Affinity chromatography of beta-glucosidase and endo-beta-glucanase from Aspergillus niger on concanavalin A-Sepharose: implications for cellulase component purification and immobilization

Affinity chromatography of a commercial preparation of beta-glucosidase from Aspergillus niger using concanavalin A-Sepharose (CAS) was employed as a means of purifying this glycoprotein. However, mannose (up to 1.08 M) was ineffective as an eluent of this enzyme from CAS, as were several other sugars and their derivatives, including 0.5 M glucose. Also, washing the CAS: beta-glucosidase complex with buffer at pH 3.5 in the absence of MnCl2 and CaCl2 (required to preserve the binding activity of concanavalin A below pH 5.0) did not result in elution of this enzyme. On the contrary, endoglucanase activity present in a crude cellulase complex (A. niger) which bound to CAS could be eluted by mannose (0.5-0.7 M) and was fractionated into at least two components. The CAS: beta-glucosidase complex hydrolyzed cellobiose to glucose and possessed an activity of 2,158 units/g dry CAS. It could be used, therefore, for continuous cellobiose hydrolysis without leakage of enzyme from the support.     

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.     

A constitutive, plasma-membrane bound β-glucosidase in Trichoderma reesei

Abstract Plasma membranes of Trichoderma reesei QM 9414, isolated from protoplasts by means of the concanavalin A procedure, contained β-glucosidase activity, which appeared constitutively upon growth on glucose. The enzyme had a pH optimum around 6, and was active on p -nitrophenyl-β- d -glucoside, cellobiose and sophorose ( K _m 0.7, 3.9 and 3.1 mM, respectively). Glucose was only weakly inhibitory ( K _i 7 mM). Treatment of the plasma membranes with Triton X-100, Tween 80 or digitonin solubilized more than 60% of the membrane-bound β-glucosidase activity. The enzyme so solubilized exhibited an M _r of 70 000 ± 5000 and an isoelectric point at pH 8.2 ± 0.3.     

Enzymatic hydrolysis of cellulose to glucose lung immobilized β-glucosidase

The production of sugars by enzymatic hydrolysis of cellulose is a multistep process which includes conversion of the intermediate cellobiose to glucose by β-glucosidase. Aside from its role as an intermediate, cellobiose inhibits the endoglucanase components of typical cellulase enzyme systems. Because these enzyme systems often contain insufficient concentrations of β-glucosidase to prevent accumulation of inhibitory cellobiose, this research investigated the use of supplemental immobilized β-glucosidase to increase yield of glucose. Immobilized β-glucosidase from Aspergillus phoenicis was produced by sorption at controlled-pore alumina with about 90% activity retention. The product lost only about 10% of the original activity during an on-stream reaction period of 500 hr with cellobiose as substrate; maximum activity occurred near pH 3.5 and the apparent activation energy was about 11 kcal/mol. The immobilized β-glucosidase was used together with Trichoderma reesei cellulase to hydrolyze cellulosic materials, such as Solka Floc, corn stove and exploded wood. Increased yields of glucose and greater conversions of cellobiose of glucose were observed when the reaction systems contained supplemental immobilized β-glucosidase.     

Purification and characterization of ββ-glucosidase from Aspergillus niger strain 322

An extracellular β-glucosidase enzyme was purified from the fungus Aspergillus niger strain 322 . The molecular mass of the enzyme was estimated to be 64 kDa by SDS gel electrophoresis. Optimal pH and temperature for β-glucosidase were 5·5 and 50 °C, respectively. Purified enzyme was stable up to 50 °C and pH between 2·0 and 5·5. The K_m was 0·1 mmol l⁻¹ for cellobiose. Enzyme activity was inhibited by several divalent metal ions.     

Production, purification and properties of endoglucanase from a newly isolated strain of Mucor circinelloides

A newly isolated strain of the fungus, Mucor circinelloides (NRRL 26519), when grown on lactose, cellobiose, or Sigmacell 50 produces complete cellulase (endoglucanase, cellobiohydrolase, and β-glucosidase) system. The extracellular endoglucanase (EG) was purified to homogeneity from the culture supernatant by ethanol precipitation (75%, v/v), CM Bio-Gel A column chromatography, and Bio-Gel A-0.5 m gel filtration. The purified EG (specific activity 43.33 U/mg protein) was a monomeric protein with a molecular weight of 27 000. The optimum temperature and pH for the action of the enzyme were at 55 °C and 4.0–6.0, respectively. The purified enzyme was fully stable at pH 4.0–7.0 and temperature up to 60 °C. It hydrolysed carboxymethyl cellulose and insoluble cellulose substrates (Avicel, Solka-floc, and Sigmacell 50) to soluble cellodextrins. No glucose, cellobiose, and short chain cellooligosaccarides were formed from these substrates. The purified EG could not degrade oat spelt xylan and larch wood xylan. It bound to Avicell, Solka-floc, and Sigmacell 50 at pH 5.0 and the bound enzyme was released by changing the pH to 8.0. The enzyme activity was enhanced by 27±5 and 44±14% by the addition of 5 mM MgCl₂ and 0.5 mM CoCl₂, respectively, to the reaction mixture. Comparative properties of this enzyme with other fungal EGs are presented.     

The influence of pH upon the kinetic parameters of the enzymatic hydrolysis of cellobiose with Novozym 188

We have studied experimentally within the pH range of 3.65-5.5 at 50 degrees C the hydrolysis of cellobiose with Novozym 188, a commercial product with high beta-1,4-glucosidase activity derived from Aspergillus niger. We used wide variations in the conversion to be able to apply the integral method and thus determine that there is substrate and mixed product inhibition. Whether the SES triple compound contributes to the formation of glucose does not influence the fitting of the experimental results to the theoretical model to any significant extent. We have established how pH affects the kinetic parameters and ascertained that pH 4.3 is the optimum for the conversion of cellobiose into glucose.     

Isolation and Characterization of a β-Glucosidase from a Clavispora Strain with Potential Applications in Bioethanol Production from Cellulosic Materials

We previously reported on a new yeast strain of Clavispora sp. NRRL Y-50464 that is capable of utilizing cellobiose as sole source of carbon and energy by producing sufficient native β-glucosidase enzyme activity without further enzyme supplementation for cellulosic ethanol production using simultaneous saccharification and fermentation. Eliminating the addition of external β-glucosidase reduces the cost of cellulosic ethanol production. In this study, we present results on the isolation and identification of a β-glucosidase protein from strain Y-50464. Using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and blast search of the NCBInr database (National Center for Biotechnology Information nonredundant), the protein from Y-50464 was identified as a β-glucosidase (BGL1) with a molecular weight of 93.3 kDa. The BGL1 protein was purified through multiple chromatographic steps to a 26-fold purity (K _m?=?0.355 mM [pNPG]; K _i?=?15.2 mM [glucose]), which has a specific activity of 18.4 U/mg of protein with an optimal performance temperature at 45 °C and pH of 6.0. This protein appears to be intracellular although other forms of the enzyme may exist. The fast growth rate of Y-50464 and its capability to produce sufficient β-glucosidase activity for ethanol conversion from cellobiose provide a promising means for low-cost cellulosic ethanol production through a consolidated bioprocessing development.     

Heterologous production and biochemical characterization of a new highly glucose tolerant GH1 β-glucosidase from Anoxybacillus thermarum

The enzymatic lignocellulosic biomass conversion into value-added products requires the use of enzyme-rich cocktails, including β-glucosidases that hydrolyze cellobiose and cellooligosaccharides to glucose. During hydrolysis occurs accumulation of monomers causing inhibition of some enzymes; thus, glucose/xylose tolerant β-glucosidases could overcome this drawback. The search of new tolerant enzymes showing additional properties, such as high activity, wide-pH range, and thermal stability is very relevant to improve the bioprocess. We describe a novel β-glucosidase GH1 from the thermophilic Anoxybacillus thermarum (BgAt), which stood out by the robustness combination of great glucose/xylose tolerance, thermal stability, and high Vmax. The recombinant his-tagged-BgAt was overexpressed in Escherichia coli, was purified in one step, showed a high glucose/xylose tolerance, and activity stimulation (presence of 0.4 M glucose/1.0 M xylose). The optimal activity was at 65 °C - pH 7.0. BgAt presented an extraordinary temperature stability (48 h – 50 °C), and pH stability (5.5–8.0). The novel enzyme showed outstanding Vmax values compared to other β-glucosidases. Using p-nitrophenyl-β-d-glucopyranoside as substrate the values were Vmax (7614 U/mg), and K_M (0.360 mM). These values suffer a displacement in Vmax to 14,026 U/mg (glucose), 14,886 U/mg (xylose), and K_M 0.877 mM (glucose), and 1.410 mM (xylose).     

1,3-β-Glucan synthase from Citrus phloem

1,3-β-Glucan synthase activity has been demonstrated in particulate fractions of bark extracts from Mexican lime. With respect to substrate, the enzyme kinetics did not conform to the Michaelis-Menten equation. The value of the Hill coefficient was 1.2 and S_0.5 is 1.1 mM. The enzyme had an optimum pH of 7.5. Maltose, sucrose, and especially cellobiose and glucose, were enzyme activators when tested at physiological concentrations. In the presence of 15 mM MgCl₂ the enzymic activity was stimulated at 10 μM UDP-glucose but decreased at 1 mM UDP-glucose, suggesting a minor 1,4-β-glucan synthase activity.     

Isolation and some properties of two extracellular β-glucosidases from Trichosporon adeninovorans

The yeast Trichosporon adeninovorans secretes two multiple forms of β-glucosidase at a high rate if grown in a medium containing cellobiose. Following mutagenesis a mutant strain resistant to 2-deoxy-D-glucose was selected. This strain produced more β-glucosidase activity and had acquired a strong resistance against repression by glucose. The β-glucosidases were separated one from each other by chromatography on hydroxylapatite and by gel filtration. Both enzymes have similar properties. The optimal temperature for their activity was 60 to 63°C and the enzymes displayed highest activity at pH of 4.5. The molecular weight of β-glucosidase I was found to be 570,000 and that for β-glucosidase II was 525,000. The K_m value for cellobiose was determined to be 4.1 mM for β-glucosidase I and 3.0 mM for β-glucosidase II.     

Components of cellulase from the higher termite,Nasutitermes walkeri

The cellulase from the termite Nasutitermes walkeri consists of two enzymes. Each has broad specificity with predominantly one activity. One enzyme is an endo-gb-1,4-glucanase (EC 3.2.1.4) which predominantly cleaves cellulose randomly to glucose, cellobiose and cellotriose. It hydrolyses cellotetraose to cellobiose but will not hydrolyse cellobiose or cellotriose. The second enzyme component is a β-1,4-glucosidase (EC 3.2.1.21) as its major activity is to hydrolyse cellobiose, cellotriose and cellotetraose to glucose; it has some exoglucosidase activity as glucose is the only product produced from cellulose. Its cellobiase activity is inhibited by glucono-δ-lactone.     

The influence of temperature upon the hydrolysis of cellobiose by beta-1,4-glucosidases from Aspergillus niger

We have made experimental studies into the enzymatic hydrolysis of cellobiose within the temperature range of 40 degrees C to 70 degrees C at pH 4.9, by using beta-1,4-glucosidase from Aspergillus niger. At 70 degrees C there was significant enzyme deactivation, which could be fitted to a potential deactivation model with values of n equal to 1.09 and k(d) to 0.1564 (g/l)(-0.09) min(-1), whereas the rate of hydrolysis could be fitted to the Michaelis-Menten equation. Between 40 degrees C and 60 degrees C we noted a substrate inhibition and that the CEC compound formed contributed to glucose production. The apparent activation energies had values of 4.66, 8.45, 4.82, and 3.99 kJ/mol for the kinetic constants k(a) and k(a2) the Michaelis constant and the substrate inhibition constant, respectively.     

Multiple sugar binding sites in α-glucosidase

Twenty-five analogs of d-glucose were examined as reversible inhibitors of yeast α-glucosidase (EC 3.2.1.20). The K_i values range from 0.38 mM for 6-deoxy-d-glucose (quinovose) to 1.0 M for d-lyxose at pH=6.3 (0.1 M NaCl, 25°). All the monosaccharides and the three disaccharides (maltose, isomaltose and α,α-trehalose) were found to be linear competitive inhibitors with respect to α-p-nitrophenyl glucoside (pNPG) hydrolysis. Multiple inhibition analysis reveals that there are at least three monosaccharide binding sites on the enzyme. One of these can be occupied by glucose [K_i=1.8(±0.1) mM], one by d-galactose [K_i=164(±11) mM] and one by d-mannose [K_i=120(±9) mM]. The pH dependence for glucose binding closely follows that of V/K [pK_a1=5.55(±0.15), pK_a2=6.79(±0.15)], but the binding of mannose does not. Although the glucose subsite can be occupied simultaneously with the mannose or galactose subsites in the enzyme–product complex, no transglucosylation can be detected between pNPG and either mannose or galactose. This suggests that neither of these nonglucose subsites can be occupied in a productive manner in the covalent glucosyl-enzyme intermediate.     

Purification and biochemical characterization of a transglucosilating β-glucosidase of <Emphasis Type="Italic">Stachybotrys</Emphasis> strain

The filamentous fungus Stachybotrys sp has been shown to possess a rich β-glucosidase system composed of five β-glucosidases. One of them was already purified to homogeneity and characterized. In this work, a second β-glucosidase was purified and characterized. The filamentous fungal A19 strain was fed-batch cultivated on cellulose, and its extracellular cellulases (mainly β-glucosidases) were analyzed. The purified enzyme is a monomeric protein of 78 kDa molecular weight and exhibits optimal activity at pH 6.0 and at 50°C. The kinetic parameters, K _m and V _max, on para-nitro-phenyl-β-d-glucopyranosid (p-NPG) as a substrate were, respectively, 1.846 ± 0.11 mM and 211 ± 0.08 μmol min⁻¹ ml⁻¹. One interesting feature of this enzyme is its high stability in a wide range of pH from 4 to 10. Besides its aryl β-glucosidase activity towards salicin, methylumbellypheryl-β-d-glucoside (MU-Glc), and p-NPG, it showed a true β-glucosidase activity because it splits cellobiose into two glucose monomers. This enzyme has the capacity to synthesize short oligosaccharides from cellobiose as the substrate concentration reaches 30% with a recovery of 40%. We give evidences for the involvement of a transglucosylation to synthesize cellotetraose by a sequential addition of glucose to cellotriose.     

Production of a highly glucose-tolerant extracellular β-glucosidase by three Aspergillus strains

Quercetin was the best inducer for the production of a highly glucose-tolerant, extracellular -glucosidase in Aspergillus niger and Aspergillus oryzae. The enzyme was separated from the major and common -glucosidase by gel filtration and that from Aspergillus oryzae further purified by ion-exchange chromatography. It was highly resistant to glucose inhibition (Ki= 953 mM), had a pI of 4.2, optimum pH of 4.5–6.0 and a molecular mass of 30 kDa according to gel filtration. The enzyme was active against cellobiose and alkyl glucosides.     

Characterization of a recombinant mannobiose 2-epimerase from Spirochaeta thermophila that is suggested to be a cellobiose 2-epimerase

A purified recombinant enzyme from Spirochaeta thermophila, that is suggested to be a cellobiose 2-epimerase, was a 47 kDa monomer with a specific activity of 29.2 U min^?1 for mannobiose. The epimerization activity of the recombinant enzyme for mannobiose was maximal at pH 7.0 and 60 °C with a half-life of 124 h. The enzyme exhibited a higher epimerization activity for mannose or the mannose moiety at the reducing end of β- and α-1,4-glycosyl-mannose than for glucose or the glucose moiety of β- and α-1,4-glycosyl-glucose. The enzyme was identified as a mannobiose 2-epimerase by evaluating its substrate specificity with not only glucose-containing sugars but also mannose-containing sugars. The activities of the reported cellobiose 2-epimerases from Caldicellulosiruptor saccharolyticus, Dictyoglomus turgidum and Ruminococcus marinus for mannobiose were higher than those for cellobiose, strongly suggesting that these enzymes are not cellobiose 2-epimerases but are mannobiose 2-epimerases.     

Some kinetic properties of the β- -glucosidase (cellobiase) in a commercial cellulase product from Penicillium funiculosum and its relevance in the hydrolysis of cellulose

Some kinetic parameters of the β- -glucosidase (cellobiase, β- -glucoside glucohydrolase, EC 3.2.1.21) component of Sturge Enzymes CP cellulase [see 1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] from Penicillium funiculosum have been determined. The Michaelis constants (K_m) for 4-nitrophenyl β- -glucopyranoside (4NPG) and cellobiose are 0.4 and 2.1 mM, respectively, at pH 4.0 and 50°C. -Glucose is shown to be a competitive inhibitor with inhibitor constants (K_i) of 1.7 mM when 4NPG is the substrate and 1 mM when cellobiose is the substrate. Cellobiose, at high concentrations, exhibits a substrate inhibition effect on the enzyme. -Glucono-1,5-lactone is shown to be a potent inhibitor (K_i = 8 μM; 4NPG as substrate) while -fructose exhibits little inhibition. Cellulose hydrolysis progress curves using Avicel or Solka Floc as substrates and a range of commercial cellulase preparations show that CP cellulase gives the best performance, which can be attributed to the activity of the β- -glucosidase in this preparation in maintaining the cellobiose at low concentrations during cellulose hydrolysis.     

Production,purification, and properties of β-glucosidase from Candida wickerhamii

Summary Candida wickerhamii growing on cellobiose produced -glucosidase with high activity against -nitrophenyl glucoside (PNPG) but low activity against cellobiose. -glucosidase production was constitutive, and was repressed by -glucosides and glucose. -glucosides containing an aromatic moiety in the aglycon were the best substrates for -glucosidase indicating that the enzyme is an aryl--glucosidase. A -glucosidase from C. wickerhamii cells was purified by (NH₄)₂SO₄ precipitation, dialysis, ion-exchange chromatography and gel filtration. The purified enzyme was homogeneous as shown by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme hydrolysed PNPG but not cellobiose. The K_m of the enzyme was 0.185 mM. Glucose inhibited the enzyme competitively and the K_i was 7.5 mM. The apparent molecular mass was 97,000. The optimum pH and temperature for enzyme activity were between pH 7 and 7.4 and 40°C respectively. At temperatures of 45°C and greater the enzyme was inactivated. The activation energy of the enzyme was 29.4 kJ · mol^-1.     

一株嗜热毛壳菌β-葡萄糖苷酶的分离纯化及特性

研究了液体发酵嗜热毛壳菌Chaetomium thermophile产生的β-葡萄糖苷酶的分离纯化及特性。粗酶液经硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Phenyl-Sepharose 疏水层析、Sephacryl S-100分子筛层析等步骤后获得凝胶电泳均一的β-葡萄糖苷酶。经12.5%SDS-PAGE和凝胶过滤层析方法分别测得该酶的分子量大小约为78.4kDa和81kDa。该酶反应的最适温度和pH值分别为60℃和4.5-5.0。有较好的酸稳定性和热稳定性。金属离子对β-葡萄糖苷酶的活性影响较大, 其中Ca2+对酶有激活作用, 而Ag+、Cu2+ 、Hg2+对酶有显著的抑制作用。该酶对水杨苷具有很强的底物特异性。