Rare sugar is a kind of important low-energy monosaccharide that is rarely found in nature and difficult to synthesize chemically. D-allose, a six-carbon aldose, is an important rare sugar with unique physiological functions. It is radical scavenging active and can inhibit cancer cell proliferation. To obtain D-allose, the microorganisms deriving D-psicose 3-epimerase (DPE) and L-rhamnose isomerase (L-Rhl) have drawn intense attention. In this paper, DPE fromrn Clostridium cellulolyticum H10 was cloned and expressed in Bacillus subtilis, and L-Rhl from Bacillus subtilis 168 was cloned and expressed in Escherichia coli BL21 (DE3). The obtained crude DPE and L-Rhl were then purified through a HisTrap HP affinity chromatography column and an anion-exchange chromatography column. The purified DPE and L-Rhl were employed for the production of rare sugars at last, in which DPE catalyzed D-fructose into D-psicose while L-Rhl converted D-psicose into D-allose. The conversion of D-fructose into D-psicose by DPE was 27.34%, and the conversion of D-psicose into D-allose was 34.64%.%稀少糖是自然界中含量稀少、化学合成困难的一类低热量单糖.D-阿洛糖是一种重要的稀少己醛糖,其具有减少活性自由基、抑制癌细胞增殖等独特的生理学功能.因此,以微生物发酵生产D-阿洛酮糖-3-差向异构酶(DPE)和L-鼠李糖异构酶(L-RhI)转化生产D-阿洛糖,成为近几年来国际研究的热点之一.文中分别克隆了来源于解纤维梭菌Clostridium cellulolyticum H10的DPE基因以及来源于枯草芽胞杆菌Bacillus subtilis 168的L-RhI基因,并分别使其在宿主菌B.subtilis及大肠杆菌Escherichia coli BL21 (DE3)中得到了表达.进一步利用镍亲和层析和阴离子交换色谱等手段对这两种酶进行了纯化,并对这两种纯化后酶的转化能力进行了分析测定.结果表明,以D-果糖为原料利用两种异构酶依次转化获得D-阿洛酮糖及D-阿洛糖,其两步转化效率分别为27.34％和34.64％.
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