Effect of carbon source on the biochemical properties of β-xylosidases produced by Aspergillus versicolor

The filamentous fungus Aspergillus versicolor produced large amounts of mycelial β-xylosidase activity when grown on xylan or xylose as the only carbon source. The presence of glucose drastically decreased the level of β-xylosidase activity, while cycloheximide prevented the induction of the enzymes by xylan or xylose. The β-xylosidases induced by xylose or xylan were purified by a simple protocol involving DEAE-cellulose chromatography and ammonium sulphate precipitation. The purified enzymes were acidic proteins, with carbohydrate contents of 21% for that induced by xylose, and 47% for that induced by xylan. Their apparent molecular masses, estimated by gel filtration, and optimal temperatures for β-xylosidase activities, were about 60 and 100 kDa, and 40 and 45 °C, respectively, for the enzymes induced by xylose and xylan. Xylose-induced β-xylosidase exhibited an optimum pH of 6.0, while that of the xylan-induced enzyme was 5.5. Both purified β-xylosidases exhibited also β-galactosidase, β-glucosidase and -arabinosidase activities. In addition to synthetic substrates, the enzymes hydrolysed xylobiose and xylotriose, suggesting a physiological role. K_M values for p-nitrophenyl β- -xylopyranoside were 0.32 mM, for the xylose-induced β-xylosidase, and 0.19 mM for the xylan-induced one. Xylose competitively inhibited both β-xylosidases, with K_I values of 5.3 and 2.0 mM, for the enzymes induced by xylose or xylan, respectively.     

Glycosidases in organic solvents: I. Alkyl-β-glucoside synthesis in a water-organic two-phase system

A low-water organic solvent two-phase system suitable for glycosylation of hydrophobic substrates is described. Almond β-glucosidase adsorbed on polymeric supports has been shown to catalyse alkyl-β-glucoside synthesis via a transferase reaction or through direct condensation of the glucosidic bond. High concentrations of glucosyl donors were present in the aqueous phase, while water-immiscible primary alcohols, which form the organic phase, served as acceptors of glucose. Reaction yield appeared to be thermodynamically controlled. The influence of various support materials, glucosyl donors, and glucosyl acceptors on reaction rate and product yield was investigated.     

Thermostability of β-xylosidase from Aspergillus sydowii MG49

Heating of Aspergillus β-xylosidase at 85°C ± 1°C and pH 5.5–6.0 (optimum for activity), causes irreversible, covalent thermoinactivation of the enzyme, involving oxidation of the thiol groups that are required for catalysis. Exogenous addition of cysteine, DTT, GSH and mercaptoethanol stabilizes the enzyme by extending its half-life. A similar effect is also exhibited by bivalent cations like Mg²⁺, Mn²⁺, Co²⁺, Ca²⁺and Zn²⁺ while, on the other hand Cu²⁺ accelerates thermoinactivation. Chemical modification of crude β-xylosidase with cross-linking agents like glutaraldehyde or covalent immobilization to a nonspecific protein like gelatin and BSA also enhances enzyme thermostability. These results suggest that addition of thiols and bivalent metal ions to a crude β-xylosidase preparation or immobilization/chemical modification enhances its thermal stability, thus preventing loss of catalytic activity at elevated temperatures.     

Enzymatic Synthesis of Thioglucosides Using Almond β-glucosidase

A selection of different glycosidases was screened for the glycosylation of 1-propanethiol. The β-glucosidases from almond, Aspergillus niger and Caldocellum saccharolyticum were capable of 1-propanethioglucoside (1-PTG) formation. The almond β-glucosidase showed the highest activity in this reversed hydrolysis type of reaction using glucose as glucosyl donor. Besides 1-propanethiol, also thioglucosides of 2-propanethiol and furfuryl mercaptan were formed by the almond β-glucosidase. The substrate specificity of the almond β-glucosidase with respect to thioglucosylation is restricted to primary and secondary aliphatic thiols. Once the thioglucosides are formed, they are not hydrolyzed at a significant rate by almond β-glucosidase. As a consequence the synthesis of 1-PTG could be observed at very low aglycone concentrations (0.5% v/v based on the reaction solution) and high yields (68% based on 1-PT and 41% based on glucose) were obtained. An excess of aglycone, otherwise frequently applied in reversed hydrolysis glycosylation, is therefore not necessary in the glucosylation of 1-PT.     

The Production of γ-Decalactone by Fermentation of Castor Oil

A microbial process for the production of optically-active γ-decalactone from the ricinoleic acid present as triglycerides in castor oil has been developed, γ-decalactone (γDL) is a component of some fruit flavours, being an important organoleptic component of peach flavours. Screening showed two red yeast microorganisms, Rhodotorula glutinis and Sporobolomyces odortts to be especially suitable for this biotransformation. The process involves lipase-mediated hydrolysis of the castor oil to give free ricinoleic acid, uptake of the acid by the cells and aerobic fermentation to achieve abbreviated β-oxidation of the ricinoleic acid (12-hydroxyoleic acid) into 4-hydroxydecanoic acid (4HDA), lactonisation of the acid into γ-DL, followed by solvent extraction and distillation. γ-DL broth concentrations of 0.5-1.2g · 1^-t were obtained after 3-5 days from fermentation media containing 10 g · 1^-1 castor oil, representing an 8.3-20.0% theoretical yield. Intermediates detected were consistent with the operation of the β-oxidation pathway. Appreciable amounts of novel metabolites identified as cis and trans isomers of a tetrahydrofuran (C₁₀) were also produced. Their formation from 4HDA appeared to be non-enzymic and was favoured by anaerobic conditions. Yields of γ-DL were inversely proportional to the concentration of castor oil present in the medium, indicating that substrate inhibition takes place. The highest yields of γ-DL were obtained when castor oil was present from the beginning of the fermentation, rather than when added once the fermentation had become established, demonstrating that the β-oxidation pathway and/or transport system require continual induction. Significant amounts of γ-DL were not produced from other fatty acids, including ricinelaidic acid, the trans isomer of ricinoleic acid. γ-DL formation was dramatically inhibited by antibiotic inhibitors of oxidative phosphorylation, indicating the importance of intact β-oxidation pathways, whereas inhibitors of protein synthesis and cell-wall synthesis had much less marked effects. Selective extraction of 4HDA from the fermentation broths, and of γDL from broth lactonised by heating at low pH, could be achieved by adsorption to Amberlite XAD-1 and XAD-7 resins respectively. Some product could be recovered from the exit gases of the fermenter by passing through propylene glycol traps. This pathway is unusual in that it is a rare example of the truncated β-oxidation of a fatty acid by microorganisms. This effect probably occurs because of partial inhibition of one or more enzymes of the β-oxidation pathway by the C₁₀ hydroxylated fatty acid intermediate(s) allowing intracellular accumulation of the 4HDA, followed by leakage out of the cell; although further metabolism of this C₁₀ intermediate does take place slowly.     

Enzymic Preparation of Benzyl β-D-Glucopyranoside

Benzyl β-D-glucopyranoside was prepared by an enzyme-catalysed direct reaction between D-glucose, or better cellobiose, and benzyl alcohol in the presence of a minimum amount of water. The enzyme β-glucosidase was used in the immobilized form (adsorbed onto macroporous polyethylene terephthalate or covalently bound on polyglycidyl methacrylate), enabling multiple application.     

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.     

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.     

Production of alkyl glucoside from cellooligosaccharides using yeast strains displaying Aspergillus aculeatus β-glucosidase 1

For efficient alkyl glucoside production from cellooligosaccharides, we constructed a yeast strain for alkyl glucoside synthesis by genetically inducing the display of β-glucosidase 1 (BGL1) from the filamentous fungus Aspergillus aculeatus No. F-50 on the cell surface. The localization of BGL1 on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying BGL1 catalyzed alkyl glucoside synthesis from p-nitrophenyl β-d-glucoside and primary alcohols. The highest yield of alkyl glucoside was 27.3% of the total sugar. The substrate specificities of the BGL1-displaying yeast strain and almond β-glucosidase were compared using different-chain-length cellooligosaccharides. The BGL1-displaying yeast showed efficient alkyl glucoside production from not only glucose but also cellohexaose. This yeast is applicable as a whole-cell biocatalyst for alkyl glucoside production from cellulose hydrolysates.     

Performance of Aspergillus niger B 03 β-xylosidase immobilized on polyamide membrane support

The dynamics of β-xylosidase biosynthesis from Aspergillus niger B 03 was investigated in laboratory bioreactor. Maximum xylosidase activity 5.5 U/ml was achieved after 80 h fermentation at medium pH 4.0. The isolated β-xylosidase was immobilized on polyamide membrane support and the basic characteristics of the immobilized enzyme were determined. Maximum immobilization and activity yield obtained was 30.0 and 6.8%, respectively. A shift in temperature optimum and pH optimum was observed for immobilized β-xylosidase compared to the free enzyme. Immobilized enzyme exhibited maximum activity at 45 °C and pH 4.5 while its free counterpart at 70 °C and pH 3.5, respectively. Thermal stability at 40 and 50 °C and storage stability of immobilized β-xylosidase were investigated at pH 5.0. Kinetic parameters K_m, V_max and K_i were determined for both enzyme forms. Free and immobilized β-xylosidase were tested for xylose production from birchwood xylan. The substrate was preliminarily depolymerized with xylanase to xylooligosaccharides and the amount of xylose obtained after their hydrolysis with free and immobilized β-xylosidase was determined by HPLC analysis. Continuous enzyme hydrolysis of birchwood xylan was performed with xylanase and free or immobilized β-xylosidase. The maximum extent of hydrolysis was 25 and 30% with free and immobilized enzyme, respectively. Immobilized preparation was also examined for reusability in 20 consecutive cycles at 40 °C.     

Hyperproduction of thermostable β-glucosidase by Sporotrichum (Chrysosporium) thermophile

The effect of the growth of temperature, pH, carbon source, nitrogen supplementation and inoculum size were examined in shake-flask-scale studies to determine the optimum conditions for β-glucosidases production by Sporotrichum (Chrysosporium) thermophile. Wheat bran and sugar-beet pulp were selected as the best carbon sources and (NH₄)₂SO₄, NH₄Cl and KNO₃ as the best nitrogen supplementation. Ten liter fermentations were carried out to study the kinetics of product formation. It was found that S. thermophile is able to produce high thermostable extracellular cellobiase and aryl-β-glucosidase. Very high aryl-β-glucosidase (PNPG) activities in the range from 30 to 40 U ml⁻¹ and cellobiase activities of 2,45 U ml⁻¹ in the 3-day batch fermentations were obtained. The K_m for aryl-β-glucosidase and its thermal properties were also estimated.     

Catalytic properties of the immobilized Talaromyces thermophilus β-xylosidase and its use for xylose and xylooligosaccharides production

The present study explores the efficiency of Talaromyces thermophilus β-xylosidase, in the production of xylose and xylooligosaccharides. The β-xylosidase was immobilized by different methods namely ionic binding, entrapment and covalent coupling and using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the best support material for β-xylosidase immobilization; it gave the highest immobilization and activity yields (94%, 87%, respectively) of initial activity, and also provided the highest stability, retaining 94% of its initial activity even after being recycled 25 times. Shifts in the optimal temperature and pH were observed for the immobilized β-xylosidase when compared to the free enzyme. The maximal activity obtained for the immobilized enzyme was achieved at pH 8.0 and 53 °C, whereas that for the free enzyme was obtained at pH 7.0 and 50 °C. The immobilized enzyme was more thermostable than the free β-xylosidase. We observed an increase of the K_m values of the free enzyme from 2.37 to 3.42 mM at the immobilized state. Native and immobilized β-xylosidase were found to be stimulated by Ca²⁺, Mn²⁺ and Co²⁺ and to be inhibited by Zn²⁺, Cu²⁺, Hg²⁺, Fe²⁺, EDTA and SDS. Immobilized enzyme was found to catalyze the reverse hydrolysis reaction, forming xylooligosaccharides in the presence of a high concentration of xylose. In order to examine the synergistic action of xylanase and β-xylosidase of T. thermophilus, these two enzymes were co-immobilized on chitosan. A continuous hydrolysis of 3% Oat spelt xylan at 50 °C was performed and better hydrolysis yields and higher amount of xylose was obtained.     

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.     

Enzymatic production of γ-L-glutamyltaurine through the transpeptidation reaction of γ-glutamyltranspeptidase from Escherichia coli K-12

γ-L-Glutamyltaurine is a naturally occurring peptide and known to have several physiological functions in mammals. This paper describes a new method for the enzymatic production of γ-L-glutamyltaurine from L-glutamine and taurine through the transpeptidation reaction of γ-glutamyltranspeptidase (EC 2.3.2.2) of Escherichia coli K-12. The optimum conditions for the production of γ-L-glutamyltaurine were 200 mM L-glutamine, 200 mM taurine and 0.2 U/ml γ-glutamyltranspeptidase, pH 10, and 1-h incubation at 37°C. Forty-five mM γ-L-glutamyltaurine was obtained, the yield being 22.5%. γ-L-Glutamyltaurine was purified on Dowex 1 × 8 and C₁₈ columns, and identified by means of NMR and a polarimeter.     

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.     

Influence of the microenvironment on the activity of enzymes immobilized on Teflon membranes grafted by γ-radiation

The effect of the microenvironment and immobilization method on the activity of immobilized β-galactosidase was investigated. Immobilization was done on Teflon membranes grafted with different acrylic monomers by γ-radiation and activated by two different coupling agents through the functional groups of the grafted monomers. 2-Hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA) were grafted on the membrane, and 1,6-hexamethylenediamine (HMDA) was used as a spacer. Glutaraldehyde or cyanuric chloride were used as coupling agents to bind the enzyme to the membrane. Four different catalytic membranes were obtained using the same solid support. Direct comparison between the isothermal behaviour of the biocatalyst in its free and immobilized form was carried out. In particular the dependence of the isothermal activity on the temperature and pH was studied and the kinetic parameters determined. The influence of the microenvironment on the observed activity of the four membranes was evidenced and discussed. The way of improving the yield of these catalytic membranes is discussed also.     

Selection and preliminary characterization of β-glycosidases producer Patagonian wild yeasts

Four pre-selected indigenous yeast strains belonging to Candida guilliermondii (V₂ and V₅), Candida pulcherrima (V₆) and Kloeckera apiculata (V₉), were used as β-glucosidase (βGL) and β-xylosidase (βXL) sources. The optimization of yeast culture conditions was carried out and the effects of oenological parameters on β-glycosidase activities were evaluated. C. guilliermondii V₂ and C. pulcherrima V₆ strains were selected. These strains showed intracellular (C. pulcherrima V₆) and parietal (C. guilliermondii V₂) constitutive βGL and βXL. The enzymatic activities were active at pH, glucose, ethanol and SO₂ concentrations usually found in winemaking and they were able to release monoterpenols and alcohols from grape juice glycoside extracts. Additionally, these yeast strains were not able to produce volatile acidity and off flavour. Regional ecological relevance of these species was also discussed. Our results evidence that the selected C. guilliermondii V₂ and C. pulcherrima V₆ strains have interesting oenological characteristics and allow us to think in their potential application in winemaking.     

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.     

Enzymatic synthesis of trisaccharides and alkyl β-d-glucosides by the transglycosylation reaction of β-glucosidase from Fusarium oxysporum

Purified β-glucosidase from Fusarium oxysporum catalyses hydrolysis and transglycosylation reactions. By utilizing the transglycosylation reaction, trisaccharides and alkyl β-d-glucosides were synthesized under optimal conditions in the presence of various disaccharides and alcohols. The yields of trisaccharides and alkyl β-d-glucosides were 22–37% and 10–33% of the total sugar, respectively. The enzyme retained 70–80% of its original activity in the presence of 25% (w/v) methanol, ethanol and propanol. Thus, β-glucosidase from F. oxysporum appears to be an ideal enzyme for the synthesis of useful trisaccharides and alkyl β-d-glucosides.