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1.
糖苷合成酶——— 一类新型的寡糖高效合成工具 总被引:5,自引:0,他引:5
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. 相似文献
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β-N-乙酰氨基己糖苷酶(EC.3.2.1.52)是一类重要的糖苷水解酶,在自然界中催化简单的β-N-乙酰氨基己糖苷或复杂的寡糖链、多糖链中末端N-乙酰己糖苷键的水解,在微生物、植物和动物中广泛分布,具有重要的生物学功能。某些种类的β-N-乙酰氨基己糖苷酶在一定的人为条件下水解β-N-乙酰氨基己糖苷键的同时还具有转糖基作用,能将β-N-乙酰氨基己糖基转移到不同的羟基化合物上,合成β-N-乙酰氨基己糖苷化合物,在糖链合成上具有应用的潜力。本文综述了β-N-乙酰氨基己糖苷酶的结构和催化机制、酶的生物学功能以及酶在β-N-乙酰氨基己糖苷化合物合成中的应用,以促进β-N-乙酰氨基己糖苷酶的进一步研究和开发应用。 相似文献
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本文对一种新的甜菊糖基转移酶进行了基因克隆和功能分析。获得的基因cDNA全长1419bp,编码473个氨基酸,蛋白质分子量约53.2K Da。与常见的糖基转移酶基因比较,相似性达44%以上,且具有糖基转移酶的保守序列。体外异源表达获得的融合蛋白,具有在花青素类和甜菊醇等糖基受体上转糖基的酶活性。在对一系列不同底物的酶活性进行比较后,推测这种糖基转移酶在体内参与了甜菊糖苷的合成。结果表明,具有广泛的底物活性的类黄酮类糖基转移酶,在甜菊体内不仅对类黄酮转糖基,而且在生成水溶性甜菊糖苷的过程中也扮演重要的角色。 相似文献
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采用盐析、DE 52、Q-Sepharose Fast Flow阴离子交换层析、Toyopearl Butyl 650C疏水层析以及Sephacryl S-300 HR凝胶过滤层析联用的方法, 从Leifsonia shinshuensis DICP 16菌体中纯化出一种β-木糖苷酶.分离后该酶在SDS-PAGE 上呈单一蛋白质条带, 通过SDS-PAGE和凝胶过滤层析法, 测得该酶是一个由两个分子量约为91 kD的相同亚基组成的同源二聚体.其水解对硝基苯酚木糖苷(pNPX)的最适反应温度为55°C, pH值为7.0.该木糖苷酶在45°C以下, pH 6.0~11.0之间具有很好的稳定性.在45°C, pH值为7.0的条件下, 水解pNPX的Km, Vmax分别为1.04 mmol/L, 0.095 mmol/(min·mg).研究不同的金属离子对该酶的活性影响, 发现Fe2+和Cu2+是很强的抑制剂.通过对天然木糖苷化合物的水解测试, 发现该酶可以水解人参皂苷Rb3的木糖基, 产生人参皂苷Rd, 却不能水解紫杉烷木糖苷的木糖基. 相似文献
11.
Sialidases, or neuraminidases (EC 3.2.1.18), belong to a class of glycosyl hydrolases that release terminal N-acylneuraminate residues from the glycans of glycoproteins, glycolipids, and polysaccharides. In bacteria, sialidases can
be used to scavenge sialic acids as a nutrient from various sialylated substrates or to recognize sialic acids exposed on
the surface of the host cell. Despite the fact that bacterial sialidases share many structural features, their biochemical
properties, especially their linkage and substrate specificities, vary widely. Bacterial sialidases can catalyze the hydrolysis
of terminal sialic acids linked by the α(2,3)-, α(2,6)-, or α(2,8)-linkage to a diverse range of substrates. In addition,
some of these enzymes can catalyze the transfer of sialic acids from sialoglycans to asialoglycoconjugates via a transglycosylation
reaction mechanism. Thus, some bacterial sialidases have been applied to synthesize complex sialyloligosaccharides through
chemoenzymatic approaches and to analyze the glycan structure. In this review article, the biochemical features of bacterial
sialidases and their potential applications in regioselective hydrolysis reactions as well as sialylation by transglycosylation
for the synthesis of sialylated complex glycans are discussed. 相似文献
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Teaching old enzymes new tricks: engineering and evolution of glycosidases and glycosyl transferases for improved glycoside synthesis. 总被引:2,自引:0,他引:2
The therapeutic potential of glycosides has made them an attractive target for drug development. The biological extraction and chemical synthesis of these molecules is often challenging and low yielding, thus alternative methods for the synthesis of polysaccharides are being pursued. A new class of enzymes, glycosynthases, which are nucleophile mutants of glycosidases, can perform the transglycosylation reaction without hydrolyzing the product, and thus provide a valuable resource for polysaccharide and glycan synthesis. Directed evolution of glycosynthases has expanded the repertoire of glycosidic linkages formed and the donors and acceptors (both sugar and nonsugar) that can be used by the glycosynthase. The application of new screening methods, such as FACS, to the directed evolution of glycosynthases will aid in the development of enzymes that are able to efficiently synthesize new, and therapeutically relevant glycosidic linkages. 相似文献
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Two long-standing questions about glucansucrases (EC 2.4.1.5) are how they control oligosaccharide versus polysaccharide synthesis and how they direct their glycosidic linkage specificity. This information is required for the production of tailor-made saccharides. Mutagenesis promises to be an effective tool for enzyme engineering approaches for altering the regioselectivity and acceptor substrate specificity. Therefore, we chose the most conserved motif around the transition state stabilizer in glucansucrases for a random mutagenesis of the glucansucrase GTFR of Streptococcus oralis, yielding different variants with altered reaction specificity. Modifications at position S628 achieved by saturation mutagenesis guided the reaction toward the synthesis of short chain oligosaccharides with a drastically increased yield of isomaltose (47%) or leucrose (64%). Alternatively, GTFR variant R624G/V630I/D717A exhibited a drastic switch in regioselectivity from a dextran type with mainly alpha-1,6-glucosidic linkages to a mutan type polymer with predominantly alpha-1,3-glucosidic linkages. Targeted modifications demonstrated that both mutations near the transition state stabilizer, R624G and V630I, are contributing to this alteration. It is thus shown that mutagenesis can guide the transglycosylation reaction of glucansucrase enzymes toward the synthesis of (a) various short chain oligosaccharides or (b) novel polymers with completely altered linkages, without compromising their high transglycosylation activity and efficiency. 相似文献
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Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria. 总被引:1,自引:0,他引:1
Sacha A F T van Hijum Slavko Kralj Lukasz K Ozimek Lubbert Dijkhuizen Ineke G H van Geel-Schutten 《Microbiology and molecular biology reviews》2006,70(1):157-176
Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and alpha-glucan products is reviewed. The GS and FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with alpha-amylase enzymes (family GH13), with a predicted permuted (beta/alpha)(8) barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of alpha-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize beta-fructan polymers with either beta-(2-->6) (inulin) or beta-(2-->1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed beta-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either beta-(2-->6) or beta-(2-->1) linkages, degree and type of branching, and fructan molecular mass remain to be identified. 相似文献
15.
Ondřej Kosík Richard P. Auburn Steven Russell Eva Stratilová Soňa Garajová Maria Hrmova Vladimír Farkaš 《Glycoconjugate journal》2010,27(1):79-87
Polysaccharide transglycosylases catalyze disproportionation of polysaccharide molecules by cleaving glycosidic linkages in
polysaccharide chains and transferring their cleaved portions to hydroxyl groups at the non-reducing ends of other polysaccharide
or oligosaccharide molecules. In plant cell walls, transglycosylases have a potential to catalyze both cross-linking of polysaccharide
molecules and grafting of newly arriving polysaccharide molecules into the cell wall structure during cell growth. Here we
describe a polysaccharide microarray in form of a glycochip permitting simultaneous high-throughput monitoring of multiple
transglycosylase activities in plant extracts. The glycochip, containing donor polysaccharides printed onto nitrocellulose-coated
glass slides, was incubated with crude plant extracts, along with a series of fluorophore-labelled acceptor oligosaccharides.
After removing unused labelled oligosaccharides by washing, fluorescence retained on the glycochip as a result of transglycosylase
reaction was detected with a standard microarray scanner. The glycochip assay was used to detect transglycosylase activities
in crude extracts from nasturtium (Tropaeolum majus) and mouse-ear cress (Arabidopsis thaliana). A number of previously unknown saccharide donor-acceptor pairs active in transglycosylation reactions that lead to the
formation of homo- and hetero-glycosidic conjugates, were detected. Our data provide experimental support for the existence
of diverse transglycosylase activities in crude plant extracts. 相似文献
16.
Abstract Glycosylation is considered to be an important reaction for the chemical modification of compounds with useful biological activities. Glycoside hydrolases are biotechnologically attractive enzymes which can be used in synthetic reactions for assembling glycosidic linkages with absolute stereoselectivity at an anomeric centre. Most of these enzymes are commercially available but there is great interest in the search for new biocatalysts with original catalytic characteristics. The marine environment has shown to be a very interesting source for new glycosyl hydrolases for both hydrolytic and synthetic aspects. In particular, Aplysia fasciata a marine herbivorous mollusc has been shown to be a potent producer of a library of glycoside hydrolases applied to the synthesis of glycosidic bonds. The impressive assortment of glycosidases in marine organisms clearly indicates that the potential biodiversity of these enzymes is still largely unexplored and that potential applications of biocatalysts from the sea will increase in the near future. 相似文献
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As a general strategy for determining the chemical function of the class of enzymes that cleaves glycosidic linkages with phosphate, the first mass spectrometry and direct detection assay for sugar phosphorylases has been developed and used to study the inhibition and minimal binding requirements of rabbit muscle phosphorylase b. In contrast to the currently employed assays for these enzymes that measure the nonphysiologically relevant reverse reaction of glycosidic bond synthesis and thereby require prior knowledge of not just one but two sugar components, this new method has the potential to greatly reduce the complexity in discovering the substrate specificity of a new enzyme. Certain phosphorylases can catalyze the degradation of glycogen into alpha-D-glucose-1-phosphate and are targets for the development of antidiabetic therapeutics. By electrospray ionization mass spectrometry analysis, the kinetic parameters K(m), V(max), and K(i) (for alpha/beta-D-glucose) have been determined for the rabbit muscle phosphorylase b. This enzyme accepts maltoheptaose, maltohexaose, and maltopentaose as substrates in the direction of glycogen degradation, but the tetrasaccharide maltotetraose cannot serve as a substrate for this phosphorylysis reaction. 相似文献
18.
The specificity of viral and bacterial sialidases for alpha(2-3)- and alpha(2-6)-linked sialic acids in glycoproteins 总被引:5,自引:0,他引:5
The anomeric specificity of six sialidases (Vibrio cholerae, Arthrobacter ureafaciens, Clostridium perfringens, Newcastle disease virus, fowl plague virus and influenza A2 virus sialidases) was assessed with sialylated antifreeze glycoprotein, ovine submandibular gland glycoprotein and alpha 1-acid glycoprotein, resialylated specifically in alpha(2-3) or alpha(2-6) linkage with N-acetylneuraminic acid or N-glycolylneuraminic acid using highly purified sialyltransferases. The rate of release of sialic acid from these substrates was found to correlate well with the specificity observed earlier with the same sialidases using small oligosaccharide substrates, i.e., alpha(2-3) glycosidic linkages are hydrolyzed faster than alpha(2-6) linkages, with the exception of the enzyme from A. ureafaciens. Sialidase activity was higher with N-acetylneuraminic acid when compared with N-glycolylneuraminic acid. The studies also showed that the core oligosaccharide and protein structure in glycoproteins may influence the rate of release for different glycosidic linkages. 相似文献
19.
Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria 总被引:6,自引:0,他引:6
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Sacha A. F. T. van Hijum Slavko Kralj Lukasz K. Ozimek Lubbert Dijkhuizen Ineke G. H. van Geel-Schutten 《Microbiological reviews》2006,70(1):157-176
Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and α-glucan products is reviewed. The GSand FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with α-amylase enzymes (family GH13), with a predicted permuted (β/α)8 barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of α-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize β-fructan polymers with either β-(2→6) (inulin) or β-(2→1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed β-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either β-(2→6) or β-(2→1) linkages, degree and type of branching, and fructan molecular mass remain to be identified. 相似文献
20.
Erika Fazekas Katalin Szabó Lili Kandra Gyöngyi Gyémánt 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(10):1976-1981
β-Amylase (EC 3.2.1.2), one of the main protein of the sweet potato, is an exo-working enzyme catalyzing the hydrolysis of α(1,4) glycosidic linkages in polysaccharides and removes successively maltose units from the non-reducing ends. The enzyme belongs to glycoside hydrolase GH14 family and inverts the anomeric configuration of the hydrolysis product. Multiple attack or processivity is an important property of polymer active enzymes and there is still limited information about the processivity of carbohydrate active enzymes. Action pattern and kinetic measurements of sweet potato β-amylase were made on a series of aromatic chromophor group-containing substrates (degree of polymerization DP 3-13) using HPLC method. Measured catalytic efficiencies increased with increasing DP of the substrates. Processive cleavage was observed on all substrates except the shortest pentamer. The mean number of steps without dissociation of enzyme–product complex increases with DP of substrate and reached 3.3 in case of CNPG11 indicating that processivity on longer substrates was more significant. A unique transglycosylation was observed on those substrates, which suffer processive cleavage and the substrates were re-built by the enzyme. Our results are the first presentation of a transglycosylation during an inverting glycosidase catalyzed hydrolysis. The yield of transglycosylation was remarkable high as shown in the change of the CNPG11 quantity. The CNPG11 concentration was doubled (from 0.24 to 0.54 mM) in the early phase of the reaction. 相似文献