Hao Yanling, Huang Xinyi, Mei Xiaohong, Li Ruoyu, Zhai Zhengyuan, Yin Sheng, Huang Ying, Luo Yunbo
College of Food Science and Nutritional Engineering, China Agricultural University, 17 Qing Hua East Road, Hai Dian District, Beijing 100083, PR China.
Protein Expr Purif. 2008 Aug;60(2):221-4. doi: 10.1016/j.pep.2008.04.004. Epub 2008 Apr 26.
A significant problem in production of fruit juices for human consumption is auto-clarification, where enzyme catalyzes pectin demethylation resulting in loss of the ''natural" cloudy appearance of juices. To overcome this problem, a plant inhibitor protein which blocks the action of pectin methylesterase has been used. In this paper, expression of recombinant kiwi pectin methylesterase inhibitor (PMEI) was carried out in Escherichia coli, and the target protein was expressed in the form of inclusion bodies. The expression level reached 46% of total cell protein. Then the fusion protein was purified by nickel ion metal affinity chromatography, and the purity was finally up to 98%. After refolding in GSH/GSSG redox system, recombinant PMEI not only could efficiently inhibit PMEs from eight different plants, but could remain effective inhibitor activity in the pH 3.0-10.0 and 20-40 degrees C. Thus, recombinant PMEI has potential application in the production of fruit juices product industry.
生产供人类消费的果汁时，一个重大问题是自动澄清，即酶催化果胶去甲基化，导致果汁失去“天然”的浑浊外观。为克服这一问题，人们使用了一种能阻断果胶甲酯酶作用的植物抑制蛋白。本文在大肠杆菌中表达了重组猕猴桃果胶甲酯酶抑制剂（PMEI），目标蛋白以包涵体形式表达，表达水平达到总细胞蛋白的46%。然后通过镍离子金属亲和层析纯化融合蛋白，最终纯度达到98%。在GSH/GSSG氧化还原体系中复性后，重组PMEI不仅能有效抑制来自8种不同植物的果胶甲酯酶，而且在pH 3.0 - 10.0和20 - 40℃条件下仍保持有效的抑制活性。因此，重组PMEI在果汁产品工业生产中具有潜在应用价值。
