Purification and characterization of beta-1,3-xylanase from a marine bacterium,Alcaligenes sp. XY-234

A beta-1,3-xylanase-producing bacterium, Alcaligenes sp. XY-234, was isolated from the marine environment. The organism produced endo-1,3-beta-xylanase at a high level in the culture fluid. The enzyme was purified 292-fold by ammonium sulfate precipitation and several column chromatographies. The final enzyme preparation appeared to be homogeneous on disc gel electrophoresis and SDS-PAGE with a molecular mass of 59 kDa, and the pI was 4.0. The enzyme hydrolyzed beta-1,3-xylan and larger xylooligosaccharides than xylobiose to give several xylooligosaccharides, but it could not hydrolyze xylobiose, p-nitrophenyl-beta-D-xyloside, and beta-1,4-xylan. The Km of the enzyme was 4.0 mg/ml. Optimal pH and temperature were 7.5 and 40 degrees C, respectively. It was stable from pH 6.0 to 10 and at a temperature of less than 40 degrees C. The enzyme was strongly inhibited by 1 mM HgCl(2)., AlCl(3), CuCl(2), FeCl(3), HgCl(2), Pb(CH(3)COO) (2), and N-bromosuccinimide.     

Xylan-degrading enzymes of the yeast Cryptococcus albidus. Identification and cellular localization

During growth on wood beta-1,4-xylans the yeast Cryptococcus albidus produced at least two enzymes which convert the polysaccharide to xylose catabolized by the cells. The enzyme almost completely secreted into culture fluid was identified as an endo-1,4-beta-xylanase. The function of the extracellular beta-xylanase is to hydrolyze xylan to oligosaccharides, mainly to xylobiose and xylotriose, which enter the cell where they are split by the second identified enzyme, a cell-bound beta-xylosidae (xylobiase). Aryl beta-xylosidase activity detected in the culture fluid was snown to be due to low affinity of beta-xylanase for p-nitrophenyl beta-D-xylopyranoside. This property of beta-xylanase was preserved after purification of the enzyme by chromatography on DEAE-cellulose, CM-Sephadex and Biogel A 1.5 m or Biogel P 100. Purified beta-xylanase exhibited certain microheterogeneity after polyacrylamide gel electrophoresis. Both extracellular beta-xylanase and intracellular beta-xylosidase were produced in much lower amounts by the cells grown on glucose than by the cells grown on xylan. This suggested that they are not produced constitutively. The investigated strain was not able to grow on cellulose and the crude and purified beta-xylanase were unable to hydrolyze cellulose or its soluble derivatives.     

Biochemical and catalytic properties of an endoxylanase purified from the culture filtrate of Sporotrichum thermophile

An endo-beta-1,4-xylanase (1,4-beta-D-xylan xylanoxydrolase, EC 3.2.1.8) present in culture filtrates of Sporotrichum thermophile ATCC 34628 was purified to homogeneity by Q-Sepharose and Sephacryl S-200 column chromatographies. The enzyme has a molecular mass of 25,000 Da, an isoelectric point of 6.7, and is optimally active at pH 5 and at 70 degrees C. Thin-layer chromatography (TLC) analysis showed that endo-xylanase liberates mainly xylose (Xyl) and xylobiose (Xyl2) from beechwood 4-O-methyl-D-glucuronoxylan, O-acetyl-4-O-methylglucuronoxylan and rhodymenan (a beta-(1-->4)-beta(1-->3)-xylan). Also, the enzyme releases an acidic xylo-oligosaccharide from 4-O-methyl-D-glucuronoxylan, and an isomeric xylotetraose and an isomeric xylopentaose from rhodymenan. Analysis of reaction mixtures by high performance liquid chromatography (HPLC) revealed that the enzyme cleaves preferentially the internal glycosidic bonds of xylooligosaccharides, [1-3H]-xylooligosaccharides and xylan. The enzyme also hydrolyses the 4-methylumbelliferyl glycosides of beta-xylobiose and beta-xylotriose at the second glycosidic bond adjacent to the aglycon. The endoxylanase is not active on pNPX and pNPC. The enzyme mediates a decrease in the viscosity of xylan associated with a release of only small amounts of reducing sugar. The enzyme is irreversibly inhibited by series of omega-epoxyalkyl glycosides of D-xylopyranose. The results suggest that the endoxylanase from S. thermophile has catalytic properties similar to the enzymes belonging to family 11.     

A Simple Determination Method for Herbicides,NIP and CNP in Soil Using Single Ion Monitoring Mass Spectrometry

A purified extracellular endo β-1,3-xylanase (EC 3.2.1.32) from an isolated strain, Aspergillus terreus A-07, was found to hydrolyze 1,3-xylosyl linkages only. When rhodymenan (β-1,4 and β-1.3-linked xylan) was hydrolyzed by β-1,3-xylanase (EF-6), four β-1,4-linked xylooligosaccharide fractions were produced. The main product was β-1,4-xylotriose, with trace amounts of other β-1,4-linked xylooligosaccharides. Successive degradation by β-l,4-xylosidase of the β,4-xylooligosaccharides that were produced from hydrolysis of β-1,3-xylanase on rhodymenan yielded only xylose as the final product.

We compared the action pattern of this enzyme with that of an extracellular endo β-l,4-xylanase (EC 3.2.1.8) of Streptomyces. From a mixture of products of β-1,4-xylanase hydrolysis on rhodymenan, an isomeric xylotriose was isolated by charcoal chromatography after treating with β-1.4-xylosidase. The structure of this isomeric xylotriose was elucidated by methylation analysis and its susceptibility to β-1,4-xylanase, β-1,3-xylanase, and β-1,4-xylosidase. The obtained isomeric xylotriose was identified as 3-O-β-xylopyranosyl-4-O-β-D-xylopyranosyl-D-xylose (X1→3X1→4X). It has a melting point of 224~225°C and ${\left[\alpha \right]}_{\text{D}}^{20}$(c = 1, H₂O)= —46°.     

Isolation and properties of a (1,3)-beta-D-glucanase from Ruminococcus flavefaciens

A (1,3)-beta-D-glucanase [(1,3)-beta-D-glucan-3-glucanohydrolase] from Ruminococcus flavefaciens grown on milled filter paper was purified 3,700-fold (19% yield) and appeared as a single major protein and activity band upon polyacrylamide gel electrophoresis. The enzyme did not hydrolyze 1,6-beta linkages (pustulan) or 1,3-beta linkages in glucans with frequent 1,6-beta-linkage branch points (scleroglucan). Curdlan and carboxymethylpachyman were hydrolyzed at 50% the rate of laminarin. The enzyme had a Km of 0.37 mg of laminarin per ml, a pH optimum of 6.8, and a temperature optimum of 55 degrees C and was stable to heating at 40 degrees C for 60 min. The molecular mass of the enzyme was estimated to be 26 kDa by gel filtration and 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was completely inhibited by 1 mM Hg2+, Cu2+, and KMnO4, 75% by 1 mM Ag2+, and Ni2+, and 50% by 1 mM Mn2+ and Fe3+. In a 2-h incubation with laminaridextrins (seven to nine glucose units) or curdlan and excess enzyme, the major products were glucose (30 to 37%), laminaribiose (17 to 23%), laminaritriose (18 to 28%), laminaritetraose (13 to 21%), and small amounts of large laminarioligosaccharides. With laminarihexaose and laminaripentaose, the products were equal quantities of laminaribiose and glucose (30%) and laminaritetraose and laminaritriose (18 to 21%). Laminaribiose or laminaritriose were not hydrolyzed, indicating a requirement for at least four contiguous 1,3-beta-linked glucose units for enzyme activity. The enzyme appeared to have the properties of both an exo- and an endoglucanase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and properties of a (1,3)-beta-D-glucanase from Ruminococcus flavefaciens.

A (1,3)-beta-D-glucanase [(1,3)-beta-D-glucan-3-glucanohydrolase] from Ruminococcus flavefaciens grown on milled filter paper was purified 3,700-fold (19% yield) and appeared as a single major protein and activity band upon polyacrylamide gel electrophoresis. The enzyme did not hydrolyze 1,6-beta linkages (pustulan) or 1,3-beta linkages in glucans with frequent 1,6-beta-linkage branch points (scleroglucan). Curdlan and carboxymethylpachyman were hydrolyzed at 50% the rate of laminarin. The enzyme had a Km of 0.37 mg of laminarin per ml, a pH optimum of 6.8, and a temperature optimum of 55 degrees C and was stable to heating at 40 degrees C for 60 min. The molecular mass of the enzyme was estimated to be 26 kDa by gel filtration and 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was completely inhibited by 1 mM Hg2+, Cu2+, and KMnO4, 75% by 1 mM Ag2+, and Ni2+, and 50% by 1 mM Mn2+ and Fe3+. In a 2-h incubation with laminaridextrins (seven to nine glucose units) or curdlan and excess enzyme, the major products were glucose (30 to 37%), laminaribiose (17 to 23%), laminaritriose (18 to 28%), laminaritetraose (13 to 21%), and small amounts of large laminarioligosaccharides. With laminarihexaose and laminaripentaose, the products were equal quantities of laminaribiose and glucose (30%) and laminaritetraose and laminaritriose (18 to 21%). Laminaribiose or laminaritriose were not hydrolyzed, indicating a requirement for at least four contiguous 1,3-beta-linked glucose units for enzyme activity. The enzyme appeared to have the properties of both an exo- and an endoglucanase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-Xylosidases and a nonspecific wall-bound beta-glucosidase of the yeast Cryptococcus albidus

Cryptococcus albidus grown on wood xylans possesses a soluble intracellular beta-xylosidase (EC 3.2.1.37) as an additional constituent of the xylan-degrading enzyme system of this yeast. The enzyme attacks linear 1,4-beta-xylooligosaccharides in an exo-fashion, liberating xylose from the non-reducing ends. The activity of the enzyme increases in the cells during growth on xylan and incubation with xylobiose or methyl beta-D-xylopyranoside which are the best inducers of extracellular beta-xylanase (EC 3.2.1.8). Various alkyl-,alkyl-1-thio- and aryl beta-D-xylopyranosides were excellent inducers of a different beta-xylosidase of Cryptococcus albidus. This enzyme is localized outside the plasma membrane and is principally associated with cell walls. Unlike the soluble intracellular beta-xylosidase, the wall-bound enzyme does not hydrolyze xylooligosaccharides. Evidence has been obtained that beta-xylosidase activity in the cell walls is not due to the presence of a specific aryl beta-xylosidase, but is exhibited by a nonspecific beta-glucosidase (EC 3.2.1.21) inducible by beta-D-xylopyranosides. The ratio of beta-glucosidase and beta-xylosidase activity in the cells and isolated cell walls from yeast induced by various beta-xylopyranosides and beta-glucopyranosides was very similar. Both wall-bound activities were inhibited in a similar pattern by inhibitors of beta-glucosidases, 1,5-gluconolactone and nojirimycin. This bifunctional enzyme does not bear any relationship to the utilization of xylans in Cryptococcus albidus.     

Production and Properties of Extracellular Endoxylanase from Neurospora crassa

Neurospora crassa 870 produced 14 and 0.025 U of extracellular xylanase (1,4-beta-d-xylan xylanohydrolase; EC 3.2.1.8) and beta-xylosidase (1,4-beta-xylan xylohydrolase; EC 3.2.1.37) per ml, respectively, in 4 days when commercial xylan was used as a carbon source. The effects of pH and carbon sources on xylanase production by N. crassa are discussed. Two xylanases (I and II) were purified and had pI values of 4.8 and 4.5 and molecular weights of 33,000 and 30,000. The maximum degree of hydrolysis of xylan by the extracellular culture broth was 66% in 4 h. The end products of xylan hydrolysis by xylanase I and II showed the presence of xylose, xylobiose, xylotriose, xylotetraose, xylopentose, and arabinose, indicating that they are endoxylanases capable of hydrolyzing 1,3-alpha-l-arabinofuranosyl branch points. Both xylanases showed activity toward carboxymethyl cellulose but no activity toward para-nitrophenyl-beta-d-xyloside or laminarin. Xylanase I showed appreciable activity toward para-nitrophenyl-beta-d-glucoside, whereas xylanase II was inactive.     

Biochemical characterization and antifungal activity of an endo-1,3-beta-glucanase of Paenibacillus sp. isolated from garden soil

A 44-kDa 1,3-beta-glucanase was purified from the culture medium of a Paenibacillus strain with a 28-fold increase in specific activity with 31% recovery. The purified enzyme preferentially catalyzes the hydrolysis of glucans with 1,3-beta-linkage and has an endolytic mode of action. The enzyme also showed binding activity to various insoluble polysaccharides including unhydrolyzable substrates such as xylan and cellulose. The antifungal activity of this Paenibacillus enzyme and a previously purified 1,3-beta-glucanase from Streptomyces sioyaensis were examined in this study. Both enzymes had the ability to damage the cell-wall structures of the growing mycelia of phytopathogenic fungi Pythium aphanidermatum and Rhizoctonic solani AG-4. Nonetheless, the Paenibacillus enzyme had a much stronger effect on inhibiting the growth of fungi tested.     

Purification and properties of the glycoprotein processing N-acetylglucosaminyltransferase II from plants

The presence of an N-acetylglucosaminyltransferase (GlcNAc-transferase) capable of adding a GlcNAc residue to GlcNAcMan3GlcNAc was demonstrated in mung bean seedlings. This enzyme was purified about 3400-fold by using (diethylaminoethyl)cellulose and phosphocellulose chromatographies and chromatography on Concanavalin A-Sepharose. The transferase was assayed by following the change in the migration of the [3H]mannose-labeled GlcNAc beta 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc on Bio-Gel P-4, or by incorporation of [3H]GlcNAc from UDP-[3H]GlcNAc into a neutral product, (GlcNAc)2Man3GlcNAc. Thus, the purified enzyme catalyzed the addition of a GlcNAc to that mannose linked in alpha 1,6 linkage to the beta-linked mannose. GlcNAc beta 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc was an excellent acceptor while Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc, Man alpha 1,6(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc, and Man alpha 1,6(Man apha 1,3)Man alpha 1,6[GlcNAcMan alpha 1,3]Man beta 1,4GlcNAc were not acceptors. Methylation analysis and enzymatic digestions showed that both terminal GlcNAc residues on (GlcNAc)2Man3GlcNAc were attached to the mannoses in beta 1,2 linkages. The GlcNAc transferase had an almost absolute requirement for divalent cation, with Mn2+ being best at 2-3 mM. Mn2+ could not be replaced by Mg2+ or Ca2+, but Cd2+ showed some activity. The enzyme was also markedly stimulated by the presence of detergent and showed optimum activity at 0.15% Triton X-100. The Km for UDP-GlcNAc was found to be 18 microM and that for GlcNAcMan3GlcNAc about 16 microM.     

Enzymatic synthesis of poly-N-acetyllactosamines as potential substrates for endo-beta-galactosidase-catalyzed hydrolytic and transglycosylation reactions

Enzymatic synthesis of GlcNAc-terminated poly-N-acetyllactosamine beta-glycosides GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(n)Galbeta1,4GlcNAcbeta-pNP (n=1-4) was demonstrated using a transglycosylation reaction of Escherichia freundii endo-beta-galactosidase. The enzyme catalyzed a transglycosylation reaction on GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (1), which served both as a donor and an acceptor, and converted 1 into p-nitrophenyl beta-glycosides GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(1)Galbeta1,4GlcNAcbeta-pNP (2), GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(2)Galbeta1,4GlcNAcbeta-pNP (3), GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(3)Galbeta1,4GlcNAcbeta-pNP (4) and GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(4)Galbeta1,4GlcNAcbeta-pNP (5). When 2 was used as an initial substrate, it led to the preferential synthesis of nonasaccharide beta-glycoside 4 to heptasaccharide beta-glycoside 3. This suggests that 4 is directly synthesized by transferring the tetrasaccharide unit GlcNAcbeta1,3Galbeta1,4GlcNAcbeta1,3Gal to nonreducing end GlcNAc residue of 2 itself. The efficiency of production of poly-N-acetyllactosamines by E. freundii endo-beta-galactosidase was significantly enhanced by the addition of BSA and by a low-temperature condition. Resulting 2 and 3 were shown to be useful for studying endo-beta-galactosidase-catalyzed hydrolytic and transglycosylation reactions.     

Purification and properties of a 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli.

A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite. The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan. The enzyme had a broad pH optimum with maximum activity at approx. pH 6.0 and a temperature optimum of 50 degrees C. The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B. succinogenes. The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis. The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively. The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%). Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products. The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis. The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B. subtilis and the similarity with the 1,4-beta-glucanase in B. succinogenes.     

Purification and characterization of a chitinase from Amycolatopsis orientalis with N-acetyllactosamine-repeating unit releasing activity

We report a novel enzyme from the culture filtrate of Amycolatopsis orientalis, that endoglycosidically releases an N-acetyllactosamine-repeating unit (Galbeta1,4GlcNAcbeta1,3Galbeta1,4GlcNAc, LN2) from a synthetic chromogenic substrate Galbeta1,4GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (1). The enzyme activity was purified by 80% saturated ammonium sulfate precipitation followed by gel filtration and affinity chromatography. The enzyme splits 1, Galbeta1,4GlcNAcbeta-pNP (2), GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (3), and GlcNAcbeta1,4GlcNAcbeta-pNP (4) into the corresponding oligosaccharides and p-nitrophenol. The catalytic efficiencies (k(cat)/K(m)) for compounds 1, 2, and 4 were 0.6, 0.05, and 13, respectively. Compound 4 acts as a fairly good substrate for the enzyme, and LN2-releasing activity was inhibited by 4 and GlcNAcbeta1,4GlcNAcbeta1,4GlcNAcbeta-pNP (7), indicating that this enzyme activity is derived from a kind of chitinase. The enzyme hydrolyzed 1 by a mechanism leading to retention of the anomeric configuration. This is the first report of a N-acetyllactosamine-repeating unit releasing enzyme.     

Partial purification of inositol polyphosphate 1-phosphomonoesterase with characterization of its substrates and products by nuclear magnetic resonance spectroscopy

A study of the enzyme activities that degrade Ins(1,3,4)P3 in rat brain showed that it was dephosphorylated primarily by a Mg2+-dependent inositol polyphosphate 1-phosphomonoesterase to Ins(3,4)P2 and then to Ins(3)P by a 4-phosphomonoesterase. A less active enzyme activity with the properties of a 4-phosphomonoesterase that converted Ins(1,3,4)P3 to Ins(1,3)P2 was also detected. The inositol polyphosphate 1-phosphomonoesterase was separated from the 4-phosphomonoesterase and the inositol monophosphate phosphomonoesterase by chromatography on phosphocellulose, DE-52 anion exchange and hydroxylapatite columns. Kinetic characterization of the partially purified inositol polyphosphate 1-phosphomonoesterase indicated that both Ins(1,3,4)P3 and Ins(1,4)P2 were substrates with apparent Km values of 0.9 microM and 0.7 microM, respectively. Either substrate was a competitive inhibitor of the other substrate and dephosphorylation of both substrates was directly inhibited by Li+ in an uncompetitive manner. These data strongly suggest that a single enzyme dephosphorylates both Ins(1,3,4)P3 and Ins(1,4)P2. The 4-phosphomonoesterase that dephosphorylated Ins(3,4)P2 to Ins(3)P was insensitive to Mg2+ and Li+ and was probably the same enzyme that degraded Ins(1,3,4)P3 to Ins(1,3)P2. The isomeric configurations of the major inositol polyphosphates formed from the degradation of Ins(1,3,4,5)P4 were determined using 1H- and 31P-NMR spectroscopy, and confirmation of the structures assigned to Ins(1,3,4,5)P4, Ins(1,3,4)P3 and Ins(3,4)P2 was obtained.     

Modification of the carbohydrate composition of sulfite pulp by purified and characterized beta-xylanase and beta-xylosidase of Aureobasidium pullulans

Both beta-xylanase and beta-xylosidase were purified to homogeneity from a xylose-grown culture of Aureobasidium pullulans. Cellular distribution studies of enzyme activities revealed that beta-xylanase was an extracellular enzyme, during both the exponential and stationary phases, whereas beta-xylosidase was mostly periplasmic associated. The beta-xylanase exhibited very high specificity for xylan extracted from Eucalyptus grandis dissolving pulp, whereas the beta-xylosidase was only active on p-nitrophenyl xyloside and xylobiose. Comparison of kcat/Km ratios showed that the beta-xylanase hydrolyzed xylan from dissolving pulp 1.3, 2.1, and 2. 3 times more efficiently than Eucalyptus hemicellulose B, Eucalyptus hemicellulose A, and larchwood xylan, respectively. The beta-xylosidase exhibited a transxylosylation reaction during the hydrolysis of xylobiose. When applied on acid sulfite pulp, both enzymes released xylose and hydrolyzed xylan to a different extent. Although beta-xylosidase (0.4 U/g pulp) liberated more xylose from pulp than beta-xylanase (4.7 U/g pulp), it was responsible for only 3% of xylan solubilization. Treatment of pulp with beta-xylanase liberated 51.7 microgram of xylose/g and hydrolyzed 10% of xylan. The two enzymes acted additively on pulp and removed 12% of pulp xylan. A synergistic effect in terms of release of xylose from pulp was observed when the enzyme mixture of beta-xylanase and beta-xylosidase was supplemented with beta-mannanase. However, this did not result in further enzymatic degradation of pulp xylan. Both beta-xylanase and beta-xylosidase altered the carbohydrate composition of sulfite pulp by increasing the relative cellulose content at the expense of reduced hemicellulose content of pulp.     

Characterisation of a novel Bacillus sp. SJ-10 β-1,3–1,4-glucanase isolated from jeotgal, a traditional Korean fermented fish

A novel β-1,3–1,4-glucanase gene was identified in Bacillus sp. SJ-10 (KCCM 90078) isolated from jeotgal, a traditional Korean fermented fish. We analysed the β-1,3–1,4-glucanase gene sequence and examined the recombinant enzyme. The open reading frame of the gene encoded 244 amino acids. The sequence was not identical to any β-glucanases deposited in GenBank. The gene was cloned into pET22b(+) and expressed in Escherichia coli BL21. Purification of recombinant β-1,3–1,4-glucanase was conducted by affinity chromatography using a Ni-NTA column. Enzyme specificity of β-1,3–1,4-glucanase was confirmed based on substrate specificity. The optimal temperature and pH of the purified enzyme towards barley β-glucan were 50 °C and pH 6, respectively. More than 80 % of activity was retained at temperatures of 30–70 °C and pH values of 4–9, which differed from all other bacterial β-1,3–1,4-glucanases. The degradation products of barley β-glucan by β-1,3–1,4-glucanase were analysed using thin-layer chromatography, and ultimately glucose was produced by treatment with cellobiase.     

Structure of beta-1,3-xylooligosaccharides generated from Caulerpa racemosa var. laete-virens beta-1,3-xylan by the action of beta-1,3-xylanase

Recently we reported the molecular cloning and characterization of a novel beta-1,3-xylanase from the marine bacterium Vibrio sp. AX-4 [Kiyohara et al. (2005) Biochem. J. 388, 949-957]. We report here the structural analysis of oligosaccharides generated from beta-1,3-xylan of a siphonous green alga, Caulerpa racemosa var. laete-virens, by the action of beta-1,3-xylanase. The enzyme degraded the polysaccharide producing oligosaccharides with different R(f)s on TLC (EX2-EX5). Sugar component, linkage, and MALDI-TOF-MS analyses revealed that EX2 and EX3 were Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,3-Xyl, respectively. On the other hand, EX4 was a mixture of Glc-1,3-Xyl-1,3-Xyl, Xyl-1,4-Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,4-Xyl, while EX5 was a mixture of tetra-saccharides containing 3-substitued Glc in addition to the same components of EX4. Branching was not likely present in EXOs prepared from the polysaccharide by the enzyme. These results strongly suggest that the C. racemosa beta-1,3-xylan is a linear heteropolysaccharide containing 1,3-Glc and 1,4-Xyl both of which are thought to be located within a beta-1,3-Xyl chain and linked via covalent bonds. This report indicates the usefulness of the enzyme for the structural analysis of beta-1,3-xylan.     

Novel inducers of the xylan-degrading enzyme system of Cryptococcus albidus

A series of compounds structurally related to xylan and 1,4-beta-xylobiose were tested as inducers of the xylan-degrading enzyme system of Cryptococcus albidus. Washed, glucose-grown cells were incubated with alpha- and beta-linked xylobioses, 4-O-beta-D-xylopyranosyl-L-arabinopyranose, 3-O-beta-D-xylopyranosyl-xylobiose, 6-O-beta-D-xylopyranosyl-cellobiose, cellobiose, and methyl beta-D-xylopyranoside. All alpha-xylobioses and cellobiose were inactive as inducers of the xylan-degrading enzyme system. Other compounds served as inducers of varying efficiency, depending on their concentration in the induction medium and the time of incubation of cells. The most rapid response of the cells, i.e., the shortest induction period of beta-xyloside permease, beta-xylosidase (EC 3.2.1.37), and beta-xylanase (EC 3.2.1.8), was observed with 1,4-beta-xylobiose, which was the most efficient inducer at low concentrations (0.1 to 0.2 mM). At higher concentrations (2 to 10 mM) and after long incubations, the highest enzyme yields were obtained with 1,2-beta-xylobiose. The results represent a new example of efficient induction of polysaccharide-degrading enzyme systems by positional isomers of dimers derived from the polysaccharide.     

Impact and efficiency of GH10 and GH11 thermostable endoxylanases on wheat bran and alkali-extractable arabinoxylans

The results of a comparative study of two thermostable (1-->4)-beta-xylan endoxylanases using a multi-technical approach indicate that a GH11 xylanase is more useful than a GH10 xylanase for the upgrading of wheat bran into soluble oligosaccharides. Both enzymes liberated complex mixtures of xylooligosaccharides. 13C NMR analysis provided evidence that xylanases cause the co-solubilisation of beta-glucan, which is a result of cell-wall disassembly. The simultaneous use of both xylanases did not result in a synergistic action on wheat bran arabinoxylans, but instead led to the production of a product mixture whose profile resembled that produced by the action of the GH10 xylanase alone. Upon treatment with either xylanase, the diferulic acid levels in residual bran were unaltered, whereas content in ferulic and p-coumaric acids were unequally decreased. With regard to the major differences between the enzymes, the products resulting from the action of the GH10 xylanase were smaller in size than those produced by the GH11 xylanase, indicating a higher proportion of cleavage sites for the GH10 xylanase. The comparison of the kinetic parameters of each xylanase using various alkali-extractable arabinoxylans indicated that the GH10 xylanase was most active on soluble arabinoxylans. In contrast, probably because GH11 xylanase can better penetrate the cell-wall network, this enzyme was more efficient than the GH10 xylanase in the hydrolysis of wheat bran. Indeed the former enzyme displayed a nearly 2-fold higher affinity and a 6.8-fold higher turnover rate in the presence of this important by-product of the milling industry.