嗜热古细菌淀粉普鲁兰酶双功能催化机制的解析与水解活性的定向进化

It is very urgent to find new amylases with high optimal temperature and high hydrolytic activity towards α-1,6-glycosidic linkages for one-step enzymatic conversion of normal cereal starch into linear maltodextrins. In our previous research, two thermophilic archaeal amylopullulanases, SMApu and CMApu, with different action patterns towards starch were heterologously expressed in Escherichia coli. Their cyclodextrin-hydrolysing activities were revealed for the first time, resulting in the finding of the sixth known activity in glycoside hydrolase family 57. These two enzymes could respectively hydrolyze potato starch and cyclodextrins into amylodextrins and linear malto oligosaccharides at 90-105oC. In this research, crystal structure of one amylopullulanase and biochemical properties of mutants will be determined. The relationships between enzymatic amino acid residues, hydrolytic activities, and molecular structures will be analyzed. The bifunctional catalytic mechanisms of amylopullulanase towards starch and cyclodextrins will be further elucidated. The structure-based site-directed or random mutagenesis and high throughput screening will be performed in order to obtain the corresponding mutant gene encoding amylopullulanase with high amylopectin-hydrolysing activity. The genetically engineered GRAS micro-organism will be constructed by co-expression of amylopullulanase mutant and Bacillus licheniformis α-amylase. The complex enzymes with high hydrolytic activities from the fermentation will improve the capacities of one-step enzymatic conversion of normal cereal starch into linear maltodextrins.

筛选最适催化反应温度高、水解α-1,6葡萄糖苷键活力高的淀粉酶，对于将普通谷物淀粉一步转化为线性麦芽糊精，具有至关重要的作用。在以前的研究中，我们用大肠杆菌表达了二个对淀粉作用方式不同的、嗜热古细菌淀粉普鲁兰酶SMApu和CMApu；发现他们也具有环糊精水解活性，将葡萄糖苷水解酶57家族的已知活性由五种增加到六种。证实他们在90-105℃，可以分别将土豆淀粉和环糊精转化为直链麦芽糊精和直链麦芽寡糖。本项目拟测定嗜热古细菌淀粉普鲁兰酶的晶体结构和突变体生化特性，分析酶分子中氨基酸残基、水解活性和酶分子结构之间变化的关系，以阐明嗜热古细菌淀粉普鲁兰酶对淀粉和环糊精的分子作用机制；在此基础上，对酶基因进行多点突变，高通量筛选具有较高支链淀粉水解活性的突变酶基因，并与地衣芽孢杆菌α淀粉酶共表达于GRAS微生物，经发酵产生高活力的复合淀粉酶，以进一步提高酶法转化普通谷物淀粉、制备线性麦芽糊精的能力。

发掘超嗜热古细菌中具有极高最适催化反应温度和极端热稳定性的淀粉普鲁兰酶，阐明催化机制，提高酶活性，已成为本领域迫切需要解决的问题。本项目采用大肠杆菌和枯草芽孢杆菌分别异源表达了Thermofilum pendens的淀粉普鲁兰酶TpApu和Staphylothermus marinus的淀粉普鲁兰酶SmApu。纯化的TpApu在pH3.5和95-100℃时表现出最大的活性，是迄今报道的极端嗜热嗜酸的淀粉普鲁兰酶，适用于高温脱除淀粉分支，制备高消化的线性糊精。将来源安全的SmApu以80-240 U/g底物与100 g /L的玉米淀粉在沸水浴中保温2小时。淀粉中慢消化淀粉的含量从17.4显著增加到42.7％。这种高温酶法制备高消化线性糊精和慢消化淀粉的技术，实现了谷物淀粉的同步糊化和水解，具有反应速度快、底物粘度低、不易被微生物污染等多种优势。采用冷冻电镜、解析了淀粉普鲁兰酶SmApu的晶体结构，分辨率达到2.9Å。结果显示，SmApu在溶液中，通过C端部分区域相互作用，形成8聚体。采用定点突变，替换活性中心入口处的氨基酸，获得突变体SmApu-F395A、SmApu-F395S、SmApu-F510A和SmApu-F510S。对酶催化反应动力学参数进行测定显示，所有的突变体都失去了环糊精水解活性，但仍保留支链淀粉脱支活性，首次证明了第395位和第510位苯丙氨酸残基是结合底物的氨基酸。通过定点突变提高淀粉普鲁兰酶支链淀粉脱支活性。删除N端110个氨基酸残基、或者删除C端100个氨基酸残基的突变体，均失去了酶活性，表明上述区域对于维系酶分子的稳定性是至关重要的。