糖苷合成酶——— 一类新型的寡糖高效合成工具

寡糖是哺乳动物细胞表面糖蛋白和糖脂以及微生物来源的生理活性物质的要素之一,其应用于医药的巨大潜能至今还没有得到充分体现,主要原因是合成足够于临床使用的寡糖非常困难.传统的化学法和酶法在大规模合成寡糖方面都有一定局限性.近年来,分子生物学技术大大推动了糖苷酶合成寡糖的研究,将糖苷酶催化中心亲核体氨基酸定点突变为非亲核体氨基酸,导致酶的原有水解活性丧失,只催化糖苷键合成反应,寡糖产量最高可达99%,人工产生了一类新酶——糖苷合成酶(glycosynthases),随后又产生了硫代糖苷酶(thioglycoligases)和硫代糖苷合成酶(thioglycosynthases).糖苷合成酶的高通量筛选可用双质粒系统和酵母三杂交系统进行,其活性的进一步改进可通过亲核体氨基酸位点不同氨基酸取代、其他位点氨基酸突变、反应条件优化等方法进行,其区域选择性的改变或增强可通过改变糖基受体分子达到.糖苷合成酶作为一种新型高效的生物催化剂,对寡糖的工业化合成有着重要意义,它的出现对糖生物学的发展必将起到巨大的推动作用.

Glycosynthases: a Novel Efficient Synthetic Tool for Oligosaccharides

Oligosaccharides are one of the essential physiological constituents of glycoproteins and glycolipids on mammalian cell surfaces and microbial metabolites. They have considerable potential as therapeutics but are only now slowly assuming this important role. One of the reasons for their slow development has been the considerable difficulty in synthesizing oligosaccharides on the scale necessary for their clinical evaluation. Classical chemical and enzymatic methods both have limitations in synthesizing large-scale oligosaccharides. In recent years, the rapid progress on molecular biotechnology has promoted the development of retaining glycosidases in oligosaccharides synthesis, which led to the production of a novel class of enzymatic activities termed the glycosynthases. These new enzymes are retaining glycosidase mutants in which the catalytic nucleophile has been converted to a non-nucleophilic residue,synthesizing oligosaccharides in high yields ( the highest yields reach 99%) without any hydrolysis. Furthermore thioglycoligases and thioglycosynthases have been developed subsequently in the past three years. Glycosynthases can be screened in high-throughput assay by the two-plasmid system and the yeast three-hybid system respectively. Their activity can be significantly enhanced by substituting alternative residues for nucleophile, additional random mutations and optimizing reaction conditions. Their regioselectivity can be modified through changes in receptors.