Synthesis and glycosidase inhibitory activity of some N-substituted 5a-carba-β-fuco- and β-galactopyranosylamines, and selected derivatives

In the course of chemical modification of -fucosidase inhibitors of 5a-carba-fucopyranosylamine type, an N-dodecyl derivative of the enantiomer 6-deoxy-5a-carba-β-D-galactopyranosylamine demonstrated very strong inhibition of β-galactosidase and β-glucosidase. This finding led us to synthesize corresponding 6-hydroxy compounds, in order to elucidate structure–activity relationships for inhibitors of this type. Among four N-alkyl-5a-carba-β-D-galactopyranosylamines prepared, the N-octyl derivative could be demonstrated to possess moderate activity toward - and β-galactosidases, and β-glucosidase.     

Separation and properties of β-galactosidase, β-glucosidase, β-glucuronidase and n-acetyl-β-glucosaminidase from rat kidney

1. The activities of β-galactosidase, β-glucosidase, β-glucuronidase and N-acetyl-β-glucosaminidase from rat kidney have been compared when 4-methylumbelliferyl glycosides are used as substrates. 2. Separation by gel electrophoresis at pH7·0 indicated slow- and fast-moving components of rat-kidney β-galactosidase. 3. The fast-moving component is also associated with the total β-glucosidase activity and inhibition experiments indicate that a single enzyme species is responsible for both activities. 4. DEAE-cellulose chromatography and filtration on Sephadex gels suggests that the β-glucosidase component is a small acidic molecule, of molecular weight approx. 40000–50000, with optimum pH5·5–6·0 for β-galactosidase and β-glucosidase activities. 5. The major β-galactosidase component has low electrophoretic mobility, a calculated molecular weight of 80000 and optimum pH3·7.     

Evidence Against the Involvement of Galactosidase or Glucosidase in Auxin- or Acid-stimulated Growth

Research on the mode of action of auxin in the promotion of growth has shown that auxin treatment leads to hydrogen ion secretion and wall acidification. It has recently been reported that auxin stimulates cell wall β-galactosidase activity in Avena coleoptiles, presumably by causing cell wall acidification, since the pH optimum for the enzyme is about 5.0. It has been suggested that enhancement of β-galactosidase and/or other glycosidase activity mediates growth promotion by auxin or low pH. This hypothesis was tested by examining the effect of inhibitors of β-galactosidase and β-glucosidase. Severe inhibition of measureable β-galactosidase or β-glucosidase activity was found to have no effect on auxin- or acid-promoted growth. It is concluded that neither β-galactosidase nor β-glucosidase plays an important role in short term growth promotion by auxin or acid. The data do not rule out the possibility that some other cell wall glycosidase is involved in auxin or acid action.     

The Involvement of Glycosidases in the Cell Wall Metabolism of Suspension-cultured Acer pseudoplatanus Cells

Several glycosidases have been isolated from suspensioncultured sycamore (Acer pseudoplatanus) cells. These include an α-galactosidase, an α-mannosidase, a β-N-acetyl-glucosaminidase, a β-glucosidase, and two β-galactosidases. The pH optimum of each of these enzymes was determined. The pH optima, together with inhibition studies, suggest that each observed glycosidase activity represents a separate enzyme. Three of these enzymes, β-glucosidase, α-galactosidase, and one of the β-galactosidases, have been shown to be associated with the cell surface. The enzyme activities associated with the cell surface were shown to possess the ability to degrade to a limited extent isolated sycamore cell walls. It was found that the activities of β-glucosidase and of one of the β-galactosidases increase as the cells go through a period of growth and decrease as cell growth ceases.     

Effect of carbon source on the biochemical properties of β-xylosidases produced by Aspergillus versicolor

The filamentous fungus Aspergillus versicolor produced large amounts of mycelial β-xylosidase activity when grown on xylan or xylose as the only carbon source. The presence of glucose drastically decreased the level of β-xylosidase activity, while cycloheximide prevented the induction of the enzymes by xylan or xylose. The β-xylosidases induced by xylose or xylan were purified by a simple protocol involving DEAE-cellulose chromatography and ammonium sulphate precipitation. The purified enzymes were acidic proteins, with carbohydrate contents of 21% for that induced by xylose, and 47% for that induced by xylan. Their apparent molecular masses, estimated by gel filtration, and optimal temperatures for β-xylosidase activities, were about 60 and 100 kDa, and 40 and 45 °C, respectively, for the enzymes induced by xylose and xylan. Xylose-induced β-xylosidase exhibited an optimum pH of 6.0, while that of the xylan-induced enzyme was 5.5. Both purified β-xylosidases exhibited also β-galactosidase, β-glucosidase and -arabinosidase activities. In addition to synthetic substrates, the enzymes hydrolysed xylobiose and xylotriose, suggesting a physiological role. K_M values for p-nitrophenyl β- -xylopyranoside were 0.32 mM, for the xylose-induced β-xylosidase, and 0.19 mM for the xylan-induced one. Xylose competitively inhibited both β-xylosidases, with K_I values of 5.3 and 2.0 mM, for the enzymes induced by xylose or xylan, respectively.     

Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by cross-linking with transglutaminase

Thermostable β-glucosidase from Sulfolobus shibatae was immobilized on silica gel modified or not modified with 3-aminopropyl-triethoxysilane using transglutaminase as a cross-linking factor. Obtained preparations had specific activity of 3883 U/g of the support, when measured at 70 °C using o-nitrophenyl β-d-galactopyranoside (GalβoNp) as substrate. The highest immobilization yield of the enzyme was achieved at pH 5.0 in reaction media. The most active preparations of immobilized β-glucosidase were obtained at a transglutaminase concentration of 40 mg/ml at 50 °C. The immobilization was almost completely terminated after 100 min of the reaction and prolonged time of this process did not cause considerable changes of the activity of the preparations. The immobilization did not influence considerably on optimum pH and temperature of GalβoNp hydrolysis catalyzed by the investigated enzyme (98 °C, pH 5.5). The broad substrate specifity and properties of the thermostable β-glucosidase from S. shibatae immobilized on silica-gel indicate its suitability for hydrolysis of lactose during whey processing.     

A cDNA cloned from Physarum polycephalum encodes new type of family 3 beta-glucosidase that is a fusion protein containing a calx-beta motif

The microplasmodia of Physarum polycephalum express three types of β-glucosidases: secretory enzyme, a soluble cytoplasmic enzyme and a membrane-bound enzyme. We are interested in the physiological role of three enzymes. We report the sequence of cDNA for membrane β-glucosidase 1, which consists of 3825 nucleotides that includes an open reading frame encoding 1248 amino acids. The molecular weight of membrane β-glucosidase 1 was calculated to be 131,843 based on the predicted amino acid composition. Glycosyl hydrolase family 3 N-terminal and C-terminal domains were found within the N-terminal half of the membrane β-glucosidase 1 sequence and were highly homologous with the primary structures of fungal β-glucosidases. Notably, the C-terminal half of membrane β-glucosidase 1 contains two calx-β motifs, which are known to be Ca²⁺ binding domains in the Drosophila Na⁺/Ca²⁺ exchanger; an RGD sequence, which is known to be a cell attachment sequence; and a transmembrane region. In this way, Physarum membrane β-glucosidase 1 differs from all previously identified family 3 β-glucosidases. In addition to cDNA for membrane β-glucosidase 1, two other distinctly different mRNAs were also isolated. Two sequences were largely identical to cDNA for membrane β-glucosidase 1, but included a long insert sequence having a stop codon, leading to truncation of their products, which could account for other β-glucosidase forms occurred in Physarum poycephalum.

Thus, the membrane β-glucosidase is a new type family 3 enzyme fused with the Calx-β domain. We propose that Calx-β domain may modulate the β-glucosidase activity in response to changes in the Ca²⁺ concentration.     

Relation of glycosidases to bean hypocotyl growth

The enzymes β-glucosidase, α-glucosidase, β-galactosidase, α-galactosidase, and β-xylosidase were detected in Phaseolus vulgaris L. var. Red Kidney bean hypocotyl tissue throughout the first 13 days of development with p-nitrophenyl glycosides as substrates. Activities of all enzymes except β-glucosidase declined as a function of increasing tissue age. In contrast, β-glucosidase activity increased rapidly 3 days after imbibition to a maximal activity at 5 days and then subsided to one-third the maximum by day 7. This activity peak immediately preceded the logarithmic phase of hypocotyl growth. This enzyme is strongly associated with cell walls during extraction, suggesting that it is wall-bound in situ. Various polysaccharide substrates were used to evaluate the specificity of this enzyme.     

In vitro evaluation of feed-grade enzyme activity at pH levels simulating various parts of the avian digestive tract

The activities of β-glucanase, xylanase, amylase, α-galactosidase and protease were measured at their published optimum pH levels and at pH levels of 3.0, 6.0, 6.5, 7.0 and 7.5 to simulate pH levels of the gizzard, the diet, the crop, and the proximal and distal parts of small intestine, respectively. The activity of β-glucanase was determined by measuring reducing sugars after incubation of β-glucan. Xylanase activity was assayed by measuring xylose after hydrolysis of xylan. The activity of amylase was measured through hydrolysis of soluble starch. The assay of α-galactosidase was based on a hydrolysis of p-nitrophenyl-α-d-galactoside followed by measurement of liberated p-nitrophenol. The activity of protease was assayed by measuring tyrosine after enzymatic hydrolysis of casein. β-Glucanase had high activity at pH levels of 3.0–7.0. Xylanase had no enzyme activity at pH 3.0, but had high activity at pH levels of 6.0–7.0. Amylase had high activity at pH levels of 6.0 and 6.5 but had no or very low activity at pH 3.0, 7.0 and 7.5. α-Galactosidase had high activity at pH 6, but not at other pH levels tested. Protease had either no or very low activity at all pH levels except at pH 3.0. These results suggest that the pH levels commonly found in the avian digestive tract may be a limiting factor for maximum activity of the exogenous enzymes, such as amylase, α-galactosidase and protease.     

Transglycosylation Catalyzed by Almond β-glucosidase and Cloned Pichia etchellsii β-glucosidase II using Glycosylasparagine Mimetics as Novel Acceptors

The stability of almond β-glucosidase in five different organic media was evaluated. After 1 hour of incubation at 30°C, the enzyme retained 95, 91, 81, 74 and 56% relative activity in aqueous solutions [30% (v/v)] of dioxane, DMSO, DMF, acetone and acetonitrile, respectively. Transglucosylation involving p-nitrophenyl β-D-glucopyranoside as donor and β-1-N-acetamido-D-glucopyranose, which is a glycosylasparagine mimic, as acceptor was explored under different reaction conditions using almond βglucosidase and cloned Pichia etchellsii β-glucosidase II. The yield of disaccharides obtained in both reactions turned out to be 3%. Both enzymes catalyzed the formation of (1→3)- as well as (1→6)- regioisomeric disaccharides, the former being the major product in cloned β-glucosidase II reaction while the latter predominated in the almond enzyme catalyzed reaction. Use of β-1-N-acetamido-D-mannopyranose and β-1-N-acetamido-2-acetamido-2-deoxy-D-glucopyranose as acceptors in almond β-glucosidase catalyzed reactions, however, did not afford any disaccharide products revealing the high acceptor specificity of this enzyme.     

Biochemical characterisation of digestive proteases and carbohydrases of the pistachio fruit hull borer,Arimania komaroffi Ragonot (Lepidoptera: Pyralidae)

Pistachio fruit hull borer, Arimania komaroffi Ragonot (Lep.: Pyralidae), is one the most important pests of pistachio in Iran. The larvae spin web as well as bore into young fruits, and the infested fruits fall off the trees. The second-generation adult moths appear in August and September, and their offspring feed on the fruit hull. Results indicated the presence of α-amylase, α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase and some proteases in the digestive tract of the pest. Highest activities of α-amylase, α-glucosidase, β-glucosidase, α-galactosidase and β-galactosidase were at pH 10, 7, 7, 6 and pH 6, respectively. Highest activities of trypsin, chymotrypsin and elastase of larval midgut were at pH 11. Zymogram analysis of α-amylase, α-glucosidase, β-glucosidase, tryptic, chymotryptic and elastase using native-PAGE revealed 1, 1, 2, 3, 3 and 2 bands of activity respectively, in A. komaroffi. One band was disappeared in the presence of the inhibitor TLCK, but no further inhibition by the inhibitors TPCK was observed. The results can be of help for designing new strategies for controlling the pistachio fruit hull borer based on natural proteases and carbohydrase inhibitors.     

Enzymatic synthesis of disaccharide-serine and peptide conjugates

We describe enzymatic transglycosylations between an appropriate glycosyl donor and galactosyl (or glucosyl)-serine and -peptide conjugates to obtain diglycosyl-serine or -peptide derivatives. The reactions are catalyzed by β-galactosidase (from E. coli or from Aspergillus oryzae) and β-glucosidase (from Almonds). The enzymatic reactions give, preferentially, β(1 å6) linked diglycosyl-serine (or -peptide) conjugates. However, in the case of the digalactosyl derivatives, β(1 å3) linkages are mainly observed. By changing the source of the enzyme (E. coli or Aspergillus oryzae) the regioselectivity can be reversed for these digalactosyl derivatives. Deprotection of the aminoacid of the diglycosyl-peptides under mild conditions is also described.     

Enzymatic Synthesis of Thioglucosides Using Almond β-glucosidase

A selection of different glycosidases was screened for the glycosylation of 1-propanethiol. The β-glucosidases from almond, Aspergillus niger and Caldocellum saccharolyticum were capable of 1-propanethioglucoside (1-PTG) formation. The almond β-glucosidase showed the highest activity in this reversed hydrolysis type of reaction using glucose as glucosyl donor. Besides 1-propanethiol, also thioglucosides of 2-propanethiol and furfuryl mercaptan were formed by the almond β-glucosidase. The substrate specificity of the almond β-glucosidase with respect to thioglucosylation is restricted to primary and secondary aliphatic thiols. Once the thioglucosides are formed, they are not hydrolyzed at a significant rate by almond β-glucosidase. As a consequence the synthesis of 1-PTG could be observed at very low aglycone concentrations (0.5% v/v based on the reaction solution) and high yields (68% based on 1-PT and 41% based on glucose) were obtained. An excess of aglycone, otherwise frequently applied in reversed hydrolysis glycosylation, is therefore not necessary in the glucosylation of 1-PT.     

Inhibition of glycosidases by aldonolactones of corresponding configuration: The C-4- and C-6-specificity of β-glucosidase and β-galactosidase

1. In barley, β-glucosidase and β-galactosidase are separate enzymes. The former also displays β-d-fucosidase activity. 2. In the limpet, Patella vulgata, β-glucosidase activity is associated with the β-d-fucosidase, previously shown to be a separate entity from the β-galactosidase also present. 3. Almond emulsin presents all three activities as a single enzyme. Each is equally inhibited by glucono-, galactono- and d-fucono-lactone. 4. In rat epididymis, there is no significant β-glucosidase activity, nor is there appreciable inhibition of the β-galactosidase and β-d-fucosidase activities of the preparation by gluconolactone.     

Production of phenolic antioxidants by the solid-state bioconversion of pineapple waste mixed with soy flour using Rhizopus oligosporus

The ability of Rhizopus oligosporus to produce enhanced levels of free phenolics from pineapple residue mixed with soy flour as potential nitrogen source was investigated. Concurrently, phenolic-linked β-glucosidase and the antioxidant activity of the extracts were followed. Two treatments were studied: (A) 9 g of pineapple residue and 1 g of soy flour (P9); (B) 5 g of pineapple residue and 5 g of soy flour (P5). The increase of water extractable phenolics was 39.3% for P9 treatment and 79.4% for P5 treatment. During early stages of growth high antioxidant activity, low phenolic content and low β-glucosidase specific activity was observed. High antioxidant activity was likely due to the presence of insoluble polymeric phenolics, know for their high antioxidant activity. A marked decrease of the antioxidant activity of P5 treatment during late stages of growth was observed due to likely formation of free soluble phenolics. The moderate total phenolics content and high β-glucosidase specific activity of P9 treatment in late stages is likely the consequence of low nitrogen content in this treatment. The bioconversion of pineapple residue by R. oligosporus leads to enhanced levels of phenolic compounds, mainly for P5 treatment. This approach offers a novel strategy to enhance the value of pineapple wastes.     

鳞杯伞α-半乳糖苷酶的提取纯化及酶学性质研究

采用离子交换层析和凝胶过滤层析对鳞杯伞子实体中的α-半乳糖苷酶进行纯化,得到了一种分子量为50 kDa的α-半乳糖苷酶,命名为CSG。纯化后的CSG纯化倍数为891.46倍,比活力为54.78 U/mg,得率为0.71%。通过BLAST比对液相色谱-串联质谱(LC-MS/MS)获得其肽段,发现其为GH27家族的α-半乳糖苷酶。CSG的最适pH为3.0,最适温度为50 ℃。在酸性范围pH 2.2-7.0和温度范围4-30 ℃有较好的稳定性。Mn²⁺、Cd²⁺、Cu²⁺对CSG有较强的抑制作用。半乳糖和蜜二糖对CSG的抑制类型为混合型抑制。化学修饰剂N-溴代琥珀酰亚胺显著降低CSG的活力,碳二亚胺对CSG具有显著的激活作用。该酶具有良好的蛋白酶抗性,且对棉子糖家族寡糖(RFOs)、瓜尔豆胶和赤槐豆胶均表现出良好的水解作用。     

Cellulase production during growth of Talaromyces emersonii CBS 814.70 on lactose containing media

The thermophilic fungus Talaromyces emersonii CBS 814.70 is capable of growth on lactose containing media. The cell protein produced towards the end of growth on that substrate is similar to those levels produced during growth of the organism on cellulose. During growth of the organism on lactose, cellulase is secreted into the medium. Analysis of the components of the cellulase system shows that both β-glucosidase and endoglucanase enzymes are produced. Levels of β-glucosidase produced during growth of the organism on lactose are well in excess of levels of that enzyme produced at any time during growth of the organism on cellulose, and we have shown that the form of that enzyme produced during growth on lactose is β-glucosidase III (BG-III). Analysis of the forms of endoglucanase indicates that not all forms of enzyme produced during growth on cellulose are produced during growth on lactose. β-Galactosidase activity was found to be present in the mycelial associated fraction, though our evidence suggests that this may simply be an incidental activity of the cell associated form of β-glucosidase IV (BG-IV).     

Activity of digestive proteinases and carbohydrases in the alimentary tract of the fig leaf roller,Choreutis nemorana Hübner (Lepidoptera: Choreutidae)

.The fig leaf roller or Fig-tree Skeletoniser, Choreutis nemorana (Lep.: Choreutidae), is a destructive pest of fig trees found in some fig-growing areas of Iran. The larvae feed on the upper level of leaves, near the main vein. In this study, digestive carbohydrases including α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase and proteinases including trypsin, chymotrypsin and elastase were investigated. The results showed that the carbohydrases were present in the alimentary tracts of the pest. Optimum pH for α-glucosidase and β-glucosidase activity was at pH 6.0 and 7.0, respectively. Maximum activity of α-galactosidase and β-galactosidase occurred at pH 6.0. Total proteolitic activity against the substrate azocasein was optimally occurred at pH 10.0. The greatest activity of trypsin, chymotrypsin and elastase was determined at pH 10.0, 11.0 and 11.0, respectively. Zymogram analyses using nitrocellulose membrane revealed two trypsin isoforms in which one of them was completely inhibited by Soybean Kunitz inhibitor and the other was notably inhibited.