mM For Cellobiose Research Articles

Structural insights and functional characterization of a novel β-glucosidase derived from Thermotoga profunda

β-Glucosidase is a crucial cellulase, as its activity determines the efficiency of cellulose hydrolysis into glucose. This study addresses the functional and structural characteristics of Thermotoga profunda β-glucosidase (Tp-BGL). Tp-BGL exhibited a Km of 0.3798 mM for p-nitrophenyl-β-d-glucopyranoside (pNPGlc) and 4.44 mM for cellobiose, with kcat/Km of 1211.16 and 4.18 s−1 mM−1, respectively. In addition, Tp-BGL showed significant pH adaptability and thermal stability, with a Tm of 85.7 °C and retaining >90 % of its activity after incubation at 80 °C for 90 min. The crystal structure of Tp-BGL was resolved at 1.95 Å resolution, and reveals a typical TIM barrel structure. Comparative structural analysis highlighted that the major distinction between Tp-BGL and the other glucosidases lies in their loop regions.

Heterologous expression and biochemical studies of a thermostable glucose tolerant β-glucosidase from Methylococcus capsulatus (bath strain)

Glucose inhibition of β-glucosidase (BG) is a bottleneck in biomass hydrolysis. In this study, a glucose resistant GH1 β-glucosidase gene- Mbgl from Methylococcus capsulatus (bath strain) was cloned and overexpressed in E.coli. The Ni-NTA affinity purified Mbgl displayed an optimum temperature of 70°C and optimum pH was 6.0. The calculated KM of the enzyme was 48.6mM and 0.12mM for cellobiose and 4-Nitrophenyl β-d-glucopyranoside (PNPG) respectively. PNPG hydrolysis in presence of various glucose concentrations showed that the enzyme was stimulated by ∼2.2 fold at 50mM glucose and was not inhibited up to 450–500mM glucose. Homology modeling and structural comparisons of Mbgl with a glucose tolerant β-glucosidase of Humicola insolens (HiBG) revealed that the Mbgl has a much broader active site unlike to a deep and narrow active site pocket of HiBG. The difference in active site shape reflects on an alternative mechanism of glucose tolerance in Mbgl. Supplementing a commercial cellulase enzyme mixture CTec with Mbgl in the hydrolysis of the pretreated rice straw enhanced the glucose yield by 10–15%. In addition, Mbgl was also stable in organic solvents, detergents and oxidative conditions which would be advantageous for biotechnological applications.

Cloning and characterization of the glucooligosaccharide catabolic pathway beta-glucan glucohydrolase and cellobiose phosphorylase in the marine hyperthermophile Thermotoga neapolitana.

Characterization in Thermotoga neapolitana of a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-beta-D-glucan glucohydrolase (GghA) and cellobiose phosphorylase (CbpA), and the apparent absence of a cellobiohydrolase (Cbh) suggest a nonconventional pathway for glucan utilization in Thermotogales. GghA purified from T. neapolitana is a 52.5-kDa family 1 glycosyl hydrolase with optimal activity at pH 6.5 and 95 degrees C. GghA releases glucose from soluble glucooligomers, with a preference for longer oligomers: k(cat)/K(m) values are 155.2, 76.0, and 9.9 mM(-1) s(-1) for cellotetraose, cellotriose, and cellobiose, respectively. GghA has broad substrate specificity, with specific activities of 236 U/mg towards cellobiose and 251 U/mg towards lactose. With p-nitrophenyl-beta-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrate- and end product-directed activation. Its capacity for transglycosylation is a factor in this activation. Cloning of gghA revealed a contiguous upstream gene (cbpA) encoding a 93.5-kDa cellobiose phosphorylase. Recombinant CbpA has optimal activity at pH 5.0 and 85 degrees C. It has specific activity of 11.8 U/mg and a K(m) of 1.42 mM for cellobiose, but shows no activity towards other disaccharides or cellotriose. With its single substrate specificity and low K(m) for cellobiose (compared to GghA's K(m) of 28.6 mM), CbpA may be the primary enzyme for attacking cellobiose in Thermotoga spp. By phosphorolysis of cellobiose, CbpA releases one activated glucosyl molecule while conserving one ATP molecule per disaccharide. CbpA is the first hyperthermophilic cellobiose phosphorylase to be characterized.

Strain variability and the effects of organic compounds on the growth of the chemolithotrophic bacterium Thiobacillus ferrooxidans.

The effects of naturally-occurring organic compounds on ferrous iron oxidation by the bacterium Thiobacillus ferrooxidans were examined with a view to using these compounds to treat or prevent acid mine/rock drainage. The compounds glucose, cellobiose, galacturonic acid, and citric acid were added to the growth medium of five different strains of the bacterium and growth studies were done to determine whether or not strain differences existed with respect to organic compound sensitivity. The effects of these compounds were compared to the effects of sodium dodecyl sulfate (SDS) an anionic detergent. Each of the compounds tested had an inhibitory effect on the strains of the bacterium and sensitivity to these compounds was strain dependent. All strains appeared to be equally susceptible to SDS. Inhibitory concentrations ranged from 70 mM to >280 mM for glucose, 7.5 mM to 150 mM for cellobiose, 20 mM to 230 mM for galacturonic acid, and 50 mM to 130 mM for citric acid. SDS effectively inhibited iron oxidation for all strains at a concentration of 0.3 mM, the lowest concentration tested. Some naturally-occurring organic compounds, therefore, might be candidates for the growth control of T. ferrooxidans.

Purification, characterization, and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana.

Two thermostable endocellulases, CelA and CelB, were purified from Thermotoga neapolitana. CelA (molecular mass, 29 kDa; pI 4.6) is optimally active at pH 6.0 at 95 degreesC, while CelB (molecular mass, 30 kDa; pI 4.1) has a broader optimal pH range (pH 6.0 to 6.6) at 106 degreesC. Both enzymes are characterized by a high level of activity (high Vmax value and low apparent Km value) with carboxymethyl cellulose; the specific activities of CelA and CelB are 1,219 and 1,536 U/mg, respectively. With p-nitrophenyl cellobioside the Vmax values of CelA and CelB are 69.2 and 18.4 U/mg, respectively, while the Km values are 0.97 and 0.3 mM, respectively. The major end products of cellulose hydrolysis, glucose and cellobiose, competitively inhibit CelA, and CelB. The Ki values for CelA are 0.44 M for glucose and 2.5 mM for cellobiose; the Ki values for CelB are 0.2 M for glucose and 1.16 mM for cellobiose. CelB preferentially cleaves larger cellooligomers, producing cellobiose as the end product; it also exhibits significant transglycosylation activity. This enzyme is highly thermostable and has half-lives of 130 min at 106 degreesC and 26 min at 110 degreesC. A single clone encoding the celA and celB genes was identified by screening a T. neapolitana genomic library in Escherichia coli. The celA gene encodes a 257-amino-acid protein, while celB encodes a 274-amino-acid protein. Both proteins belong to family 12 of the glycosyl hydrolases, and the two proteins are 60% similar to each other. Northern blots of T. neapolitana mRNA revealed that celA and celB are monocistronic messages, and both genes are inducible by cellobiose and are repressed by glucose.

Purification and Characterization of a Hexose Oxidase with Excellent Strengthening Effects in Bread

ABSTRACTHexose oxidase (EC 1.1.3.5) (HOX) was purified 51‐fold from the red algae Chondrus crispus, by several chromatography methods, including hydrophobic interaction, chelating Sepharose, anion exchange, gel filtration, and chromatofocusing. Purified HOX was subjected to native PAGE and activity staining with nitroblue tetrazolium. For HOX electroeluted out of the gel and digested with endoproteinase Lys‐C, the internal peptide sequence determined was: D‐P‐G‐Y‐I‐V‐I‐D‐V‐N‐A‐G‐T‐(V or P)‐D‐K‐P‐D‐P‐X. The molecular mass, determined by gel filtration, was 126 kDa, versus 65 kDa determined by SDS‐PAGE. The pI was determined to 4.64 and 4.79 as a double band on an isoelectrofocusing gel. Km was determined to 2.7 mM for D‐glucose, 3.6 mM for D‐galactose, 20.2 mM for cellobiose, 43.7 mM for maltose, 90.3 mM for lactose, 102 mM for xylose, and 531 mM for arabinose. The oxidation of thiol groups in gluten was determined by using Ellman's reagent: 5,5′‐dithiobis (2‐nitrobenzoic acid). The effect of HOX was compared to that of glucose oxidase. Both enzymes caused a dose‐responsive reduction in the free thiol groups. Extensigraph measurements and baking tests confirmed that HOX caused increased dough strength and increased bread volume more efficiently than glucose oxidase used in the same dosage.

A Combined Cellobiose Oxidase/Glucose Oxidase Biosensor for HPLC Determination On-Line of Glucose and Soluble Cellodextrines

Highly specific biosensors can be prepared by immobilizing flavin-containing oxidases in a redox polymer on an electrode surface. By combining one glucose oxidase and one cellobiose oxidase electrode in a flow cell we have made a sensor for flow-injection analysis, or postcolumn quantification, of glucose, cellobiose, and higher cellodextrines in an HPLC system. Samples of different concentrations of glucose and cellobiose, separately or mixed, were injected into the mobile phase and the current response was recorded simultaneously from both electrodes. The recorded response peak heights could be used for calibration curves. The usable measuring ranges were roughly 50 μM-50 mM for glucose and 5 μM-80 mM for cellobiose. Soluble cellodextrines, Glc1-6, could be separated on a C18 column by isocratic elution and detected by the sensor.

Beta-glucosidase from Penicillium purpurogenum: purification and properties.

beta-Glucosidase was purified from the culture supernatant of Penicillium purpurogenum. The purified enzyme was homogeneous on both nondenaturing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The enzyme is a monomeric glycoprotein with M(r) of 90,000 as determined by gel filtration on Bio-Gel P-300 and SDS-polyacrylamide gels. Two enzyme forms were resolved by chromatofocusing and isoelectric focusing, and the pI values obtained with both methods were 4.2 (major form) and 6.0. The major form was characterised further. Enzyme activity was optimal at pH 3.5 and at 60 degrees C. The enzyme was stable in the pH range 2.5-9.5 for 24 h at 4 degrees C. Kinetic analysis gave Kms of 0.8 mM for cellobiose and 85 microM for p-nitrophenyl-beta-D-glucopyranoside. The enzyme hydrolyses a wide range of substrates including aryl-beta-glucosides, cellobiose, and amygdalin. Glucose inhibits competitively and glucono-delta-lactone is a mixed inhibitor of the enzyme.

Purification and characterization of extracellular ?-glucosidase from Myceliophthora thermophila

The extracellular β-glucosidase has been purified from culture broth of Myceliophthora thermophila ATCC 48104 grown on crystalline cellulose. The enzyme was purified approximately 30-fold by (NH4)2SO4 precipitation and column chromatography on DEAE-Sephadex A-50, Sephadex G-200 and DEAE-Sephadex A-50. The molecular mass of the enzyme was estimated to be about 120 kD by both sodium dodecyl sulphate gel electrophoresis and gel filtration chromatography. It displayed optimal activity at pH 4.8 and 60°C. The purified enzyme in the absence of substrate was stable up to 60°C and pH between 4.5 and 5.5. The enzyme hydrolysed p-nitrophenyl-β-D-glucoside, cellobiose and salicin but not carboxymethyl cellulose or crystalline cellulose. The K m of the enzyme was 1.6MM for p-nitrophenyl-β-D-glucoside and 8.0MM for cellobiose. D-Glucose was a competitive inhibitor of the enzyme with a K of 22.5MM. Enzyme K activity was inhibited by HgCl2, FeSO4, CuSO4, EDTA, sodium dodecyl sulphate, p-chloromercurobenzoate and iodoacetamide and was stimulated by 2-mercaptoethanol, dithiothreitol and glutathione. Ethanol up to 1.7 M had no effect on the enzyme activity.

Kinetic study of a cellobiase purified from Neocallimastix frontalis EB188

A cellobiase was purified from the culture supernatant of Neocallimastix frontalis EB188. This enzyme possessed a molecular weight of 85,000 and an isoelectric point of 6.95. The enzyme rapidly hydrolyzed cellobiose, p-nitrophenyl (pNP) beta-D-glucopyranoside (pNPG) and cellotriose and slowly hydrolyzed cellopentaose and salicin. The enzyme did not hydrolyze pNP alpha-D-glucopyranoside or pNP beta-D-cellobioside. Substrate inhibition was observed when cellobiose or pNPG were used as the substrates and glucose production was measured. The kinetic parameters were: K = 0.053 mM, V = 5.88 U/mg of protein and Ki = 0.95 mM for cellobiose; K = 0.36 mM, V = 1.05 U/mg and Ki = 8.86 mM for pNPG. Substrate inhibition was not detected during the hydrolysis of pNPG when pNP production was measured. The kinetic parameters for pNPG were: K = 0.67 mM and V = 1.49 U/mg of protein. The presence of an enzyme.glucose.substrate complex and transglucosylation was evident during the catalysis. Glucose, cellobiose, glucono-delta-lactone, galactose, lactose, maltose and salicin acted as competitive inhibitors during the hydrolysis of pNPG with the apparent inhibition constants (Kis) of 4.8 mM, 0.035 mM, 0.062 mM, 28.5 mM, 0.38 mM, 15.0 mm and 31.0 mM, respectively.

A thermostable ?-glucosidase isolated from a bacterial species of the genus Thermus

An extremely thermophilic aerobic bacterium which produced β-glucosidase was isolated from soil collected at the Yudanaka hot spring in Japan. It was identified as belonging to the genus Thermus. Production of β-glucosidase by this bacterium was stimulated by the addition of cellobiose or laminaribiose to the medium. The optimum pH and temperature of the enzyme were 4.5–6.5 and 85° C respectively. The enzyme was stable in the pH range of 4.5–7.0 at 70° C for 2 h and the half-life at 75° C was 5 days. The Km value of the enzyme for p-nitrophenyl-β-d-glucopyranoside, determined at 70° C in 0.1 M sodium phosphate buffer (pH 6.5), was 0.28 mM while the Km was 2.0 mM for cellobiose. The enzyme effectively hydrolysed cellobiose at 70° C and the conversion yields of cellobiose to glucose were 95%, 93% and 90% at substrate concentrations of 5%, 10% and 15%, respectively.

Enzymatic hydrolysis of lignocellulosic biomass from Onopordum nervosum

Some properties of the cellulolytic complex obtained from Trichoderma reesei QM 9414 grown on Solka floc as carbon source and its ability to hydrolyze the lignocellulosic biomass of Onopordum nervosum Boiss were studied. The optimum enzyme activity was found at temperatures between 50 and 55 degrees C and pH ranging from 4.3 to 4.8. Hydrolysis of 4-nitropnenyl-beta-D-glucopyranoside (4-NPG) and cellobiose by the beta-glucosidase of the complex, showed competitive inhibition by glucose with a K(i) value of 0.8 mM for 4-NPG and 2. 56 mM for cellobiose. Enzymatic hydrolysis yield of Onopordum nervosum, evaluated as glucose production after 48 h, showed a threefold increase by pretreating the lignocellulosic substrate with alkali. When the loss of glucose incurred by de pretreatment was taken into account, a 160% increase in the final cellulose to glucose conversion was found to be due to the pretreatment.

Purification and properties of an extracellular ?-glucosidase from the cellulolytic thermophile Clostridium stercorarium

Clostridium stercorarium cultures grown on cellobiose contain both an extracellular and a cell-bound β-glucosidase activity. A substantial portion of the cell-bound enzyme could be extracted by osmotic shock, suggesting a periplasmic localization. The β-glucosidase present in culture supernatants was purified to homogeneity. It was found to be identical in all aspects tested with the cell-bound β-glucosidase. The enzyme exists as a monomer with an apparent molecular weight of 85.000 (SDS-PAGE) and a pI of 4.8. It shows optimal activity as pH 5.5 and 65° C. Thiol groups are essential for enzyme activity. In the presence of reducing agents and divalent cations the half-life of the purified enzyme was more than 5 h at 60°C. The enzyme hydrolyses at different rates a wide range of substrates including aryl-β-glucosides, cellobiose, and disordered cellulose. K m values were determined as 0.8 mM for p-nitrophenyl-β-glucoside (PNPG) and 33 mM for cellobiose. The cellular localization and the substrate specificity pattern are consistent with a dual role of the C. stercorarium β-glucosidase in cellulose saccharification: (1) Cleavage of cellobiose formed by exoglucanase and (2) degradation of cellodextrins produced by endoglucanase action.

Continuous glucose production using encapsulated mycelial-associated ?-glucosidase from Trichoderma E58

Cellobiose and salicin were continuously hydrolysed in a packed bed reactor containing Trichoderma sp. E-58, encapsulated in calcium alginate beads. Continuous β-glucosidase activities were measured using inlet substrate concentrations of 10 mM and 35 mM for cellobiose and salicin respectively. Maximal activities achieved using the described immobilization procedure were between 70 and 90 μmoles substrate reacted per minute per liter of bead volume. Immobilized mycelial-associated β-glucosidase activity was shown to have a half-life of greater than 1000 hours when operating continuously at 50°C.

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

AbstractThe 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 Ki 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.

Purification and Properties of β-1,4-Oligoglucan: Orthophosphate Glucosyltransferase from Clostridium thermocellum

Abstract β-1,4-Oligoglucan:orthophosphate glucosyltransferase from Clostridium thermocellum has been purified nearly 450-fold by a procedure which removes all known interfering enzymes. The enzyme catalyzes the phosphorolysis of cellotriose, cellotetraose, cellopentaose, and cellohexaose. Although cellobiose is an excellent glucosyl acceptor, the enzyme is unable to catalyze either the synthesis or the phosphorolysis of this disaccharide. The product formed in a reaction mixture with α-d-glucose-1-P as the glucosyl donor and cellobiose as the glucosyl acceptor has been isolated and identified as cellotriose. The enzyme is active on the following glucosyl acceptors: cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, cellobiitol, 4-O-β-d-glucosyl-d-altrose, 4-O-β-d-glucosyl-2-deoxy-d-glucose, 4-O-β-d-glucosyl-d-mannose, 4-O-β-d-glucosyl-d-xylose, and laminaritriose. The enzyme catalyzes the arsenolysis of cellotriose; however, no exchange occurs between glucose-1-P and Pi or arsenate. The apparent Km values are 1.2 mm for cellobiose and 4.7 mm for glucose-1-P. The apparent Km value is about 1.0 mm for cellotriose, cellotetraose, and cellopentaose, and the apparent Km value is 0.37 mm for cellohexaose. The apparent Km value for Pi, determined during the phosphorolysis of different cellodextrins, is 0.13 mm with cellotriose, 0.19 mm with cellotetraose, 0.24 mm with cellopentaose, and 0.26 mm with cellohexaose. The pH optimum for the synthesis of cellotriose is about 7.5. The purified enzyme is inactive in the absence of thiol compounds such as cysteine or dithiothreitol. Sodium sulfite activates the enzyme, but it is not as effective as cysteine or dithiothreitol. Pyridoxal 5-phosphate, adenine nucleotides, sugar phosphates, and d-glucose had no effect on the activity of the enzyme. There appears to be no requirement for any dissociable cofactors.

Purification and Specificity of Cellobiose Phosphorylase from Clostridium thermocellum

Abstract A nonspecific cellobiose phosphorylase from Clostridium thermocellum, which has been purified over 100-fold, is active on 10 different glucosyl acceptors, d-glucose, 2-deoxyglucose, 6-deoxyglucose, d-glucosamine, d-mannose, d-altrose, l-galactose, l-fucose, d-arabinose, and d-xylose. Following electrophoresis on polyacrylamide gels, the activity with various acceptors occurs at the location of the major protein component, indicating that a single enzyme catalyzes these reactions. The pH optimum is about 6.5 with d-xylose as an acceptor. The relative Vmax and apparent Km values are 202 and 9.2 mm for 6-deoxyglucose, 100 and 35 mm for d-xylose, 53 and 73 mm for 2-deoxyglucose, 31 and 9.5 mm for d-glucosamine, 22 and 85 mm for d-mannose, 9 and 240 mm for d-arabinose and 8 and 160 mm for l-fucose. The Ki for d-glucose is 1.2 mm. The apparent Km value is 7.3 mm for cellobiose and 2.9 mm for Pi.