环糊精葡萄糖基转移酶催化制备2-氧-α-D-吡喃葡萄糖基-L-抗坏血酸中的关键问题研究

2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) has a broad prospect of application in industries including food, pharmacy and chemistry for its improved stability than L-ascorbic acid (L-AA) and its maintenance of physiological function for L-AA. In previous work, the applicants found that the poor affinity of receptor, competitive inhibition of by-product small molecular sugars and low utilization efficiency of the branch component of the substrate starch act as the major limitation in the preparation of AA-2G with cyclodextrin glycosyltransferase (CGTase) using starch as glycosyl donor. According to these factors, this project aims to perform directed selection and function reconstruction on the foundation of specific transglycosylation mechanism of CGTase using L-AA as a receptor; to study the catalysis mechanism and function reconstruction of isomaltosyl glucosaccharide-forming enzyme (IMGase), which attenuates the inhibition effect of by-product small molecular sugars on CGTase by converting them into AA-2G; to explore the collaborate mechanism and regulation strategy of CGTase, IMGase, and starch debranching enzyme as a combination, based on the complexity of the starch substrate and versatility of products in the reaction process; to illustrate the molecular foundations of enzymatic preparation of AA-2G. This project not only contributes theoretical and technological foundations in the promotion of AA-2G manufacturing, but also of great academic significance for extensive understanding the CGTase structure-function relationship and multi-enzyme-catalysis regulation.

2-氧-α-D-吡喃葡萄糖基-L-抗坏血酸（AA-2G）可克服L-抗坏血酸（L-AA）稳定性差的缺点并保持其生理功效，在食品、医药和化工等领域应用前景广阔。我们前期发现受体亲和性差、副产物小分子糖竞争性抑制和淀粉分支组分利用率低是制约环糊精葡萄糖基转移酶（CGTase）以淀粉为糖基供体制备AA-2G的关键因素。针对这些问题，本项目将在解析L-AA为受体的CGTase转糖基反应特异性机制基础上开展酶定向筛选和功能改造；研究可利用副产物小分子糖以削弱其对CGTase抑制的α-异麦芽糖基葡萄糖基糖合成酶（IMGase）催化机制并优化其功能；针对淀粉底物复杂性和中间产物多样性，探索CGTase、IMGase和淀粉脱支酶协同反应体系作用机制及调控策略，阐明酶法制备AA-2G的分子基础，从而为推进AA-2G工业化进程奠定理论技术基础，还为深入理解CGTase结构功能关系和多酶催化调控机制提供借鉴作用。

2-氧-α-D-吡喃葡萄糖基-L-抗坏血酸（AA-2G）是一种L-抗坏血酸的糖基化衍生物，在食品工业领域应用前景广阔。本项目通过数据库挖掘获得了AA-2G合成能力较强的Bacillus stearothermophilus CGTase。根据CGTase家族受体结合区域同源性和差异性，通过定点突变和随机突变结合的方式进一步优化酶性能，获得了AA-2G转化率提升7.3%的突变体K228R/M230L，动力学分析表明其对L-AA受体Km降低35%。将CGTase与AA-2G4进行对接，发现突变体在+1亚位点处Arg228和L-AA之间增加两个氢键，同时His229与L-AA之间的氢键作用增强，均有利于提高CGTase对L-AA的亲和性；通过随机突变获得CGTase产量提升的突变体I641T、K647E、I631T，其酶产量分别为野生酶的2.5倍、2.4倍、1.7倍。此外，研究还发现突变体K647E在CGTase家族中对于提升酶产量具有普遍适用性。研究了CGTase在枯草芽孢杆菌中的重组表达，通过启动子和信号肽筛选以及N末端替换，使重组菌3L罐发酵酶活达到249.3 U/mL，是出发菌株摇瓶发酵酶活的39倍。将AA-2G制备用酶普鲁兰酶在枯草芽孢杆菌中表达，通过启动子筛选和宿主菌蛋白酶含量优化等策略，使普鲁兰酶3-L罐发酵酶活达到8037.9 U/mL，为目前资料报道最高水平的3.83倍；将上述获得的酶以总量100g/L的马铃薯淀粉和L-AA为底物制备AA-2G，通过多酶复配协同调控与优化，在获得的最优条件下，AA-2G产量达到37.0 g/L。为了提高生产强度，进一步采用了高浓度底物麦芽糊精500g/L和L-AA 250g/L制备AA-2G，产量达到211 g/L，为国内外报道的最高水平。进一步通过分析反应过程中AA-2Gn分布情况可以看出，突变体催化产物中AA-2G1-2比例增加，AA-2G3基本不变，AA-2G4-7比例降低，推测可能是由于突变体受体亚位点对L-AA亲和性提高，有利于AA-2Gn作为糖基供体发生反应，进而提高底物利用率和AA-2G产量。本项目对于CGTase功能认知和改造以及AA-2G规模化制备具有积极的推进作用。

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