Purification and properties of the endo-1,4-β-glucanase III from Ruminococcus albus

Endoglucanase III (EGIII) was purified from Ruminococcus albus culture supernatant. An enzyme having a molecular weight of 53,000 was stabilized by mercaptoethanol and inhibited by sulfhydryl group-blocking reagents, and exhibited its highest CMC-degrading activity of pH 5.7 and 55°C. The enzyme hydrolyzed cellobiose (G2) and cellotriose (G3) only negligibly, but significantly hydrolyzed cellotetraose (G4), cellopentaose (G5) and cellohexaose (G6). The major hydrolysis reactions conducted by the enzyme were G4→2G2, G5→G2+G3, G6→G2+G4 and G6→2G3. The V_max values of these reactions increased remarkably while the K_m values decreased significantly with an increase in degree of polymerization of the substrate.     

Purification and characterization of endo-β-1,3-1,4-d-glucanase activity from Bacillus licheniformis

Summary An extracellular -glucanase from Bacillus licheniformis has been isolated and characterized. Isolation has been performed by salting out and gel filtration chromatography, yielding a homogeneous active component with a molecular mass of 27 000–28 000 daltons and an isoelectric point of 4.7. In addition to being quite a thermostable protein (optimal temperature 55°C) the enzyme is active under a wide range of conditions including pH (4.0–10.5), and in the presence of a large number of metal ions, sodium dodecylsulphate and ethylenediaminetetraacetate. The simple purification procedure and useful properties make this enzyme suitable for brewing processes.     

Purification and properties of the Endo-1,4-β-glucanase from Ruminococcus albus and its gene product in Escherichia coli

Endoglucanases, EGI and EgI, were produced from the same Ruminococcus albus gene in R. albus and recombinant Escherichia coli, respectively. EGI was purified from R. albus culture supernatant and EgI was extracted from the transformant E. coli (JM101/pURA1) and purified. The purified enzymes EGI and EgI revealed maximum endoglucanase activity at a same pH of 6.8 and a temperature of 37°C. Both enzymes were stable at temperatures below 30°C. In addition, about 10% of their original activities were conserved even after boiling for 10 min. Amino acid sequences of both enzymes at the N-terminal (Ala-Ala-Asp-Glu-Ser-Glu-Thr-Glu-Asn-Val-Pro-Val-Ser-Gln-Thr-His--) were consistent with each other. The antiserum against EgI reacted with both EgI and EGI, indicating that both their protein moieties were the same immunologically. However, the molecular size of EGI (43,000) was larger than that of EgI (39,000) due to the presence of sugar moiety. The specific activity (54 units/mg) of EGI was almost double that (27 units/mg) of EgI. EGI was immunologically different from the endoglucanase purified in the previous paper [Ohmiya et al.: Carbohydrate Res., 166, 145–155 (1987)].     

Purification and properties of endo-1,4-β-xylanase from Trichosporon cutaneum

Summary The endoxylanase (1,4-D-xylan xylanohydrolase, EC 3.2.1.8) was purified 3,7 fold from the culture filtrate of the yeast Trichosporon cutaneum grown on oathusk xylan. The final enzyme preparation gave a single protein band on disc gel electrophoresis and has a molecular weight of approx. 45000. The enzyme has a pH optimum of 5.0 and a temperatur optimum of 50°C. Patterns of hydrolysis demonstrate that this xylanase is an endo-splitting enzyme able to break down xylans at random giving xylobiose, xylotriose and xylose as the main end-products. Since the enzyme seems not to be capable of liberating L-arabinose from arabino-xylan branched arabinose-containing xylooligosaccharides are formed, too. This enzyme contains carbohydrates in a noncovalent manner, indicating that this extracellular xylanase, is not a glycoprotein.     

Purification and Characterization of endo-(1→4)-β-xylanase fromGeotrichum candidum 3C

A method of purification of endo-( 1 → 4)-β-xylanase (endoxylanase; EC 3.2.1.8) from the culture liquid ofGeotrichum candidum 3C, grown for three days, is described. The enzyme, purified 23-fold, had a specific activity of 32.6 U per mg protein (yield, 14.4%). Endoxylanase was shown to be homogeneous by SDS-PAGE (molecular weight, 60 to 67 kDa). With carboxymethyl xylan as the substrate, the optimum activity (determined viscosimetrically) was recorded at pH 4.0 (pI 3.4). The enzyme retained stability at pH 3.0-4.5 and 30–45°C for 1 h. With xylan from birch wood, the hydrolytic activity of the enzyme (ability to saccharify the substrate) was maximum at 50°C. In 72 h of exposure to 0.2 mg/ml endoxylanase, the extent of saccharification of xylans from birch wood, rye grain, and wheat straw amounted to 10,12, and 7.7%, respectively. At 0.4 mg/ml, the extent of saccharification of birch wood xylan was as high as 20%. In the case of birch wood xylan, the initial hydrolysis products were xylooligosaccharides with degrees of polymerization in excess of four; the end products were represented by xylobiose, xylotriose, xylose, and acid xylooligosaccharides.     

Cyclic (1→2)-β-d-Glucan-hydrolyzing Enzymes from Acremonium sp. 15 Purification and Some Properties of Endo-( 1 → 2)-α-d-glucanase and β-d-Glucosidase

Acremonium sp. 15 a fungus isolated from soil, produces an extracellular enzyme system degrading cyclic (1→2)-β-d-glucan. This enzyme was found to be a mixture of endo-(1→2)-β-d-glucanase and β-d-glucosidase. The (1→2)-β-d-glucanase was purified to homogeneity shown by disc-electrophoresis after SP-Sephadex column chromatography, Sephadex G-75 gel filtration, and rechromatography on SP-Sephadex. The molecular weight of the enzyme was 3.6 × 10⁴ by SDS-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was pH 9.6. The enzyme was most active at pH 4.0—4.5, and stable up to 40°C in 20 mm acetate buffer (pH 5.0) for 2 hr of incubation. This enzyme hydrolyzed only (l→2)-β-d-glucan and did not hydrolyze laminaran, curdlan, or CM-cellulose. The hydrolysis products from cyclic (1→2)-β-d-glucan were mainly sophorose.

The β-d-glucosidase was purified about 4000-fold. The rate of hydrolysis of the substrates by this β-d-glucosidase decreased in the following order: β-nitrophenyl-β-d-glucoside, sophorose, phenyl-β-d-glucoside, laminaribiose, and salicin. This enzyme has strong transfer action even at the low concentration of 0.75 mm substrate.     

Purification and characterization of (1→3, 1→4)-β-glucan endohydrolases from germinated wheat (Triticum aestivum)

A (13, 14)--glucan 4-glucanohydrolase [(13, 14)--glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (13, 14)--glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (13, 14)--glucanase isoenzyme EI from barley. The complete primary structure of the wheat (13, 14)--glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)⁺ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH₂-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32085 and a predicted pI of 8.1. The other cDNA, designated LW1, carries a 109 nucleotide pair sequence at its 5 end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.     

Purification and properties of an extracellular endo-1,4-β-glucanase fromLenzites trabea

An extracellular endo-1,4--glucanase (EC 3.2.1.4) has been isolated and purified from the culture solution of the basidiomyceteLenzites trabea grown on glucose and cellulose. Besides-glucosidase activity (EC 3.2.1.21) no evidence for C₁-activity (EC 3.2.1.91) in the culture solution was found.The endoglucanase has been purified in a four-step procedure including chromatography on Sepharose 6-B and DEAE-Sephadex A-50, adsorption on hydroxylapatite and gel filtration on Bio-Gel P-100. The enzyme showed maximum activity at pH 4.4 and 70°C. A molecular weight of 29000 Daltons was estimated by calibration on Bio-Gel P-100. The enzyme hydrolyses carboxymethyl cellulose (CMC) as well as xylan.List of Abbreviations CMC carboxymethyl cellulose - D.S. degree of substitution - D.P. degree of polymerisation - MW molecular weight     

Purification and partial characterization of an endo-(1→3)-β-D-glucanase fromEuphausia superba Dana (Antarctic Krill)

Summary We have found out that cell-free extracts from frozen krill decompose many oligo-and polysacharides, particularly with (13)--and (14)--linkages. Two individual proteins have very high activity with laminaran as the substrate. One of them has been isolated and purified 980-fold. Polyacrylamide-gel electrophoresis of purified preparation of krill (13)--glucanase [(13)--D-glucan glucanohydrolase, EC 3.2.1.6] demonstrated that it was slightly contaminated by one protein band inactive in laminaran hydrolysis. Studies on the hydrolysis of different substrates showed that the enzyme was able to break down only (13)--D-linkages by an endo-splitting mechanism. Glucono--lactone and heavy metal ions such as Hg²⁺ inhibited enzyme activity. The activity of the endo-(13)--glucanase of krill strictly depended on free thiol groups in a enzyme molecule. The Michaelis constant value for laminaran was 0.063 mg/ml. Optimal determined temperature was 65°C and optimal pH 5.0. Because of this enzyme's strong interaction with concavalin A-Sepharose it is suggested that it might be a glycoprotein.This work was supported by the Institute of Ecology of Polish Academy of Sciences as a part MR I/29 programme     

Purification and characterization of endo-1,4-β-xylanase from Paecilomyces varioti Bainier

An endo-xylanase (1,4-β-d-xylanxylanohydrolase EC 3.2.1.8) was isolated from the culture filtrate of Paecilomyces varioti Bainier. The enzyme was purified 3.2 fold with a 60% yield by gel filtration and ion exchange chromatography. The purified enzyme had a molecular weight of 25,000 with a sedimentation coefficient of 2.2 S. The isoelectric point of the enzyme was 3.9. The enzyme was obtained in crystalline form. The optimum pH range was 5.5–7.0 and the temperature, 65°C. The Michaelis constant was 2.5 mg larchwood xylan/ml. The enzyme was found to degrade xylan by an endo mechanism producing arabinose, xylobiose, xylo- and arabinosylxylo-oligosaccharides, during the initial stages of hydrolysis. On prolonged incubation, xylotriose, arabinosylxylotriose and xylobiose were the major products with traces of xylotetraose, xylose and arabinose.     

An endo-(1→6-β-D-glucanase from Mucor hiemalis

An endo-(1→6)-β-D-glucanase (EC 3.2.1), isolated from the culture filtrate of Mucor hiemalis, was purified by ammonium sulphate fractionation and gel filtration. The homogeneity of the enzyme was confirmed by disc electrophoresis. The enzyme had a wide range of temperature and pH stability, high substrate specificity, and an action pattern of the endo-type.     

Substrate subsite recognition of the xyloglucan endo-transglycosylase or xyloglucan-specific endo-(1→4)-β-d-glucanase from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds

We have investigated the substrate subsite recognition requirement of the xyloglucan endo-transglycosylase/xyloglucan-specific endo-(14)--d-glucanase (NXET) from the cotyledons of nasturtium seedlings. Seed xyloglucans are composed almost entirely of the Glc₄ subunits XXXG, XLXG, XXLG and XLLG, where G represents an unsubstituted glucose residue, X a xylose-substituted glucose residue and L a galactosyl-xylose-substituted glucose residue. Thus in the xyloglucan sequence shown below, the xylose (Xyl) residues at the backbone glucose (Glc) residues numbered — 3,— 2, + 2 and + 3 may be galactose-substituted, and NXET cleaves between the unsubstituted glucose at — 1 and the xylose-substituted glucose at + 1, which never carries a galactosyl substituent. We have isolated the xyloglucan oligosaccharides XXXGXXXG and XLLGXLLG from NXET digests of tamarind seed xyloglucan, have modified them enzymatically using a pure xyloglucan oligosaccharide-specific -xylosidase from nasturtium seeds to give GXXGXXXG and GLLGXLLG, and have identified and compared the products of NXET action on XXXGXXXG, GXXGXXXG, XLLGXLLG and GLLGXLLG. We have also compared the molar proportions of XXXG, XLXG, XXLG and XLLG in native tamarind and nasturtium seed xyloglucans with those in NXET digests of these polysaccharides. Using these and existing data we have demonstrated that NXET action does not require xylosesubstitution at glucose residues — 4, — 2, + 1 and + 3 and that xylose substitution at + 2, is a requirement. There may also be a requirement for xylose substitution at — 3. We have demonstrated also that galactosyl substitution of a xylose residue at + 1 prevents, and at — 2 modifies, chain-cleavage. A partial model for the minimum substrate binding requirement of NXET is proposed.Abbreviations G unsubstituted glucose residue - X xylose-substituted glucose residue - L galactosylxylose-substituted glucose residue - F fucosyl-galactosylxylose-substituted glucose residue - Gal galactose - Glc glucose - HPAE high-performance anion-exchange chromatography - NXET nasturtium xyloglucan endo-transglycosylase or xyloglucan-specific endo-(14)--d-glucanase - Xyl xylose This work was funded jointly by Unilever UK and the Department of Trade and Industry (UK) via the LINK initiative Agro-Food Quality.     

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 Partial Characterization of an Endo-(1→3, 1→4)-β-glucanase from Rice,Oryza sativa L.

(1→3, 1→4)-β-Glucanase (EC 3.2.1.73), with a molecular weight of 34, 000 and an isoelectric point of 4.9, was purified to homogeneity from extracts of fresh rice bran. The enzyme specifically hydrolyzed (1→3, 1→4)-β-glucans such as barley β-glucan and lichenans, but laminarins and CM-cellulose were not substrates. Endproduct analysis using barley β-glucan as the substrate suggested that the enzyme is an endo-type (1→3, 1→4)-β-glucanase.     

Purification and properties of a novel β-galactosidase or exo-(1 → 4)-β-d-galactanase from the cotyledons of germinated Lupinus angustifolius L. seeds

The main polysaccharide component of the thickened cell walls in the storage parenchyma of Lupinus angustifolius L. cotyledons is a linear (1 4)--linked d-galactan, which is mobilised after germination (L.A. Crawshaw and J.S.G Reid, 1984, Planta 160, 449–454). The isolation from the germinated cotyledons of a -d-galactosidase or exo-(1 4)--d-galactanase with a high specificity for the lupin galactan is described. The enzyme, purified using diethylaminoethyl-cellulose, carboxymethyl-cellulose and affinity chromatography on lactose-agarose, gave two bands (major 60 kDa, minor 45 kDa) on sodium dodecyl sulphate-gel electrophoresis, and two similar bands on isoelectric focusing (major, pI 7.0, minor pI 6.7, both apparently possessing enzyme activity). The minor component cross-reacted with an antiserum raised against, and affinity-purified on, the major band. Both components had a common N-terminal sequence. The minor component was probably a degradation product of the major one. The enzyme had limited -galactosidase action, catalysing the hydrolysis of p-nitrophenyl--d-galactopyranoside and (1 4)- and (1 6)--linked galactobioses. Lactose [-d-galactopyranosyl-(1 4)-d-glucose] was hydrolysed only very slowly and methyl--d-galactopyranoside not at all. Lupin galactan was hydrolysed rapidly and extensively to galactose, whereas other cell-wall polysaccharides (xyloglucan and arabinogalactan) with terminal non-reducing -d-galactopyranosyl residues were not substrates. A linear (1 4)--linked galactopentaose was hydrolysed efficiently to the tetraose plus galactose, but further sequential removals of galactose to give the tetraose and lower homologues occurred more slowly. Galactose, -galactonolactone and Cu⁺² were inhibitory. No endo--d-galactanase activity was detected in lupin cotyledonary extracts, whereas exo-galactanase activity varied pari passu with galactan mobilisation. Exo-galactanase protein was detected, by Western immunoblotting of cotyledon extracts, just before the activity could be assayed and then increased and decreased in step with the enzyme activity. The exo-galactanase is clearly a key enzyme in galactan mobilisation and may be the sole activity involved in depolymerising the dominant (1 4)--galactan component of the cell wall.Abbreviations CM carboxymethyl - DEAE diethylaminoethyl - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TLC thin-layer chromatography We wish to thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for the award of a studentship to M.S. Buckeridge, and the Government of São Paulo State, Brazil for granting him leave of absence. We are grateful to Dr. Amanda Heyller (Unilever Research Laboratory, Colworth House, Bedford, UK) for N-terminal sequence determinations, to Dr. Stuart Wilson (Stirling) for preparing gelatin SDS-gels and to Cristina Fanutti (Stirling) for purifying the xyloglucan oligosaccharide.     

Purification and characterisation of two endo-(1→3)-β-d-glucanases from Telescopium telescopium

The crystalline style of the gastropod Telescopium telescopium contains two (1→3)-β-d-glucanases and a β-d-glucosidase. The two glucanases (I and II) have been purified and shown to be endo-enzymes. Both enzymes attack laminarin, carboxymethylpachyman, and lichenin, but have no action towards carboxymethyl-cellulose. The main products of hydrolysis of laminarin are d-glucose and β-(1→3)-linked oligosaccharides of d.p. 2, 3, and 4. Glucanases I and II are similar to each other, although they differ in molecular weight and kinetic properties.     

Biochemical and catalytic properties of endo-1,4-β-xylanases from Thermomyces lanuginosus (wild and mutant strains)

Endoxylanases from the thermophilic fungus, Thermomyces lanuginosus ATCC 44008 (cellulase free wild and mutant strains), were purified to homogeneity by anion-exchange and molecular-sieve chromatographic methods. The purified enzymes were monomers with molecular masses of 22 kDa (wild type) and 24 kDa (mutant), estimated by SDS-PAGE and gel filtration. As glycoproteins, the purified enzymes had 0.74% (wild type) and 11.8% (mutant) carbohydrate contents, and pI values of 5.8 and 6, respectively. The optimal pH and temperature values of wild type xylanase were determined to be pH 7 and 60 °C, whereas pH 6.7 and 70 °C, were optimal for the purified mutant enzyme (K _m and V _max values of 3.7 mg ml^–1 and 670 mol min^–1 xylose compared to the kinetic values of the purified wild type xylanase –5.1 mg ml^–1 and 385 mol min^–1 xylose). Inhibition studies suggested the possible involvement of histidine, tryptophan residues and carboxylic groups in the binding or catalysis.     

Production and properties of carboxymethylcellulase (endo-1,4-β-glucanase) fromCurvularia lunata

An extracellular carboxymethylcellulase (endo-1,4--glucanase) fromCurvularia lunata, grown at 30°C with an initial pH of 6.0, had optimal activity at pH 4.8 and 50°C. The enzyme was unstable above 50°C. The enzyme had aK _m for carboxymethylcellulose of 0.97 g/l and aV _max of 5.4 IU/ml.