定向分子进化提高极端热稳定麦芽糖淀粉酶延缓大米饭回生作用的研究

Retrogradation of gelatinized starch resulting in a decreased sensory quality of cooked rice has been recognized as the major bottleneck for the development of instant rice. In our previous research, two extremely thermostable maltogenic amylases from archaea with different action patterns toward starch successfully retarded the retrogradation of cooked rice when they were used in the cooking period of raw rice. In this project, two strategies of directed evolution and secretory overexpression in Bacillus subtilis will be employed to obtain extremely thermostable maltogenic amylase with safe-to-eat and high activity of retarding starch retrogradation. Based on the size and amino acid profiles of active site pockets of two archaeal maltogenic amylases, site-directed mutagenesis will be performed to replace acceptor-binding amino acids for increasing the activities of transglycosylation facilitating the conversion of long side chain of amylopectin into short chain, and reduce the size of active site pocket for increasing the substrate preference for amylose over amylopectin. High throughput screening method will be developed to obtain the high retrogradation retarding enzymes with the catalytic properties of two parent enzymes from DNA shuffling library constructed using ssDNA of two archaeal maltogenic amylases. The modification of amylose and amylopectin of rice with the wide and mutant enzymes will be determined. The effect of the wide and mutant enzymes on thermodynamic properties and crystal structure of starch will be analyzed. The relationships between the molecular structures of enzyme-treated rice starch and the retrogradation properties of rice meal will be constructed. The action patterns of extremely thermostable maltogenic amylases towards rice starch will be further elucidated.

糊化淀粉的回生,降低大米饭的感官品质，是方便米饭类食品发展的瓶颈。在以前研究中，我们将二个热稳定性最高、但对淀粉作用方式不同的古细菌麦芽糖淀粉酶，直接加入蒸煮中的大米，降低了米饭低温贮存时的回生。本项目拟采用酶的二种定向分子进化策略和芽孢杆菌宿主，获得安全性高、用量少、抑制回生作用更强的极端热稳定麦芽糖淀粉酶。根据酶和低回生淀粉的结构特点，对酶基因定点突变"替换结合受体底物氨基酸"和"减小酶活性中心入口尺寸"，增加酶催化支链淀粉形成短支链的转糖基化活性和选择性地降解直链淀粉的活性；以二个麦芽糖淀粉酶的混合基因片段，建立DNA改组文库，高通量筛选具有二个母酶优点的抗回生突变酶；测定野生酶和突变酶对大米直链淀粉和支链淀粉的修饰方式；测定野生酶和突变酶对大米淀粉热力学特性与回生结晶结构的影响；建立酶所引起大米淀粉分子结构变化与米饭回生的关系；进一步阐明嗜热古细菌麦芽糖淀粉酶对大米淀粉的作用机制。

采用淀粉酶，延缓大米饭的回生，能够提高方便米饭、紫菜包饭、寿司、粽子、米线等多种大米食品的感官品质。但是，目前这些细菌来源淀粉酶的最适催化反应温度较低、热稳定性较差。使用这些淀粉酶延缓大米饭的回生，一般是将淀粉酶与生大米粒加入常温水中浸泡或者提高温度浸泡，然后再蒸煮；或者，在生大米粒蒸煮后，再降温，然后加淀粉酶处理。这二种加酶方式，所需要的反应时间长，处理不均匀，而且容易受到微生物的污染。 . 本项目预先将极端热稳定麦芽糖淀粉酶加入到生大米粒与水的混合物中，在生大米粒蒸煮的高温环境中，对大米淀粉进行分子修饰，对米粒的处理均匀，不易被微生物污染，所得到的大米饭经室温冷却、4℃贮存28天，具有相对较低的硬度。. 基因随机突变提高古细菌麦芽糖淀粉酶对淀粉的水解活性。野生型TfMA对直链淀粉kcat/Km值是0.98 s−1 mL mg−1，而突变型TfMA-V430I对直链淀粉kcat/Km值是5.87 s−1 mL mg−1，催化效率提高6.00倍。野生型TfMA的比活力是3.58U mg−1；而突变型TfMA-V430I的比活力是4.63U mg−1，淀粉水解活性提高1.30倍。. 基因定点突变确定古细菌麦芽糖淀粉酶中关键氨基酸对酶活性的影响。野生型TfMA的γ-CD水解活性是535.10U/mg；突变型TfMA-G407E的γ-CD水解活性是2.13U/mg，水解活性下降251.22倍。. 枯草芽孢杆菌表达古细菌麦芽糖淀粉酶。使用穿梭质粒pUBRTAmy，将基因Smar_0613插入到启动子AmyR2下游，构建重组质粒pUBRTAmy-Smar0613，再转化Bacillus subtilis ISW1214，然后培养纯化得到比活力为66.96 U/mg 的SMMA；是大肠杆菌表达SMMA比活力的2倍多。. 古细菌麦芽糖淀粉酶改变大米淀粉的结构、显著延缓大米淀粉的回生。在50mM pH 5.5的乙酸钠缓冲液和0.5% (w/v) 大米淀粉中，加入3.5U/g starch的TfMA，在95℃保温120min；这种酶处理的淀粉在4℃贮存21天，回生速率显著下降；直链淀粉含量由由16.6%下降到8.6%，支链淀粉含量保持不变，而短分支对长分支的比例增加，是产生低回生的部分原因。