Purification and properties of two endo-1,4-beta-xylanases from Irpex lacteus (Polyporus tulipiferae)

Two different endo-1,4-beta-xylanases [1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8], named Xylanases I and III, were purified to homogeneity by gel filtration and ion exchange column chromatography from Driselase, a commercial enzyme preparation from Irpex lacteus (Polyporus tulipiferae). The purified enzymes were found to be homogeneous on polyacrylamide disc electrophoresis and their specific activities toward xylan were increased approximately 28.7 and 19.8 times, respectively. The activities of each enzyme were considerably inhibited by Hg2+, Ag+, and Mn2+. Their molecular weights were estimated to be approximately 38,000 and 62,000 by gel filtration and sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis, respectively. Their carbohydrate contents were 2.5% and 8.0% as glucose, and their amino acid composition patterns resembled each other, showing high contents of acidic amino acids, serine, threonine, alanine, and glycine. Both enzymes were most active at pH 6.0 but Xylanase I was more stable as to pH. Their optimum temperatures were 60 degrees C and 70 degrees C, respectively. Xylanase I split up to 34.5% of larchwood xylan whereas Xylanase III split only 18.9% of it. The products with the former were mainly xylose (X1), xylobiose (X2), and xylotriose (X3), whereas X2 and X3 were the main products with the latter. Both enzymes did not hydrolyze X2. Xylanase I produced almost equal quantities of X1 and X2 from X3, while Xylanase III did not attack this substrate. Both enzymes showed no activity toward glycans, other than xylan, such as starch, pachyman and Avicel (microcrystalline cellulose), except the almost one twentieth activity of Xylanase III toward sodium carboxymethyl cellulose (CMC).     

Sensitive detection of endo-1,4-beta-glucanases and endo-1,4-beta-xylanases in gels

A simple, highly sensitive zymogram technique for detection of endo-1,4-beta-glucanases and endo-1,4-beta-xylanases in polyacrylamide gels after electrophoresis or isoelectric focusing was developed. The detection employs transparent agar replicas containing soluble covalently dyed polysaccharides, hydroxyethylcellulose dyed with Ostazin brilliant red H-3B and beechwood 4-O-methyl-D-glucurono-D-xylan dyed with Remazol brilliant blue R, as the respective substrates. The high sensitivity of the detection is achieved by selective removal of depolymerized dyed substrates from the agar replicas by solvents which neither solubilize nor precipitate the original nondegraded dyed polysaccharides present in the agar gel.     

Soluble chromogenic substrates for the assay of endo-1,4-beta-xylanases and endo-1,4-beta-glucanases

New soluble chromogenic substrates were prepared for specific and rapid assays of endo-1,4-beta-xylanases and endo-1,4-beta-glucanases. A soluble beechwood 4-O-methyl-D-glucurono-D-xylan was dyed with Remazol brilliant blue R, and hydroxyethylcellulose was coupled to Ostazin brilliant red H-3B. The assays are based on photometric measurements of the enzyme-released dyed fragments soluble in the presence of organic solvents which precipitate the original substrates and their high-molecular-weight fractions. The assays are advantageous for rapid analyses of large amount of samples and also permit evaluation of the activities of both enzymes in the presence of exo-beta-glycanases and beta-glycosidases, at a high level of reducing compounds and viable cells, on the cell surface and on cell membranes and organelles.     

Purification and characterization of a new endo-1,4-beta-D-glucanase from Beltraniella portoricensis

A new endoglucanase, designated BCE1, produced by Beltraniella portoricensis, was purified from the culture supernatant. The N-terminal amino acid sequence suggests that BCE1 belongs to family 45 glycoside hydrolase (family 45 endoglucanase). The molecular mass of BCE1 was found to be 40 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for the carboxymethyl cellulase (CMCase) activity of BCE1 was 4.5, and the optimum temperature was 55 degrees C. Among family 45 endoglucanases, RCE1 and RCE2 from Rhizopus oryzae, PCE1 from Phycomyces nitens, and EGL3 and EGL4 from Humicola grisea, BCE1 was most resistant to anionic surfactant and oxidizing agent. These results indicate that BCE1 might prove to be a useful enzyme in the detergent industry.     

Fractionation and purification of endo-1,4-beta-xylanases and exo-1,4-beta-xylosidases of Aspergillus niger]

Two endo-1,4-beta-zylanases (m. w. 24,000 and 41,000) and six exo-1,4-beta-xylosidases, differing in their molecular weights and isoelectric points, were found in a xylanase preparation from Aspergillus niger, using different methods of fractionation. An electrophoretically homogeneous exo-1,4-beta-xylosidase (m. w. 30,000) purified 120-fold, with pI 4.6, having optimal effect on methyl-beta-D-xyloside at pH 3.0 was obtained. Exo-1,4-beta-xylosidase splits off xylose from the ends of the xylan chains at xylotriose, xylobiose and methyl-beta-D-xyloside and is characterized by a high transglycosilase activity. An electrophoretically homogeneous endo-1,4-beta-xylanase (m. w. 24,000) purified 250-fold, with pI 4.2 and optimal effect on carboxymethylxylan at pH 4.2 was isolated. Endo-1,4-beta-xylanase splits arabinoglucuronoxylan to form xylooligosaccharides; however, it does not hydrolyze xylobiose.     

Purification,characterization and cloning of an endo-1,4-B-glucanase fromRuminococcus sp.

Endoglucanase ofRuminococcus sp. is composed of seven active protein components when chromatographed on an ion exchange column (Q-Sepharose). Component I (endoglucanase A) did not bind to the column and was purified to homogeneity by molecular sieve chromatography. It had a mol. wt. of 22 000. Component II was fractionated into two active protein peaks (endoglucanase B and C) having mol. wt. of 225 000 and 10 000. The endoglucanase A had high affinity for CMC (Km 8 mg/ml). The temperature optimum of all three endoglucanase was between 40–45°C. The gene encoding for endolucanase activity was cloned inE. coli HB101 with pBR322. A 4.3 kilobaseBamH1 fragment encoding endoglucanase was hybridized toRuminococcus chromosomal DNA.     

Purification and properties of an endo-1,4-beta-glucanase from Clostridium josui.

An enzyme active against carboxymethyl cellulose (CMC) was purified from the stationary-phase-culture supernatant of Clostridium josui grown in a medium containing ball-milled cellulose. The purification in the presence of 6 M urea yielded homogeneous enzyme after an approximately 50-fold increase in specific activity and a 13% yield. The enzyme had a molecular mass of 45 kilodaltons. The optimal temperature and pH of the enzyme against CMC were 60 degrees C and 6.8, respectively. The enzyme hydrolyzed cellotetraose, cellopentaose, and cellohexaose to cellobiose and cellotriose but did not hydrolyze cellobiose or cellotriose. A microcrystalline cellulose, Avicel, was also hydrolyzed significantly, but the extent of hydrolysis was remarkably less than that of CMC. On the basis of these results, the enzyme purified here is one of the endo-1,4-beta-glucanases. The N-terminal amino acid sequence of the enzyme is Tyr-Asp-Ala-Ser-Leu-Lys-Pro-Asn-Leu-Gln-Ile-Pro-Gln-Lys-Asn-Ile-Pro-Asn- Asn-Asp-Ala-Val-Asn-Ile-Lys.     

Purification and characterization of an extracellular endo-1,4-β-xylanase from Fusarium oxysporum f. sp. melonis

Abstract Fusarium oxysporum f. sp. melonis produces extracellular endo-1,4-β-xylanase and β-xylosidase when grown in shaken culture at 26°C in a mineral salts medium containing oat spelt xylan and glucose as carbon sources. Endo-1,4-β-xylanase was purified 251 times from 5-day-old culture filtrates, by Sephacryl S-200, ion exchange and gel filtration HPLC. The purified sample yielded a single band in SDS polyacrylamide gels with a molecular mass of 80 kDa on electrophoretic mobility and 83 kDa by gel filtration behavior. High activity of the endo-1,4-β-xylanase against xylan was observed between 5 and 8 pH, and between 40 and 60°C, the optimum pH and temperature being 5.0 and 50°C, respectively. Kinetic properties of the enzyme are similar to those of other fungal xylanases, showing high affinity towards oat spelt xylan with a K _m of 1 mM expressed as xylose equivalent.     

Purification and characterization of an endo-1,4-beta-glucanase from Neisseria sicca SB that hydrolyzes beta-1,4 linkages in cellulose acetate

An enzyme catalyzing hydrolysis of beta-1,4 bonds in cellulose acetate was purified 18.3-fold to electrophoretic homogeneity from a culture supernatant of Neisseria sicca SB, which can assimilate cellulose acetate as the sole carbon and energy source. The molecular mass of the enzyme was 41 kDa and the isoelectric point was 4.8. The pH and temperature optima of the enzyme were 6.0-7.0 and 60 degrees C. The enzyme catalyzed hydrolysis of water-soluble cellulose acetate (degree of substitution, 0.88) and carboxymethyl cellulose. The Km and Vmax for water-soluble cellulose acetate and carboxymethyl cellulose were 0.242% and 2.24 micromol/min/mg, and 2.28% and 12.8 micromol/min/mg, respectively. It is estimated that the enzyme is a kind of endo-1,4-beta-glucanase (EC 3.2.1.4) from the substrate specificity and hydrolysis products of cellooligosaccharides. The enzyme and cellulose acetate esterase from Neisseria sicca SB degraded water-insoluble cellulose acetate by synergistic action.     

Enzymatic hydrolysis of water-soluble wheat arabinoxylan. 1. Synergy between alpha-L-arabinofuranosidases,endo-1,4-beta-xylanases,and beta-xylosidase activities

Hydrolysis of arabinoxylan is an important prerequisite for improved utilization of wheat hemicellulose in the ethanol fermentation industry. This study investigates the individual and combined efficiencies of three commercial, cellulytic and hemicellulytic enzyme preparations, Celluclast 1.5 L, Ultraflo L, and Viscozyme L, in catalyzing the liberation of arabinose and xylose from water-soluble wheat arabinoxylan. Ultraflo L was the best enzyme preparation for releasing arabinose, liberating 53 wt% of the theoretical maximum after 48 h of reaction (10 wt% enzyme/substrate ratio, 40 degrees C, pH 6). Celluclast 1.5 L was superior to the other enzyme preparations in releasing xylose, liberating 26 wt% of the theoretical maximum after 48 h of reaction (10 wt% enzyme/substrate ratio, 50 degrees C, pH 5). The 50:50 mixtures of the enzyme preparations showed no synergistic cooperation in arabinose release, but a synergistic interaction in xylose release was found between Ultraflo L and Celluclast 1.5 L. On the basis of high-performance anion exchange chromatography (HPAEC) analysis of the hydrolysates after enzymatic reaction, we propose that the observed synergism between Celluclast 1.5 L and Ultraflo L is the result of positive interaction between alpha-L-arabinofuranosidase and endo-1,4-beta-xylanase activities present in Ultraflo L that released arabinose, xylobiose and xylotriose, and beta-xylosidase activities in Celluclast 1.5 L, capable of catalyzing the hydrolysis of xylobiose and xylotriose to xylose.     

Purification and partial characterization οf a new β-xylosidase from Humicola grisea var. thermoidea

Summary The thermophilic fungus Humicola grisea var. thermoidea produces a mycelium-associated β-xylosidase activity when grown in liquid-state cultures on media containing oat spelt xylan as the carbon source. The β-xylosidase was purified to apparent homogeneity by gel filtration and anion exchange chromatography. Its molecular weight was 37 and 50 kDa, as determined by MALDI/TOF mass spectrometry and SDS-PAGE, respectively. The purified enzyme exhibited maximum activity at 55 °C and pH 6.5. It was also active at pH 8.8, retaining 60% of its activity after 6 h of incubation at 50 °C. β-xylosidase was strongly inactivated by NBS and slightly activated by DTT and β-mercaptoethanol. The enzyme was highly specific for PNPX as the substrate. The purified β-xylosidase showed K _m and V _max values of 1.37 mM and 12.98 IU ml⁻¹, respectively.     

Purification and properties of an acid endo-1,4-beta-glucanase from Bacillus sp. KSM-330

A novel acid cellulase (endo-1,4-beta-glucanase, EC 3.2.1.4) was found in a culture of Bacillus sp. KSM-330 isolated from soil. One-step chromatography on a column of CM-Bio-Gel A yielded a homogeneous enzyme, as determined by silver staining of both sodium dodecyl sulphate (SDS) and nondenaturing gels. The enzyme had a molecular mass of 42 kDa, as determined by SDS-polyacrylamide gel electrophoresis. The isoelectric point was higher than pH 10. The N-terminal amino acid sequence of the enzyme was Val-Ala-Lys-Glu-Met-Lys-Pro-Phe-Pro-Gln-Gln-Val-Asn-Tyr-Ser-Gly-Ile-Leu- Lys-Pro . This enzyme had an optimum pH for activity of 5.2, being active over an extremely narrow range of pH values, from 4.2 to 6.9; below and above these pH values no activity was detectable. The optimum temperature at pH 5.2 was around 45 degrees C. The enzyme efficiently hydrolysed carboxymethylcellulose (CMC) and lichenan, but more crystalline forms of cellulose, curdlan, laminarin, 4-nitrophenyl-beta-D-glucopyranoside and 4-nitrophenyl-beta-D-cellobioside were barely hydrolysed. The enzymic activity was inhibited by Hg2+ but was not affected by other inhibitors of thiol enzymes, such as 4-chloromercuribenzoate. N-ethylmaleimide and monoiodoacetate. N-Bromosuccinimide abolished the enzymic activity, and CMC protected the enzyme from inactivation by this tryptophan-specific oxidant. It is suggested that a tryptophan residue(s) is involved in the mechanism of action of the Bacillus cellulase and that the inhibition of enzymic activity by Hg2+ is ascribable to interactions with the tryptophan residue(s) rather than with thiol group(s).     

Purification and biochemical characterization of β-xylosidase from Humicola grisea var. thermoidea

Abstract β-d-Xylosidase production was maximal for Humicola grisea var. thermoidea grown on xylan as the sole carbon source. The main β-d-xylosidase activity was localised in the periplasm. β-Xylosidase was purified from crude extracts by heat treatment, ammonium sulfate precipitation and chromatography on DEAE-cellulose and Sephadex G-100. The purified enzyme was a monomer of molecular mass estimated to be 43 kDa by SDS-PAGE and gel filtration. Optima of pH and temperature were 6.0 and 50 °C, respectively. The enzyme activity was stimulated by Ca²⁺, Fe²⁺, and Mg²⁺. The purified β-xylosidase did not exhibit xylanase, carboxymethylcelullase, galactosidase, glucosidase, fucosidase or arabinosidase activities. The purified β-xylosidase hydrolysed xylobiose and xylo-oligosaccharides of up to five monosaccharide units. The enzyme had a K _m of 0.49 mM for p -nitrophenyl- β -d-xylopyranoside and was not inhibited by its product, xylose.     

Purification and properties of an endo-1,4-beta-glucanase translated from a Clostridium josui gene in Escherichia coli.

An endoglucanase encoded by a gene of Clostridium josui was expressed in Escherichia coli and purified. The homogeneous enzyme, with a molecular weight of 39,000, revealed maximum endoglucanase activity at pH 7.2 to 7.5 and a temperature of 65 to 70 degrees C. The enzyme was stable at a temperature lower than 45 degrees C (the growth temperature of the bacterium) in the range of pH 4.5 to 9.0. The amino acid sequence of the enzyme at the N terminus was Val-Glu-Glu-Asp-Ser-Ser-His-Leu-Ile-Thr-Asn-Gln-Ala-Lys-Lys----. The enzyme hydrolyzed cellotetraose to cellobiose and then transferred cellobiose to the residual cellotetraose. The resulting cellohexaose was cleaved to cellotriose.     

Purification and characterization of two 1,4-beta-xylan endohydrolases from the rumen fungus Neocallimastix frontalis.

Two beta-endoxylanases produced by Neocallimastix frontalis have been purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. Xylanase I is a nonglycosylated protein with an apparent molecular mass of 45 kDa. Xylanase II is a glycoprotein with an apparent molecular mass of 70 kDa. The pH optima of these enzymes were 5.5 and 6, respectively, and the temperature optimum was 55 degrees C for each enzyme. The endo mode of action of the enzymes was revealed by thin-layer chromatography of xylan hydrolysates. Antibodies raised against each purified protein exhibited no cross-reaction, confirming the biochemical specificities of the enzymes. Both enzymes exhibited carboxymethyl cellulase activity, and xylanase I was absorbed on crystalline cellulose, indicating that these enzymes might belong to the F family of beta-1,4-glycanases.     

Purification and characterization of thermostable endo-1,5-alpha-L-arabinase from a strain of Bacillus thermodenitrificans

A strain of a thermophilic bacterium, tentatively designated Bacillus thermodenitrificans TS-3, with arabinan-degrading activity was isolated. It produced an endo-arabinase (ABN) (EC 3.2.1.99) and two arabinofuranosidases (EC 3.2.1.55) extracellularly when grown at 60 degrees C on a medium containing sugar beet arabinan. The ABN (tentatively called an ABN-TS) was purified 7,417-fold by anion-exchange, hydrophobic, size exclusion, and hydroxyapatite chromatographies. The molecular mass of ABN-TS was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was pH 4.5. The enzyme was observed to be more thermostable than known ABNs; it had a half-life of 4 h at 75 degrees C. The enzyme had optimal activity at 70 degrees C and pH 6.0. The enzyme had apparent K(m) values of 8.5 and 45 mg/ml and apparent V(max) values of 1.6 and 1.1 mmol/min/mg of protein against debranched arabinan (alpha-1,5-arabinan) and arabinan, respectively. The enzyme had no pectin-releasing activity (protopectinase activity) from sugar beet protopectin, differing from an ABN (protopectinase-C) from mesophilic Bacillus subtilis IFO 3134. The pattern of degradation of debranched arabinan by ABN-TS indicated that the enzyme was an endo-acting enzyme and the main end products were arabinobiose and arabinose. The results of preliminary experiments indicated that the culture filtrate of strain TS-3 is suitable for L-arabinose production from sugar beet pulp at high temperature.     

Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca, Ampullaria crossean

Two novel endo-β-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50℃ and 60 ℃; for EG45 it was 50 ℃. The analysis on the stability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 ℃, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 ℃ for 24 h. However, less than 10% residual activity of EG45 was detected at 50 ℃. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan （from oat spelt） and Sigmacell 101, and they were inactive to p-nitrophenyl-β-D-cellobioside, salicin and starch.     

Purification and characterization of an extracellular alpha-D-glucuronidase from Phlebia radiata

Alpha-D-glucuronidase was isolated from the culture filtrate of Phlebia radiata grown on wheat bran and purified to homogeneity by chromatographic methods. The final enzymic preparation was purified 65-fold with an activity yield of 58%; it showed a high level of specific activity (over 23,000 nkat/mg protein). The molecular and hydrolytic properties of the purified enzyme were studied. The secreted alpha-glucuronidase had a molecular weight of 110 kDa, as established by gel permeation chromatography (GP HPLC), had a determined pI just below 4.4, and was stable at pH 5.5 for prolonged times. The carbohydrate content in protein molecules was found to be 15%. The activity of alpha-D-glucuronidase peaked at pH 3,8 and 60 degrees C with aldouronic acids preparation as the substrate. The Michaelis-Menten constant (K(m)), the maximum reaction velocity (V(max)), and the activation energy (E(a)) were 0.18 mM, 0.13 microM/min and 5.91 kJ/mol, respectively. The alpha-glucuronidase was active mainly on small substituted xylooligomers. When this enzyme was used with endoxylanase for the degradation of oat xylan, synergistic effects were observed.