α-转移葡萄糖苷酶的转苷作用机理探究

通过用高效液相色谱(HPLC)全程跟踪转苷反应过程的方法,对α-转移葡萄糖苷酶将麦芽糖转变为异麦芽糖的酶促反应作用机理进行了探讨。结果表明,在转化过程中α-转移葡萄糖苷酶先把麦芽糖的α-1,4糖苷键打断,分解为两个葡萄糖单元,然后再通过α-1,6糖苷键连接的方式重新键合,完成从麦芽糖到异麦芽糖的异构化过程。而由麦芽糖生成异麦芽糖的整个转苷过程是在分子内进行的。与此同时,由麦芽糖分解出来的部分葡萄糖单元通过分子间作用的方式与麦芽糖或异麦芽糖发生反应,生成潘糖或者异麦芽三糖。     

EVIDENCE FOR THE PRESENCE OF MALTASE AND α-GLUCOSIDASE ISOENZYMES IN BARLEY

An examination of the development of α-glucosidase and maltase activities (as measured by the hydrolysis of p-nitrophenyl α-D-glucoside and maltose respectively) indicated that two genotypes of Glacier barley had the same general pattern of enzyme development. However, the development of α-glucosidase activity followed a different course from that of maltase activity suggesting that separate enzyme proteins are involved in hydrolysing these substrates. Further evidence that separate enzyme proteins were responsible for hydrolysis of maltose and p-nitrophenyl α-glucoside was obtained by column chromatography of extracts of germinated barley which indicated the presence of two maltases and two α-glucosidases. The maltases and the α-glucosidases differed in molecular weight, pH of optimum activity and in thermostability. When isomaltose was used as a substrate the optimum pH and behaviour on gel chromatography were coincident with that of maltase activity but different from the α-glucosidases.     

Purification and Characterization of a Maltotriogenic α-Amylase I and a Maltogenic α-Amylase II Capable of Cleaving α-1,6-Bonds in Amylopectin

Extracellular α-amylases I and II, produced by a facultative thermophile Bacillus thermoamyloliquefaciens KP 1071 capable of growing at 30–66°C, were purified to homogeneity. α-Amylase I consisted of a single polypeptide with methionine residue at the NH₂-terminus. α-Amylase II consisted of two equivalent polypeptides each comprising a methionine at the NH₂-terminus. α-Amylase I hydrolyzed endotypically α-1,4-bonds in glycogen, amylopectin and β-limit dextrin, but not their α-1,6-bonds. α-Amylase II degraded amylopectin and β-limit dextrin in exo-fashion by cleaving preferentially α-maltose units from the non-reducing ends and hydrolyzing their α-1,6-branch points. α-Amylase II hydrolyzed maltotriose, phenyl-α-maltoside, α- and β-cyclodextrins and pullulan, whereas α-amylase I had no activity for all these sugars. α-Amylases I and II hydrolyzed maltotetraose, maltopentaose, α-limit dextrin and amylose, but they were inactive for maltose, isomaltose and panose. It was suggested that α-amylase I is the most thermostable type of hitherto known maltotriogenic endo-acting α-amylases, and α-amylase II is the first maltogenic exo-acting α-amylase able to split α-1,6-bonds in amylopectin.     

Production and Characterization of Branched Oligosaccharides from Liquefied Starch by the Action of B. Licheniformis Amylase

A branched oligosaccharides (BOS) mixture was produced from liquefied starch solution using a maltogenic amylase of Bacillus licheniformis (BLMA). The BOS mixture was produced by both α-1,4-bond hydrolyzing and α-1,6-transglycosylation activities of BLMA, and it contained 58.3% of various branched oligosaccharides. Small branched oligosaccharides such as isomaltose, isopanose, and panose were identified in the mixture by various analyses including high performance ion-chromatography (HPIC). Major branched DP4 and DP5 molecules in the mixture were determined as 6²-O-α-maltosylmaltose, 6³-O-α-maltosyl-maltotriose and 6²-O-α-maltotriosyl-maltose, respectively. Time course study of BOS production suggested that the hydrolysis and transglycosylation reactions catalyzed by BLMA were coupled. BLMA was likely to transfer a sugar moiety hydrolyzed from a non-reducing end of maltooligosaccharide, mainly maltose, to another moiety of sugar via the formation of α-1,6-linkage. Immobilization of BLMA was attempted as an effort to achieve a continuous process for BOS production. the immobilized enzyme showed improved thermal stability and slight loss of enzyme activity was observed during repeated usage.     

Characterization of Three Fungal Isomaltases Belonging to Glycoside Hydrolase Family 13 That Do not Show Transglycosylation Activity

α-1,6-Glucosidase (isomaltase) belongs to glycoside hydrolase (GH) families 13 and 31. Genes encoding 3 isomaltases belonging to GH family 13 were cloned from filamentous fungi, Aspergillus oryzae (agl1), A. niger (agdC),and Fusarium oxysporum (foagl1), and expressed in Escherichia coli. The enzymes hydrolyzed isomaltose and α-glucosides preferentially at a neutral pH, but did not recognize maltose, trehalose, and dextran. The activity of AgdC and Agl1 was inhibited in the presence of 1 % glucose, while Foagl1 was more tolerant to glucose than the other two enzymes were. The three fungal isomaltases did not show transglycosylation when isomaltose was used as the substrate and a similar result was observed for AgdC and Agl1 when p-nitrophenyl-α-glucoside was used as the substrate.     

Kinetics of Reactions with Amyloglucosidase and their Relevance to Industrial Applications

The two types of intramolecular bonds existing in native starch, the α-1,4 and the α-1,6 linkage, are shown to have a largely different free enthalpy of hydrolysis. Hence, on incubation of glucose or a dextrin with an enzyme containing α-1,4 as well as α-1,6 degrading activities, a situation finally results in which the isomaltose concentration is higher by a factor of six than the maltose concentration. These observations are, in combination with simple mass action law relationships, applied to the modelling of the degradation of dextrins by the enzyme amyloglucosidase. The model is shown to account for the known transient top in DE (dextrose equivalent) on incubation of dextrins with an amyloglucosidase. Furthermore, increasing the dry solids content of the dextrins solution is shown to have a pronounced influence on the DE versus time curves. The influence of the relative activities of the α-1,4 and the α-1,6 degradation activities is discussed. The effect of immobilization on the performance of the enzyme is analyzed on a qualitative base.     

Purification and Characterization of Extracellular Isoamylase from Flavobacterium sp

An extracellular isoamylase from Flavobacterium sp., was purified by fractionation with ammonium sulfate, DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose column chromatography. Single band of the debranching activity of the purified enzyme was detected by polyacrylamide gel electrophoresis. The enzyme efficiently hydrolyzed α-1,6-glucosidic linkage of glycogen and amylopectin and formed amylose chains, but did not hydrolyze pullulan. The enzyme released maltotriose from ß-limit dextrin of waxy maize amylopectin and glycogen, but no detectable maltose and glucose. Action of the isoamylase is similar to other microbial isoamylases but its physical properties are different.     

Comparison of the antioxidant and pro‐oxidant activities of broccoli amino acids with those of common food additives

The antioxidant and pro‐oxidant activities of broccoli amino acids were compared with those of common food additives. In decreasing order, the data showed that Asp, SMC, GABA, Glu, Gln, Pro, Phe, Leu, Lys, Arg, Asn, Val, Ile, His, Ser, Gly, Orn and Ala, when dissolved in water at concentrations of 0.5 and 0.05 mM , partially inhibited damage to deoxyribose in the presence of ferric‐EDTA and H₂O₂. In contrast, Tyr and Thr acted as pro‐oxidants in this system. The amino acids present in broccoli had no hydrogen peroxide‐scavenging effect. When dissolved in water, methanol or ethanol, SMC, Glu, Thr, Gln, Ser, GABA, Pro, Ala, Ile, Phe, Asp, Orn and Tyr inhibited lipid peroxidation. However, Asn, Val, Arg, Leu, Lys, His and Gly were not effective in decreasing peroxidation at concentrations of 0.5 and 0.05 mM . Asp > SMC > Ala > Phe > Hys > Orn > Gln = Ser > Lys > Leu = GABA = Gly > Tyr > Arg = Thr > Val > Asn > Pro > Ile > Glu (p < 0.025) showed scavenging activity towards hypochlorous acid, protecting α₁‐antiproteinase against inactivation. In this paper it has been established that some amino acids premixed with propyl gallate increase its hypochlorous acid‐scavenging capacity, while other amino acids have an additive effect with propyl gallate, permitting smaller quantities of propyl gallate to be used as food additives in some products which contain these amino acids. © 2001 Society of Chemical Industry     

A Novel α-Glucosidase of the Glycoside Hydrolase Family 31 from Aspergillus sojae

We characterized an α-glucosidase belonging to the glycoside hydrolase family 31 from Aspergillus sojae. The α-glucosidase gene was cloned using the whole genome sequence of A. sojae, and the recombinant enzyme was expressed in Aspergillus nidulans. The enzyme was purified using affinity chromatography. The enzyme showed an optimum pH of 5.5 and was stable between pH 6.0 and 10.0. The optimum temperature was approximately 55 °C. The enzyme was stable up to 50 °C, but lost its activity at 70 °C. The enzyme acted on a broad range of maltooligosaccharides and isomaltooligosaccharides, soluble starch, and dextran, and released glucose from these substrates. When maltose was used as substrate, the enzyme catalyzed transglucosylation to produce oligosaccharides consisting of α-1,6-glucosidic linkages as the major products. The transglucosylation pattern with maltopentaose was also analyzed, indicating that the enzyme mainly produced oligosaccharides with molecular weights higher than that of maltopentaose and containing continuous α-1,6-glucosidic linkages. These results demonstrate that the enzyme is a novel α-glucosidase that acts on both maltooligosaccharides and isomaltooligosaccharides, and efficiently produces oligosaccharides containing continuous α-1,6-glucosidic linkages.     

Characterization of Pullulanase and α-Amylase Activities of a Thermus sp. AMD33

Thermostable Thermus sp. AMD 33 pullulanases (I and II) capable of cleaving α-1,6-links in pullulan as well as α-1,4-glucosidic linkages in amylose were purified to electrophoretically homogeneous states. Relative molecular masses and pI values were determined as 135,000 (I and II) by SDS-PAGE and 4.2 (I) and 4.3 (II) by isoelectric focusing, respectively. The pullulanase and α-amylase activities of the purified enzyme II responded similarly to temperature and pH, with optima at 70°C and pH 5.5–6.0. Both activities were activated by Ca²⁺ and inhibited by Hg²⁺, Fe³⁺, NBS, DBS, SDS and urea to almost the same extent. Both activities were also inhibited competitively by CDs. Enzyme II catalyzed the hydrolysis of α-1,6-glucosidic linkages in maltosyl- and maltotriosyl-α-CD as well as that of α-1,4-bonds in amylose and related linear malto-oligosaccarides larger than maltotriose, but exhibited no action on panose, isopanose or glucosyl α-CD.     

ISOLATION AND CHARACTERISATION OF THE AMYLOTIC SYSTEM OF SCHWANNIOMYCES CASTELLII

The amylolytic system of Schwanniomyces castellii has been isolated and purified by means of ultrafiltration followed by polyacrylamide gel electrophoresis. Both α-amylase and glucoamylase were purified. α-Amylase activity was stable from pH 5·5 to 6·5 and glucoamylase activity was stable at a more acidic range of pH 4·2 to 5·5. The optimal temperature of α-amylase activity was between 30 and 40°C with rapid deactivation at 70°C. The optimal temperature of glucoamylase was 40 to 50°C with rapid decline of activity at 60°C. The K_m of α-amylase with soluble starch as the substrate was 1·15 mg/ml and the K_m of glucoamylase with the same substrate was 10·31 mg/ml. Glucoamylase was able to hydrolyze α-1, 4 and α-1,6 glucosidic linkages, as demonstrated by its ability to hydrolyse maltose and isomaltose respectively, whereas α-amylase could hydrolyse α-1,4 glucosidic linkages only. α-Amylase was shown to be a glycoprotein, whereas no carbohydrates were associated with glucoamylase.     

Relative distribution of free amino acids in buckwheat

The most abundant amino acid in the sprouts of common buckwheat (CB) was Val (40%), followed by Tyr (28%), whereas Val accounted for 62% in tatary buckwheat (TB). The buckwheat stem and root commonly contained Gln (40–42% in stem; 30–37% in root). Thus, soluble amino nitrogen source is used for Gln in buckwheat. The main difference of amino acid distribution in 3 tissues between CB and TB was Tyr in sprouts. A low level of Tyr in TB was presumably resulted from the conversion to other phenolic metabolites. The content of essential free amino acids in TB sprout was 53% higher than that in CB. Thus, the TB sprouts are beneficial to the human nutrition.     

小麦胚水溶性糖蛋白(WGWSGP)的化学组成及结构分析

为研究小麦胚水溶性糖蛋白(WGWSGP)的化学组成和结构,采用化学比色法、氨基酸自动分析和气相色谱等方法分别对其蛋白质和糖的含量和组成进行了检测;并通过β-消去、紫外扫描、红外光谱和核磁共振等对其结构进行了初步分析。分析结果表明:WGWSGP中糖的含量高达43.6%,并且甘露糖是主要中性糖组分,其含量占中性糖总量的94%;WGWSGP含有多种人体所必需的氨基酸,其中天冬氨酸、谷氨酸、甘氨酸、缬氨酸的含量较高,不含蛋氨酸。WGWSGP中糖与蛋白的肽链之间的连接点类型是O-糖肽键;聚糖是以α糖苷键相连,寡糖链的糖苷键构型为α型吡喃糖;寡糖链的α-D-甘露糖吡喃糖残基上的C-2、C-3位或C-4位羟基没有发生取代;样品中含有己糖醛酸和6-位脱氧糖的甲基存在。     

Relationships Between Structure and Activity in the α-Amylase Family of Starch-metabolising Enzymes

α-Amylases are known to be multidomain proteins, i.e., the molecules consist of several folding units. Each α-amylase is believed, however, to have a catalytic domain consisting, of a barrel of eight parallel α-strands surrounded by eight α-strands. with an extra helix inserted after the sixth γ-strands. The α-strands and helices alternate along the polypeptide chain and are linked together by irregular loops. Amino acid residues situated on the loops joining the C-terminal end of each α-strand to the N-terminal end of the following helix make up the active site of the enzymes. A similar structure has been found in cyclodextrin glucanotransfcrases and it is now believed that such a (α/α)8-barrel also constitutes the catalytic domain of enzymes active on α-1.6-glucosidic bonds, and of enzymes with dual specificity for both α-1.4- and α-1.6- bonds. Knowledge of the three-dimensional structure of α-amylases and cyclodextrin glucanotransferase has made possible identification of structural features important for enzymic activity and specificity. By analogy, some general conclusions are reached concerning pullulanase, isoamylase. oligo-1,6-glucosidase, neopullulanase and branching enzymes.     

Prediction of sugars and acids in Chinese rice wine by mid-infrared spectroscopy

The use of Fourier transform mid-infrared spectroscopy (FT-MIR) for the rapid determination of sugars and acids in Chinese rice wine was presented in this study. Calibration models were developed by partial least squares regression (PLSR) for eleven parameters related to sugar content and acidity—namely, total sugar, non-sugar solid, glucose, maltose, isomaltotriose, isomaltose, panose, total acid, amino acid nitrogen, pH and lactic acid. In the calibration step, most of the parameters were accurately determined, obtaining regression coefficients of calibration (r_cal) ranging from 0.821 to 0.991. In validation, regression coefficients of validation (r_val) obtained for most parameters were higher than 0.85. Unsatisfactory predictions were obtained for isomaltotriose and isomaltose with r_val being 0.488 and 0.716, respectively. The residual predictive deviation (RPD) values were also higher than or close to 2.0 for all the parameters except for isomaltose and isomaltotriose. Overall, the results indicate that MIR spectroscopy can be applied to the quality determination of Chinese rice wine.     

An active-site mutation causes enhanced reactivity and altered regiospecificity of transglucosylation catalyzed by the Bacillus sp. SAM1606 alpha-glucosidase

Bacillus sp. SAM1606 alpha-glucosidase catalyzes the transglucosylation of sucrose to produce three regioisomers of the glucosylsucroses, with theanderose (6-O(G)-glucosylsucrose) as the most abundant transfer product. To find the active-site amino acid residues which can affect the reactivity and regiospecificity of the glucosyl transfer, 16 mutants with amino acid substitutions near the active site were allowed to react with 1.75 M sucrose at 60 degrees C, pH 6.0, and the course of transglucosylation as well as the product specificity were analyzed. The sites of the amino acid substitutions were selected by comparing the conserved amino acid sequences located near the active site of the SAM1606 enzyme with those of the Bacillus oligo-1,6-glucosidases (O16G), which have very high amino acid sequence similarities near the active site but have a distinct substrate specificity. The results showed that, among the mutated SAM1606 enzymes examined, only the mutants with substitution of Gly273 with Pro showed an altered reactivity and specificity of transglucosylation; these mutants exhibited a significantly enhanced initial velocity of glucosyl transfer, yielding isomelezitose (6-O(F)-glucosylsucrose) instead of theanderose as the major transfer product. These results indicate that the substitution of Gly273 with Pro critically governs the enhanced reactivity and altered specificity of the transglucosylation. The notion that the amino acid residue at this position is the determinant of the glucosyl-transfer specificity was further confirmed by observation that the Bacillus cereus O16G, which has a proline at the corresponding position, produced isomelezitose as the major transfer product during transglucosylation with sucrose.     

Effect of salting and roasting on the carbohydrates and proteins of Iranian pistachio kernels

The free sugars which make up 2.7% of Iranian pistachio cv Badami were identified as fructose, glucose, sucrose, maltose, isomaltose, cellobiose, raffinose and stachyose. Both protein and free amino acids contained the same seventeen amino acids but lacked tryptophan, asparagine and glutamine. After salting and roasting, total available carbohydrates, total starches and dextrins, and total free sugars all decreased compared to controls, as did a number of individual sugars, especially the reducing sugars. While free amino acids had severely decreased, total protein amino acids and individual protein amino acid were not affected at all. The decreases in reducing sugars and in free amino acids may be due in part to their taking part in Maillard reactions.     

Grape skin extract inhibits mammalian intestinal α-glucosidase activity and suppresses postprandial glycemic response in streptozocin-treated mice

Intestinal α-glucosidases are the key enzymes responsible for starch digestion and absorption and their inhibition has been proven effective in both preventing and treating diabetes through improvement of postprandial hyperglycaemia. This study, for the first time, identified that a Norton grape skin extract (GSE) significantly inhibited mammalian intestinal α-glucosidases but not other digestive enzymes including structurally relevant pancreatic α-amylase. Norton GSE inhibited rat intestinal α-glucosidases through a competitive mode with an IC₅₀ of 0.384 mg/ml. Further animal study revealed that the oral intake of Norton GSE (400 mg/kg) significantly reduced postprandial blood glucose by 30.9% in the streptozocin-treated male C57BL/6 J mice following starch challenge. These findings suggest that Norton GSE may have a unique property of suppressing postprandial blood glucose through a mechanism involving the inhibition of α-glucosidases, thereby providing a novel dietary opportunity for diabetes management.     

Rheinheimera sp.QH胞外角蛋白酶KerQH2的生物信息学分析

目的:预测海洋菌Rheinheimera sp.QH分泌的角蛋白酶KerQH2的结构特征及与底物的互作位点,为深入研究KerQH2对角蛋白底物的降解机理提供理论依据。方法:应用生物信息学技术对KerQH2的结构特征、作用于角蛋白底物的可能位点及关键氨基酸等进行分析。结果:KerQH2为S8家族丝氨酸蛋白酶,其分子表面无规则卷曲占比较高,α-螺旋与β-折叠占比较低且多位于酶分子内部。KerQH2具有保守的催化三联体（Asp8、His41和Ser197）,催化三联体区域表面静电势为中性。KerQH2催化结构域中含有大量保守的极性氨基酸及芳香族氨基酸,与底物结合有关的序列富含Gly和Ala。KerQH2主要切割疏水性氨基酸Val(V)/ILe(I)和极性氨基酸Cys(C)/Gln(Q)之间形成的肽键。结论:角蛋白酶KerQH2的特殊结构有助于其降解结构复杂角蛋白分子,这可能是海洋菌对海洋寡营养环境的一种适应性。     

Kombinierte enzymatische Stärkehydrolyse

Combined Enzymatic Starch Hydrolysis. From researches so far there comes out that glucoamylase AMG 300 L® and pullulanase Promozyme 200 L® when used in quantities the same as in preparation of Dextrozyme 225/75 L® Novo at an action on liquified starch by means of α-amylase after 48 h of saccharification already (similarly like Dextrozyme®) are able to get up to 98 DE. Chromatographic analysis proved that glucoamylase AMG 300 L Novo® and succouring it pullulanase Promozyme 200 L® are working most effectively when both enzymes are added to the liquified starch medium simultaneously. From this comes out that pullulanase hydrolyzes better α-1,6 bonds in lowmolecular dextrins than in oligosaccharides G₄ to G₇ formed at previous action of glucoamylase. At an optimum ratio of glucoamylase and pullulanase in relation to the dissolved starch after 8 h of the hydrolysis there are neither iso-sugars (isomaltose, panose), no oligosaccharides higher than G₅ and no dextrins. At the solution of the starch by α-amylase and its hydrolysis by enzymatic preparation Fungamyl 800 L Novo®, at doses 0,02–0,08% to d. s. of starch, already after 8 h the reaction of hydrolysis contents of 36–62% maltose in dry substance of hydrolyzates are reached with only traces of glucose.