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.

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.

Production of extracellular beta-mannanases by yeasts and yeast-like microorganisms.

A new screening method for simultaneous detection of endo-beta-1,4-mannanase and endo-beta-1,4-xylanase producing microorganisms is described. Two differently dyed substrates Ostazin Brilliant Red-galactomannan and Remazol Brilliant Blue-xylan were incorporated into the same agar media. Decolorizing of one or both substrates around the cell colonies indicates secretion of the corresponding enzyme(s). The method was used to screen 449 yeasts and yeast-like microorganisms belonging to 68 different genera. The secretion of endo-beta-1,4-mannanases and/or endo-beta-1,4-xylanases was found within 10 genera (42 positive strains out of 261 tested). A low frequency of occurrence of endo-beta-1,4-mannanases was observed within the genera Cryptococcus (1 positive strain out of 15 tested), Geotrichum (1 of 6) and Pichia (1 of 35). The highest frequency of occurrence of endo-beta-1,4-mannanases was found within the genera Stephanoascus (2 of 2) and Aureobasidium (14 of 14). Strains hydrolyzing Ostazin Brilliant Red-galactomannan were cultivated in liquid media containing 1% locust bean gum. The best producers of extracellualr endo-beta-1,4-mannanases were found to be the strains of Aureobasidium pullulans.

Stereochemistry of the hydrolysis of glycosidic linkage by endo-beta-1,4-xylanases of Trichoderma reesei.

Methyl beta-D-xylotrioside was used as a non-reducing substrate to investigate the stereochemistry of hydrolysis of beta-1,4-xylopyranosidic linkage by purified endo-beta-1,4-xylanases (EC 3.2.1.8) of Trichoderma reesei, employing 1H NMR spectroscopy. The fungus produces one acidic species (pI 4.8-5.5), designated as EXI, and one alkaline species (pI 8.5-9.0), designated as EXII. Both enzymes were found to cleave the xylotrioside predominantly to methyl beta-D-xyloside and xylobiose. Monitoring of the intensity of the H-1 signals of alpha- and beta-xylobiose during the time course of hydrolysis clearly showed that both enzymes liberate the beta-anomer of xylobiose, i.e. a product with anomeric configuration identical with that of the cleaved glycosidic linkage. This means that both EXI and EXII belong to the so-called retaining glycanases that utilize the double displacement reaction mechanism of hydrolysis.

Simultaneous production and purification of Bacillus subtilis alpha-amylase.

Production of alpha-amylase with B. subtilis CCM 2722 in an aqueous two-phase polyethylene glycol/dextran system integrated with product purification by affinity chromatography on crosslinked starch during cultivation was studied. The medium was drawn from the bioreactor to the external settler during fermentation. After phase separation in the settler the dextran-rich bottom phase with cells was returned to the bioreactor. The PEG-rich top phase was pumped to the column with crosslinked starch and returned to the bioreactor after alpha-amylase adsorption. The same volumetric productivities, 0.53 U/mL/h, were reached in both batch and described process, but total productivity of the latter method was much higher owing to shortening upstream and downstream processing time. The enzyme of 98% homogenity in 95% yield was obtained after its elution from the column.