Purification and characterization of a β-glucosidase fromAspergillus niger

The high-molar mass from of β-glucosidase fromAspergillus niger strain NIAB280 was purified to homogeneity with a 46-fold increase in purification by a combination of ammonium sulfate precipitation, hydrophobic interaction, ion-exchange and gel-filtration chromatography. The native and subunit molar mass was 330 and 110 kDa, respectively. The pH and temperature optima were 4.6–5.3 and 70°C, respectively. TheK _m andk _cat for 4-nitrophenyl β-d-glucopyranoside at 40°C and pH 5 were 1.11 mmol/L and 4000/min, respectively. The enzyme was activated by low and inhibited by high concentrations of NaCl. Ammonium sulfate inhibited the enzyme. Thermolysin periodically inhibited and activated the enzyme during the course of reaction and after 150 min of proteinase treatment only 10% activity was lost with concomitant degradation of the enzyme into ten low-molar-mass active bands. When subjected to 0–9 mol/L transverse urea-gradient-PAGE for 105 min at 12°C, the nonpurified β-glucosidase showed two major bands which denatured at 4 and 8 mol/L urea, respectively, with half-lives of 73 min.     

Properties of β-glucosidase purified from Aspergillus niger mutants USDB 0827 and USDB 0828

Summary Two extracellular -glucosidases (EC 3.2.1.21) were isolated from Aspergillus niger USDB 0827 and A. niger USDB 0828, and their physical and kinetic properties studied. Both enzymes were very similar in terms of molecular size (230000 Da), pH optimum (pH 4.6), temperature optimum (65° C), stability at high temperatures and substrate preferences. They were capable of hydrolysing -linked disaccharides, phenyl -d-glucoside, p-nitrophenyl -d-glucoside (PNPG), o-nitrophenyl -d-glucoside, salicin and methyl -d-glucoside but lacked activity towards -linked disaccharides, a range of p-nitrophenyl monoglycosides and p-nitrophenyl diglycosides. Both -glucosidases were better at hydrolysing cellobiose than cellotriose, cellotetraose or cellopentaose. For both enzymes, glucose showed competitive inhibition with PNPG as substrate but had no effect with cellobiose. However, the two -glucosidases differed in inhibition by glucono-1,5-lactone and affinity for cellobiose. -Glucosidase from A. niger USDB 0827 also gave lower specific activity, and was more susceptible to metal ions (Ag⁺, Fe²⁺ and Fe³⁺) inhibition than that of A. niger USDB 0828. Correspondence to: Y. K. Hoh     

The stability of extracellular β-glucosidase fromAspergillus niger is significantly enhanced by non-covalently attached polysaccharides

The removal of noncovalently bound polysaccharide coating from the extracellular enzymes ofAspergillus niger, by the technique of compartmental electrophoresis, had a very dramatic effect on the stability of β-glucosidase. The polysaccharide-β-glucosidase complex was extremely resistant to proteinases and far more stable against urea and temperature as compared with polysaccharide-free β-glucosidase. The β-glucosidase-polysaccharide complex was 18-, 36-, 40-, and 82-fold more stable against chymotrypsin, 3 mol/L urea, total thermal denaturation and irreversible thermal denaturation, respectively, as compared with polysaccharide-free β-glucosidase. The activation energy of polysaccharide-complexed β-glucosidase (55 kJ/mol) was lower than polysaccharide-free enzyme (61 kJ/mol), indicating a slight activation of the enzyme by the polysaccharide. No significant difference could be detected in the specificity constant (V/K _m) for 4-nitrophenyl β-d-glucopyranoside between polysaccharide-free and polysaccharide-complexed β-glucosidase. We suggest that the function of these polysaccharides secreted by fungi includingA. niger might be to protect the extracellular enzymes from proteolytic degradation, hence increasing their life span.     

Properties of an intracellular β-glucosidase purified from the cellobiose-fermenting yeast Candida wickerhamii

An intracellular -glucosidase was isolated from the cellobiose-fermenting yeast, Candida wickerhamii. Production of the enzyme was stimulated under aerobic growth, with the highest level of production in a medium containing cellobiose as a carbohydrate source. The molecular mass of the purified protein was approximately 94 kDa. It appeared to exist as a dimeric structure with a native molecular mass of about 180 kDa. The optimal pH ranged from 6.0 to 6.5 with p-nitrophenyl -d-glucopyranoside (NpGlc) as a substrate. The optimal temperature for short-term (15-min) assays was 35°C, while temperature-stability analysis revealed that the enzyme was labile at temperatures of 28° C and above. Using NpGlc as a substrate, the enzyme was estimated to have a K _m of 0.28 mM and a V _max of 525 mol product min^–1 mg protein^–1. Similar to the extracellular -glucosidase produced by C. wickerhamii, this enzyme resisted end-product inhibition by glucose, retaining 58% of its activity at 100 mM glucose. The activity of the enzyme was highest against aryl -1,4-glucosides. However, p-nitrophenyl xylopyranoside, lactose, cellobiose, and trehalose also served as substrates for the purified protein. Activity of the enzyme was stimulated by long-chain n-alkanols and inhibited by ethanol, 2-propanol, and 2-butanol. The amino acid sequence, obtained by Edman degradation analysis, suggests that this -glucosidase is related to the family-3 glycosyl hydrolases.     

Optimization of β-glucosidase production from Aspergillus niger

本研究对Aspergillus niger Glu05生产β-葡萄糖苷酶的培养基组分及培养条件进行了优化.优化后的培养基组成和培养条件分别为:麸皮4％,tryptone 4％,1μmol MnSO4,1μmol NaCl,KH2PO40.2％,oH自然,摇床转速250 r/min,培养温度30℃,培养周期5d.优化后发酵液中酶活力达到44.11 IU/mL,与初始的产酶水平32.87 IU/mL相比,提高了36％.     

In situ immobilized β-glucosidase fromAspergillus phoenicis QM 329

Summary Aspergillus phoenicis QM 329 was found to grow in the shape of beads in shake flasks and in an air-lift fermentor. Initial culture pH, pH profile during cultivation, carbon source, inoculum size and fermentor configuration influenced the retainment of the -glucosidase activty in the mycelium. Glucose and soluble starch produced beads with the highest activity and the best stability. Glucose-derived beads were more homogeneous in size and shape than the starch-derived beads. These beads kept their integrity for 10 d at 50° C. After 48 h hydrolysis of 50 g/l cellobiose 75% of the initial enzyme activity remained. The beads could be air-dried and alcohol-sterilized with only minor loss of activity. The size of the beads could be controlled by varying the size of the conidia inoculum. In an air-lift fermentor with a working volume of 1500 ml,900 ml beads with an average diameter of 2 mm (estimated to 37–45,000 beads) were produced in less than 3 d with glucose as carbon source. The beads held a -glucosidase activity of 0.140 IU/bead determined with the pNP assay.     

Synthesis of alkyl β-glucosides from cellobiose with Aspergillus niger β-glucosidase II

An extracellular -glucosidase II of Aspergillus niger catalyzed the synthesis of methyl -glucoside and ethyl -glucoside with 5.0% (v/v) cellobiose as glucosyl donor in a biphasic media containing 20% (v/v) methanol and 30% (v/v) ethanol, respectively. The maximum yield of methyl -glucoside and ethyl -glucoside was 83% (mol/mol; 12 mg/ ml) and 53% (mol/mol; 5.5 mg/ml), based on cellobiose consumed. © Rapid Science Ltd. 1998     

Over-production of β-glucosidase by Aspergillus niger mutant from lignocellulosic biomass

The maximum yield of -glucosidase by A. niger KK2 mutant, grown on the basal medium for 7 days, was 514 I U g^–1 ground rice straw, and was about twice those obtained from wheat straw or bran by previous researchers. Optimal activity of -glucosidase was at 60–70 °C and pH 4.8.     

Purification of β-N-acetyl-d-glucosaminidase fromAspergillus niger using thermostabilization and heat as the initial step

A β-N-acetyl-d-glucosaminidase (EC 3.2.1.30) produced byAspergillus niger 419, was completely inactivated after heating 15 min at 65°C in 100 mM sodium phosphate buffer pH 7. The presence of 10% of polypropyleneglycol 1025 induced the thermal stability of the enzyme, the activity remaining unchanged after heating 60 min at 65°C. When this thermal treatment was used as the initial step of purification, the protein content of the crude extract was reduced by 98% without loss of the total initial enzymatic activity of the sample and a purification factor of 61. As the second and third step of purification DEAE-Sephacel, and Sephadex-G150 column chromatography were used, respectively. The final purification factor was 230 with a yield of 76%.     

Synthesis of cello-oligosaccharides from cellobiose with β-glucosidase II from Aspergillus niger

An extracellular -glucosidase II of Aspergillus niger catalysed the synthesis of cello-oligosaccharides from cellobiose (15%, w/v). The enzyme was stable at and below 4°C for at least 230 days and also stable at 30°C with the presence of 2.0% (w/v) cellobiose. The maximum yield of cello-oligosaccharides was about 30% (mol/mol), based on cellobiose (130 mg/mL) consumed. © Rapid Science Ltd. 1998     

Comparative physico-kinetic properties of a homogenous purified β-glucosidase from Withania somnifera leaf

β-glucosidase from Withania somnifera (Solanaceae) leaf has been purified to homogeneity and characterized for its physico-kinetic properties. The enzyme purification was achieved through a sequence of gel filtration and ion-exchange column chromatography, and PAGE revealed the homogeneity purification status of the enzyme. The properties of the enzyme included an acidic pH optima (4.8), alkaline pI (8.7), meso-thermostabity, monomeric structure with subunit molecular weight of about 50 kDa, high affinity for substrate (K _m) for pNPG (0.19 mM) and high (105,263 M^?1 s^?1) catalytic efficiency (K _cat/K _m). The mesostable enzyme had a stringent substrate specificity restricted only to β-linked gluco-conjugate. The enzyme is optimally active at 40 °C with 12.4 kcal Mol^?1 activation energy, and was highly sensitive to d-gluconic acid lactone inhibition (94 % at 1 mM) with an apparent K _i 0.21 mM. The enzyme could catalyze transglucosylation of geraniol with pNPG as glucosyl donor, but not with cellobiose. Some of the physico-kinetic properties were noted to be novel when comprehensively compared with its counterparts from plant, animal and microbial counterparts. Nevertheless, the catalytic and other features of the enzyme were relatively closer to Oryza sativa among plants and Talaromyces thermophillus among fungi. Significance of building-up of a library of novel plant β-glucosidases for structural investigation to understand naturally evolved mechanistics of catalysis has been indicated.     

Reaction mechanism of isomaltooligosaccharides synthesis by α-glucosidase fromAspergillus carbonarious

Summary An -glucosidase fromAspergillus carbonarious CCRC 30414 was employed for investigating the enzymatic synthesis of isomaltooligosaccharides from maltose. The enzyme transferred a glucose unit from the nonreducing end of maltose and other -linked glucosyl oligosaccharides to glucose and other glucosyl oligosaccharides which function as accepting co-substrates. The transfer of a glucose unit occurs most frequently to the 6-OH position of the nonreducing end of acceptor, but transfer to 4-OH position also occurs. Treatment of 30 % (w/v) maltose with the enzyme under optimum conditions afforded more than 50% isomaltooligosaccharides.     

Solid state fermentation for cellulases and β-glucosidase production by Aspergillus niger

Production of cellulases and β-glucosidase was studied using locally-isolated Aspergillus niger on various cheap sources of cellulose like bagasse, corn corbs, computer cards and sawdust, by solid state fermentation (SSF) and by liquid state fermentation (LSF). Enzyme activities were increased about 30–80% by SSF in comparison with conventional LSF. Enzyme production was further improved by various pretreatments, making cellulosic material easily accessible. The best results were obtained with 5 M NaOH treatment.     

Structural analyses of new tri- and tetrasaccharides produced from disaccharides by transglycosylation of purified Trichoderma viride β-glucosidase

A new -glucosidase was partially purified from Trichoderma viride cellulase. This -glucosidase catalyzed a transglycosylation reaction of cellobiose to give -D-Glc-(16)--D-Glc-(14)-D-Glc (1, yield: 18.8%) and -D-Glc-(16)--D-Glc-(16)--D-Glc-(14)-D-Glc (2, 3.7%), regioselectively. Furthermore, the enzyme regioselectively converted laminaribiose and gentiobiose into -D-Glc-(16)--D-Glc-(13)-D-Glc (3, 15.3%) and -D-Glc-(16)--D-Glc-(16)-D-Glc (4, 20.2%), respectively. The structures (1–4) of the products were determined by ¹H and ¹³C NMR spectroscopies. This high regio- and stereoselectively of the -glucosidase could be applied for oligosaccharide synthesis.     

Synthetic inhibitors of glucocerebroside β-glucosidase

The glucocerebrosidase of human placenta was studied with various potential inhibitors. Several compounds that resemble the lipoidal product of enzyme action, ceramide, proved to be excellent inhibitors, acting by mixed modes (primarily noncompetitively). These were N-decyl-dl-erythro-3-phenyl-2-amino-l, 3-propanediol and several p-substituted derivatives. These compounds were also highly effective in rat spleen toward glucocerebroside and p-nitrophenyl β-glucoside as substrates. The compounds were inactive toward the analogous enzyme, galactocerebrosidase of rat brain, and were slightly stimulatory toward the rat brain enzyme which makes galactocerebroside. Longer and shorter N-alkyl groups proved to be less effective. Decanoic acid amides of phenylaminopropanediol and related compounds proved to be relatively inert, although some were stimulatory. Deoxycorticosterone β-glucoside was a moderately effective noncompetitive inhibitor and is apparently hydrolyzed by a different glucosidase. p-Nitrophenyl β-glucoside was also a moderately effective inhibitor, acting by mixed modes. p-Chloromercuribenzenesulfonate was a good inhibitor, presumably acting on a sensitive cysteine residue. It is concluded that cerebrosidase contains two sensitive sites, one catalytic and the other allosteric, each containing an important anionic group and able to bind glucosides and ceramide-like compounds.     

Production of β-glucosidase byCladosporium resinae

Summary Cladosporium resinae QM 7998 produced high activities of extracellular and constitutive -glucosidase when grown on a variety of sugars or cellulose. Starch and ribose induced enzyme synthesis several fold.Cladosporium resinae could utilize agricultural waste residues for growth and -glucosidase production. The initial pH of the medium had a marked effect on enzyme prowduction and optimum pH was between 4.0 and 5.0 depending on the assay method. Mixed culturing ofC. resinae with yeasts, viz.Saccharomyces cerevisiae andCandida utilis, increased the -glucosidase production while that with other fungi decreased the enzyme yield. The- glucosidase preparation fromC. resinae significantly increased the saccharification of rice and wheat straw (untreated or delignified) withTrichoderma reesei QM 9414 cellulase preparation.

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Résumé Cladosporium resinae QM 7998 produit des concentrations élevées de -glucosidase tant extracellulaire que constitutive lorsqu'elle croît sur une variété de sucres ou sur la cellulose. On a trouvé que l'amidon et le ribose augmentent de plusieurs fois la quantité d'enzyme synthétisée.Cladosporium resinae peut utiliser des résidus agricoles pour sa croissance et pour la production de -glucosidase. Le pH initial du milieu exerce un effet marqué sur la production d'enzyme et le pH optimum est compris entre 4.0 et 5.0 selon les conditions de l'essai. La croissance mixte deCladosporium resinae avec diverses levures, notammentSaccharomyces cerevisiae etCandida utilis, augmente la production de -glucosidase tandis que celle avec d'autres moisissures diminue le rendement en enzyme. La -glucosidase deCladosporium resinae augmente de manière significative la saccharification des pailles de riz et de froment (non-traitées ou délignifiées) traités par la cellulase deTrichoderma reesei QM 9414.

     

Properties of a β-(1→4)-glucan hydrolase from Aspergillus niger

1. A beta-(1-->4)-glucan hydrolase prepared from Aspergillus niger, as described by Clarke & Stone (1965a), showed a pH optimum in the range 4.5-6 and K(m) 0.25% when acting on a cellulose dextrin sulphate substrate. 2. The hydrolase rapidly decreased the specific viscosity of carboxymethylcellulose with a small increase in the production of reducing sugars. The identity of the products of hydrolysis of cellotetraose, cellopentaose and their reduced analogues indicate a preferential cleavage of non-terminal glucosidic linkages. The enzyme may be described as beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4). 3. In addition to carboxymethylcellulose, cellulose dextrins, cellopentaose and cellotetraose the enzyme fraction hydrolysed lichenin, oat and barley glucans, ivory-nut mannan and a glucomannan from Konjak flour. No hydrolysis of wheat-straw beta-(1-->4)-xylan, Lupinus albus beta-(1-->4)-galactan, pneumococcal type III polysaccharide, chitin, hyaluronic acid, laminarin, pachydextrins, carboxymethylpachyman or beta-(1-->3)-oligoglucosides was detected. 4. The hydrolase showed no transglycosylase activity from cellodextrin or cellopentaose substrates to glucose or methanol acceptors. 5. The hydrolysis of cellodextrins was inhibited completely by 1.0mm-Hg(2+), 0.7mm-phenylmercuric nitrate and 1.0mm-iodine.     

Distinctive Properties of β-Glucosidases and Related Enzymes Derived from a Commercial Aspergillus niger Cellulase

Eight highly purified β-glucosidases from Aspergillus niger were compared enzymatically, chemically, and immunologically. Ultraviolet spectra, pH-activity responses, substrate specificities, thermal stabilities, kinetic changes in the viscosity of substrate, Michaelis-Menten parameters, adsorption characteristics on cellulose, and exclusion characteristics on dextran gels were determined. The data indicate that the several components represent distinctly different enzymes in terms of mode of attack on substrate. The concept of partial denaturation of a single enzyme precursor is unable to explain the heterogeneity observed. Comparison of the effect of pH on hydrolysis of carboxymethylcellulose and cellohexaose suggests that a negative charge center on the substrate has a pronounced inhibitory effect on the enzymes.