【作者】 刘彬；

【导师】 王静云；

【作者基本信息】 大连理工大学 ， 生物化工， 2009， 硕士

【摘要】 本文对实验室筛选的两株高产耐热菊粉酶的Bacillus smithii菌株,优化条件下进行产酶发酵,分离纯化得到耐热菊粉酶,研究其酶学性质,应用化学修饰的方法表征影响耐热菊粉酶活性的关键氨基酸组成,探讨其催化作用机理和耐热机制。优化培养Bacillus smithii T4,离心收集发酵上清液,通过硫酸铵分级沉淀、透析脱盐、Sepharose Q-FF、DEAE-Sepharose CL-6B离子交换层析和Superdex 75凝胶过滤层析得到纯化的菊粉酶,SDS-PAGE检测为单一条带,亚基分子量约为45.5KD,比酶活为881.6 IU/mg。酶学性质研究表明:该酶催化作用的最适温度为60℃,60℃和70℃的半衰期分别为8h和5h;最适pH为5.0,在pH4.5-7稳定性较好;以菊粉为底物时,米氏常数Km值为2.73mmol/L,没有转化酶活性,表明来源Bacillus smithii T4菊粉酶的底物专一性较好。而来源于Bacillus smithii T7耐热菊粉酶,比来源于Bacillus smithii T4菊粉酶催化效率高,比酶活可达1283.9 IU/mg,而且耐热性能更好。采用化学修饰法研究Bacillus smithii T7产耐热菊粉酶的酶活中心氨基酸组成:发现PMSF(苯甲基磺酰氟)、2,3-丁二酮、NAI(N-乙酰基咪唑)、IAA(碘代乙酸)、DTT(二硫苏糖醇)修饰对菊粉酶活力影响不大,说明丝氨酸、精氨酸、酪氨酸以及赖氨酸均不是酶催化反应所必需氨基酸,且酶活中心不存在二硫键;DEPC(焦碳酸二乙酯)和EDCI(碳二亚胺盐酸盐)修饰菊粉酶的结果表明:菊粉酶的催化活性中心存在一个组氨酸残基和一个谷氨酸(天冬氨酸)残基,且组氨酸残基参与了菊粉酶的底物结合过程,而谷氨酸(天冬氨酸)残基只参与催化水解过程,推测Bacillus smithii T7产耐热菊粉酶的催化机理是经典酸碱催化机制;利用NBS(N-溴代琥珀酰亚胺)修饰菊粉酶及Tsou’s plot分析,发现菊粉酶中有两个色氨酸残基为酶活性必需基团,而且色氨酸修饰前后,菊粉酶耐热性的变化表明色氨酸构成的疏水环境可能对Bacillus smithii T7产菊粉酶的耐热性起重要作用。更多还原

【Abstract】 In this paper,thermostable inulinases from two newly-isolated thermophilic strain of Bacillus smithii in our lab were purified and characterized.The key amino acid residues participating in the active site of the thermostable inulinase were also investigated by the method of chemical modifications and the catalytic mechanism and heat-resistant mechanism was proposed.With respect to the purification of inulinase,the fermental liquid of Bacillus smithii T4 was centrifugated at 4℃and the supernatant was collected as crude enzyme,and then the purified inulinase was achieved through ammonium sulphate fractional precipitation,dialysis desalination,Sepharose Q-FF,DEAE-Sepharose CL-6B and Superdex 75 chromatography. The purified inulinase was identified as a single band by SDS-PAGE and the subunit molecular weight was about 45.5KD.The specific activity was more than 881.6 IU/mg.The characterization of inulinase from Bacillus smithii T4 showed that the optimum temperature for enzyme activity is 60℃,and the half life of the inulinase is 8h and 5h at 60℃and 70℃,respectively.The optimum pH for this inulinase is 5.0 and stable at pH range of 4.5-7.The lower Km(2.73mM) of the inulinase for inulin indicated that the inulinase from Bacillus smithii T4 has better substrate specificity and no invertase activity.When compared with the inulinase from Bacillus smithii T7,it was found that inulinase from T7 has higher catalytic efficiency and heat-resistance than the inulinase from Bacillus smithii T4.The specific activity of inulinase from Bacillus srnithii T7 is up to 1283.9 IU/mg.The amino acid residues participating in the active site of the thermostable inulinase from Bacillus smithii T7 were investigated by chemical modifications.The results showed that serine,tyrosine,lysine and arginine residues of inulinase did not take part in the catalysis of enzyme.There was also non-involvement of disulfide bond in catalysis.The chemical modification of inulinase by DEPC and carbodiimide showed that one histidine residue might play a key role in substrate binding and catalysis,and one carboxylate residue located in the active site may act as a nucleophile base in the cleavage of substrate.The Tsou’s plot analysis indicated that the inactivation of the enzyme by N-bromosuccinimide(NBS) is dependent upon the modification of two essential tryptophan residues.Moreover,tryptophan residues could play an important role in the thermal stabilization of the active site conformation at higher temperatures as a result of hydrophobic environment.更多还原