Chiang Chung-Jen, Wang Jen-You, Chen Po-Ting, Chao Yun-Peng
Department of Medical Laboratory Science and Biotechnology, China Medical University, No. 91, Hsueh-Shih Road, Taichung, 40402, Taiwan.
Appl Microbiol Biotechnol. 2009 Mar;82(3):445-51. doi: 10.1007/s00253-008-1772-z. Epub 2008 Nov 19.
Levan is a homopolymer of fructose which can be produced by the transfructosylation reaction of levansucrase (EC 2.4.1.10) from sucrose. In particular, levan synthesized by Zymomonas mobilis has found a wide and potential application in the food and pharmaceutical industry. In this study, the immobilization of Z. mobilis levansucrae (encoded by levU) was attempted for repeated production of levan. By fusion levU with the chitin-binding domain (ChBD), the hybrid protein was overproduced in a soluble form in Escherichia coli. After direct absorption of the protein mixture from E. coli onto chitin beads, levansucrase tagged with ChBD was found to specifically attach to the affinity matrix. Subsequent analysis indicated that the linkage between the enzyme and chitin beads was substantially stable. Furthermore, with 20% sucrose, the production of levan was enhanced by 60% to reach 83 g/l using the immobilized levansucrase as compared to that by the free counterpart. This production yield accounts for 41.5% conversion yield (g/g) on the basis of sucrose. After all, a total production of levan with 480 g/l was obtained by recycling of the immobilized enzyme for seven times. It is apparent that this approach offers a promising way for levan production by Z. mobilis levansucrase immobilized on chitin beads.
果聚糖是一种果糖同聚物，可通过来自蔗糖的果聚糖蔗糖酶（EC 2.4.1.10）的转果糖基化反应产生。特别地，运动发酵单胞菌合成的果聚糖已在食品和制药工业中发现了广泛且有潜力的应用。在本研究中，尝试固定化运动发酵单胞菌果聚糖蔗糖酶（由levU编码）以重复生产果聚糖。通过将levU与几丁质结合结构域（ChBD）融合，杂合蛋白在大肠杆菌中以可溶形式过量表达。在将来自大肠杆菌的蛋白质混合物直接吸附到几丁质珠上后，发现带有ChBD标签的果聚糖蔗糖酶特异性附着于亲和基质。随后的分析表明酶与几丁质珠之间的连接相当稳定。此外，使用固定化果聚糖蔗糖酶时，在20%蔗糖存在下，果聚糖产量比游离酶提高了60%，达到83 g/l。基于蔗糖，该产量占转化率（g/g）的41.5%。毕竟，通过将固定化酶循环使用七次，总共获得了480 g/l的果聚糖产量。显然，这种方法为通过固定在几丁质珠上的运动发酵单胞菌果聚糖蔗糖酶生产果聚糖提供了一种有前景的途径。
