利用重组大肠杆菌合成几丁寡糖的研究

Biosynthesis of Chitooligosaccharides by Recombinant Escherichia coli

摘要:

提取根瘤菌 Mesorhizobium. loti基因组，克隆编码N-乙酰氨基葡萄糖转移酶nodC基因，插入质粒pUC19的lac启动子的下游，构建并筛选出能够合成几丁寡糖的重组大肠杆菌DC_L-3。利用优化的MMYNG培养基，重组大肠杆菌DC_L-3在10L发酵罐中培养26h后，培养液菌体浓度测定OD=10.8，几丁寡糖得率达到526mg/L。收集重组细菌的细胞并煮沸破碎，利用活性炭的吸附和P4凝胶层析对几丁寡糖产物进行分离纯化。纯化产物的液质分析（LC-ESI-MS）结果表明：主要寡糖产物为几丁四糖（m/z, 831［M+H］⁺）和几丁五糖（m/z, 1034［M+H］⁺）。

Abstract:

Acetyl-N-glucosaminyltransferase gene (nodC) was successfully cloned to Escherichia colifrom Mesorhizobium loti. The recombinant E.coli harboring nodC gene was able to synthesize chitooligosaccharides (COs) in MMYNG medium. In optimized condition, a yield of 526 mg/L was obtained after 26 h cultivation in 10 L bioreactor. COs concentration reached up to 4.5% of the cell dry weight. The COs products were purified by charcoal adsorption and Bio-gel P4 chromatography, penta-N-acetylchitopentaose （m/z, 1034［M+H］⁺）and tetra-N-acetylchitopentaose （m/z, 831［M+H］⁺） were identified as the dominating COs product using the method of liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS).