基于支链淀粉building blocks构建优质BE突变酶定向修饰淀粉调控机制的研究

Currently, it is one of the hotest research points that enzymic method is being utilized to modify starches instead of chemical method existing certain safety problems at home and abroad. However, different internal structure of starches can produce different modified starches using enzymic method, leading to lower application stability. So, how to purposefully modify different starch chain sturctures using enzymic method to prepare green modified starches with desired and stable qualities has become an urgent issue to be resolved. In this study, in order to establish and select the excellently mutated branching enzyme (BE) with high enzymatic activity, heat stability and product specificity, the site-directed mutagenesis will firstly be carried out to mutate four amino acid residues located at the active center in BE from E. coli BL21(DE3)/pET20b-BE identified in our previous research. Secondly, the key point of starch internal structure- the smallest building blocks of amylopectin will be as a breakthrough point, the optimal structure parameters of building blocks that are positively correlated with the excellent physicochemical and slowly digestible properties of starches will be determined, and they will be as an important theoretical basis for starches modified by using the excellently mutated BE. Finally, on the basis of the optimal structure parameters of building blocks, the key regulation factors in enzymatic reaction will be exploited and located, and then the regulatory mechanism that starches can be directionally converted to target product specificity possessing optimal building blocks by using mutated BE will be established. It will supply a theoretical and technological foundation for preparing green modified starches with the desired quality.

酶法替代存在一定安全性问题的化学法对淀粉改性是目前国内外研究热点之一。然而不同淀粉内部结构引起酶改性淀粉生成的产物差异性大，导致其应用稳定性差。因此，如何利用酶法目的性修饰不同淀粉链结构以生产预期的、品质稳定的改性淀粉是尚需解决的核心问题。本项目拟对已鉴定的E. coli BL21(DE3)/pET20b-BE工程菌的分支酶（BE）催化活性中心处四种氨基酸残基进行定点突变，筛选出酶活性、热稳定性和产物特异性高的优质BE突变酶；以淀粉内部结构关键环节—支链淀粉最小构建单元building blocks为切入点，确定与淀粉良好物化性能和缓慢消化特性呈正相关性building blocks结构参数，并以此参数为理论依据，挖掘和锁定酶促反应关键调控因素，构建优质BE突变酶改性淀粉定向转化生成具有良好building blocks目标产物特异性调控机制，为制备预期绿色改性淀粉提供理论依据和实践基础。

利用生物酶法替代存在一定安全性问题的化学法对淀粉进行改性已成为目前国内外研究热点。若要分支酶（BE）或葡萄糖基转移酶（TG）对淀粉链进行目的性修饰，需要解决的关键环节是支链淀粉最小构建单元building blocks，而在building blocks上引入新分支点则是解决问题的有效策略。然而，BE 或/和TG酶如何从building blocks水平上目的性修饰淀粉链结构以定向转化生成目标产物特异性调控机制尚未明确。因此，本项目首先利用筛选出的优质BE或/和TG酶对谷物淀粉（玉米、小麦、大米、糯米）和根茎类淀粉（马铃薯和红薯）进行结构修饰，确定与淀粉良好物化加工性能和缓慢消化特性呈正相关性的良好building blocks结构参数如下：与淀粉溶解度和溶胀性呈正相关性的building blocks线性链长为DP 6-9；与淀粉热稳定性呈正相关性的building blocks线性链长为DP≥24；与淀粉粘度呈正相关性的building blocks线性链长为DP6-12；与淀粉老化呈正相关性的building blocks线性链长为DP≥18；与缓慢消化特性呈正相关性的building blocks线性链长为DP 9-11；与淀粉膜疏水性呈正相关性的building blocks线性链长为DP 6-12；与淀粉膜疏水性呈正相关性的building blocks线性链长为DP 6-12；与淀粉膜拉伸强度和断裂伸长率呈正相关性的building blocks线性链长为DP≥37。并以此结构参数为重要理论依据，挖掘并锁定优质BE或/和TG酶酶促反应的关键调控因素，主要是酶解温度、酶解时间和酶添加量。即BE酶：温度60℃、时间20h和酶添加量308U/g；TG酶：温度55℃、时间12h和酶添加量9088U/g。与原淀粉相比，酶改性淀粉颗粒溶解度和溶胀度显著增高了100%以上，淀粉糊化温度约降低了6℃左右，淀粉糊粘度增加了40-60%；糊化峰值温度降低到56℃左右；淀粉糊在4℃储藏一周，其老化度约从60%显著降低到36%；缓慢消化淀粉和抗性淀粉含量分别从25%和8%显著增加到48%和30%。酶改性淀粉具有显著增加的抗性淀粉含量（煮熟前84%，煮熟后38%）。最终构建了BE或/和TG酶改性淀粉定向转化生成具有良好building blocks的目标产物特异性调控机制