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.     

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.     

Preparation of Fine Magnetic Particles and Application for Enzyme Immobilization

Fine magnetic particles (ferrofluid) were prepared from a co-precipitation method by oxidation of Fe²⁺ with nitrite. The particles were activated with (3-aminopropyl)triethoxysilane in toluene and the activated particles were combined with some enzymes by using glutaraldehyde. Enzyme-immobilized magnetic particles were between 4-70 nm and the size could be changed corresponding to the ratio of the amount of Fe²⁺ to that of nitrite. In the immobilization of β-glucosidase, activity yield was 83% and 168 mg protein was immobilized per g magnetite. Other enzymes or proteins could be immobilized at the level between about 70 and 200mg/g support. Immobilized β-glucosidase was stable at 4°C. Magnetic particles immobilized with β-glucosidase responded quickly to the magnetic field and “ON-OFF” control of the enzyme reaction was possible.     

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.     

Physicochemical properties of pretreated poplar feedstocks during simultaneous saccharification and fermentation

Physicochemical properties of native and dilute acid pretreated (0.6% H₂SO₄, 10 min, and either 170°C or 180°C) poplar were investigated before and during simultaneous saccharification and fermentation (SSF). SSF duration was 5 days and employed Trichoderma reesei cellulases and Saccharomyces cerevisiae fermentation. Chemical composition (glucan, xylan, lignin), enzyme-accessible surface area (based on solute exclusion), crystallinity index, particle size distribution, particle shape, and enzyme adsorption (cellulase, β-glucosidase) were compared to cellulose conversion. Cellulose conversion varied from 8% for native poplar to 78% for the 180°C-pretreated poplar. The physicochemical properties of native poplar changed little during SSF. In contrast, the physicochemical properties of the 180°C-pretreated feedstock changed markedly. Enzyme-accessible surface area and β-glucosidase adsorption increased by 83% and 65%, respectively, as cellulose was removed from the feedstock. Crystallinity index and particle size (large fraction) decreased by 65% and 93%, respectively. Cellulase adsorption per unit weight increased initially (+45%) followed by a slight decrease (−13%). The same trends were observed, although to a lesser extent, for 170°C-pretreated feedstock.     

Characterization of the Cellulase Complex of Penicillium echinulatum

New cellulases from a strain of Penicillium echinulatum were characterized for their filter paper activity and β-glucosidase activity. Both activities showed maximum values between pH 4 and 5. With citrate buffer, activities were slightly higher than in acetate buffer of the same pH. Thermal stability of both activities was good up to 55°C. Filter paper activity was significantly reduced at higher temperatures.     

Enzyme markers and isolation of the microvillar and perimicrovillar membranes of Dysdercus peruvianus (hemiptera: pyrrhocoridae) midgut cells

The midgut of Dysdercus peruvianus is divided into four sections (V₁-V₄). All the cells have microvilli ensheathed by a lipoprotein membrane (perimicrovillar membrane) extending toward the lumen as narrow tubes with dead ends. Subcellular fractionation of V₁ and V₂ tissue in isotonic and hypotonic conditions showed that -glucosidase is associated with membranous structures larger than those associated with β-glucosidase. The /β-glucosidase activity ratio is 34 ± 4 in V₁ tissue and 170 ± 10 in membranes recovered from the V₁ luminal contents. These membranes are resolved in sucrose gradients into low density (1.087 ± 0.001 g/cm³) -glucosidase-carrying membranes (/β-glucosidase activity ratio of 330±30) and high density (1.132 ± 0.002g/cm³) β-glucosidase-carrying-membranes. Low-density membranes have 1090 ± 60 μg lipid/mg protein and apparently are not contaminated by high-density ones (electron micrographs). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that membranes recovered from V₁ luminal contents are composed mainly of a-glucosidase-rich membranes. The data suggest that -glucosidase-rich membranes are perimicrovillar membranes which may be partly lost into luminal contents on dissection, with densities and lipid/protein ratios similar to that of myelin sheaths, in accordance with previous freeze-fracture data. β-Glucosidase-rich membranes are probably microvillar membranes with densities increased by the presence of associated portasomes.     

A digestive beta-glucosidase from the silkworm, Bombyx mori: cDNA cloning, expression and enzymatic characterization

A digestive β-glucosidase cDNA was cloned from the silkworm, Bombyx mori. The B. mori β-glucosidase cDNA contains an open reading frame of 1473 bp encoding 491 amino acid residues. The B. mori β-glucosidase possesses the amino acid residues involved in catalysis and substrate binding conserved in glycosyl hydrolase family 1. Southern blot analysis of genomic DNA suggested the B. mori β-glucosidase to be a single gene. Northern blot analysis of B. mori β-glucosidase gene confirmed larval midgut-specific expression. The B. mori β-glucosidase mRNA expression in larval midgut was detectable only during feeding period, whereas its expression was downregulated during starvation. The B. mori β-glucosidase cDNA was expressed as a 57-kDa polypeptide in baculovirus-infected insect Sf9 cells, and the recombinant β-glucosidase was active on cellobiose and lactose, but not active on salicin, indicating that the B. mori β-glucosidase possesses the characteristics of the Class 2 enzyme. The enzyme activity of the purified recombinant β-glucosidase expressed in baculovirus-infected insect cells was approximately 665 U per μg of recombinant B. mori β-glucosidase. The purified recombinant B. mori β-glucosidase showed the highest activity at 35 °C and pH 6.0, and were stable at 50 °C at least for 10 min. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that the recombinant B. mori β-glucosidase is N-glycosylated, but the carbohydrate moieties are not essential for enzyme activity.     

Properties of endogenous β-glucosidase of a Saccharomyces cerevisiae strain isolated from Sicilian musts and wines

Three hundred sixty-one yeast strains (80 of which ascribable to Saccharomyces cerevisiae) were isolated from Sicilian musts and wines with the purpose of looking for β-glucosidase (βG, EC 3.2.1.21) activity. Of these, the AL 41 strain had highest endogenous βG activity and was identified as belonging to the species S. cerevisiae by biochemical and molecular methods. This enzyme was subsequently characterized. It had optimum effect at pH 3.5–4.0, whilst optimum temperature was 20 °C, compatible with typical wine-cellar conditions; it was not inhibited by ethanol, at concentrations of 12–14%, or fructose and glucose. The βG was also characterised in terms of the kinetic parameters K_m (2.55 mM) and V_max (1.71 U mg⁻¹ of protein). Finally, it remained stable for at least 35 days in model solutions of must and wine.     

The temperature and pH properties of the extracellular hemicellulose-Degrading enzymes of aureobasidium pullulans NRRL Y 2311-1

Aureobasidium pullulans grown on arabinoxylan accumulates β-xylanase, p-nitrophenyl xylosidase, - -arabinofuranosidase and acetyl esterase activity in the culture fluid. The pH and temperature optima of these arabinoxylan-degrading enzymes were determined. The temperature optima of β-xylanase and p-nitrophenyl xylosidase were between 45 and 50°C whereas the optima for acetyl esterase and - -arabinofuranosidase were 55 and 60°C, respectively. β-xylanase, p-nitrophenyl xylosidase and - -arabinofuranosidase were stable over 3 h at 35°C, 35°C and 60°C, respectively, whereas acetyl esterase remained stable at 55°C for h. The enzymes were inactivated at higher temperatures. The pH optima for β-xylanase, p-nitrophenyl xylosidase and - -arabinofuranosidase were pH 4·0, between 4·0 and 7·0 and 5·0, respectively. β-xylanase, p-nitrophenyl xylosidase, - -arabinofuranosidase and acetyl esterase were most stable at pH 5·0 4·0–5·0, 6·0 and 5·0–6·0, respectively. The most suitable conditions for the use of the enzymes together to hydrolyze arabinoxylan would be 35 °C and pH 5.     

Temperature and carbon source affect the production and secretion of a thermostable β-xylosidase by Aspergillus fumigatus

The effect of the temperature of growth and carbon source on the production and secretion of β-xylosidase (EC 3.2.1.37) by the thermotolerant fungi Aspergillus fumigatus was studied in submerged cultures. In cultures developed at optimal temperature (30 °C), the enzyme was predominantly cell-bound, while in cultures developed at higher temperature (42 °C), the β-xylosidase activity was predominantly found in the cell-free filtrates. The use of corn cob powder instead of xylan as substrate increased considerably the secretion of enzyme. The highest level of extracellular β-xylosidase (45 U/ml or 360 U/mg protein) was obtained in 3% corn cob cultures grown at 42 °C for 72 h. The partially purified enzyme was active and stable at high temperatures. The presence of high titres of β-xylosidase activity in association with xylanase in the culture filtrates enhanced the efficiency of the pulp hydrolysis process.     

Properties of endogenous β-glucosidase of a Pichia anomala strain isolated from Sicilian musts and wines

The AL 112 strain, isolated from 361 yeast strains in Sicilian musts and wines, has been identified by biochemical and molecular methods as belonging to Pichia anomala, and your endogenous β-glucosidase (βG, EC 3.2.1.21) subsequently characterised. This strain not only has extremely high specific productivity of βG, but above all shows arabinosidase (Ara, EC 3.2.1.55) activity, essential for aroma enhancement of wine. βG from Al 112 is activated by ethanol at the concentrations typically found in wine; it is not inhibited by fructose, whilst glucose, a non-competitive inhibitor, despite lowering activity, actually protects the enzyme from factors that could damage it. It has an optimum temperature of 20 °C, compatible with typical cellar conditions, and stability in model must-wine and wine solutions ≥40 days.     

Cellobiose phosphorylase from Cellulomonas uda: gene cloning and expression in Escherichia coli, and application of the recombinant enzyme in a ‘glycosynthase-type’ reaction

We have cloned and sequenced the gene encoding cellobiose phosphorylase from Cellulomonas uda and report high yield production in Escherichia coli of a functional recombinant enzyme containing an N-terminal metal affinity fusion peptide. Use of heterologous gene expression increases the space-time yield of active phosphorylase by three orders of magnitude, compared to production of the enzyme with the natural organism. The full-length phosphorylase is a 91.3 kDa protein that consists of 821 amino acids and whose primary structure shares significant residue identity with different members of glycosyltransferase family 36. Purified enzyme was obtained in 39% overall yield by using copper-chelate and hydroxyapatite chromatographies. A comparative steady-state kinetic analysis for enzymatic reactions in the directions of phosphorolysis and synthesis of cellobiose at 30 °C and pH 6.6 demonstrates that the catalytic properties of the natural enzyme are retained completely in the recombinant cellobiose phosphorylase. The ability of the phosphorylase to utilize - -glucose 1-fluoride (G1F) as alternate glucosyl donor in place of - -glucose 1-phosphate (G1P) is exploited for the synthesis of β-1,4-glucosides under thermodynamic control in close to 100% yield.     

Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol

Wheat straw consists of 48.57 ± 0.30% cellulose and 27.70 ± 0.12% hemicellulose on dry solid (DS) basis and has the potential to serve as a low cost feedstock for production of ethanol. Dilute acid pretreatment at varied temperature and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from wheat straw (7.83%, w/v, DS) by dilute H₂SO₄ (0.75%, v/v) pretreatment and enzymatic saccharification (45 °C, pH 5.0, 72 h) using cellulase, β-glucosidase, xylanase and esterase was 565 ± 10 mg/g. Under this condition, no measurable quantities of furfural and hydroxymethyl furfural were produced. The yield of ethanol (per litre) from acid pretreated enzyme saccharified wheat straw (78.3 g) hydrolyzate by recombinant Escherichia coli strain FBR5 was 19 ± 1 g with a yield of 0.24 g/g DS. Detoxification of the acid and enzyme treated wheat straw hydrolyzate by overliming reduced the fermentation time from 118 to 39 h in the case of separate hydrolysis and fermentation (35 °C, pH 6.5), and increased the ethanol yield from 13 ± 2 to 17 ± 0 g/l and decreased the fermentation time from 136 to 112 h in the case of simultaneous saccharification and fermentation (35 °C, pH 6.0).     

Production, purification and properties of γ-glutamyltranspeptidase from a newly isolated Bacillus subtilis NX-2

Production, purification and properties of γ-glutamyltranspeptidase from a newly isolated Bacillus subtilis NX-2 was investigated. At the optimum conditions for enzyme formation, a high level, 3.2 U/ml of γ-GTP was obtained. The extracellular γ-GTP from this strain was purified 111.15-fold to homogeneity from the culture supernatant by acetone precipitation, hydrophobic interaction chromatography and ion exchange chromatography. The purified enzyme was a heterodimer consisting of one large subunit (43 kDa) and one small subunit (32 kDa), and exhibited high activity at 40–60 °C, pH 8.0. It preferred basic amino acids as γ-glutamyl acceptor in transpeptidation, and the stereochemistry of the γ-glutamyl acceptor had no influence on the enzyme activity, which was different from other γ-GTPs reported. Furthermore, it was proved that γ-GTP of this strain could catalyze the transfer of l-glutamine to glycylglycine to synthesize Gln–Gly–Gly, which was promising for the synthesis of valuable γ-glutamyl peptides.     

The influence of pH on the viscous behavior of starch-poly(ethylene-co-acrylic acid) complexes

Starch-poly (ethylene-co-acrylic acid) (EAA) complexes were prepared by jet-cooking mixtures of either cornstarch, waxy cornstarch or high amylose cornstarch with aqueous ammonia dispersions of EAA (4% EAA based on the weight of starch). Viscosities (η) were determined at temperatures ranging from 80°C to 22°C, and plots of log η versus 1/T (K⁻¹) were prepared. When cooked with EAA, cornstarch and waxy cornstarch showed major changes in viscous behavior between 50°C and 60°C. Above 50–60°C, viscosity increased markedly with a reduction in temperature; however, viscosity increased slowly below 50–60°C with an apparent activation energy for the process approximating that of water itself. The temperature dependence of the measured viscosity from 80°C to 60°C could be attributed to the large increase in size and complexity of the flowing particles as individual amylopectin molecules were bound together by complexed EAA. Apparently, complexing is essentially complete at 50°C. When high amylose cornstarch was cooked in the absence of EAA, retrogradation produced a sharp increase in log η at temperatures below about 50°C. However, if EAA is present, association between amylose molecules apparently takes place via complex formation rather than retrogradation, since log η increases sharply at about 70–80°C. Also, in contrast to cornstarch and waxy cornstarch, log η versus 1/T plots for high amylose cornstarch did not level off at low temperatures. In general, viscosities increased with the pH of the system, particularly when η was measured at high temperatures. This could result from improved complexing ability of EAA under high pH conditions, possibly due to reduced micelle size and maximum extension of polymer chains from micelle surfaces.     

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.     

Structure and physicochemical properties of barley non-starch polysaccharides — I. Water-extractable β-glucans and arabinoxylans

Water-soluble non-starch polysaccharides were extracted from a Canadian malting barley (cv. Harrington) by sequential treatment with water at 40 °C (WE40) and 65 °C (WE65). The yields were 1.4 and 1.3% (w/w), respectively, of the dry barley grist. The WE40 extract was composed of 82.5% glucose, 8.9% xylose, and 7.0% arabiose residues, whereas WE65 contained 93.3% Glc, 3.3% Xyl, and 2.5% Ara. Only minute amounts of mannose and galactose residues were found in either fraction. Both extracts were further fractionated by stepwise (NH₄)₂SO₄ precipitation into several polysaccharide populations. Subfractions from both extracts, obtained up to 45% saturation with (NH₄)₂SO₄, contained mostly β-glucans, whereas subfractions precipitated at increasing saturation levels of (NH₄)₂SO₄ (45–100%) contained progressively more arabinoxylans and less β-glucans. Compared to WE40, the WE65 extract was enriched in β-glucan populations with higher molecular size, higher limiting viscosity values, and higher content of β-(1 → 4) linkages. The ratio of tri-/ tetrasaccharide oligomers was also higher in β-glucans extracted at 65 °C than those extracted at 40 °C. Arabinoxylans in both extracts, WE40 and WE65, were highly substituted and contained large proportions of doubly substituted xylose residues.