淀粉通道蛋白对淀粉颗粒酶解的影响机制

Starch is depolymerized by enzymes to produce converted products in industry. Previous studies provided evidence that channels of cereal starch granules connect the granular central cavity to external environment. The present of proteins in channels was found in cereal starch granules later. The pattern of digestion of native starch granules is greatly influenced by the existence of pores and channels. When starch granules are incubated with amylolytic enzymes, the enzymes migrate inside the starch granules through the channels and initiate hydrolysis leading to an inside-out pattern of digestion. Although the amorphous regions have been generally thought to be easily digested, the amorphous region and crystalline region are digested simultaneously. Our previous studies suggested that the rates of hydrolysis are related to the content of proteins in starch granules. Therefore, it was hypothesized that the granule-associated protein could affect the binding of α-amylase and amyloglucosidase to substrates, leading to a slower enzyme reaction. The enzyme reaction might be related to the molecular structure of protein in starch channels. In this study, we chose eight different genetic rice starches, including starch channel mutant starch as research material. The rice starches were subjected to α-amylase and amyloglucosidase with limited hydrolysis. The effect of granule-associated protein on the structural, physicochemical and functional properties of rice starch during enzyme hydrolysis will be investigated. The relationship between structural properties of granule-associated protein and the rate of hydrolysis will be developed. This study has broad implications for understanding the mechanism of starch enzyme hydrolysis.

淀粉糖是淀粉深加工的主要产品，如何高效利用生淀粉制糖是业界亟待解决的重要问题。淀粉酶能够穿过谷物淀粉通道到达内部空洞，以淀粉通道作为反应起点，遵循由内而外的同步消解模式。我们的前期研究显示，尽管淀粉颗粒中蛋白质含量极低，却在淀粉颗粒酶解中具有非常重要的功能和贡献，但其作用机制尚未完全明确。受以上现象启发，本课题组提出淀粉通道蛋白对淀粉颗粒酶解的影响机制假说：在酶解过程中，淀粉颗粒中蛋白质如同淀粉分子的天然保护屏障，阻止淀粉分子进入淀粉酶的活性位点，淀粉通道蛋白的分子结构与淀粉酶解反应密切相关。本项目拟选择８种不同基因型大米淀粉（包括淀粉通道突变体）作为原料，通过α-淀粉酶和葡萄糖苷酶的单一或复合作用对淀粉颗粒进行有限酶解，从淀粉分子结构-壳层-淀粉颗粒三个结构层次，探讨淀粉通道蛋白对淀粉化学精细结构与物理结构的影响机制，建立淀粉通道蛋白分子结构与酶解反应之间的关系，为制糖工业提供理论基础。

由于淀粉颗粒中的蛋白质含量极低（0.1%-0.5%），研究者通常认为其对淀粉性质影响较小，导致对其研究内容缺乏。本项目观察到淀粉颗粒结合蛋白分布于淀粉颗粒的表面和蛋白，称为颗粒表面蛋白和颗粒通道蛋白，并提取鉴定了蜡质，低直链和高直链大米淀粉中淀粉颗粒结合蛋白，发现了10种主要的差异表达蛋白，包括14-3-3-like蛋白和核糖体蛋白。利用淀粉颗粒不同层次上的蛋白分子对β-巯基乙醇敏感度的差异性，选择性去除通道蛋白和结合蛋白，建立原淀粉颗粒、通道蛋白缺失型和结合蛋白缺失型模型，研究模型的结构性质和体外消化率。结果表明，通道蛋白的提取导致直链淀粉的浸出，溶胀力和溶解度增加，表观结晶度，最终粘度，糊化温度，储能模量和损耗模量下降。蛋白分子对淀粉的糊化焓，双螺旋结构和氢键强度没有显着影响，而通道蛋白在物理化学变化中比表面蛋白具有更重要的作用。通道蛋白和结合蛋白的剥离导致快速消化淀粉，缓慢消化淀粉和抗性淀粉含量的不同变化。可能的机制与淀粉酶进入淀粉颗粒的可及性和蛋白分子的结构特性有关，从而进行α-淀粉酶和淀粉葡糖苷酶有限酶解，研究蛋白对酶解前后淀粉的化学精细结构与物理结构的影响机制。α-淀粉酶水解开始于淀粉通道和腔的水平和垂直方向的扩大，去除蛋白大大提高水解速率，通过加速水解过程来促进或增强相对结晶度的变化。不含通道蛋白的淀粉颗粒与α-淀粉酶的结合效率更高，可以加速α-淀粉酶向颗粒的渗透并增加对底物的吸收。结合蛋白的去除提高了淀粉葡糖苷酶的结合能力和水解率，加速了相对结晶度的变化和结晶区域的破坏。但去除通道蛋白后，淀粉葡糖苷酶仅结合到颗粒表面并降低水解速率。研究表明，水解速率的变化程度不是由大米淀粉的通道蛋白含量决定的，通道蛋白在延缓大米淀粉的淀粉酶水解方面比表面蛋白具有更重要的作用。通道蛋白不仅仅是淀粉颗粒表面的天然屏障，在α-淀粉酶和淀粉葡糖苷酶水解淀粉颗粒时表现出明显不同的影响机制。