【摘要】 谷氨酸棒杆菌CCTCCM201005能合成一种以半乳糖醛酸为主要结构单元的蛋白聚糖类生物絮凝剂(命名为REA 11).为了研究该聚合物的生物合成途径,首先构建了谷氨酸棒杆菌生物合成REA 11的假设途径,然后从(1)中间代谢产物的添加及相关途径关键酶活性的检测和(2)胞内中间代谢产物的检测两个方面来验证该代谢途径的合理性.研究表明,在培养基中添加代谢途径的中间产物UDP 葡萄糖,可显著提高REA 11的絮凝活性,并且UDP 半乳糖差向酶和UDP 半乳糖脱氢酶的比酶活也分别提高了200%和50%;以不同底物为碳源,UDP 葡萄糖焦磷酸化酶、UDP 半乳糖差向酶和UDP 半乳糖脱氢酶的比酶活与REA 11产量的相关系数可分别达到0.75,0.89,0.97.此外,利用HPLC检测出REA 11合成途径中3种关键中间产物UDP 葡萄糖、UDP 半乳糖和UDP 葡萄糖醛酸.由此证明,所构建的REA 11生物合成途径基本合理.更多还原

【Abstract】 Corynebacterium glutamicum CCTCC M201005 produces a novel polysaccharideprotein bioflocculant REA11 consisting of galacturonic acid as the main structure unit. A hypothetical biosynthetic pathway of REA11in C. glutamicum CCTCC M201005 was therefore proposed. The rationality of the biosynthetic pathway was demonstrated by (1) analyzing the response upon addition of UDPglucose to the culture medium; (2) correlating the activities of several key enzymes involved in the pathway with the yields of polygalacturonic acid; and (3) detecting the existence of several key intermediates in the pathway. It was shown that polygalacturonic acid production, UDPgalactose epimerase activity, and UDPgalactose dehydrogenase activity were improved 24%, 200%, and 50%, respectively, upon UDPglucose addition. In addition, it was found the activities of UDPglucose pyrophosphorylase (R2=0.97), UDPgalactose epimerase (R2=0.75) and UDPgalactose dehydrogenase (R2=0.89) were well correlated with the yields of polygalacturonic acid when different sugars were used as a sole carbon source. Furthermore, the key intermediates in the hypothetical biosynthetic pathway, such as UDPglucose, UDPgalactose and UDPglucuronic acid, were detected in the cellfree extract. 更多还原