Thermobifida fusca异淀粉酶的作用机制分析

Mechanism Analysis of Thermobifida fusca Isoamylase

异淀粉酶可水解支链淀粉的α-1,6糖苷键,为淀粉彻底水解所必须.使用仪器分析方法推断此类酶作用机制的研究国内未见报道.Thermobifida fusca异淀粉酶经镍离子亲和色谱纯化,以玉米支链淀粉（maize amylopeetin,AP）为底物,结合体积排阻色谱、高效阴离子交换色谱-脉冲安培检测器及氢谱核磁共振等仪器分析方法研究其作用机制.结果表明：AP分子的相对分子质量为2 812 000,水解反应2h后生成两类葡聚糖组分,相对分子质量分别为760 000和7.2 000,比例为1∶4.4;IAM随机水解支链淀粉分子的α-1,6糖苷键,并可切断带有2-3个葡萄糖残基的侧链分支点生成麦芽糖和麦芽三糖,而对α-1,4糖苷键无作用.完全水解支链淀粉生成具有α或β型还原末端的直链淀粉链或麦芽寡糖,产物聚合度（degree of polymerization,DP）主要在15～35之间.IAM作用机理的研究可为其工业化应用奠定基础.

Isoamylase is able to cleave α-1,6-linkages in amylopectin,which is essential for starch saccharification.Research on mechanism of Thermobifidafusca isoamylase （IAM） by instrumental analysis methods has not been reported.In this study,IAM was purified by affinity chromatography and used to hydrolyze amylopectin from maize （AP）.Results of size exclusion chromatography （SEC） indicated that IAM hydrolyzed both inner and outer branching linkages of amylopectin.The polymer structure was completely destroyed by IAM and the mean molecular weight （Mw） decreased from 2 812 000 to 760 000 and 7.2 000 after 2 h,the ratio of large glucans to the small ones was 1∶ 4.4.The population of starch fragments released was characterized by high performance anion exchange chromatography with pulsed amperometric detector （HPAEC-PAD）,the degree of polymerization （DP） of final hydrolysates distributed mainly in 15～35,released maltose and maltotriose demonstrated that the smallest units hydrolysed by IAM were side chains with 2～3glucose residues.1H nuclear magnetic resonance （1H-NMR） spectrum showed that IAM specifically and completely cleaved α-1,6 glycosidic linkages and produced amylose chains or maltoligosaccharides with α-or β-anomeric reducing ends.The mechanism analysis of IAM could lay the foundation for its applications.