环糊精酶法合成反应中产物抑制的机制及调控研究

Strong product inhibition in enzymatic reaction for cyclodextrin synthesis from starch by cyclodextrin glycosyltransferase (CGTase) has resulted in relatively low yield and high production cost of cyclodextrin, so it is particularly important to regulate products inhibition. The item focuses on the CGTases. Firstly, the inhibition modes for different types of cyclodextrin will be investigated by the kinetic equations to find the regular pattern of product inhibition in enzymatic reaction for cyclodextrin synthesis. Then, the primary structure characteristics of CGTase related to cyclodextrin product inhibition will be analyzed by structural alignment, computer simulation, and site-directed mutagenesis. Effects of important amino acid residues on product inhibition are subsequently studied. Finally, the CGTase mutants can be constructed to lower product inhibition and enhance cyclodextrin production. In addition, Based on these researches, regulation strategies for product inhibition in enzymatic reaction for cyclodextrin synthesis are summarized, which helps to significantly enhance the cyclodextrin yield and reduce the production cost, and then provide theoretical basis and practice guidance for regulating products inhibition in other amylase-catalyzed reactions.

由于环糊精葡萄糖基转移酶(CGT酶)作用淀粉合成环糊精反应中存在较强的产物抑制，造成环糊精得率较低、生产成本偏高，因此，着力于对产物抑制进行调控显得尤为重要。本项目以CGT酶为研究对象，首先，通过动力学方程分析不同类型环糊精引发产物抑制的作用方式，比较其差异，探究不同来源CGT酶催化反应中产物抑制的规律；然后，利用结构分析、计算机模拟和定点突变等技术，解析与CGT酶产物抑制相关的一级结构特征，阐明重要氨基酸残基对产物抑制产生影响的规律及机制；最后，构建具有较低产物抑制、能高产环糊精的CGT酶突变体，总结出基于CGT酶本身的产物抑制调控策略，从根本上提高环糊精得率、降低生产成本，并为其他淀粉酶促反应产物抑制调控提供理论基础和实践指导。

随着环糊精在食品、医药、环保等领域的广泛应用，其产量已满足不了需求，而酶法制备环糊精过程中的产物抑制是环糊精工业化生产中一个很大的阻碍。在环糊精酶法生产过程中，随着反应的进行，产物环糊精逐渐增多，容易与CGT酶结合，使其活性受到严重损失，极大地影响了环糊精的产率，进而阻碍了环糊精的生产，因此，如何降低环糊精生产过程中的产物抑制成为一个亟待解决的问题。由于不同来源的CGT酶可能有不一样的产物抑制类型，本项目选取2种来源的CGT酶为对象，分别是来源于Paenibacillus macerans JFB05-01的α-CGT酶和来源于Bacillus circulans STB01的β-CGT酶，对其进行动力学分析，通过米氏方程计算抑制常数分析了不同类型环糊精引发产物抑制的作用方式，探寻出不同环糊精对两种酶不同的抑制规律，发现α-、β-和γ-环糊精对α-CGT酶均为竞争性抑制，而对β-CGT酶均为线性混合型抑制。采用结构分析、计算机模拟技术解析了与CGT酶产物抑制相关的一级结构特征，并通过基因单突变、双突变、多突变等技术阐明了重要氨基酸残基对产物抑制产生影响的规律及机制，发现麦芽糖基结合位点2处的氨基酸对CGT酶的产物抑制影响较大。获得了具有较低产物抑制、能高产环糊精的CGT酶突变体：A599N、A599N/Y633A、MBS2 (β→α)、N603D/L600R、N603D/L600E以及MBS2-Bc DF9R，总结出基于CGT酶本身的产物抑制调控策略，可为其他淀粉酶促反应产物抑制调控提供理论基础和实践指导。