TreY酶在阿卡波糖及其结构类似物组分C竞争性合成中的调节机制研究

Acarbose, a pseudotetrasaccharide, is clinically and widely used in the treatment of type II diabetes mellitus. However, a series of structure analogue is always accompanied with the fermentation of acarbose. Especially, the concentration of component C (Acarviosy-1,4Glc-1,1-Glc) can reach a high level, and its molecule structure is highly similar to acarbose, which causes the acarbose purification become very difficult. On the basis of the previous investigation on the industrial acarbose fermentation by Actinoplanes sp. A56, it was observed that the concentration of osmolality had a significant effect on the biosynthesis of acarbose and acarbose. Although approximately 5000 mg/l of acarbose was obtained under the optimal conditions for industrial Actinoplanes sp. A56 fermentation, the yield of component C could highly reach 530 mg/L. Based on this fact, with the key enzyme TreY involved in component C bisosynthesis pathway as the object of study, this project aims to illuminate the regulation mechanism on the competitive biosynthesis between acarbose and component C. Firstly, the temporal dynamic changes of TreY activity and component C yield in Actinoplanes sp. A56 were investigated under the fermentation conditions with various osmolality concnetrations. After the enzyme TreY was induced and purified form the gene engineering bacteria, the crystal structures variation of the enzyme TreY was determined under binding with the reaction substrate maltoriose, and the enzyme kinetics of TreY was further analyzed in the case of various osmolality concnetrations. In a word, the purpose of this project is to theoretically explain the abatement law of component C biosynthesis, which will further provide a new thinking for establishing a control strategy for industrial Actinoplanes sp. A56 fermentation, resulting in a higher acarbose yield and a lower component C biosynthesis.

阿卡波糖系用于Ⅱ型糖尿病治疗的假性四糖物质，其发酵过程会伴随一系列结构类似物的生成。尤其以组分C的生成量大且因其结构与阿卡波糖极为类似，给分离纯化带来了极大困难。本课题组以自行筛选到的游动放线菌A56进行工业化发酵时发现，渗透压会对阿卡波糖及组分C的产量产生显著影响，尽管阿卡波糖产量可达5000mg/L,但组分C含量也高达530 mg/L。为此，本项目以催化组分C合成的关键酶TreY酶为研究对象，首先研究游动放线菌A56在不同渗透压浓度下的TreY酶酶活及组分C的时序性动态变化，并通过纯化基因工程菌诱导表达的TreY酶，研究TreY酶与反应底物麦芽三糖结合前后的空间构象变化规律，以及不同渗透压控制浓度下TreY酶酶反应动力学特征，揭示TreY酶在阿卡波糖及组分C竞争性合成中的调节机制。本研究旨在从理论上阐释组分C的消减规律，以期为建立阿卡波糖高产、组分C低产的工业化发酵工艺提供新的思路。

游动放线菌A56发酵过程借助补加麦芽糖所维持的高渗条件能显著提高阿卡波糖产率，同时组分C的产量会大幅度下降，然而这一机理尚不清晰。为此，本项目对游动放线菌A56在高渗胁迫下的菌体代谢调节机制以及TreY酶的响应特征进行了研究。. 1）研究结果表明：在高渗条件下，游动放线菌A56生长会受到显著抑制，中心代谢途径的效率会显著下降，但阿卡波糖的产量显著上升，同时副产物——组分C的合成会受到抑制，而TreY酶的活性也会受到高渗条件的强烈抑制。. 2）采用基于GC-MS的代谢组学研究平台揭示了游动放线菌在不同渗透压条件下的菌体代谢轮廓变化，试验结果表明，渗透压的升高会抑制游动放线菌胞内TCA循环的效率，进而促使更多的代谢流向HMP途径转移，进而促进阿卡波糖的合成。. 3）进行了TreY酶的分离和纯化，研究了该酶最适反应pH值及pH稳定性、最适反应温度及热稳定性等酶学参数。 . 4）研究了不同渗透压条件下TreY酶的反应动力学特征常数（Km 和Vmax），结果表明TreY酶的活力在高渗条件下会受到显著抑制；采用圆二色光谱初步研究了不同渗透压条件下TreY酶的二级结构变化，结果表明阿卡维基转移酶酶蛋白为典型的α/β型蛋白结构，渗透压的升高会显著降低阿卡维基转移酶α-螺旋比例并提高β-折叠比例。. 本课题的研究为阐述TreY酶在不同渗透压条件下的反应特性，进而为降低组分C合成提供了理论依据。.