Showing papers on "Xylanase published in 1985"

Two types of xylanases of alkalophilic Bacillus sp. No. C-125

Abstract: One alkalophilic Bacillus sp. strain C-125 (FERM No. 7344) was isolated from soil. From this organism, two types of xylanases, designated xylanase A and xylanase N, were purified by an ammonium sul...

Production, purification, and properties of thermostable xylanase from Clostridium stercorarium

Abstract: The production of xylanase and endoglucanase in Clostridium stercorarium has been studied in different conditions. Activities were higher when the organism was grown on xylan and cellulose than on soluble substrates. Catabolite repression of xylanase synthesis occurred when glucose and other readily metabolizable substrates were added during growth on cellulose. Three endoxylanases, A, B, and C, from culture filtrate were purified to homogeneity. Most of the properties of xylanases A, B, and C were similar (optimum pH in the range of 5.5–7.0 at 65 °C; isoelectric pH, 4.4–4.5; Km values of 2.9–3.7 mg/mL). The enzymes were inactivated by Hg2+ and p-chloromercuribenzoate but slight inhibition was obtained with more specific thiol reagents such as 5,5′-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide. Carbohydrate content (3–19%) and half-life (2 min 30 s to 90 min) varied. Patterns of hydrolysis demonstrate that the three enzymes are endo-splitting enzymes able to break down xylan at random giving xylobio...

Studies on the extracellular xylanase activity of some thermophilic actinomycetes

Abstract: An agar plate-clearing assay was used to screen 37 thermophilic actinomycete strains for extracellular xylanase production. The xylanase activity in culture supernatants of strains representing Saccharomonospora viridis and three Thermomonospora spp. was characterised by measurement of reducing sugar released from oat spelt xylan and analysis of degradation products by thin-layer chromatography. In all four species, xylanase activity was optimal within the temperature range 60–75°C and between pH 5 and pH 8. While culture supernatants of Thermomonospora strains incubated at 70°C for 60 min retained >80% of their activity, that of S. viridis was almost, totally inactivated.

Xylanolytic Activity of Clostridium acetobutylicum.

Abstract: Of 20 strains of Clostridium spp. screened, 17 hydrolyzed larch wood xylan. Two strains of Clostridium acetobutylicum, NRRL B527 and ATCC 824, hydrolyzed xylan but failed to grow on solid media with larch xylan as the sole carbon source; however, strain ATCC 824 was subsequently found to grow on xylan under specified conditions in a chemostat. These two strains possessed cellulolytic activity and were therefore selected for further studies. In cellobiose-limited continuous cultures, strain NRRL B527 produced maximum xylanase activity at pH 5.2. Strain ATCC 824 produced higher xylanase, xylopyranosidase, and arabinofuranosidase activities in chemostat culture with xylose than with any other soluble carbon source as the limiting nutrient. The activities of these enzymes were markedly reduced when the cells were grown in the presence of excess glucose. The xylanase showed maximum activity at pH 5.8 to 6.0 and 65°C. The enzyme was stable on the alkaline side of pH 5.2 but was unstable below this pH value. The extracellular xylanolytic activity from strain ATCC 824 hydrolyzed 12% of the larch wood xylan during a 24-h incubation period, yielding xylose, xylobiose, and xylotriose as the major hydrolysis products. Strain ATCC 824, after being induced to grow in batch culture in xylan medium supplemented with a low concentration of xylose, failed to grow reproducibly in unsupplemented xylan medium. A mutant obtained by mutagenesis with ethyl methanesulfonate was able to grow reproducibly in batch culture on xylan. Both the parent strain and the mutant were able to grow with xylan as the sole source of carbohydrate in continuous culture with the pH maintained at either 5.2 or 6.0. Under these conditions, the cells utilized approximately 50% of the xylan. Images

Isolation and culture of a thermophilic fungus, Melanocarpus albomyces, and factors influencing the production and activity of xylanase

Abstract: Summary: An uncommon thermophilic fungus, Melanocarpus albomyces, was isolated from soil and compost by incubating samples in a glucose/sorbose/asparagine liquid medium, followed by enrichment culture in medium containing sugarcane bagasse as carbon source. The culture filtrate protein of the fungus grown in the presence of bagasse or xylose hydrolysed xylan and some other polysaccharides but cellulose was not hydrolysed. High extracellular xylanase (EC 3.2.1.8) activity was produced by cultures grown on xylose or hemicellulosic materials. The enzyme was induced in glucose-grown washed mycelia in response to addition of xylose or xylan but not by alkyl or aryl β-D-xylosides. Cultures produced higher enzyme yields in shaken flasks than in a fermenter. Gel-filtration chromatography of culture filtrate protein showed the presence of two isoenzymes of xylanase, whose relative proportions varied with the carbon source used for growth. The extent of hydrolysis of heteroxylans or the hemicellulosic fraction of bagasse by culture filtrate protein preparations was greater when the cultures had been grown on bagasse rather than xylose as the inducing substrate. The activity of xylanase preparations was increased when an exogenous β-glucosidase was added.

Xylanase production in solid-state fermentation: a study of its properties

Abstract: Xylanase production by Aspergillus niger van Tieghem was studied in solid-state cultivation. The screening of substrates was carried out in column incubators aerated with humidified air at 30°C. Results of physiological studies showed that the best yield of xylanase was 2500 U/g dry matter on a mixture of straw+bran 1:1 at 70% of moisture content.

Molecular cloning and expression of the xylanase gene of alkalophilic Bacillus sp. strain C-125 in Escherichia coli.

Abstract: The gene for xylanase A of alkalophilic Bacillus sp. strain C-125 was cloned in Escherichia coli with pBR322. The plasmid pCX311 contained 2.6- and 2.0-kilobase-pair HindIII fragments. The characteristics of the purified pCX311-encoded xylanase were the same as those of purified xylanase A from alkalophilic Bacillus sp. strain C-125.

Expression of a xylanase gene of Bacillus pumilus in Escherichia coli and Bacillus subtilis

Abstract: A hybrid plasmid, pOXN29 (10.4 Mdal), coding the xylanase (xynA) and β-xylosidase (xynB) genes of Bacillus pumilus IPO was constructed by the ligation of pBR322 and a 7.7 Mdal PstI-fragment of chromosomal DNA as reported in our previous paper (Panbangred et al. 1983). A deletion plasmid of pOXN29, pOXN293 (9.2 Mdal), which contains xynA and xynB, was ligated with pUB110 at an EcoRI site, and used to transform B. subtilis MI111. Two selected clones of B. subtilis as xylanase hyper-producers contained plasmids pOXW11 (4.2 Mdal) and pOXW12 (4.0 Mdal), both consisting of only pUB110, xynA, and its flanking regions, as the result of spontaneous deletion. These B. subtilis clones produced 2.7–3.0 times as much xylanase as B. pumilus. Escherichia coli and B. subtilis clones harbouring the hybrid plasmids synthesized xylanase and β-xylosidase constitutively, whereas both enzymes were induced by xylose in B. pumilus. Xylanase synthesized by B. subtilis harbouring pOXW11 or pOXW12 was excreted into the medium like that of B. pumilus IPO, but xylanase synthesized in E. coli harbouring pOXN29, 293 or pOXW1 coding xynA was intracellular. In a previous investigation (Panbangred et al. 1983), xylanase was found to be located in the cytoplasm, not the periplasm nor the membrane fraction in E. coli cells harbouring pOXN29 derivatives. In spite of the abnormal location of xylanase synthesized in E. coli, the signal peptide was processed in the same way as in B. pumilus, with the same molecular weight and the same amino terminal sequences of xylanase prepared from E. coli cells and B. pumilus culture fluid.

Effects of aromatic compounds on hemicellulose-degrading enzymes in Aspergillus japonicus.

Abstract: Aspergillus japonicus was grown in the presence of various aromatic compounds at 0.1 and 1 mg/mL, and extracellular xylanase and arabanase activities were measured. Some of the aromatic compounds tested, especially at the higher concentration, suppressed the appearance of hemicellulase activities, expressed as xylanase or arabanase. Vanillin at 1 mg/mL in the presence of either xylan or araban completely suppressed growth, and guaiacol and p-coumaric acid markedly inhibited the growth of A. japonicus. The effects of the aromatic compounds on the activity of crude enzyme preparations were also ascertained. In vitro arabanase activity was affected more than xylanase activity.

Isolation and partial characterization of a mutant of Penicillium funiculosum for the saccharification of straw.

Abstract: Clearing of agar plates containing ball-milled, delignified straw has been used for screening mutants of Penicillium funiculosum IMI 87160 III. The effects of glycerol and a number of sugars on the clearing were investigated for selecting derepressed mutants. The ..beta..-glucosidase synthesis by one such mutant, C22c, in shake flasks containing straw was not repressed by 5% glycerol, whereas activities on filter paper, CM-cellulose, and p-nitrophenyl-..beta..-xylosidase were only partially derepressed; xylanase was extensively derepressed. The evidence for separate control of the enzymes involved in the solubilization of straw is discussed. 23 references.

Kinetic characterization of the extracellular xylanases of Thermomonospora sp.

Abstract: This study deals with characterizing the extracellular xylanases produced by a strain of the thermophilic bacterial genus Thermomonospora. Supernatant from centrifuged fermentation broth was used as a crude enzyme preparation. From pH 5.5 to pH 7.7 the temperature optimum based on a 10-min assay of activity was 80°C. The crude enzyme had a half-life of approximately 1 month when stored at 55°C at pH 6.5. The enzyme produced a mixture of xylose oligomers from xylan, with xylobiose occuring in greatest quantity on a molar basis. Only trace quantities of xylose were produced by this hydrolysis.

Purification and properties of two endo-1,4-beta-xylanases from Irpex lacteus (Polyporus tulipiferae).

Abstract: Two different endo-1,4-beta-xylanases [1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8], named Xylanases I and III, were purified to homogeneity by gel filtration and ion exchange column chromatography from Driselase, a commercial enzyme preparation from Irpex lacteus (Polyporus tulipiferae). The purified enzymes were found to be homogeneous on polyacrylamide disc electrophoresis and their specific activities toward xylan were increased approximately 28.7 and 19.8 times, respectively. The activities of each enzyme were considerably inhibited by Hg2+, Ag+, and Mn2+. Their molecular weights were estimated to be approximately 38,000 and 62,000 by gel filtration and sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis, respectively. Their carbohydrate contents were 2.5% and 8.0% as glucose, and their amino acid composition patterns resembled each other, showing high contents of acidic amino acids, serine, threonine, alanine, and glycine. Both enzymes were most active at pH 6.0 but Xylanase I was more stable as to pH. Their optimum temperatures were 60 degrees C and 70 degrees C, respectively. Xylanase I split up to 34.5% of larchwood xylan whereas Xylanase III split only 18.9% of it. The products with the former were mainly xylose (X1), xylobiose (X2), and xylotriose (X3), whereas X2 and X3 were the main products with the latter. Both enzymes did not hydrolyze X2. Xylanase I produced almost equal quantities of X1 and X2 from X3, while Xylanase III did not attack this substrate. Both enzymes showed no activity toward glycans, other than xylan, such as starch, pachyman and Avicel (microcrystalline cellulose), except the almost one twentieth activity of Xylanase III toward sodium carboxymethyl cellulose (CMC).

Butanediol production from cellulose and hemicellulose by Klebsiella pneumoniae grown in sequential coculture with Trichoderma harzianum.

Abstract: The bioconversion of cellulose and hemicellulose substrates to 2,3-butanediol by a sequential coculture approach was investigated with the cellulolytic fungus Trichoderma harzianum E58 and the fermentative bacterium Klebsiella pneumoniae. Vogel medium optimal for the production of the cellulolytic and xylanolytic enzymes of the fungus was found to be inhibitory to butanediol fermentation. This inhibition appeared to be due to a synergistic effect of various ingredients, particularly the salts, present in the fungal medium. The removal or replacement of such ingredients from Vogel medium led to the relief of fermentation inhibition, but the treatments also resulted in a significant decrease in fungal enzyme production. Resting cells of K. pneumoniae could be used for butanediol production in the fungal medium, indicating that the inhibitory effect on solvent production under such conditions was due to the indirect result of growth inhibition of the bacterial cells. The resting-cell approach could be combined with a fed-batch system for the direct conversion of 8 to 10% (wt/vol) of Solka-Floc or aspenwood xylan to butanediol at over 30% of the theoretical conversion efficiencies.

Measurement of xylanase activity with insoluble xylan substrate

Abstract: Insoluble xylan was prepared from ground birch (Betula pubescens) pulp by alkali extraction and precipitation with ethanol. The only sugar detected after acid hydrolysis of the preparation was xylose. The insoluble xylan was used as substrate in a nephelometric assay to determine the xylanase (EC 3.2.1.8, 1,4-beta-D-xylan xylanohydrolase and EC 3.2.1.37, 1,4-beta-D-xylan xylohydrolase) activities of Aspergillus and Trichoderma enzymes. The nephelometric method is reliable in evaluating xylanase hydrolysis of insoluble xylan.

Glucanase gene diversity in prokaryotic and eukaryotic organisms.

Abstract: A number of bacteria and eukaryotes produce extracellular enzymes that degrade various types of polysaccharides including the glucans starch, cellulose and hemicellulose (xylan). The similarities in the modes of expression and specificity of enzyme classes, such as amylase, cellulose and xylanase, suggest common genetic origins for particular activities. Our determination of the extent of similarity between these glucanases suggests that such data may be of very limited use in describing the early evolution of these proteins. The great diversity of these proteins does allow identification of their most highly conserved (and presumably functionally important) regions.

Survey and electrophoretical separation of the glycosidases of Rhagium inquisitor (Coleoptera: Cerambycidae) larvae

Abstract: 1. 1. A Rhagium inquisitor larval preparation contains glycosidases attacking diverse oligosaccharides, heterosides and polysaccharides. There are α- and β-d-glucosidase, α- and β-d-galactosidase, α- and β-d-mannosidase, β-d-xylosidase, α-l-fucosidase, but neither α-d-xylosidase nor β-l-fucosidase. There are maltase, trehalase, cellobiase, lactase and sucrase, amylase, cellulase, laminarinase, lichenase, xylanase, mannanase and polygalacturonase. 2. 2. The diversified glycosidase equipment of the R. inquisitor larva makes it thus capable of best utilization of the carbohydrates reserve of the poured phloem where it lives and on which it feeds. 3. 3. Utilizing continuous gradient polyacrylamide gel electrophoresis, several glycosidase species were shown up in the larval preparation: a maltase, an amylase, a sucrase, two trehalases, a β-d-glucosidase, an α- and two β-d-galactosidases, an α- and a β-d-mannosidase, a β-d-xylosidase, an α-l-fucosidase, two laminarinases, two lichenases, a xylanase and a mannanase. 4. 4. Maltase and sucrase are not the same enzyme. Trehalase 1 hydrolyses neither maltose nor sucrose. β-d-Glucosidase and β-d-galactosidase 2 are not the same enzyme. The β-d-glucosidase is active on p-nitrophenyl-β-d-glucopyranoside, salicin and cellobiose; it is a cellobiase of wide specificity. β-d-Galactosidase 2 is a lactase, while the former appears as not attacking lactose. The amylase is an endoamylase. Laminarinase 1, xylanase and mannanase are random-cleaving enzymes. 5. 5. Molecular weight estimations are: below 35,000 for amylase, β-d-galactosidase 2, lichenase 2, xylanase and mannanase, 35,000 for sucrase, β-d-glucosidase and β-d-xylosidase, 45,000 for α-d-galactosidase and laminarinase 2, 70,000 for β-d-galactosidse 1, 85,000 for maltase, 120,000 for laminarinase 1, 130,000 for β-d-mannosidase, 140,000 for lichenase 1, 200,000 for α-d-mannosidase and α-l-fucosidase, 210,000 for trehalase 1.

Molecular Cloning and Expression of a Xylanase Gene of Alkalophilic Aeromonas sp. no. 212 in Escherichia coli

Abstract: Summary: A gene coding for a xylanase activity of alkalophilic Aeromonas sp. no. 212 (ATCC 31085) was cloned in Escherichia coli HB101 with pBR322. Plasmid pAX1 was isolated from transformants producing xylanase, and the xylanase gene was located in a 6.0 kb Hind III fragment. The pAX1-encoded xylanase activity in E. coli HB101 was about 80 times higher than that of xylanase L in alkalophilic Aeromonas sp. no. 212. About 40% of the enzyme activity was observed in the periplasmic space of E. coli HB101. The pAX1-encoded xylanase had the same enzymic properties as those of xylanase L produced by alkalophilic Aeromonas sp. no. 212, but its molecular weight was lower (135000 vs 145000, as estimated by SDS polyacrylamide gel electrophoresis).

An Inducible Xylanase of the Mushroom Termitomyces clypeatus Differing from the Xylanase/Amylase Produced in Dextrin Medium

Abstract: Summary: The mushroom Termitomyces clypeatus produces a single endoxylanase (1,4-β-D-xylan xylano-hydrolase, EC 3.2.1.8) in the presence of either dextrin or xylan as sole source of carbon. The enzymes produced in the two conditions are different. The enzyme induced by xylan has been purified 67-fold from the culture filtrate of T. clypeatus. The enzyme preparation gave a single protein band on SDS-PAGE, corresponding to a molecular weight of about 24000. The enzyme has an isoelectric point at pH 4·0 and acts on arabinoxylan and arabinogalactan, but not amylopectin or galactomannan. It shows maximum activity on xylan (1,4-β-linked D-xylopyranose units) at pH 3·5 and 55°C and is fairly stable up to 60°C. The K m for xylan is 4 mg ml-1. Hg2+, Fe2+ and Ag+ are the most potent inhibitors of the enzyme. The pH optimum and molecular weight of this inducible xylanase differ from those of the enzyme produced by the same organism grown in dextrin medium.

Purification and Partial Characterization of Alkaline Xylanase from Escherichia coli Carrying pCX311

Abstract: Xylanase produced by E. coli HB101 carrying plasmid pCX311, which contains the xylanase A gene of alkalophilic Bacillus sp. strain C-125, was purified by ammonium sulfate precipitation, DEAE-cellulose column chromatography and Sephadex G-75 gel filtration. The purified enzyme had a molecular weight of 43, 000. The pH and temperature optima for its activity were 6-10 and 70°C, respectively. The enzyme retained full activity after incubation at 50°C for 10min. These enzymatic properties of the xylanase were almost the same as those of xylanase A. But this enzyme was less stable than xylanase A at low pHs. Furthermore, we could purify a larger amount of alkaline xylanase from E. coli than from alkalophilic Bacillus sp. strain C-125.

The localization and activity of Cellulomonas xylanase on sugarcane bagasse pith

Abstract: RODR~GUEZ, H., A. ENRIQUEZ, and 0. VOLFOVA. 1985. The localization and activity of Cellulomonas xylanase on sugarcane bagasse pith. Can. J. Microbiol. 31: 754-756. Xylanase activity was found both free and bound to the cells and (or) the substrate during the first growth phase of Cellulornonas on bagasse pith. At the same time, rapid degradation of the bagasse hemicellulose was observed. The enzymes became bound mainly during the second growth phase, during which hemicellulose degradation was still detected, although at a lower rate. Intracellular xylanase activity was about one-tenth of the activity detected outside the cell during this phase. At the end of growth, 89% of the bagasse hemicellulose had been consumed. RODRIGUEZ, H., A. ENR~QUEZ et 0. VOLFOVA. 1985. The localization and activity of Cellulornonas xylanase on sugarcane

A simple method for xylanase preparation used for the hydrolysis and fermentation of hemicellulose to butanediol

Abstract: When culture filtrates ofTrichodermaharzianum E58 were concentrated by passage through an ultrafiltration membrane with a molecular weight exclusion limit of 10,000 Daltons, 80% of the original xylanase activity was recovered in the ultrafiltrates. Culture filtrates and ultrafiltrates which were concentrated by rotary evaporation contained inhibitors which restricted the fermentation of the hemicellulose-derived sugars to 2,3-butanediol. A simple solvent-exchange treatment of the ultrafiltrates could effectively concentrate the xylanases as well as remove the fermentation inhibitors.

Selective excretion of alkaline xylanase by escherichia coli carrying PCX311

Abstract: Plasmid pCX311, which we constructed, has two HindlU DNA fragments (2.6 kbp and 2.0 kbp) of alkalophilic Bacillus sp. strain C-125 in the HindlU site of pBR322.These two fragments were essential not only for the xylanase production but also for the excretion of periplasmic proteins. The cloned 4.6 kbp fragment encodes some components that made the outer membrane of E. coli permeable. Some proteins such as xylanase and ²-lactamase were excreted, but alkaline phosphatase was not excreted into the culture broth.