The inhibition of β-glucosidase activity in Trichoderma reesei C30 cellulase by derivatives and isomers of glucose

The inhibition of β-glucosidase in Trichoderma reesei C30 cellulase by D -glucose, its isomers, and derivatives was studied using cellobiose and ρ-nitrophenyl-β-glucoside (PNPG) as substrates for determining enzyme activity. The enzymatic hydrolysis of both substrates was inhibited competitively by glucose with approximate K_i values of 0.5mM and 8.7mM for cellobiose and PNPG as substrate, respectively. This inhibition by glucose was maximal at pH 4.8, and no inhibition was observed at pH 6.5 and above. The α anomer of glucose inhibited β-glucosidase to a greater extent than did the β form. Compared with D -glucose, L -glucose, D -glucose-6-phosphate, and D -glucose-1-phosphate inhibited the enzyme to a much lesser extent, unlike D -glucose-L -cysteine which was almost as inhibitory as glucose itself when cellobiose was used as substrate. Fructose (2?100mM) was found to be a poor inhibitor of the enzyme. It is suggested that high rates of cellobiose hydrolysis catalyzed by β-glucosidase may be prolonged by converting the reaction product glucose to fructose using a suitable preparation of glucose isomerase.     

The Structure of M-Gtfi,An Inhibitor of Glucosyltransferase from S. Mutans,And Its Inhibitory Relationship with Other Sulfate Ester-Containing Inhibitors

Abstract

M-GTFI, an inhibitor of glucosyltransferase from S. mutans was produced by Micromonospora narashinoensis strain No. 731. The isolation procedure for M-GTFI was improved and established for spectro-scopic analyses, and some properties of the inhibitor were investigated. The structure of M-GTFI was shown to be trisodium [2-sulphonato-9-undecenyll-oxacyclotriacont-3-en-2-one, 16, 18-his sulp-honate. The chemical structure of M-GTFI was therefore similar to that of izumenolide which is a β-lactamase inhibitor containing sulfate ester groups in its molecule.

The inhibitory characteristics of M-GTFI were parallel to that of other inhibitory compounds containing sulphate esters but the spectrum of activity was wider.     

Purification and characterization of a novel β-glucosidase from Aspergillus flavus and its application in saccharification of soybean meal

Abstract

Aspergillus flavus has been regarded as a potential candidate for its production of industrial enzymes, but the details of β-glucosidase from this strain is very limited. In herein, we first reported a novel β-glucosidase (AfBglA) with the molecular mass of 94.2?kDa from A. flavus. AfBglA was optimally active at pH 4.5 and 60?°C and is stable between pH 3.5 and 9.0 and at a temperature of up to 55?°C for 30?min remaining more than 90% of its initial activity. It showed an excellent tolerance to Trypsin, Pepsin, Compound Protease, and Flavourzyme and its activity was not inhibited by specific certain cations. AfBglA displayed broad substrate specificity, it acted on all tested pNP-glycosides and barley glucan, indicating this novel β-glucosidase exhibited a β-1, 3-1, 4-glucanase activity. Moreover, the AfBglA could effectively hydrolyze the soybean meal suspension into glucose and exhibit a strong tolerance to the inhibition of glucose at a concentration of 20.0?g/L during the saccharification. The maximum amount of the glucose obtained by AfBglA corresponded to 67.0?g/kg soybean meal. All of these properties mentioned above indicated that the AfBglA possibly attractive for food and feed industry and saccharification of cellulolytic materials.     

A Novel Phospholipase C Inhibitor,S-PLI Produced by Streptomyces Sp. Strain No. A-6288

Abstract

S-PLI, an inhibitor of phospholipase C (PLC) produced by Strepromyces sp. strain No. 6288, was purified from the culture filtrate by salting-out with solid ammonium sulfate, column chromatography on CM-cellulose and gel filtration on Sephadex G-75. The molecular weight of S-PLI was estimated to be 65,000 by SDS-polyacrylamide gel electrophoresis. The inhibitor was found to be a glycoprotein with a composition of 609 amino acids and 19 glucose residues having an isoelectric point at 7.8. S-PLI was stable from pH 3 to 10 at 37°C and up to 40° at pH 6.0. The inhibitory activity showed pH-and temperature-dependence with a maximum around pH 7.0 at 50°C. S-PLI inhibited phospholipase C in a competitive manner (K_i value; 9.5 × 10-⁶ mM), but did not inhibit S-Hemolysin, phospholipase A₂, phospholipase B, phospholipase D and phosphatases. S-PLI is the first reported example of a glycoproteinaceous inhibitor of microbial origin which is able to specifically inhibit phospholipase C.     

Heavy Metal Effects on β-Glucosidase Activity Influenced by pH and Buffer Systems

Abstract

Inhibition of β-glucosidase activity by Cu(II), Zn(II) and Ni(II) was investigated as a function of pH and buffer type. Both factors were found to exert a strong effect on the activity of the enzyme. All three of the investigated heavy metals inhibited the enzyme activity in acetate buffer. At metal concentrations of 0.6 mM, Zn and Ni reduced the enzyme activity by 25-30% under optimal pH conditions (pH 5-5.2). Under the same conditions, Cu showed an even more pronounced inhibitory effect than Zn and Ni. In presence of 0.6 mM Cu, the enzyme activity was lowered by more than 90% in comparison to metal free systems. In contrast to these results, no enzyme inhibition was observed in citrate buffer, even in the presence of 1 mM Cu.

The inhibition of β-glucosidase activity by Cu increased with increasing pH. Inhibition by Zn and Ni was less pH-dependent in the observed pH range (pH 4-5.5). Copper caused a distinct shift in the pH optimum of enzyme activity, whereas this was not the case for Zn or Ni. The effects of buffer and pH on enzyme inhibition by Cu, Zn and Ni were successfully described using a chemical speciation model, based on the assumption that enzyme activity depends on the protonation of the amino acids at the reactive site and that enzyme activity is inhibited by complexation of the reactive sites by the heavy metal cations. The results show the importance of taking chemical conditions and speciation into account when investigating the effect of heavy metal cations on biological systems.     

Characteristics of M-GTFI, a new inhibitor of Streptococcus mutans glucosyltransferase

M-GTFI, originally screened as an inhibitor of Streptococcus mutans glucosyltransferase, strongly inhibited alpha-glucosidase, in a non-competitive manner especially when the synthetic substrate p-nitrophenyl-alpha-D-glucopyranoside was used. It also inhibited beta-glucosidase, beta-amylase and, to a lesser extent, beta-glucuronidase. The inhibitor was stable in neutral and alkaline pH ranges and dependency of the inhibition on pH and temperature was not observed. Some proteinases and polysaccharides-hydrolyzing enzymes as well as human saliva did not inactivate the inhibitor. There was a correlation between the release of sulfate anions from the inhibitor molecule on incubation with HCl (0.2 N) at 100 degrees C and loss of inhibitory properties of the molecule. It is suggested that the presence of sulfate ester linkages in the inhibitor molecule play an important role in the inhibition process.     

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.     

Identification of β-glucosidase 1 as a biomarker and its high expression in hepatocellular carcinoma is associated with resistance to chemotherapy drugs

Context and objective: Long-term prognosis of hepatocellular carcinoma (HCC) patients is challenging, and novel biomarkers are needed to predict patient risk and serve as potential therapeutic target.

Results: We found β-glucosidase 1 is significantly overexpressed and activated in primary HCC tissue and multiple HCC cell lines. β-Glucosidase 1 expression is associated with predicting prognosis of HCC patients under chemotherapy. Silencing β-glucosidase 1 inhibits growth and survival of HCC cells, with preferential inhibitory effects on high β-glucosidase 1-expressing cells. Combination of chemo drug with β-glucosidase 1 inhibitor sensitized HCC cells to chemotherapy.

Conclusion: Our data support β-glucosidase 1 as a HCC biomarker due to its prognosis significance.     

Design,synthesis and α-glucosidase inhibition study of novel embelin derivatives

Abstract

Embelin is a naturally occurring para-benzoquinone isolated from Embelia ribes (Burm. f.) of the Myrsinaceae family. It was first discovered to have potent inhibitory activity (IC₅₀ = 4.2?μM) against α-glucosidase in this study. Then, four series of novel embelin derivatives were designed, prepared and evaluated in α-glucosidase inhibition assays. The results show that most of the embelin derivatives synthesised are effective α-glucosidase inhibitors, with IC₅₀ values at the micromolar level, especially 10d, 12d, and 15d, the IC₅₀ values of which are 1.8, 3.3, and 3.6?μM, respectively. Structure–activity relationship (SAR) studies suggest that hydroxyl groups in the 2/5-position of para-benzoquinone are very important, and long-chain substituents in the 3-position are highly preferred. Moreover, the inhibition mechanism and kinetics studies reveal that all of 10d, 12d, 15d, and embelin are reversible and mixed-type inhibitors. Furthermore, docking experiments were carried out to study the interactions between 10d and 15d with α-glucosidase.     

Studies on α-Glucosidase in Rice

Two kinds of αglucosidase which were homogeneous in disc electrophoretic and ultra-centrifugal analysis were isolated from rice seeds by means of ammonium sulfate fractionation and CM-cellulose, Sephadex G–100 and DEAE-cellulose column chromatography and designated as α-glucosidase I and α-glucosidase II.

Both α-glucosidases hydrolyzed maltose and soluble starch to glucose and showed same optimal pH (4.0) on the both substrates. In addition, both enzymes acted on various α-linked gluco-oligosaccharides and soluble starch but little or not on α-linked hetero-glucosides and α-l,6-glucan (dextran).

Activity of the enzymes on maltose and soluble starch was inhibited by Tris and erythritol. α-Glucosidase II was more sensitive to the inhibitors than α-glucosidase I.

Km value for maltose was 1.1 mM for α-glucosidase I and 2.0 mM for α-glucosidase II.     

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.     

Characterization of nimbidiol as a potent intestinal disaccharidase and glucoamylase inhibitor present in Azadirachta indica (neem) useful for the treatment of diabetes

Azadirachta indica, used in antidiabetic herbal drugs, was reported to contain α-glucosidase inhibitor. Bioassay guided purification characterized the inhibitor as nimbidiol (a diterpenoid), present in root and stem-bark of the tree. Nimbidiol inhibited intestinal (mammalian) maltase-glucoamylase, sucrase-isomaltase, lactase, trehalase and fungal α-glucosidases. Nimbidiol showed a mixed competitive inhibition on intestinal carbohydrases. IC₅₀, Ki and Ki′ (µM) were 1.35 ± 0.12, 0.08 ± 0.01, 0.25 ± 0.11, respectively, for maltase-glucoamylase (maltotetraose as substrate). Nimbidiol was more potent inhibitor of isomaltase (IC₅₀ 0.85 ± 0.035 µM), lactase (IC₅₀ 20 ± 1.33 µM) and trehalase (IC₅₀ 30 ± 1.75 µM) than acarbose, voglibose, salacinol, kotalanol and mangiferin. Ki and Ki′ values (µM) for intestinal sucrase were 0.7 ± 0.12 and 1.44 ± 0.65, respectively. Development of nimbidiol as an antidiabetic drug appears to be promising because of broad inhibition spectrum of intestinal glucosidases and easy synthesis of the molecule.     

The bgl1 gene of Trichoderma reesei QM 9414 encodes an extracellular, cellulose-inducible β-glucosidase involved in cellulase induction by sophorose

We have investigated the effect of disruption of the bgl1-(β-glucosidase l-encoding) gene of Trichoderma reesei on the formation of other β-glucosidase activities and on the induction of cellulases. To this end the bgl1 locus was disrupted by insertion of the Aspergillus nidulans amdS (acetamidase-encoding) gene. The bgl1-disrupted strain did not produce the 75kDa extracellular β-glucosidase on cellulose or lactose, but still formed β-glucosidase activity on glucose, cellobiose, xylan or β-1,3-glucan, suggesting that the enzyme(s) exhibiting this β-glucosidase activity is (are) not encoded by bgl1. The cellulose-inducer sophorose induced the bgl1-encoded β-glucosidase, whereas the remaining β-glucosidase activity was induced by methyl-β-D-glucoside. The bgl1-gene product was mainly secreted into the medium, whereas the other β-glucosidase activity was mainly associated with the cells. A bgl1-multicopy strain formed higher amounts of cellulases than the parent strain. Nonsaturating concentrations of sophorose efficiently induced cellobiohydrolase I formation in the bgl1-multicopy strain, but less efficiently in the bgl1-disrupted strain. The multicopy strain and the parent strain were comparably efficient at saturating sophorose concentrations. The β-glucosidase inhibitor nojirimycin strongly inhibited induction in all strains. These data suggest that the bgl1-encoded β-glucosidase is not identical to the plasma-membrane-bound, constitutive, methyl-β-glucoside inducible β-glucosidase, but represents an extracellular cellulose-induced enzyme. Both enzymes contribute to rapid induction of cellulases by modifying the inducer sophorose.     

Production of cellulases by Thermomucor indicae-seudaticae: characterization of a thermophilic β-glucosidase

Abstract

The current study evaluated the production and characterization of β-glucosidase by the thermophilic fungus Thermomucor indicae-seudaticae in solid-state fermentation of wheat bran. Isolated fungi have significant amounts of β-glucosidase, an enzyme that may be applied to different industrial processes, such as the production of fuels, food, and other chemical compounds. Maximal enzyme activity occurred in pH 3.5–4.5 and at 70?°C. The enzyme exhibited high thermostability, for 1?h, up to 60?°C, and good tolerance to glucose (10?mM) and ethanol (10%). The optimization of fermentative parameters on the production of β-glucosidase was carried out by evaluating the best supplementary nutrient source, pH of nutrient solution, initial substrate moisture and fermentation temperature. The optimization of the above fermentation parameters increased enzyme activity by 120.0%. The highest enzymatic activity (164.0?U/g) occurred with wheat bran containing 70% initial moisture, supplemented with 1.0% (NH₄)₂SO₄ solution at pH 5.5–6.0 and fungus incubated at 40?°C. A more detailed study of β-glucosidase suggested that Sulfur is an important component of the main amino acid present in this enzyme. The enhancer of the enzyme activity occurred when the fungus was grown on wheat bran supplemented with a sulfur-containing solution. In fact, increasing the concentration of sulfur in the solution increased its activity.     

Overexpression of a multifunctional β-glucosidase gene from thermophilic archaeon Sulfolobus solfataricus in transgenic tobacco could facilitate glucose release and its use as a reporter

The β-glucosidase, which hydrolyzes the β(1–4) glucosidic linkage of disaccharides, oligosaccharides and glucose-substituted molecules, has been used in many biotechnological applications. The current commercial source of β-glucosidase is mainly microbial fermentation. Plants have been developed as bioreactors to produce various kinds of proteins including β-glucosidase because of the potential low cost. Sulfolobus solfataricus is a thermoacidophilic archaeon that can grow optimally at high temperature, around 80 °C, and pH 2–4. We overexpressed the β-glucosidase gene from S. solfataricus in transgenic tobacco via Agrobacteria-mediated transformation. Three transgenic tobacco lines with β-glucosidase gene expression driven by the rbcS promoter were obtained, and the recombinant proteins were accumulated in chloroplasts, endoplasmic reticulum and vacuoles up to 1%, 0.6% and 0.3% of total soluble protein, respectively. By stacking the transgenes via crossing distinct transgenic events, the level of β-glucosidase in plants could further increase. The plant-expressed β-glucosidase had optimal activity at 80 °C and pH 5–6. In addition, the plant-expressed β-glucosidase showed high thermostability; on heat pre-treatment at 80 °C for 2 h, approximately 70% residual activity remained. Furthermore, wind-dried leaf tissues of transgenic plants showed good stability in short-term storage at room temperature, with β-glucosidase activity of about 80% still remaining after 1 week of storage as compared with fresh leaf. Furthermore, we demonstrated the possibility of using the archaebacterial β-glucosidase gene as a reporter in plants based on alternative β-galactosidase activity.

     

A highly glucose-tolerant GH1 β-glucosidase with greater conversion rate of soybean isoflavones in monogastric animals

In the feed industry, β-glucosidase has been widely used in the conversion of inactive and bounded soybean isoflavones into active aglycones. However, the conversion is frequently inhibited by the high concentration of intestinal glucose in monogastric animals. In this study, a GH1 β-glucosidase (AsBG1) with high specific activity, thermostability and glucose tolerance (IC₅₀ = 800 mM) was identified. It showed great glucose tolerance against substrates with hydrophobic aryl ligands (such as pNPG and soy isoflavones). Using soybean meal as the substrate, AsBG1 exhibited higher hydrolysis efficiency than the GH3 counterpart Bgl3A with or without the presence of glucose in the reaction system. Furthermore, it is the first time to find that the endogenous β-glucosidase of soybean meal, mostly belonging to GH3, plays a role in the hydrolysis of soybean isoflavones and is highly sensitive to glucose. These findings lead to a conclusion that the GH1 rather than GH3 β-glucosidase has prosperous application advantages in the conversion of soybean isoflavones in the feed industry.

     

Comparative Study of β-Glucosidase from Cotyledons and Fruits of Cucumber, Cucumis sativus

This study was conducted to compare the β-glucosidase of cotyledons and fruits of Cucumis sativus L. cv. Chipper. The concentration of the enzyme was followed throughout the growth period of each organ. The greatest concentration of the enzyme did not correspond with the most rapid period of growth. Each enzyme was characterized kinetically. The Michaelis constant of the cotyledon β-glucosidase for p-NO₂-phenyl-β-D-gluco-pyranoside was 1.57 mM, and was 0.35 mM for the fruit enzyme. The enzymes from the two sources also differed in affinity for glucono-1,5-lactone, a competitive inhibitor of β-glucosidases, susceptibility to inhibition by saccharides, and heat stability. The two organs apparently contain different forms of β-glucosidase.     

Partial Purification and Characterization of Naegleria fowleriβ-Glucosidase1

Naegleria fowleri cells, grown axenically, contain high levels of β-D-glucosidase which catalyzes the hydrolysis of 4-methylumbelliferyl-β-D-glucopyranoside (4MUGlc) (K_m, 0.9 mM), octyl-β-D-glucoside (K_m, 0.17 mM), and p-nitrophenyl-β-D-glucopyranoside at relative rates of 1.00, 2.88, and 1.16, respectively (substrate concentration, 3.0 mM). When the amebae are subjected to freeze-thawing, sonication, and centrifugation (100,000 g, 1 h), 85% of the β-glucosidase activity appears in the supernatant fraction. The β-glucosidase was purified 40-fold (34% yield) using a combination of chromatographic steps involving DE-52 cellulose, concanavalin A-Sepharose, and hydroxylapatite followed by isoelectric focusing. The predominant soluble β-D-galactosidase activity in the Naegleria extract copurifies with the β-D-glucosidase; the two activities have the same isoelectric point (pI, 6.9), similar heat stabilities, are both inhibited by lactobionic acid (K_i, 0.40 mM), and exhibit optima at pH 4.5, indicating that they are probably the same enzyme. The Naegleriaβ-D-glucosidase has an apparent molecular weight of 66,000, a Stokes radius of 25 Å, and a sedimentation coefficient of 4.2S. The β-glucosidase is not inhibited by conduritol β-epoxide or galactosylsphingosine but is completely inhibited by 1.25 mM bromo conduritol β-epoxide. The latter compound, when present in the growth medium, inhibits the growth of the organism and profoundly alters its ultrastructure, the main effect being the apparent inhibition of cytokinesis and the generation of multinucleate cells. The issue of the role of the β-glucosidase in the metabolism of the ameba and its possible role in pathogenic mechanisms are discussed.     

Inhibition of Growth of Callus Cells by Degradation Products of Dehydroascorbic Acid

Candida pelliculosa var. acetaetherius was found to produce a β-glucosidase intracellularly. The enzyme was purified 200-fold by fractionation with ammonium sulfate and chromatography on Sephadex G-100 and DEAE Sepharose CL-6B. After polyacrylamide gel electrophoresis of the final fraction, one protein band corresponding to β-glucosidase was detected. The molecular weights determined by SDS-PAGE and by Sephacryl S-300 chromatography were 90,000 and 360,000, respectively, suggesting that the enzyme was a tetramer. The enzyme was a glycoprotein and its isoelectric point was at pH 4.9. It’s optimum pH and temperature were 6.5 and 50°C, respectively. The enzyme activity was inhibited by Zn^{2 +}, Hg^{2 +}, Cu^{2 +}, Co^{2 +}, p-chloromercuribenzoate, and glucose. Inhibition by glucose was competitive with a Ki value of 6.5 mm. Specificity studies for substrates indicated that the enzyme was specific for the p-configuration of sugars. Km values measured at 50°C were 0.5 mm for p-nitrophenyl-β-glucoside and 37 mm for cellobiose.     

Catalytic and thermodynamic properties of β-glucosidases produced by Lichtheimia corymbifera and Byssochlamys spectabilis

Abstract

The objective of the present study was to optimize parameters for the cultivation of Lichtheimia corymbifera (mesophilic) and Byssochlamys spectabilis (thermophilic) for the production of β-glucosidases and to compare the catalytic and thermodynamic properties of the partially purified enzymes. The maximum amount of β-glucosidase produced by L. corymbifera was 39?U/g dry substrate (or 3.9?U/mL), and that by B. spectabilis was 77?U/g (or 7.7?U/mL). The optimum pH and temperature were 4.5 and 55?°C and 4.0 and 50?°C for the enzyme from L. corymbifera and B. spectabilis, respectively. β-Glucosidase produced by L. corymbifera was stable at pH 4.0–7.5, whereas the enzyme from B. spectabilis was stable at pH 4.0–6.0. Regarding the thermostability, β-glucosidase produced by B. spectabilis remained stable for 1?h at 50?°C, and that from L. corymbifera was active for 1?h at 45?°C. Determination of thermodynamic parameters confirmed the greater thermostability of the enzyme produced by the thermophilic fungus B. spectabilis, which showed higher values of ΔH, activation energy for denaturation (E_a), and half-life t_(1/2). The enzymes were stable in the presence of ethanol and were competitively inhibited by glucose. These characteristics contribute to their use in the simultaneous saccharification and fermentation of vegetable biomass.