The role of the glucuronoxylan carboxyl groups in the action of endoxylanases of three glycoside hydrolase families: A study with two substrate mutants.

BACKGROUND: Bacterial appendage-dependent GH30 glucuronoxylan hydrolases recognize the substrate through an ionic interaction of a conserved positively charged arginine with the carboxyl group of 4-O-methyl-d-glucuronic acid. One of the options to verify this interaction is preparation of enzyme mutants. An alternative approach is a chemical modification of the substrate, glucuronoxylan, in which the free carboxyl group in all residues of MeGlcA is eliminated. METHODS: In this work the carboxyl groups of 4-O-methyl-d-glucuronic acid residues of an alkali extracted beechwood xylan were esterified with methanol. A water-soluble fraction of the polysaccharide methyl ester was converted by NaBH4 reduction to the second soluble derivative, 4-O-methylglucoxylan. Specific activities of several endoxylanases (EXs) of GH families 10, 11 and 30 were determined on glucuronoxylan, and its two new uncharged derivatives. RESULTS: Elimination of the free carboxyl group from the polysaccharide did not influence activities of GH10 EXs, but resulted in 50% decrease of specific activity of GH11 EXs, and led to more than 300-fold reduction of specific activity of Erwinia chrysanthemi GH30 xylanase. CONCLUSIONS: These results confirm the crucial role of the interactions between GH30 xylanases and the MeGlcA carboxyl group for efficient cleavage of the polysaccharide. Analysis of the hydrolysis products by TLC and MS confirmed that all three types of xylanases hydrolyzed uncharged glucuronoxylans similarly as the original one. SIGNIFICANCE: The uncharged glucuronoxylan derivatives will be useful to differentiate GH30 xylanases with various degree of selectivity for glucuronoxylan, including fungal enzymes without the conserved arginine.

Crystallization and preliminary X-ray diffraction analysis of the glucuronoyl esterase catalytic domain from Hypocrea jecorina.

The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was overexpresssed, purified and crystallized by the sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. The crystals belonged to space group P2(1)2(1)2(1) and X-ray diffraction data were collected to 1.9 A resolution. This is the first enzyme with glucoronoyl esterase activity to be crystallized; its structure will be valuable in lignocellulose-degradation research.

Purification and characterization of a type B feruloyl esterase (StFAE-A) from the thermophilic fungus Sporotrichum thermophile.

A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity. The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0+/-1.5 kDa, with a mass of 33+/-1 kDa on SDS-PAGE. The pI of the enzyme was estimated by cation-exchange chromatofocusing to be at pH 3.1. The enzyme activity was optimal at pH 6.0 and 55-60 degrees C. The purified esterase was stable at the pH range 5.0-7.0. The enzyme retained 70% of activity after 7 h at 50 degrees C and lost 50% of its activity after 45 min at 55 degrees C and after 12 min at 60 degrees C. Determination of k(cat)/ K(m) revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively. No activity on methyl sinapinate was detected. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and it hydrolyzed 4-nitrophenyl 5- O- trans-feruloyl-alpha- l-arabinofuranoside (NPh-5-Fe-Ara f) 2-fold more efficiently than NPh-2-Fe-Ara f. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from S. thermophile (a maximum of 34% total ferulic acid released after 1 h incubation). StFAE-A by itself could release FA, but at a level almost 47-fold lower than that obtained in the presence of xylanase. The potential of StFAE-A for the synthesis of various phenolic acid esters was tested using a ternary water-organic mixture consisting of n-hexane, 1-butanol and water as a reaction system.

Purification and characterization of a feruloyl esterase from Fusarium oxysporum catalyzing esterification of phenolic acids in ternary water-organic solvent mixtures.

An extracellular feruloyl esterase (FAE-II) from the culture filtrates of Fusarium oxysporum F3 was purified to homogeneity by SP-Sepharose, t-butyl-HIC and Sephacryl S-200 column chromatography. The protein corresponded to molecular mass and pI values of 27 kDa and 9.9, respectively. The enzyme was optimally active at pH 7 and 45 degrees C. The purified esterase was fully stable at pH 7.0-9.0 and temperature up to 45 degrees C after 1 h incubation. Determination of k(cat)/K(m) revealed that the enzyme hydrolysed methyl sinapinate 6, 21 and 40 times more efficiently than methyl ferulate, methyl coumarate and methyl caffeate, respectively. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 but inactive to the C-2 positions of arabinofuranose such as 4-nitrophenyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and 4-nitrophenyl 2-O-trans-feruloyl-alpha-L-arabinofuranoside. In the presence of Sporotrichum thermophile xylanase, there was a significant release of ferulic acid from destarched wheat bran by FAE-II, indicating a synergistic interaction between FAE-II and S. thermophile xylanase. FAE-II by itself could release only little ferulic acid from destarched wheat bran. The potential of FAE-II for the synthesis of various phenolic acid esters was tested using as a reaction system a surfactantless microemulsion formed in ternary mixture consisting of n-hexane, 1-propanol and water.

Enzymic alpha-galactosylation of a cyclic glucotetrasaccharide derived from alternan.

Alternanase catalyzes the hydrolysis of alternan, an alpha-(1-->3)-alpha-(1-->6)-D-glucan produced by Leuconostoc mesenteroides, resulting in the formation of a cyclic tetramer cyclo -->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->(2) (cGlc(4)). Two alpha-galactosidases, one from coffee bean and the other produced by a fungus, currently described as Thermomyces lanuginosus, were found to catalyze an efficient 6-O-alpha-D-galactopyranosylation of cGlc(4). The attachment of a nonreducing alpha-D-galactopyranosyl residue to the cGlc(4) molecule opens new possibilities for future applications of the cyclic tetramer, since the D-galactopyranosyl residue can be easily modified by D-galactose oxidase to introduce a reactive aldehyde group. The results also extend our knowledge about the synthetic potential of T. lanuginosus alpha-galactosidase.

Purification and characterization of alpha-galactosidase from a thermophilic fungus Thermomyces lanuginosus.

An extracellular alpha-galactosidase was purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of a mannanolytic strain of the thermophilic fungus Thermomyces lanuginosus. Molecular mass of the enzyme is 57 kDa. The pure enzyme which has a glycoprotein nature, afforded several forms on IEF, indicating its microheterogeneity. Isoelectric point of the major form was 5.2. Enzyme is the most active against aryl alpha-D-galactosides but efficiently hydrolyzed alpha-glycosidically linked non-reducing terminal galactopyranosyl residues occurring in natural substrates such as melibiose, raffinose, stachyose, and fragments of galactomannan. In addition, the enzyme is able to catalyze efficient degalactosylation of polymeric galactomannans leading to precipitation of the polymers. Stereochemical course of hydrolysis of two substrates, 4-nitrophenyl alpha-galactopyranoside and galactosyl(1)mannotriose, followed by (1)H NMR spectroscopy, pointed out the alpha-anomer of D-galactose was the primary product of hydrolysis from which the beta-anomer was formed by mutarotation. Hence the enzyme is a retaining glycosyl hydrolase. In accord with its retaining character the enzyme catalyzed transgalactosylation from 4-nitrophenyl alpha-galactopyranoside as a glycosyl donor. Amino acid sequence alignment of N-terminal and two internal sequences suggested that the enzyme is a member of family 27 of glycosyl hydrolases.

X-ray structure determination and modeling of the cyclic tetrasaccharide cyclo.

The cyclic tetrasaccharide cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] is the major compound obtained by the action of endo-alternases on the alternan polysaccharide. Crystals of this cyclo-tetra-glucose belong to the orthorhombic space group P2(1)2(1)2(1) with a = 7.620(5), b = 12.450(5) and c = 34.800(5) A. The asymmetric unit contains one tetrasaccharide together with five water molecules. The tetrasaccharide adopts a plate-like overall shape with a very shallow depression on one side. The shape is not fully symmetrical and this is clearly apparent on comparing the (phi, psi) torsion angles of the two alpha-(1-->6) linkages. There is almost 10 degrees differences in phi and more than 20 degrees differences in psi. The hydrogen bond network is asymmetric, with a single intramolecular hydrogen bond: O-2 of glucose ring 1 being the donor to O-2 of glucose ring 3. These two hydroxyl groups are located below the ring and their orientation, dictated by this hydrogen bond, makes the floor of the plate. Among the five water molecules, one located above the center of the plate occupies perfectly the shallow depression in the plate shape formed by the tetrasaccharide. Molecular dynamics simulation of the tetrasaccharide in explicit water allows rationalization of the discrepancies observed between the X-ray structures and data obtained previously by NMR.

A chromogenic substrate for a beta-xylosidase-coupled assay of alpha-glucuronidase.

4-Nitrophenyl 2-(4-O-methyl-alpha-d-glucopyranuronosyl)-beta-d-xylopyranoside obtained on deesterification of 4-nitrophenyl 2-O-(methyl 4-O-methyl-alpha-d-glucopyranosyluronate)-beta-d-xylopyranoside (Hirsch et al., Carbohydr. Res. 310, 145-149, 1998) was found to be an excellent substrate for the measurement of hemicellulolytic alpha-glucuronidase activity. A new precise alpha-glucuronidase assay was developed by coupling the alpha-glucuronidase-catalyzed formation of 4-nitrophenyl beta-d-xylopyranoside with its efficient hydrolysis by beta-xylosidase. A recombinant strain of Saccharomyces cerevisiae, harboring and expressing the beta-xylosidase gene xlnD of Aspergillus niger under control of the alcohol dehydrogenase II promoter on a multicopy plasmid, was used as a source of beta-xylosidase. The activity values of beta-xylosidase in the assay required to achieve a steady-state rate of 4-nitrophenol formation shortly after starting the alpha-glucuronidase reaction were obtained both experimentally and by calculation using the kinetics of coupled enzyme reactions.

Effects of purified endo-beta-1,4-xylanases of family 10 and 11 and acetyl xylan esterases on eucalypt sulfite dissolving pulp.

Sulfite dissolving pulp from Eucalyptus grandis contained approximately 3.8% O-acetyl-4-O-methylglucuronoxylan with a molar ratio of xylose:4-O-methylglucuronic acid:acetyl group close to 13.6:1:6.2. The effects produced by purified endo-xylanases from two different glycosyl hydrolase families (family 10 and 11) as well as acetyl xylan esterases were examined and assessed on pulp in relation to their bleaching abilities. The purified endo-xylanases hydrolyzed only a limited portion (less than 30%) of the acetylglucuronoxylan present in the pulp. The enzymes of family 10 produced acetylated xylobiose and xylotriose whereas acetylated xylobiose was not observed among the products released from the pulp by the family 11 xylanases. The esterases however were not capable of deacetylating the acetylated aldouronic acids generated by the xylanases. Regardless of the different mode of action of the endo-xylanases on dissolving pulp, their effect on pulp bleaching was not related to the amount and nature of sugars generated or the glycosyl hydrolase family. No additional brightness gain was obtained when endo-xylanases were used in conjunction with acetyl xylan esterases, suggesting that the latter do not play an important role in biobleaching of eucalypt sulfite dissolving pulps.

Relatedness of Thermomyces lanuginosus strains producing a thermostable xylanase.

Properties of an endo-beta-xylanase produced by a locally isolated Thermomyces lanuginosus strain SSBP was compared to seven other T. lanuginosus strains isolated from different geographical regions. Strain SSBP produced the highest xylanase activity of 59600 nkat ml(-1) when cultivated on corn cobs (maize) medium, whereas the seven other strains produced xylanase activities ranging from 6000 to 32000 nkat ml(-1). No cellulase activity was produced by the strains. Despite the variability in the production of xylanase, little difference in the other characteristics of the strains could be found. The optimal temperature and pH for xylanase production by the strains was either 40 or 50 degrees C and between pH 6 and 7, respectively. Optimal xylanase activity of the strains was observed at 70 degrees C and at pH 6 or 6.5. Culture supernatant analysis by SDS-PAGE and isoelectric focusing PAGE of all strains revealed the presence of a single 24.7 kDa and pI 3.9 xylanase. Phylogenetic analysis by PCR amplification and sequencing of the internal transcribed spacer of nuclear rRNA repeat units and 5.8S rDNA revealed no strain diversity. However, random amplified polymorphic DNA analysis pointed to greater diversity and with one primer (5'-GCCCGACGCG-3'), a relationship was established between xylanase levels and the RAPD pattern.

Inverting character of alpha-glucuronidase A from Aspergillus tubingensis.

Alpha-glucuronidase A from Aspergillus tubingensis was found to be capable of liberating 4-O-methyl-D-glucuronic acid (MeGlcA) only from those beechwood glucuronoxylan fragments in which the acid is attached to the non-reducing terminal xylopyranosyl residue. Reduced aldotetrauronic acid, 4-O-methyl-D-glucuronosyl-alpha-1,2-D-xylopyranosyl-beta-1,4-xylopyranosyl-beta-1,4-xylitol, was found to be a suitable substrate to follow the stereochemical course of the hydrolytic reaction catalyzed by the purified enzyme. The configuration of the liberated MeGlcA was followed in a D(2)O reaction mixture by (1)H-NMR spectroscopy. It was unambiguously established that MeGlcA was released from the substrate as its beta-anomer from which the alpha-anomer was formed on mutarotation. This result represents the first experimental evidence for the inverting character of a microbial alpha-glucuronidase, a member of glycosyl hydrolase family 67 (EC 3.1.1.139).

Biochemical and catalytic properties of an endoxylanase purified from the culture filtrate of Thermomyces lanuginosus ATCC 46882.

An endoxylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) from the culture filtrates of T. lanuginosus ATCC 46882 was purified to homogeneity by DEAE-Sepharose and Bio-Gel P-30 column chromatographies. The purified endoxylanase had a specific activity of 888.8 mumol min-1 mg-1 protein and accounted for approximately 30% of the total protein secreted by this fungus. The molecular mass of native (non-denatured) and denatured endoxylanase were 26.3 and 25.7 kD as, respectively. Endoxylanase had a pI of 3.7 and was optimally active between pH 6.0-6.5 and at 75 degrees C. The enzyme showed > 50% of its original activity between pH 5.5-9.0 and at 85 degrees C. The pH and temperature stability studies revealed that this endoxylanase was almost completely stable between pH 5.0-9.0 and up to 60 degrees C for 5 h and at pH 10.0 up to 55 degrees C for 5 h. Thin-layer chromatography (TLC) analysis showed that endoxylanase released mainly xylose (Xyl) and xylobiose (Xyl2) from beechwood 4-O-methyl-D-glucuronoxylan, O-acetyl-4-O-methyl-D-glucuronoxylan and rhodymenan (a beta-(1-->3)-beta(1-->4)-xylan). Also, the enzyme released an acidic xylo-oligosaccharide from 4-O-methyl-D-glucuronoxylan, and an isomeric xylotetraose and an isomeric xylopentaose from rhodymenan. The enzyme hydrolysed [1-3H]-xylo-oligosaccharides in an endofashion, but the hydrolysis of [1-3H]-xylotriose appeared to proceed via transglycosylation. since the xylobiose was the predominant product. Endoxylanase was not active on pNPX and pNPC at 40 and 100 mM for up to 6 h, but showed some activity toward pNPX at 100 mM after 20-24 h. The results suggested that the endoxylanase from T. lanuginosus belongs to family 11.

Substrate binding and catalytic mechanism of a barley beta-D-Glucosidase/(1,4)-beta-D-glucan exohydrolase.

A beta-glucosidase, designated isoenzyme betaII, from germinated barley (Hordeum vulgare L.) hydrolyzes aryl-beta-glucosides and shares a high level of amino acid sequence similarity with beta-glucosidases of diverse origin. It releases glucose from the non-reducing termini of cellodextrins with catalytic efficiency factors, kcat/Km, that increase approximately 9-fold as the degree of polymerization of these substrates increases from 2 to 6. Thus, the enzyme has a specificity and action pattern characteristic of both beta-glucosidases (EC 3.2.1.21) and the polysaccharide exohydrolase, (1,4)-beta-glucan glucohydrolase (EC 3.2.1.74). At high concentrations (100 mM) of 4-nitrophenyl beta-glucoside, beta-glucosidase isoenzyme betaII catalyzes glycosyl transfer reactions, which generate 4-nitrophenyl-beta-laminaribioside, -cellobioside, and -gentiobioside. Subsite mapping with cellooligosaccharides indicates that the barley beta-glucosidase isoenzyme betaII has six substrate-binding subsites, each of which binds an individual beta-glucosyl residue. Amino acid residues Glu181 and Glu391 are identified as the probable catalytic acid and catalytic nucleophile, respectively. The enzyme is a family 1 glycoside hydrolase that is likely to adopt a (beta/alpha)8 barrel fold and in which the catalytic amino acid residues appear to be located at the bottom of a funnel-shaped pocket in the enzyme.

Endo-beta-1,4-xylanase families: differences in catalytic properties.

Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 (formerly F) and 11 (formerly G) differ in their action on 4-O-methyl-D-glucurono-D-xylan and rhodymenan, a beta-1,3-beta-1,4-xylan. Two high molecular mass EXs (family 10), the Cryptococcus albidus EX and XlnA of Streptomyces lividans, liberate from glucuronoxylan aldotetrauronic acid as the shortest acidic fragment, and from rhodymenan an isomeric xylotriose of the structure Xyl beta 1-3Xyl beta 1-4Xyl as the shortest fragment containing a beta-1,3-linkage. Low molecular mass EXs (family 11), such as the Trichoderma reesei enzymes and XlnB and XlnC of S. lividans, liberate from glucuronoxylan an aldopentauronic acid as the shortest fragment, and from rhodymenan an isomeric xylotetraose as the shortest fragment containing a beta-1,3-linkage. The structure of the oligosaccharides was established by: NMR spectroscopy, mass spectrometry of per-O-methylated compounds and enzymic hydrolysis by beta-xylosidase and EX, followed by analysis of products by chromatography. The structures of the fragments define in the polysaccharides the linkages attacked and non-attacked by the enzymes. EXs of family 10 require a lower number of unsubstituted consecutive beta-1,4-xylopyranosyl units in the main chain and a lower number of consecutive beta-1,4-xylopyranosyl linkages in rhodymenan than EXs of family 11. These results, together with a greater catalytic versatility of EXs of family 10, suggest that EXs of family 10 have substrate binding sites smaller than those of EXs of family 11. This suggestion is in agreement with the finding that EXs of family 10 show higher affinity for shorter linear beta-1,4-xylooligosaccharides than EXs of family 11. The results are discussed with relevant literature data to understand better the structure-function relationship in this group of glycanases.

The beta-D-xylosidase of Trichoderma reesei is a multifunctional beta-D-xylan xylohydrolase.

An extracellular multifunctional beta-D-xylan xylohydrolase, previously described as beta-xylosidase, was purified from Trichoderma reesei RUT C-30 to physical homogeneity. The active enzyme was a 100 (+/-5) kDa glycosylated monomer that exhibited a pl of 4.7. Its activity was optimal at pH 4 and it was stable between pH 3 and 6. Its temperature-stability was moderate (70 degrees zero of activity remaining after 60 min at 50 degrees C) and optimal activity was observed at 60 degrees C. It is capable of hydrolysing beta-1.4-xylo-oligosaccharides [degree of polymerization (DP) 2-7], the apparent Vmax increasing with increasing chain length. The enzyme also attacked debranched beech-wood (Lenzing) xylan and 4-O-methylglucuronoxylan, forming xylose as the only end product. The K(m) for xylan was 0.7 g/l. For this reason we consider the enzyme to be a beta-D-xylan xylohydrolase. The enzyme also exhibits alpha-L-arabinofuranosidase activity on 4-nitrophenyl alpha-L-arabinofuranoside, and evidence is presented that this is not caused by an impurity in the enzyme preparation. The beta-D-xylan xylohydrolase exhibits glycosyltransferase activity with xylo-oligosaccharides and at high concentrations of 4-nitrophenyl beta-D-xylopyranoside (4-Nph-beta-Xyl). The enzyme hydrolyses beta-1, 4-linkages preferentially to beta-1,3-linkages, and beta-1,2-linked xylo-oligosaccharides are not hydrolysed at all. The enzyme liberates terminal beta-1,4-xylopyranose residues linked to a 2-O-substituted xylopyranose residue, but not that linked to a 3-O-substituted xylopyranose residue. The enzyme does not attack methyl, methyl 1-thio-benzyl or butyl l-thio-beta-D-xylopyranosides and 4-naphthyl, 2-naphthyl and phenyl beta-D-xylopyranosides.

Action of acetylxylan esterase from Trichoderma reesei on acetylated methyl glycosides.

Substrate specificity of purified acetylxylan esterase (AcXE) from Trichoderma reesei was investigated on partially and fully acetylated methyl glycopyranosides. Methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside was deacetylated at positions 2 and 3, yielding methyl 4-O-acetyl-beta-D-xylopyranoside in almost 90% yield. Methyl 2,3-di-O-acetyl beta-D-xylopyranoside was deacetylated at a rate similar to the fully acetylated derivative. The other two diacetates (2,4- and 3,4-), which have a free hydroxyl group at either position 3 or 2, were deacetylated one order of magnitude more rapidly. Thus the second acetyl group is rapidly released from position 3 or 2 after the first acetyl group is removed from position 2 or 3. The results strongly imply that in degradation of partially acetylated beta-1,4-linked xylans, the enzyme deacetylates monoacetylated xylopyranosyl residues more readily than di-O-acetylated residues. The T. reesei AcXE attacked acetylated methyl beta-D-glucopyranosides and beta-D-mannopyranosides in a manner similar to the xylopyranosides.

Substrate specificity of acetylxylan esterase from Schizophyllum commune: mode of action on acetylated carbohydrates.

Substrate specificity of a purified acetylxylan esterase from Schizophyllum commune was investigated on a variety of methyl per-O-acetyl glycopyranosides, methyl di-O-acetyl-beta-D-xylopyranosides and acetylated polysaccharides. The enzyme preferentially deacetylated the 3-position of methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside. Removal of the 3-acetyl group from the xylopyranoside was accompanied by a slower deacetylation at positions 2 and 4. A similarly slower, accompanying deacetylation occurred primarily at position 2 with the glucopyranoside. Such specificity corresponds well to the expected function of the esterase in acetylxylan degradation. Of the three possible diacetates of methyl beta-D-xylopyranoside, the 3,4-diacetate was found to be the most rapidly deacetylated. Unexpectedly, products of its deacetylation were a mixture of 2- and 4-monoacetate. The formation of the methyl 2-O-acetyl-beta-D-xylopyranoside involved an enzyme-mediated acetyl group transfer because the rate of the enzyme-catalyzed reaction exceeded the rate of spontaneous migration of acetyl groups. This is the likely mechanism for acetyl removal from position 2 in the native substrate. The enzyme exhibited the highest regioselectivity with methyl 2,3,4,6-tetra-O-acetyl-beta-D-mannopyranoside. An 80% conversion of this substrate to methyl 4,6-di-O-acetyl-beta-D-mannopyranoside, a new mannose derivative, was achieved. In contrast to the majority of lipases and esterases exploited for regioselective deacetylation, the S. commune acetylxylan esterase did not attack the C-6 acetyl linkages in methyl hexopyranosides when other acetyl groups were available.

Substrate specificity and mode of action of acetylxylan esterase from Streptomyces lividans.

The substrate specificity of purified acetylxylan esterase (AcXE) from Streptomyces lividans was investigated on partially and fully acetylated methyl glycopyranosides. The enzyme exhibited deacetylation regioselectivity on model compounds which provided insights pertaining to its function in acetylxylan degradation. The enzyme catalyzed double deacetylation of methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside at positions 2 and 3. Two methyl xylopyranoside diacetates, which had a free hydroxyl group at position 2 or 3, i.e. the derivatives that most closely mimic monoacetylated xylopyranosyl residues in acetylxylan, were deacetylated 1 to 2 orders of magnitude faster than methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3-di-O-acetyl-beta-D-xylopyranoside. These observations explain the double deacetylation. The second acetyl group is released immediately after the first one is removed from the fully acetylated methyl beta-D-xylo- and -glucopyranoside. The results suggest that in acetylxylan degradation the enzyme rapidly deacetylates monoacetylated xylopyranosyl residues, but attacks doubly acetylated residues much more slowly. Evidence is also presented that the St. lividans enzyme could be the first real substrate-specific AcXE.

Isolation and Characterization of Microorganisms with Alternan Hydrolytic Activity

Alternan is an unusual alpha-D-glucan containing alternating (1 --> 3), (1 --> 6) linkages that exhibits remarkable resistance to enzymatic hydrolysis. The commercial potential of the polysaccharide may be enhanced by the ability to economically modify the native form into fractions of varying molecular weight. By employing isolation procedures with covalently dyed alternan as the substrate, several bacterial isolates that produced endohydrolytic activity were obtained in pure culture. The activity was confirmed by decreases in viscosity and by direct examination of the hydrolysis products with thin layer chromatography. Analysis of the hydrolysis products established that all isolates produced enzymes with identical alternan depolymerizing activity, producing a cyclic tetrasaccharide as a major product. All alternanase activity was shown to be extracellularly located. A single strain exhibited constitutive production of alternanase, while all other isolates required the presence of alternan in the growth media for enzyme production. All isolates were phenotypically similar, produced heat-resistant spores, and were tentatively identified as members of the genus Bacillus.

Inversion of configuration during hydrolysis of alpha-1,4-galacturonidic linkage by three Aspergillus polygalacturonases.

Endopolygalacturonases I and II (PGI and PGII) of Aspergillus niger and an exopolygalacturonase (ExoPG) of A. tubingensis were investigated to reveal the stereochemistry of their hydrolytic action. Reduced pentagalacturonic acid (pentaGalU-ol) and reduced trigalacturonic acid (triGalU-ol) were used as non-reducing substrates for the enzymes. The configuration of the reducing ends in the products formed in D2O reaction mixtures was followed by 1H-NMR spectroscopy. It has been unambiguously established that primary cleavage of pentaGalU-ol by both PGI and PGII leads to diGalU-ol and the beta-anomer of triGalUA. The primary products of hydrolysis of triGalUA-ol by ExoPG were diGal-ol and the beta-anomer of GalUA. Thus, all three Aspergillus polygalacturonases belong to the so-called inverting glycanases, i.e. they utilize the single displacement mechanism of hydrolysis of the glycosidic linkage.