酶法制备几种功能性低聚糖的研究进展

功能性低聚糖是一类重要的益生元,具有许多重要的生理功能,近年来受到研究人员和广大消费者的广泛关注。功能性低聚糖的制备方法主要有化学法、酶法和物理法,其中酶法制备是绿色、高效和最具发展和应用潜力的方法。本文针对不同类型功能性低聚糖的酶法制备过程差异,系统介绍了近年来国内外在低聚木糖、几丁寡糖、琼脂寡糖、低聚半乳糖和甘露寡糖等5类典型功能性低聚糖糖酶法制备方面所取得的研究进展;也总结介绍了不同类型功能性低聚糖的原料来源、结构差异以及健康功能活性等;最后对功能性低聚糖领域的基础研究的发展趋势进行了预测和展望,以期为我国功能性低聚糖的研究和实际生产应用提供参考。     

蔗糖磷酸化酶的研究进展

蔗糖磷酸化酶属于糖苷水解酶13家族,能够催化蔗糖的可逆磷酸解。利用其广泛的底物混杂性,蔗糖磷酸化酶可以将葡萄糖基转移至不同的受体合成熊果苷、甘油葡萄糖苷、低聚糖及多酚化合物的衍生物等产物,这些催化产物可广泛应用于食品、药品、化妆品等行业。随着酶催化技术和蛋白质工程的发展,蔗糖磷酸化酶受到了越来越多的关注,该酶的应用范围也得到了扩大。本文综述了近年来蔗糖磷酸化酶在酶的来源、结构、功能及应用领域等的研究进展,同时讨论了该酶的蛋白质工程改造方法与局限性,并展望了该酶可能的研究方向。     

功能性低聚糖及其生产应用

功能性食品是当今食品和营养科学中最受关注和推广的领域之一,功能性低聚糖(又称非消化性低聚糖)是功能性食品的重要组成部分。聚焦于功能性低聚糖,首先对与其相关的益生菌、益生元、合生元的基本概念进行说明,然后对功能性低聚糖的概念及种类、理化性质及生理功能、重要商品化种类的生产方式进行总结和评述,最后对功能性低聚糖领域的发展进行了展望。     

低聚乳果糖酶法制备的研究进展

低聚乳果糖是一种新型功能性低聚糖,是双歧杆菌的有效增殖因子。本文主要介绍了低聚乳果糖国内外研究状况、产品类型、安全性;重点介绍低聚乳果糖酶法制备方法,微生物来源,现有酶的改造修饰,并对制约低聚乳果糖发展的瓶颈问题简单概述。     

功能性低聚糖生产工艺的研究现状

功能性低聚糖（functional oligosaccharides）是由2—10个单糖通过糖苷键连接形成直链或支链的一类寡糖,具有低热量、抗龋齿、抗肿瘤、防治糖尿病、防止腹泻和便秘等生理作用。因具有糖类某些共同特性,可作为甜食配料,但人体肠道内不具备分解消化功能性低聚糖的酶系统,所以不被人体胃酸、胃酶降解,不在小肠吸收,直接进入大肠内为双歧杆菌所利用,是一类优良的双歧杆菌增殖因子,故又名双歧因子。功能性低聚糖包括水苏糖、棉子糖、低聚果糖、低聚木糖、低聚半乳糖、大豆低聚糖和低聚异麦芽糖等。近年来,国外上市品种有10多种,生产批量较大,我国自1996年开始才有批量生产,主要以低聚异麦芽糖、低聚果糖和大豆低聚糖为主。本文综述了不同种类的功能性低聚糖及生产工艺的现状。     

微生物酶法合成低聚半乳糖的新进展

低聚半乳糖(GOS)是目前国际上已开发的功能性低聚糖之一,其商业化产品是应用微生物β-半乳糖苷酶以乳糖为原料进行转糖基反应获得,不同来源的酶合成GOS的结构不同,转糖基效率也存在差异.天然酶合成GOS的产量一般为20%～45%,分子改造获得的人工酶能将90%的乳糖底物转化为GOS;采用两相体系或反相胶束可以在一定程度上提高GOS产量.应用填充床反应器、活塞流反应器、膜反应器可规模化合成GOS;采用色谱柱法、酶法、纳滤膜法和微生物发酵法可纯化GOS产品,去除单糖及乳糖组分,扩大其应用范围.     

酶法制备功能性纤维低聚糖的研究

研究里氏木霉(Trichoderma reesei)Rut C30纤维素酶单一组分EGI、EGII和CBHI降解纤维素的机理及纤维低聚糖酶法制备技术,进而初步研究纤维低聚糖对青春双歧杆菌的增殖作用。以内切葡聚糖酶EGII酶法制备纤维低聚糖,每克纤维素最佳酶用量1 U,最佳酶解时间90 min,制备得到的纤维低聚糖中纤维二糖、纤维三糖和纤维四糖占总糖的比例分别为43.8%、34.8%和7.9%。以纤维二糖、纤维低聚糖为C源增殖青春双歧杆菌,菌体质量浓度增殖倍数分别为2.14、2.84。     

酶法制备甲壳低聚糖研究的进展

甲壳低聚糖具有多种重要的生理活性,可作为食品添加剂、生物农药和新型药物,是低聚糖研发的新热点。与壳聚糖相比,由于多糖链缩短使其水溶性好、黏度低且容易吸收。本文对酶法制备甲壳低聚糖的催化剂种类和特性、生物反应器选用及其工艺优化的最新进展进行综述,对酶法生产甲壳低聚糖进行总结并展望未来的研发重点。     

产气肠杆菌菌体内核苷磷酸化酶的诱导表达

目的:阿糖腺苷(Ara-A)是一种广谱抗病毒药物,临床上用于治疗多种病毒性疾病.同时也是合成阿糖腺苷单磷酸(Ara-AMP)的重要原料.本课题旨在寻找一种高效酶法生产嘌呤类阿糖核苷的方法.方法:以产气肠杆菌完整细胞为酶源,研究产气肠杆菌菌体培养条件对核苷磷酸化酶的影响及其诱导性.结果:胸苷磷酸化酶、尿苷磷酸化酶和嘌呤核苷磷酸化酶均可被多种核苷、核苷酸甚至碱基诱导.胞苷或胞苷酸的添加量为15-20mmol/L,诱导时间在0-8小时均可.经胞苷和胞苷酸诱导的菌体可使酶反应时间缩短6倍,大大提高了反应效率.经诱导的菌体,在反应后仍保持较高的核苷磷酸化酶活力;而未经诱导的菌体,一次反应后即丧失大量的酶活力.结论:核苷磷酸化酶的活性可以通过诱导而提高,以此优化阿糖腺苷的生产.     

重组β-葡萄糖苷酶生产龙胆低聚糖的工艺条件优化

摘要：【目的】β-葡萄糖苷酶可用于酶法生产龙胆低聚糖。为了给龙胆低聚糖的生产提供大 量的酶来源,构建基因工程菌表达黑曲霉(CMI CC 324626)β-葡萄糖苷酶基因（bgl）并研究重组酶生产龙胆低聚糖的工艺条件。【方法】将bgl克隆到表达载体pPIC9K,转化毕赤酵母（Pichia pastoris）KM71。表达产物通过HPLC和LC-MS鉴定了其可用于生产龙胆低聚糖的转苷活性,并对酶转化葡萄糖生产龙胆低聚糖的反应条件进行了优化。【结果】实现了β-葡萄糖苷酶的过量表达。当底物葡萄糖浓度为80%,反应pH4.5,温度为60℃,加酶量为每克葡萄糖60 U,添加1 mmol/L的K+,转化周期为48 h,龙胆低聚糖累计达到最大为50 g/L。【结论】本研究是国内外首次利用重组酶酶法生产龙胆低聚糖的报道。     

糖链的化学合成

作为生物大分子之一,糖链的研究还没有像蛋白质和核酸那样深入。现阶段糖链的获得仍然存在很大的挑战,阻碍了糖生物学的发展。鉴于通过分离手段得到所需的糖链很困难,酶法合成糖链亦存在着诸多问题,因此目前化学方法合成糖链是最佳的选择。对近年来糖链的化学合成所取得的最新进展进行简要的介绍,主要包括一釜合成、固相合成和标签辅助的合成三个方面。     

Biosynthesis of lipid-linked oligosaccharides. I. Preparation of lipid-linked oligosaccharide substrates.

In order to purify the glycosyltransferases involved in the assembly of lipid-linked oligosaccharides and to be able to study the acceptor substrate specificity of these enzymes, methods were developed to prepare and purify a variety of lipid-linked oligosaccharides, differing in the structure of the oligosaccharide moiety. Thus, Man9 (GlcNAc)2-pyrophosphoryl-dolichol was prepared by isolation and enzymatic synthesis using porcine pancreatic microsomes, while Glc3Man9(GlcNAc)2-PP-dolichol was isolated from Madin-Darby canine kidney cells. Treatment of these oligosaccharide lipids with a series of selected glycosidases led to the preparation of Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6(Man alpha 1,3)Man alpha 1,6]Man beta 1,4GlcNAc beta 1,4GlcNAc-PP-dolichol; Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6]Man beta 1,4GlcNAc beta 1, 4GlcNac-PP-dolichol; and Man alpha 1,6(Man alpha 1,3)Man alpha 1, 6[Man alpha 1,3]Man beta 1,4GlcNAc-beta 1,4GlcNAc-PP-dolichol. The preparation, isolation, and characterization of each of these lipid-linked oligosaccharide substrates are described.     

Rabbit muscle phosphorylase derivatives with oligosaccharides covalently bound to the glycogen storage site

Linear maltooligosaccharides, e.g., maltoheptaose or terminal 4-O-methylmaltoheptaose, activated by cyanogen bromide, react covalently with rabbit muscle phosphorylases b and a (EC 2.4.1.1). Site-specific modification prevents further binding to glycogen and shifts the phosphorylase a tetramer-dimer equilibrium in favor of the dimer. Use was made of these properties to separate by affinity chromatography and gel filtration phosphorylase a dimers with specifically bound oligosaccharide from unspecifically modified products. The phosphorylase a-maltoheptaose derivative carries one oligosaccharide residue per monomer and can be distinguished from the native enzyme by its electrophoretic mobility in polyacrylamide gels or by affinity electrophoresis. Phosphorylase a preparations with covalently bound maltooligosaccharides are enzymatically active in the presence of a primer and alpha-D-glucopyranose 1-phosphate (glucose-1-P). Methylation of the nonreducing chain terminus of the bound oligosaccharide has no effect on glycogen synthesis. These findings exclude the participation of bound oligosaccharides in chain elongation. Purified covalent phosphorylase a-maltoheptaose complexes are stable dimers. They are no longer activated by glycogen. The properties of covalently modified phosphorylase-oligosaccharides are consistent with and provide direct evidence for the existence of a glycogen storage site in rabbit muscle phosphorylases. Covalent occupation of the storage site renders the affinity of glucose-1-P to phosphorylase a independent of modulation by glycogen, supporting the assumption that the glycogen storage site is involved in interactions with the catalytic site.     

不同处理方法对灵芝β-葡聚糖降解效果的比较研究

本研究采用酸法、碱法、酶法和微波法对灵芝β-葡聚糖进行降解,通过降解率、产物分子量变化、产物聚合度分布等指标比较了不同方法的降解效果。结果表明,微波法降解率高达94%,处理后产物的分子量明显降低,寡糖产物聚合度分布广。酶法降解率约为40%,寡糖产物中含有DP2-5的成分。酸法及碱法降解率低于20%,寡糖产物少。研究表明,与其他3种方法相比,微波法降解率高、产物丰富、操作条件易于控制,是一种简单、高效的降解灵芝β-葡聚糖、制备灵芝β-葡寡糖的方法。     

Incompletely processed LH molecules synthesized by rat gonadotrophs treated with inhibitors of oligosaccharide processing

Inhibitors of N-linked oligosaccharide processing are useful tools for studies on the biological function of the oligosaccharide structures in glycoprotein hormones. We have synthesized molecules of lutropin (LH) containing high-mannose- and hybrid-type oligosaccharides using rat gonadotroph-enriched primary cultures in the presence of castanospermine (a glucosidase I inhibitor) or swainsonine (a mannosidase II inhibitor), in order to compare the actions of these molecules with that of the hormone containing complex-type oligosaccharides in the activation of the receptor-adenylate cyclase system. Treatment of gonadotrophs with the above inhibitors caused an increase in the synthesis of highly basic LH molecules (pI 9.6-10.0), because addition of charged carbohydrate moieties to these molecules was prevented. Characterization of the oligosaccharide structure performed by enzymatic treatment (endoglycosidase H and neuraminidase) and the use of immobilized lectins (wheat germ agglutinin and Ricinus communis agglutinin-120) showed that these inhibitor-synthesized LH molecules contained high-mannose- and hybrid-type (asialo and sialylated) oligosaccharides. Their immunological properties were similar to that of complex-type oligosaccharide LH, but they had significantly higher receptor-binding ability in comparison with a sialylated complex-type oligosaccharide LH (about 12-fold) and an asialo complex-type oligosaccharide LH (about 3-fold). It was noted that the incompletely processed molecules were less potent than complex-type oligosaccharide LH in the activation of adenylate cyclase of Leydig cells, showing about 40-60% of the activity induced by the sialylated complex-type oligosaccharide molecule. The present data indicate that the inhibition of terminal processing of N-linked oligosaccharides by castanospermine and swainsonine impairs the full hormonal function of rat LH.     

Enabling methodology for the end functionalization of glycosaminoglycan oligosaccharides

The chemical functionalization of glycosaminoglycans is very challenging due to their structural heterogeneity and polyanionic character; but as an enabling technology it promises rich rewards in terms of the structural and biological data it will afford. This review surveys the known methods for the preparation of glycosaminoglycan oligosaccharides and conditions for the selective functionalization of both the reducing and non-reducing ends. The synthetic merits of each approach are discussed, together with the structural modification of the glycosaminoglycan oligosaccharide which they confer. Recent applications of this methodology are highlighted, including introduction of functional labels for gel mobility shift assays and NMR studies of glycosaminoglycan-protein complexes, and synthesis of immobilised glycosaminoglycan arrays.     

Detection and isolation of oligosaccharides with Lea and Leb blood group activities by affinity chromatography using monoclonal antibodies

Affinity columns prepared by immobilizing monoclonal antibodies that specifically recognize the Lea or the Leb blood group antigens can be used for analytical or preparative isolation of oligosaccharides with the corresponding reactivities. The number of immobilized functional antibody combining sites on a column and the dissociation constants for standard oligosaccharides are determined by frontal analysis. By employing a simple approximation [K.-I. Kasai et al. (1986) J. Chromatogr. 376, 33-47] these parameters can be used to rationally design columns with properties appropriate for zonal affinity chromatography. The affinity for binding of the Lea-active oligosaccharide lacto-N-fucopentaose II (LNF II) by the anti-Lea antibody CO-514 doubles for each 8 degrees C downward shift in temperature between 37 and 4 degrees C. By zonal chromatography, Lea- or Leb-active oligosaccharides are recovered from a complex mixture of milk oligosaccharides containing more than a 20-fold molar excess of structurally similar but antigenically distinct oligosaccharides. The capacity for preparative isolation of an oligosaccharide increases in a linear fashion with the amount of antibody loaded on the solid support. The monoclonal antibodies used in these studies are products of hybridomas derived from mice immunized with human colorectal carcinoma cell lines [M. Blaszczyk et al. (1984) Arch. Biochem. Biophys. 233, 161-168]. The experiments establish that affinity chromatography applied to mixtures of oligosaccharides released by enzymatic or chemical cleavage of glycoconjugates may simplify the task of isolating and characterizing biologically interesting target antigens of monoclonal antibodies.     

Enzymatic synthesis of oligosaccharides by two glycosyl hydrolases of Sulfolobus solfataricus

The importance of carbohydrates in a variety of biological functions is the reason that interest has recently increased in these compounds as possible components of therapeutic agents. Thus, the need for a technique allowing the easy synthesis of carbohydrates and glucoconjugates is an emerging challenge for chemists and biologists involved in this field. At present, enzymatic synthesis has resulted in the most promising approach for the production of complex oligosaccharides. In this respect, the enzymological characteristics of the catalysts, in term of regioselectivity, substrate specificity, and operational stability, are of fundamental importance to improve the yields of the process and to widen the repertoire of the available products. Here, two methods of oligosaccharide synthesis performed by a glycosynthase and by an alpha-xylosidase from the hyperthermophilic archaeon Sulfolobus solfataricus are briefly reviewed. The approaches used and the biodiversity of the catalysts together are key features for their possible utilization in the synthesis of oligosaccharides.     

Monolith enzymatic microreactor at the frontier of glycomic toward a new route for the production of bioactive oligosaccharides

Recent research in the area of bioactive carbohydrates has shown the efficiency of oligosaccharides as signal molecules in a lot of biological activities. Newly observed functions of oligosaccharides and their abilities to act as specific regulatory molecules on various organisms have been more and more described. A successful development of these bioactive molecules in future needs efficient processes for specific oligosaccharides production. To exploit them for putative industrial scale up processes, two main strategies are currently investigated: the synthesis (chemical or bioconversion processes) and the polysaccharide cleavage (chemical, physical or biological processes). Nevertheless, if new manufacturing biotechnologies have considerably increased the development of these functional molecules, the main drawback limiting their biological applications is the complexity to engender specific glycosidic structures for specific activities. In the recent years, new enzymatic reactors have been developed, allowing the automatic synthesis of oligosaccharide structures. This review focuses on the knowledge in the area of bioactive oligosaccharides and gives the main processes employed to generate them for industrial applications with challenges of monolith microreactors.     

Enzymatic catalysis in crystals of Escherichia coli maltodextrin phosphorylase

The bacterial enzyme maltodextrin phosphorylase (MalP) catalyses the phosphorolysis of an alpha-1,4-glycosidic bond in maltodextrins, removing the non-reducing glucosyl residues of linear oligosaccharides as glucose-1-phosphate (Glc1P). In contrast to the well-studied muscle glycogen phosphorylase (GP), MalP exhibits no allosteric properties and has a higher affinity for linear oligosaccharides than GP. We have used MalP as a model system to study catalysis in the crystal in the direction of maltodextrin synthesis. The 2.0A crystal structure of the MalP/Glc1P binary complex shows that the Glc1P substrate adopts a conformation seen previously with both inactive and active forms of mammalian GP, with the phosphate group not in close contact with the 5'-phosphate group of the essential pyridoxal phosphate (PLP) cofactor. In the active MalP enzyme, the residue Arg569 stabilizes the negative-charged Glc1P, whereas in the inactive form of GP this key residue is held away from the catalytic site by loop 280s and an allosteric transition of the mammalian enzyme is required for activation. The comparison between MalP structures shows that His377, through a hydrogen bond with the 6-hydroxyl group of Glc1P substrate, triggers a conformational change of the 380s loop. This mobile region folds over the catalytic site and contributes to the specific recognition of the oligosaccharide and to the synergism between substrates in promoting the formation of the MalP ternary complex. The structures solved after the diffusion of oligosaccharides (either maltotetraose, G4 or maltopentaose, G5) into MalP/Glc1P crystals show the formation of phosphate and elongation of the oligosaccharide chain. These structures, refined at 1.8A and at 2.2A, confirm that only when an oligosaccharide is bound to the catalytic site will Glc1P bend its phosphate group down so it can contact the PLP 5' phosphate group and promote catalysis. The relatively large oligosaccharide substrates can diffuse quickly into the MalP/Glc1P crystals and the enzymatic reaction can occur without significant crystal damage. These structures obtained before and after catalysis have been used as frames of a molecular movie. This movie reveals the relative positions of substrates in the catalytic channel and shows a minimal movement of the protein, involving mainly Arg569, which tracks the substrate phosphate group.