白水解酶抗性 ,因此可用于生物控制领域。更多还原

【Abstract】 An extracellular chitinase secreted byBacillus brevis was purified to homogeneity by a combination of ammonium sulfate precipitation, Phenyl-Sepharose hydrophobic-interaction chromatography and DEAE anion-exchange chromatography. On SDS-polyacrylamide gel electrophoresis analysis, the purified enzyme showed a mass of 85 kD even in the presence of β-mercaptoethanol, but shifted to 48 kD when heated in boiling water or treated with 8 mol/L urea at 50 ℃ for 10 min. The depolymerization of subunits was accompanied with the loss of chitinase activity, and removing denaturing factors by dialysis could restore the dimer structure and enzymatic activity. The enzyme had an isoelectric point of 5.5 and an optimal temperature of 60 ℃, and was most active at pH 8.0. The enzymatic activity was stable at pH 6—10, and inhibited by Ag+. Ten N-terminal amino acids were determined to be AVSNSKIIGY, demonstrating that the purified enzyme was a novel one. The hydrolysis pattern of the purified enzyme indicated that the chitinase was an endochitinase. The extraordinary thermo-stability and high resistance to proteolysis provide the enzyme with a good prospect to be used as a new tool for biocontrol.更多还原