Biomedical subjects

M Tenkanen

Publications and source records attributed to M Tenkanen.

31 records · Page 2Linked to original sources

PubMed1997

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.

Endo-1,4-beta Xylanases↗

PubMed1997

Action of Trichoderma reesei mannanase on galactoglucomannan in pine kraft pulp.

The di-, tri- and tetrasaccharides formed during Trichoderma reesei endo-beta-D-mannanase treatment of pine kraft pulp were studied. The oligosaccharides in the hydrolysate were fractionated using size-exclusion, anion exchange and activated carbon chromatography. The primary sequence of the purified oligomers was determined by two-dimensional NMR techniques. The T. reesei mannanase cleaves the beta-1,4-glycosidic linkage of D-mannosyl residues attached either to D-mannose or D-glucose. The D-mannosyl residue may also be substituted by a D-galactosyl group. The main disaccharide produced was mannobiose, but a significant amount of 4-O-beta-D-glucopyranosyl-D-mannopyranose (GlcMan) was also produced. After extensive hydrolysis the main trisaccharides produced were 4-O-beta-D-mannopyranosyl-[6-O-alpha-galactopyranosyl]-D-mannopyranose (Gal1Man2) and 4-O-beta-D-glucopyranosyl-4-O-beta-D-glucopyranosyl-D-mannopyranose (Glc2Man). Some mannotriose 4-O-beta-D-glucopyranosyl-4-O-beta-D-mannopyra-nosyl-D-manno pyranose (GlcMan2) and 4-O-beta-D-glucopyranosyl-[6-O-alpha-galactopyranosyl]-D-mannopyranose (Gal1GlcMan) were also detected in the hydrolysate. The structures of two tetrasaccharides were studied. They appeared to be 4-O-beta-D-glucopyranosyl-4-O-beta-D-glucopyranosyl-4-O-beta-D- glucopyranosyl-D-mannopyranose (Glc3Man) and 4-O-beta-D-glucopyranosyl-4-O-beta-D-mannopyranosyl-4-O-beta-D -glucopyranosyl-D-mannopyranose (GlcManGlcMan). According to the results obtained, the galactoglucomannan in pine contains regions in which two or three glucose units are linked together, which further means that it may contain regions with several successive mannose residues. The galactose side groups were found to be attached only to mannose.

Hydrolysis↗

PubMed1997

Hemicellulases in the bleaching of chemical pulps.

Hemicellulase-aided bleaching is the first full-scale biotechnical application in the pulp and paper industry which truly exploits the unique specificity and safety of biocatalysts. Hemicellulases are used to modify the structure of xylan and glucomannan in pulp fibers in order to enhance the chemical delignification. This technology can be combined with various types of kraft pulping processes and bleaching sequences. The aims of the enzymatic treatment depend on the actual mill conditions, and may be related to environmental demands, reduction of chemical costs, or maintenance or even improvement of product quality. The technology is applied on the mill scale in several countries. This review describes the principles of the enzyme-aided bleaching, the composition of the fiber substrates, the basic enzymology involved, and the present knowledge of the mechanisms of the action of enzymes, as well as the practical results and advantages obtained on the laboratory and industrial scale.

Glycoside Hydrolases↗

PubMed1996

Effect of side groups on the action of beta-xylosidase from Trichoderma reesei against substituted xylo-oligosaccharides.

The action of beta-xylosidase from Trichoderma reesei against different substituted xylo-oligosaccharides was studied. The enzyme cleaved off all unsubstituted xylose units from the non-reducing end of 1,2-linked uronic acid substituted xylo-oligosaccharides. Surprisingly, an L-arabinofuranosyl group linked alpha-1,3 to the xylopyranosyl ring was found to protect the beta-1,4-xylosidic linkage before the substituted xylose unit from being cleaved by the beta-xylosidase. Most probably the 1,3-linked substituent sterically hinders the hydrolysis. According to the results of the present work, beta-xylosidase of T. reesei is not able to remove all unsubstituted xylose units from the non-reducing end of substituted xylo-oligosaccharides, as had been believed previously.

Chromatography, Ion Exchange↗

PubMed1996

4-O-methyl-beta-L-idopyranosyluronic acid linked to xylan from kraft pulp: isolation procedure and characterisation by NMR spectroscopy.

The tetrasaccharide 2"-O-(4-O-methyl-beta-L-idopyranosyluronic acid)xylotriose was isolated from enzymatically hydrolysed, unbleached, birch kraft pulp by anion-exchange chromatography in two steps. The primary structure of the tetrasaccharide was determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. NOE data and 3JH,H coupling constants show that the 4-O-methyl-beta-L-idopyranosyluronic acid in the tetrasaccharide is predominantly in the 1C4 chair conformation. The pKa value (3.17) for 4-O-methyliduronic acid attached beta-(1-->2) to xylose was determined from the pH-dependent chemical shift of H-5. The amount of 4-O-methyliduronic acid (0.1-0.5 mol%) in surface xylan of unbleached birch and pine kraft pulps was determined by extensive xylanase treatment and further analysis by NMR spectroscopy and high-performance anion-exchange chromatography.

Carbohydrate Conformation↗

PubMed1996

Three alpha-galactosidase genes of Trichoderma reesei cloned by expression in yeast.

Three alpha-galactosidase genes, agl1, agl2 and agl3, were isolated from a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevisiae by screening the library on plates containing the substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside. The genes agl1, agl2 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences. The deduced amino acid sequences of AGLI and AGLIII showed similarity with the alpha-galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLII showed similarity with the bacterial alpha-galactosidases of family 36. The enzymes produced by yeast were analysed for enzymatic activity against different substrates. AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p-nitrophenyl-alpha-D-galactopyranoside and the small galactose-containing oligosaccharides, melibiose and raffinose. They liberated galactose from polymeric galacto(gluco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo-1,4-beta-mannanase of T. reesei. AGLII and AGLIII showed synergy in galacto(gluco)mannan hydrolysis with the endo-1,4-beta-mannanase of T. reesei and a beta-mannosidase of Aspergillus niger. The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the alpha-galactosidase previously purified from T. reesei.

Amino Acid Sequence↗

PubMed1996

Acetyl xylan esterase from Trichoderma reesei contains an active-site serine residue and a cellulose-binding domain.

The axe1 gene encoding acetyl xylan esterase was isolated from an expression library of the filamentous fungus Trichoderma reesei using antibodies raised against the purified enzyme. Apparently axe1 codes for the two forms, pI 7 and pI 6.8, of acetyl xylan esterase previously characterized. The axe1 encodes 302 amino acids including a signal sequence and a putative propeptide. The catalytic domain has no amino acid similarity with the reported acetyl xylan esterases but has a clear similarity, especially in the active site, with fungal cutinases which are serine esterases. Similarly to serine esterases, the axe1 product was inactivated with phenylmethylsulfonyl fluoride. At its C-terminus it carries a cellulose binding domain of fungal type, which is separated from the catalytic domain by a region rich in serine, glycine, threonine and proline. The binding domain can be separated from the catalytic domain by limited proteolysis without affecting the activity of the enzyme towards acetylated xylan, but abolishing its capability to bind cellulose.

Acetylesterase↗

PubMed1996

Identification of the acidic degradation products of hexenuronic acid and characterisation of hexenuronic acid-substituted xylooligosaccharides by NMR spectroscopy.

A 4-O-methylglucuronoxylan was converted into a hexenuronoxylan at high temperature and alkalinity similar to the conditions used during kraft pulping. The hexenuronoxylan was hydrolysed with enzymes, and acidic xylooligosaccharides were separated from the hydrolysate by anion-exchange and size-exclusion chromatography. The primary structure of the two main hexenuronic acid-substituted xylooligosaccharides (a tetramer and a pentamer) was determined by two-dimensional 1H and 13C NMR spectroscopy. The 4-deoxy-hexenuronic acid is not stable under the acid hydrolysis step of conventional carbohydrate analysis. Here, we have identified the acidic degradation products of 4-deoxy-hexenuronic acid by NMR spectroscopy. Two degradation pathways were observed, both resulting in a furan derivative.

Carbohydrate Sequence↗

PubMed1996

Cloning of genes encoding alpha-L-arabinofuranosidase and beta-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae.

A cDNA expression library of Trichoderma reesei RutC-30 was constructed in the yeast Saccharomyces cerevisiae. Two genes, abf1 and bxl1, were isolated by screening the yeast library for extracellular alpha-L-arabinofuranosidase activity with the substrate p-nitrophenyl-alpha-L-arabinofuranoside. The genes abf1 and bxl1 encode 500 and 758 amino acids, respectively, including the signal sequences. The deduced amino acid sequence of ABFI displays high-level similarity to the alpha-L-arabinofuranosidase B of Aspergillus niger, and the two can form a new family of glycosyl hydrolases. The deduced amino acid sequence of BXLI shows similarities to the beta-glucosidases grouped in family 3. The yeast-produced enzymes were tested for enzymatic activities against different substrates. ABFI released L-arabinose from p-nitrophenyl-alpha-L-arabinofuranoside and arabinoxylans and showed some beta-xylosidase activity toward p-nitrophenyl-beta-D-xylopyranoside. BXLI did not release L-arabinose from arabinoxylan. It showed alpha-L-arabinofuranosidase, alpha-L-arabinopyranosidase, and beta-xylosidase activities against p-nitrophenyl-alpha-L-arabinofuranosidase, p-nitrophenyl-alpha-L-arabinopyranoside, and p-nitrophenyl-beta-D- xylopyranoside, respectively, with the last activity being the highest. It was also able to hydrolyze xylobiose and slowly release xylose from polymeric xylan. ABFI and BXLI correspond to a previously purified alpha-L-arabinofuranosidase and a beta-xylosidase from T. reesei, respectively, as confirmed by partial amino acid sequencing of the Trichoderma-produced enzymes. Both enzymes produced in yeasts displayed hydrolytic properties similar to those of the corresponding enzymes purified from T. reesei.

Amino Acid Sequence↗

PubMed1995

An acetylglucomannan esterase of Aspergillus oryzae; purification, characterization and role in the hydrolysis of O-acetyl-galactoglucomannan.

An acetyl glucomannan esterase (AGME) was purified to electrophoretic homogeneity from the culture supernatant of Aspergillus oryzae. This new enzyme had a molecular mass of 36 kDa and an isoelectric point of 4.6. It was most active in the pH range 5.0-5.5 and was stable for 24 h at 40 degrees C at pH 5.0-6.0. The purified esterase liberated acetic acid from O-acetyl-galactoglucomannan, O-acetyl-4-O- methylglucuronoxylan and alpha-naphtyl acetate. The specific activity was 10-times higher for acetylated mannan than for acetylated xylan. The enzyme was able to act on polymeric substrate but activity was clearly enhanced by addition of mannanase from Trichoderma reesei and alpha-galactosidase from guar seeds. Presence of mannanase also increased the liberation of acetic acid in long-term hydrolysis (24 h), while the addition of alpha-galactosidase had no effect. No significant synergism between these two glycanases and the previously characterized esterase of A. oryzae (FE), which is also able to deacetylate galactoglucomannan, was observed. Even though the AGME had 8-times higher specific galactomannan deacetylating activity than the FE, the maximum amount of acetic acid liberated from the polymeric galactoglucomannan by AGME was only 80% of that of FE. Both esterases clearly enhanced the action of mannanase and alpha-galactosidase in the degradation of O-acetyl-galactoglucomannan isolated from Norway spruce.

Aspergillus oryzae↗

PubMed1995

Characterisation of 4-deoxy-beta-L-threo-hex-4-enopyranosyluronic acid attached to xylan in pine kraft pulp and pulping liquor by 1H and 13C NMR spectroscopy.

A new acidic sidegroup in xylans, from both kraft pulp and pulping liquor, was identified by NMR spectroscopy. Unmodified oligosaccharides from kraft pulp xylan were obtained by enzymatic hydrolysis with xylanase (Trichoderma reesei). The acidic oligosaccharides were separated from the natural forms on an anion exchange resin. The new acidic sidegroup was identified as 4-deoxy-beta-L-threo-hex-4-enopyranosyluronic acid (hexenuronic acid) by 1H and 13C NMR spectroscopy. Hexenuronic acid is a beta-elimination product of 4-O-methylglucuronic acid and is formed during kraft pulping. HMBC and NOESY experiments showed that hexenuronic acid is attached beta-(1 --> 2) to xylose. The NOESY data further indicated that hexenuronic acid protrudes from the main xylan chain. The pKa values for hexenuronic acid (3.03) and 4-O-methylglucuronic acid (3.14) attached (1 --> 2) to xylose were determined from pH-dependent chemical shifts.

Carbohydrate Sequence↗

PubMed1994

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.

Endo-1,4-beta Xylanases↗

PubMed1990

Clinical and panoramic assessment of marginal bone loss. A cadaver study.

The purpose of this study was to investigate the reliability of panoramic tomography for the assessment of marginal alveolar bone loss. The study was conducted with 20 human cadavers, in relation to 123 of their teeth. Panoramic tomograms of the cadaver dentitions were taken using a Zonarc radiographic apparatus. Root lengths and bone losses were measured mesially and distally from the panoramic tomograms. Bone loss was also measured by surgical exposure of the marginal bone. Root length was also measured after extraction of the tooth. After allowing for the vertical magnification, radiographic bone loss values were found to be about 0.8 mm (27%) less than clinical values. This difference was statistically significant only in the incisor area. It was almost significant in the premolar region. It is therefore concluded that panoramic tomography is sufficiently reliable for clinical assessment of alveolar bone loss but that the bottom of periodontal pockets always tends to be somewhat deeper than the radiographically visible defect.

Adult↗