Purification and characterization of an extracellular β-D-xylosidase from Aspergillus carbonarius

The production of an extracellular -D-xylosidase (-D-xyloside xylohydrolase, EC 3.2.1.37) by four Aspergillus strains (A. carbonarius, A. nidulans, A. niger and A. oryzae) grown on wheat bran medium was compared. The highest amount of the enzyme was found in the culture of A. carbonarius. The -D-xylosidase from A. carbonarius was purified to homogeneity by a rapid procedure, using hydrophobic interaction chromatography, chromatofocusing and affinity chromatography. The purified enzyme possessed not only -D-xylosidase activity, but also -L-arabinosidase activity. Mixed substrate experiments revealed that a single active centre was responsible for the splitting of the corresponding synthetic substrates. The molecular weight of the purified enzyme proved to be 100,000 Da, as estimated by SDS–PAGE. The isoelectric point was at pH 4.4. The pH and temperature optima were 4.0 and 60 °C, respectively. The enzyme remained stable over a pH range of 3.5–6.5 and up to 50 °C for 30 min. The Michaelis constant for p-nitrophenyl -D-xyloside was 0.198 mM. Kinetic studies demonstrated that the lack of the C-5 hydroxylmethyl group and the configuration of the C-4 hydroxyl group on the pyranoside ring play an important role in both substrate binding and splitting.     

Purification and properties of an extracellular β-glucosidase from Lenzites trabea

Summary When culturing the cellulolytic-active Basidiomycete and brown-rot fungus Lenzites trabea A-419 in submerged culture with glucose and cellulose as a carbon source, the fungus only excreted -glucosidase (EC 3.2.1.21) and an endo-1,4--glucanase (EC 3.2.1.4).No evidence for C₁ activity (EC 3.2.1.91) was found in the culture filtrate or in the ultra concentrate. -Glucosidase could be separated from endoglucanase by chromatography on Sepharose 6-B. Further fractionation of the -glucosidase on DEAE-Sephadex A-25 resulted in a 525-fold purification. The molecular weight of the isolated -glucosidase was determined by co-chromatography on Sephadex G-200 to be 320,000 daltons. The enzyme developed maximum activities at pH 4.5 and 75°C. The enzyme does not act on crystalline cellulose or CMC, but it hydrolyzes cellotriose,-tetraose, and-pentaose to cellobiose and glucose. -glucosidase activity was strongly inhibited by the reaction product, glucose. A K_i value of 2.7×10^–3 (M) for noncompetitive inhibition was found.     

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60 °C and 4.0, respectively. The enzyme was stable for 1 h at 55 °C, showing a t₅₀ of 53 min at 60 °C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K_m of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).     

Purification and properties of an α-amylase produced by a cassava-fermenting strain ofMicrococcus luteus

An extracellular α-amylase produced by a cassava-fermenting strain ofMicrococcus luteus was purified 26-fold by gel filtration and ion-exchange chromatography. The molar mass was estimated to be approximately 56 kDa. The optimum temperature of the enzyme was 30°C, optimum pH 6.0 and optimum substrate concentration was 0.6% (W/V). Treatment of the enzyme at 70°C for 10 min resulted in 70% loss of activity. The activation energy was determined to be 34.8 kJ/mol. The activity of the enzyme was enhanced by Mg²⁺, Ca²⁺, K⁺, Na⁺ and inhibited by EDTA, KCN and citric acid. The enzyme may find some application in local food processing.     

Purification and some properties of Aspergillus pulverulentus β-xylosidase with transxylosylation capacity

Two β-xylosidases [EC 3.2.1.37], β-Xyl I (molecular mass 180 kDa, pI 4.7) and β-Xyl II (molecular mass 190 kDa, pI 3.5), derived from Aspergillus pulverulentus were separated and purified by successive chromatographies and their characterization and transxylosylation were studied. β-Xyl I and β-Xyl II were stable at temperatures up to 50°C and from pH 1.5 to 6.5 and 2.5 to 7.0, respectively, while their highest activities were in the pH ranges 2.5–3.5 and 4.0–5.0 at 60°C. Although both enzymes were strongly inhibited by N-bromosuccinimide, the inhibitory effect of HgCl₂ was not significant on either. The two enzymes exhibited different resistances against AgNO₃, glucono 1,5-lactone and nojirimycin. They were shown to have broad acceptor specificity in transferring the xylosyl residue of xylooligosaccharides to various alcohol and phenolic compound acceptors. In the presence of 25% or more 2-propanol, the synthesis of the transfer product, 2-propyl β-xyloside, was closely consistent with the theoretical yield.     

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.     

Biochemical properties of a β-mannanase and a β-xylanase produced by Ceriporiopsis subvermispora during biopulping conditions

One mannanase and one of the three xylanases produced by Ceriporiopsis subvermispora grown on Pinus taeda wood chips were characterized. A combination of ion exchange chromatography and SDS-PAGE data revealed the existence of a high-molecular-weight mannanase of 150 kDa that was active against galactoglucomannan and xylan. Its activity was optimal at pH 4.5. The K_m value with galactoglucomannan as substrate was 0.50 mg ml^?1. One xylanase with molecular mass of 79 kDa was also purified and characterized. Its activity was optimal at 60 °C and pH 8.0. Its K_m value with birchwood xylan as substrate was 1.65 mg ml^?1. Both the mannanase and the 79 kDa xylanase displayed relatively high activity on carboxymethyl cellulose. The sensitivity of the xylanase and mannanase to various salts was evaluated. None of the tested salts inhibited the xylanase, but Mn⁺², Fe⁺³, and Cu⁺² were strong inhibitors for the mannanase.     

Purification and properties of a β-N-acetylaminoglucohydrolase from malted barley

β-N-Acetylaminoglucohydrolase (β-2-acetylamino-2-deoxy-D-glucoside acetylaminodeoxyglucohydrolase, EC 3.2.1.30) was extracted from malted barley and purified. The partially purified preparation was free from α-and β-glucosidase, α- and β-galactosidase, α-mannosidase and β-mannosidase. This preparation was free from α-mannosidase only after affinity chromatography with p-amino-N-acetyl-β-D-glucosaminidine coupled to Sepharose. The enzyme was active between pH 3 and 6.5 and had a pH optimum at pH 5. A MW of 92000 was obtained by sodium dodecyl sulfate-acrylamide gel electrophoresis and a sedimentation coefficient of 4.65 was obtained from sedimentation velocity experiments. β-N-Acetylaminoglucohydrolase had a K_m of 2.5 × 10^?4 M using the p-nitrophenyl N-acetyl β-D-glucosaminidine as the substrate.     

Purification and properties of a β-glucuronidase from pig kidney

A β-glucuronidase has been isolated from pig kidney and purified 1600-fold using sodium desoxycholate precipitation, ammonium sulphate fractionation, heat treatment and chromatography on Sephadex G200, DEAE-cellulose (DE-52) and hydroxyapatite. The enzyme activity was assayed using oestrone 3-glucuronide as substrate; the final specific activity was 254 nmol oestrone/min/mg of protein. The purified enzyme showed apparent homogeneity in gel filtration and polyacrylamide gel electrophoresis. The pig kidney β-glucuronidase has a single pH optimum of 4.0–4.4 in acetate- and 5.4 in citrate-buffer; an activation energy of 16,800 cal/mol and a molecular weight of 275,000 were estimated. The K_M for oestrone 3-glucuronide was 22.6 μM. The enzyme was not inhibited by N-ethylmaleimide nor by dithioerythritol, however, it was strongly inhibited by Hg²⁺. Oestradiol-17β 3-glucuronide and oestriol 3-glucuronide acted as competitive inhibitors, whereas oestradiol-17β 17β-glucuronide, oestriol 16α-glucuronide, testosterone 17-glucuronide and cholesteryl 3-glucuronide were uncompetitive, pregnanediol 3-glucuronide was noncompetitive, and Cortisol 21-glucuronide gave a mixed type inhibition. The synthetic β-d-glucuronides of phenolphthalein, p-nitrophenol, naphthol, 6-bromo-naphthol and methylumbelliferone all inhibited the hydrolysis of oestrone 3-glucuronide; the inhibition was of a more complex type than simple competitive inhibition.     

Purification and properties ofβ-fructofuranosidase from Aspergillus japonicus

-Fructofuranosidase fromAspergillus japonicus, which produces 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose, was purified to homogeneity by fractionation with calcium acetate and ammonium sulphate and chromatography with DEAE-Cellulofine and Sephadex G-200. Its molecular size was estimated to be about 304,000 Da by gel filtration. The enzyme was a glycoprotein which contained about 20% (w/w) carbohydrate. Optimum pH for the enzymatic reaction was 5.5 to 6. The enzyme was stable over a wide pH range, from pH 4 to 9. Optimum reaction temperature for the enzyme was 60 to 65°C and it was stable below 60°C. The K_m value for sucrose was 0.21m. The enzyme was inhibited by metal ions, such as those of silver, lead and iron, and also byp-chloromercuribenzoate.     

Purification and properties of two extracellular β-lactamases from Bacillus cereus 569/H

1. When Bacillus cereus 569/H was grown in a casamino acid (casein-hydrolysate) medium containing zinc sulphate rapid production of extracellular beta-lactamase II preceded that of beta-lactamase I. 2. beta-Lactamase I was separated from beta-lactamase II by fractional precipitation with ammonium sulphate. 3. beta-Lactamase I was purified by a process involving chromatography on Celite and DEAE-cellulose and beta-lactamase II by chromatography on DEAE-cellulose after denaturation of beta-lactamase I by heat. Both enzymes were obtained in crystalline form. 4. beta-Lactamase II prepared in this way appeared to have a higher molecular weight than beta-lactamase I and required Zn(2+) as a cofactor for both cephalosporinase and penicillinase activities.     

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

Summary Clostridium stercorarium cultures grown on cellobiose contain both an extracellular and a cell-bound -glucosidase activity. A substantial portion of the cell-bound enzyme could be extracted by osmotic shock, suggesting a periplasmic localization. The -glucosidase present in culture supernatants was purified to homogeneity. It was found to be identical in all aspects tested with the cell-bound -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--glucosides, cellobiose, and disordered cellulose. K _m values were determined as 0.8 mM for p-nitrophenyl--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 -glucosidase in cellulose saccharification: (1) Cleavage of cellobiose formed by exoglucanase and (2) degradation of cellodextrins produced by endoglucanase action.     

Production of a thermostable extracellular lipase by Kluyveromyces marxianus

Kluyveromyces marxianus was grown in submerged culture in a complex medium with several potential inducers of lipolytic activity (triacylglycerols, fatty acids). The highest extracellular lipolytic enzyme production (about 80 U ml^–1 in 3 d) was obtained when the medium was supplemented with 2 g urea l^–1 plus 5 g tributyrin l^–1. Addition of surfactants (1 g l^–1) did not improve production. The lipase had a high thermal stability in aqueous solution (73% residual activity after 9 d at 50 °C, 16 min half-life time at 100 °C). It was also stable at acidic pH and showed good tolerance to organic solvents (70% residual activity after 2 d in n-hexane of cyclohexane).     

Purification and characterization of extracellular β-glucosidase from Myceliophthora thermophila

The extracellular -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--d-glucoside, cellobiose and salicin but not carboxymethyl cellulose or crystalline cellulose. The K _m of the enzyme was 1.6mm for p-nitrophenyl--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.The authors are with the Department of Microbiology, Bose Institute, 93/1, A.P.C. Road, Calcutta 700 009, India. S.K. Raha is presently with the Department of Medicine, University of Saskatchewan, Saskatoon, Canada S7N OXO.     

Purification and Characterization of Extracellular β-Xylosidase and β-Glucosidase from Aspergillus fumigatus

A β-xylosidase (β-d-xyloside xylohydrolase, EC 3.2.1.37) and β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) extracted from a wheat bran culture of Aspergillus fumigatus were purified up to 90-fold and 131-fold, respectively, by ammonium sulfate precipitation, gel filtration, ion exchange chromatography, and hydroxylapatite chromatography. Molecular weights of the β-xylosidase and β-glucosidase were 360,000 and 380,000, respectively, each consisting of four identical subunits. The isoelectric points of β-xylosidase and β-glucosidase were at pH 5.4 and 4.5, respectively. The optimum temperature for the β-xylosidase was 75°C, being stable up to 65°C for 20 min and for the β-glucosidase was 65°C, being stable up to 60°C for 20 min. The optimum pH for both enzymes was about 4.5, being stable between 2 and 8 at 50°C for 20 min. Both enzymes were inhibited by Fe³⁺, Cu²⁺, Hg²⁺, SDS, and p-chloromercuribenzoate. The apparent Michaelis constants of the β-xylosidase were 2.0 and 23.8 mM for p-nitrophenyl-β-xyloside and xylobiose, respectively, and those of the β-glucosidase were 1.4, 11.4, and 24.8 mM for p-nitrophenyl-β-glucoside, gentiobiose, and cellobiose, respectively. To produce xylose from crude xylooligosac-charides prepared by steam-explosion of cotton seed waste (DP ≤10, 53%, total sugars = 150 g/ liter), the crude enzyme from A. fumigatus (β-xylosidase activity = 14.7 units/ml, xylanase activity = 20 units/ml) could hydrolyze the substrate at 55°C and pH 4.5 resulting in almost complete conversion to xylose (160 g/liter).     

Characterization and some reaction-engineering aspects of thermostable extracellular β-galactosidase from a newBacillus species

A new strain of Bacillus sp. was isolated from a hot water spring in India. This strain generated a high activity of extracellular beta-galactosidase at 37 degrees C in shake flasks. The beta-galactosidase activity was found to increase continuously but the production rate was slower than with some other organisms reported in the literature. There were noteworthy differences in the time-domain profiles of bacterial concentration and beta-galactosidase activity when the starting concentration of substrate (glucose) was tripled from 10 g/L. These differences may be explained in terms of the relative rates of enzyme synthesis and its diffusion across the cell wall. The enzyme produced by this organism is more stable than other beta-galactosidases; its half-life is 408 h at 50 degrees C and 94 h at 55 degrees C, while the reported enzymes showed perceptible loss of activity within 2 h.     

Purification and characterization of a thermostable α-galactosidase with transglycosylation activity from Aspergillus parasiticus MTCC-2796

An extracellular thermostable α-galactosidase from Aspergillus parasiticus MTCC-2796 was purified 16.59-fold by precipitation with acetone, followed by sequential column chromatography with DEAE-Sephadex A-50 and Sephadex G-100. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found to be a monomeric protein with a molecular weight of about 67.5 kDa. The purified enzyme showed optimum activity against o-nitrophenyl-α-d-galactopyranoside (oNPG) at pH 5.0 and a temperature of 50 °C. The enzyme was thermostable, showing complete activity even after heating at 65 °C for 30 min. The enzyme showed strict substrate specificity for α-galactosides and hydrolyzed oNPG (K_m = 0.83 mM), melibiose (K_m = 2.48 mM) and raffinose (K_m = 5.83 mM). Among metal ions and reagents tested, Ca²⁺ and K⁺ enhanced the enzymatic activity, but Mg²⁺, Mn²⁺, ethylenediaminetetraacetic acid (EDTA) and 2-mercaptoethanol showed no effect, while Ag⁺, Hg²⁺ and Co²⁺ strongly inhibited the activity of the enzyme. The enzyme catalyzed the transglycosylation reaction for the synthesis of melibiose.     

Purification and characterization of a thermostable α-amylase fromBacillus stearothermophilus

A soil isolate of Bacillus stearothermophilus was found to synthesize thermostable alpha-amylase. The enzyme was purified to homogeneity by ammonium sulfate fractionation and IECC on DEAE-cellulose column. The purified enzyme was considered to be a monomeric protein with a molar mass of 64 kDa, as determined by SDS-PAGE. The enzyme showed a wide range of pH tolerance and maximum activity at pH 7.0. The temperature tolerance was up to 100 degrees C with more than 90% catalytic activity; the maximum activity was observed at 50 degrees C. Divalent metal ions exhibited inhibitory effect on the enzyme activity. However, proteinase inhibitor did not react positively.     

Catalytic and thermodynamic properties of β-glucosidases produced by Lichtheimia corymbifera and Byssochlamys spectabilis

Abstract

The objective of the present study was to optimize parameters for the cultivation of Lichtheimia corymbifera (mesophilic) and Byssochlamys spectabilis (thermophilic) for the production of β-glucosidases and to compare the catalytic and thermodynamic properties of the partially purified enzymes. The maximum amount of β-glucosidase produced by L. corymbifera was 39?U/g dry substrate (or 3.9?U/mL), and that by B. spectabilis was 77?U/g (or 7.7?U/mL). The optimum pH and temperature were 4.5 and 55?°C and 4.0 and 50?°C for the enzyme from L. corymbifera and B. spectabilis, respectively. β-Glucosidase produced by L. corymbifera was stable at pH 4.0–7.5, whereas the enzyme from B. spectabilis was stable at pH 4.0–6.0. Regarding the thermostability, β-glucosidase produced by B. spectabilis remained stable for 1?h at 50?°C, and that from L. corymbifera was active for 1?h at 45?°C. Determination of thermodynamic parameters confirmed the greater thermostability of the enzyme produced by the thermophilic fungus B. spectabilis, which showed higher values of ΔH, activation energy for denaturation (E_a), and half-life t_(1/2). The enzymes were stable in the presence of ethanol and were competitively inhibited by glucose. These characteristics contribute to their use in the simultaneous saccharification and fermentation of vegetable biomass.     

Purification and some properties of β-mannanase fromPolyporus versicolor

Multiple enzyme forms of -mannanase activity fromPolyporus versicolor were puritied to molecular homogeneity by a sequence involving DEAE Bio-Gel A chromatography, gel filtration on Sephadex G-100 and high-performance liquid chromatography using anion exchange and hydrophobic Interaction media. Overall, 7.6% of input activity was recovered in four -mannanases, A, B, C and 2A, which were purified 112.6-, 165.5-, 143.7-and 19.9-fold respectivety. The -mannanases were acidic proteins displaying isoelectric points from 3.75 to 4.6, molecular weights in the range of 33,900 to 57,500 and increasing hydrophobicity in the order of C>B>2A>A. Optimal pH and temperature for the hydrotysis of glucomannan by all activities were pH 5.5 and 65°C, respectively. All preparations exhibited activity after 30 min at 65°C, or after protease digestion. Although the response of individual enzymes to selected ions was variable, all -mannanases were inhibited in decreasing order of Hg²⁺>Cu²⁺>Zn²⁺>Mn²⁺. All activities functioned as endomannanases.

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Résumé De multiples formes enzymatiques de l'activité -mannanasique dePolyporus verslcolor ont été purifiées jusqu'à l'homogénéité moléculaire par une séquence impliquant la chromatographie sur Bio-gel DEAE A, la filtration sur gel de Sephadex G-100, et la chromatographie liquide à haute performance utilisant l'échange anionique et les milieux à interaction hydrophobique. On a récupéré en tout 7.6% de l'activité Initiale dans quatre -mannanases, A, B, C, et 2A, qui ont été purifiées respectivement 112.6, 165.5, 143.7 et 19.9 fois. Les -mannanases sont des protéines acidiques exhibant des pointsiso-électriques de 3.75 à 4.6, des poids moléculaires compris entre 33 900 et 57 500, et une hydrophobicité croîssante dans l'ordre C>B>2A>A. Les pH et température optimum pour l'hydrolyse de la glucamannane par toutes les activités sont de 5.5 et 65°C respectivement. Toutes les préparations exhibent encore une activité après 30 minutes et 65°C ou après la digestion protéolytique. Bien que la réponse individuelle des enzymes à quelques ions choisis était variable, toutes les -mannanases sont inhibées dans l'ordre décroissant: Hg²⁺>Cu²⁺>Zn²⁺>Mn²⁺. Toutes les activités fonctionnent comme endomannanases.

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This article is issued as NRCC No. 31269.