Production, characterization and properties of polysaccharide depolymerizing enzymes from a strain ofCurvularia inaequalis

Xylanase, β-glucosidase, β-xylosidase, endoglucanase and polygalacturonase production fromCurvularia inaequalis was carried out by means of solid-state and submerged fermentation using different carbon sources. β-Glucosidase. β-xylosidase, polygalacturonase and xylanase produced by the microorganisms were characterized. β-Glucosidase presented optimum activity at pH 5.5 whereas xylanase, poly-galacturonase and β-xylosidase activities were optimal at pH 5.0. Maximal activity of β-glucosidase was determined at 60°C, β-xylosidase at 70°C, and polygalacturonase and xylanase at 55°C. These enzymes were stable at acidic to neutral pH and at 40–45 °C. The crude enzyme solution was studied for the hydrolysis of agricultural residues.     

β-Xylosidase and xylanase characterization and production by Streptomyces sp. CH-M-1035

Extracellular xylanase activity and cell-bound β-xylosidase production by a selected strain of Streptomyces sp. CH-M-1035 was characterized during growth on three xylans, sugar cane bagasse pith and lemon peel as sole carbon source. The cell-bound β-xylosidase and extracellular endoxylanase had pH optima of 6·0 and 5·0, and temperature optima of 50°C and 60°C, respectively. The highest level of β-xylosidase activity was obtained when Streptomyces sp. CH-M-1035 was grown on larchwood xylan, whereas the maximal endoxylanase production was found on lemon peel. Reducing sugars accumulated in the culture media when Streptomyces sp. CH-M-1035 was grown on xylans, but not on agroindustrial residues.     

Effects of additives on the activity of Bacillus sp. β-xylanase

Xylan hydrolysis activity of β-xylanase from Bacillus sp. increased up to 65% in the presence of sugars and up to 44% in the presence of surfactants. Metal ions and EDTA inhibited the activity while the addition of cell extract, containing β-xylosidase, increased xylan hydrolysis by 38%.     

Xylanases of anaerobic fungus <Emphasis Type="Italic">Anaeromyces mucronatus</Emphasis>

The anaerobic fungus Anaeromyces mucronatus KF8 grown in batch culture on M10 medium with rumen fluid and microcrystalline cellulose as carbon source produced a broad range of enzymes requisite for degradation of plant structural and storage saccharides including cellulase, endoglucanase, xylanase, α-xylosidase, β-xylosidase, α-glucosidase, β-glucosidase, β-galactosidase, mannosidase, cellobiohydrolase, amylase, laminarinase, pectinase and pectate lyase. These enzymes were detected in both the intra- and extracellular fractions, but production into the medium was prevalent with the exception of intracellular β-xylosidase, chitinases, N-acetylglucosaminidase, and lipase. Xylanase activity was predominant among the polysaccharide hydrolases. Extracellular production of xylanase was stimulated by the presence of cellobiose and oat spelt xylan. Zymogram of xylanases of strain KF8 grown on different carbon sources revealed several isoforms of xylanases with approximate molar masses ranging from 26 to 130 kDa.     

Identification of thermostable β-xylosidase activities produced by <Emphasis Type="Italic">Aspergillus brasiliensis</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Twenty Aspergillus strains were evaluated for production of extracellular cellulolytic and xylanolytic activities. Aspergillus brasiliensis, A. niger and A. japonicus produced the highest xylanase activities with the A. brasiliensis and A. niger strains producing thermostable β-xylosidases. The β-xylosidase activities of the A. brasiliensis and A. niger strains had similar temperature and pH optima at 75°C and pH 5 and retained 62% and 99%, respectively, of these activities over 1 h at 60°C. At 75°C, these values were 38 and 44%, respectively. Whereas A. niger is a well known enzyme producer, this is the first report of xylanase and thermostable β-xylosidase production from the newly identified, non-ochratoxin-producing species A. brasiliensis.     

Induction of xylanase and β-xylosidase in Cellulomonas flavigena growing on different carbon sources

When Cellulomonas flavigena CDBB-531 was grown on glucose, xylose, glycerol, solka floc, sugarcane bagasse or xylan, xylanase activity was found only in the fermentation broth, while -xylosidase activity was always associated with the cells. Both enzymes were inducible, sugar-cane bagasse was the best inducer, solka floc and avicel were moderately good, while xylan was poor. A synergistic effect on xylanase and -xylosidase synthesis was observed when cellulose and hemicellulose were used together as carbon sources. When this strain was grown on glucose, cellobiose, arabinose or xylose, only low levels of both enzymes were detected. These results indicate that xylanase and -xylosidase were carbon-source-repressed by readily metabolizable substrates. The effect of glycerol on enzymes that were already induced was studied. The addition of glycerol caused a significant decrease in the levels of xylanases, while -xylosidase activity remained unchanged.     

Characterization of a bifunctional xylanase/endoglucanase from yak rumen microorganisms

A new gene, RuCelA, encoding a bifunctional xylanase/endoglucanase, was cloned from a metagenomic library of yak rumen microorganisms. RuCelA showed activity against xylan and carboxymethylcellulose (CMC), suggesting bifunctional xylanase/endoglucanase activity. The optimal conditions for xylanase and endoglucanase activities were 65°C, pH 7.0 and 50°C, pH 5.0, respectively. In addition, the presence of Co⁺ and Co²⁺ can greatly improve RuCelA's endoglucanase activity, while inhibits its xylanase activity. Further examination of substrate preference showed a higher activity against barley glucan and lichenin than against xylan and CMC. Using xylan and barley glucan as substrates, RuCelA displayed obvious synergistic effects with β-1,4-xylosidase and β-1,4-glucosidase. Generation of soluble oligosaccharides from lignocellulose is the key step in bioethanol production, and it is greatly notable that RuCelA can produce xylo-oligosaccharides and cello-oligosaccharides in the continuous saccharification of pretreated rice straw, which can be further degraded into fermentable sugars. Therefore, the bifunctional RuCelA distinguishes itself as an ideal candidate for industrial applications.     

The regulation of xylanolytic enzyme formation by Butyrivibrio fibrisolvens NCFB 2249

Butyrivibrio fibrisolvens NCFB 2249 formed xylan-degrading enzymes on a wide range of carbohydrate growth substrates. The specific activities of α-L-arabinofuranosidase and β-D-xylosidase were increased (up 20-fold) after growth on xylan or xylose-containing saccharides. Xylose was not an effective substrate for xylanase production although its formation was induced on xylobiose and higher DP xylose-containing saccharides. Acetyl esterase activity was also highest after growth on xylan. The synthesis of xylanase and β-xylosidase was repressed by glucose and hemicellulosic pentoses and although α-L-arabinofuranosidase formation was also subject to catabolite regulation, xylose did not repress its synthesis.     

Purification and biochemical characterization of β-xylosidase from Humicola grisea var. thermoidea

Abstract β-d-Xylosidase production was maximal for Humicola grisea var. thermoidea grown on xylan as the sole carbon source. The main β-d-xylosidase activity was localised in the periplasm. β-Xylosidase was purified from crude extracts by heat treatment, ammonium sulfate precipitation and chromatography on DEAE-cellulose and Sephadex G-100. The purified enzyme was a monomer of molecular mass estimated to be 43 kDa by SDS-PAGE and gel filtration. Optima of pH and temperature were 6.0 and 50 °C, respectively. The enzyme activity was stimulated by Ca²⁺, Fe²⁺, and Mg²⁺. The purified β-xylosidase did not exhibit xylanase, carboxymethylcelullase, galactosidase, glucosidase, fucosidase or arabinosidase activities. The purified β-xylosidase hydrolysed xylobiose and xylo-oligosaccharides of up to five monosaccharide units. The enzyme had a K _m of 0.49 mM for p -nitrophenyl- β -d-xylopyranoside and was not inhibited by its product, xylose.     

Molecular cloning of the genes for xylan degradation of Bacillus pumilus and their expression in Escherichia coli

Summary The 7.7 Mdal PstI fragment of Bacillus pumilus IPO containing genes for xylan degradation, xylanase, and -xylosidase was inserted at the PstI site of pBR322 and cloned in E. coli C600. The hybrid plasmid thus formed was named pOXN29. The amount of xylanase and -xylosidase expressed in E. coli harboring pOXN29 was about 6% and 20% of the activity produced by the donor, B. pumilus. The reverse orientation of the inserted fragment resulted respectively in 5 times and 50 times increases in xylanase and -xylosidase productivities. Both enzymes expressed in E. coli transformants were shown to be indistinguishable from those of B. pumilus by immunological and chemical criteria. Digestion of pOXN29 with BglII produced two fragments; one was 6.7 Mdal in size and contained the whole pBR322 and the -xylosidase gene, and the other was 3.7 Mdal and coded for xylanase. Analysis of enzymes expressed in the transformant cells indicated that neither enzyme was secreted into the culture medium, periplasm nor membrane bound, although xylanase but not -xylosidase, was secreted into the medium in a B. pumilus culture.     

Xylan-hydrolysing enzymes of the yeast Pichia stipitis

Summary Two xylanolytic enzymes, xylanase and -xylosidase from the yeast Pichia stipitis were purified to homogeneity and characterized. Both enzymes are secreted into the culture medium upon growth on xylan. The xylanase is a glycoprotein with an approximate molecular mass of 43 kDa. The N-linked carbohydrate content was estimated to be 26% by endoglycosidase H digestion. The -xylosidase protein has a molecular mass of 37 kDa as determined by sodium dodecyl sulphate gel electrophoresis. Synthesis of xylanase was found to be inducible by xylan and repressible by xylose and glucose. By contrast, -xylosidase is synthesized constitutively to a considerable degree. The purified -xylosidase is able to hydrolyse aryl--D-glucosides with an even higher rate than -xylosides. Thus, this enzyme may not be a specific component of the xylan-degrading system of P. stipitis. Offprint requests to: M. Ciriary     

High activity xylanase from Aspergillus sydowii MG49 during growth on jute stalk lignocellulose

Aspergillus sydowil MG49 produced 33.0 U mg^-1 of extracellular xylanase activity when grown in liquid state fermeniation (LSF) with 1% ground jute stalk as the sole carbon source compared to 56.0 U mg^-1 when pure xylan was used. Optimum time-course and pH for maximum enzyme production were 144 h and 4.0 respectively. The culture filtrate was devoid of any cellulase and β-xylosidase activity. The xylanase exhibited optimum activity at 60°C and pH 5.5. Partially-fermented jute stalk could be recycled at least twice for xylanase production, exhibiting 25.8 and 17.4 U mg^-1 activity in two later consecutive cycles respectively.     

Production of xylanolytic enzymes during growth on pulverized grass by Aspergillus ochraceus-42

Xylanase and β-xylosidase with activity of 6.46 U mg^-1 and 0.500 U mg^-1, respectively, were produced extracellularly by Aspergillus ochraceus during growth on pulverized grass in liquid state fermentation, compared to 9.3 U mg^-1 and 0.74 U mg^-1 when pure xylan was used. The culture filtrate was devoid of any cellulase activity. Xylanolytic enzymes were produced optimally in 144 h of incubation on 1% pulverized grass, pH 6.5. About 8.43% (w/w) sugars were liberated from alkali-treated grass in 6 h by the synergistic effect of xylanolytic enzymes. The half-lives for xylanase and β-xylosidase at 50°C were 210 min and 300 min, respectively, and half-life increased with the increase in protein concentration. Both mono- and divalent cations, especially K⁺ and Zn²⁺, exhibited a profound effect on the rate of enzyme saccharification.     

Regulation of β-xylosidase formation by xylose in Trichoderma reesei

The soft-rot fungus Trichoderma reesei forms -xylosidase (EC 3.2.1.37) activity during cultivation on xylan and xylose, but not on glucose. When mycelia precultivated on glycerol were washed and transferred to fresh medium without a carbon and nitrogen source, -xylosidase formation was induced by xylan, xylobiose and xylose. A supply of 4 mm xylose and a pH of 2.5 provided optimal conditions for induction. -Xylosidase accounted for the major portion of total extracellular protein under these conditions, and could be purified to physical homogeneity by a single anion exchange chromatography step. A recombinant strain of T. reesei that carries multiple copies of the homologous xylanase II-encoding gene has a six-fold increased xylanase activity, but forms comparable -xylosidase activities. This shows that the rate of xylan hydrolysis has no effect on the induction of -xylosidase. Methyl--d-xyloside inhibited -xylosidase competitively and was a weak -xylosidase inducer. The induction by xylobiose and xylan was strongly enhanced by the simultaneous addition of methyl--d-xylosidese and xylan or xylobiose. The results suggest that a slow supply of xylose is a trigger for -xylosidase induction.     

Production and characterization of cellulolytic and xylanolytic enzymes from the ligninolytic white-rot fungus Phanerochaete chrysosporium grown on sugarcane bagasse

The ligninolytic white-rot fungus Phanerochaete chrysosporium BKM-F-1767 produced extracellular cellulolytic enzymes (carboxymethylcellulase, CMCase and -glucosidase) and xylanolytic enzymes (xylanase and -xylosidase) in liquid medium containing 1.0% sugarcane bagasse with or without 1.0% glucose. The changes in pH and soluble protein content were monitored in the culture filtrates. The results obtained showed that the pH decreased after 3 days and then increased. The soluble protein content increased and reached the maximum value after 12 days. The results showed that the activities of enzymes were higher in the case of sugarcane bagasse without glucose. The characterization study indicated that the optimum pH values were 4.6, 4.2, 5.0 and 5.0 for CMCase, -glucosidase, xylanase and -xylosidase, respectively and the optimum temperatures were 60, 70, 65 and 60 °C for the investigated enzymes, respectively. The results showed also that after prolonged heating (5 h) at 60 °C, CMCase, -glucosidase, xylanase and -xylosidase retained 81.2, 86.8, 51.5 and 27.4% activity, respectively.     

Induction of xylanase by β-methylxyloside in Cryptococcus albidus

Abstract The compound β-methylxyloside (β-MX) was found to induce the production of extracellular xylanase (EC 3.2.1.8) by the yeast Cryptococcus albidus . The induction of xylanase by β-MX requires de novo protein synthesis and proceeds via de novo accumulation of translatable mRNA coding for xylanase as demonstrated by the addition of cycloheximide. In vitro translation of cellular messenger RNA followed by immunoprecipitation with xylanase-specific antibodies reveals that the stimulation leads to the appearance of xylanase-coding mRNA. In vivo labeling experiments show that the β-MX induces specifically the 48 kDa xylanase, and that the addition of xylose in the culture medium reverses the β-MX action by suppressing completely the production of xylanase by the cells.     

Extracellular xylanolytic enzymes ofPaecilomyces varioti

Summary Paecilomyces varioti produced an extracellular xylanase and B-xylosidase when cultured in a medium containing xylan and corn steep liquor. Xylose (2%, w/v) totally inhibited production of both enzymes. The enzymes were purified and both had a pH optimum of 4.0. The xylanase had a molecular weight of 20,000, an isoelectric point of 5.2 and was inactive on all substrates tested except xylan. The -xylosidase, a glycoprotein, had a molecular weight of 67,000, an isoelectric point of 4.0 and had highest activity on p-nitrophenyl--D-xyloside. The xylanase had a Km of 49.5 mg/ml for xylan and the -xylosidase had a Km of 5.4 mM for p-nitrophenyl--D-xyloside.     

Thermolabile xylanase of the Antarctic yeast Cryptococcus adeliae: production and properties

Xylanase production by the Antarctic psychrophilic yeast Cryptococcus adeliae was increased 4.3 fold by optimizing the culture medium composition using statistical designs. The optimized medium containing 24.2 g l⁻¹ xylan and 10.2 g l⁻¹ yeast extract and having an initial pH of 7.5 yielded xylanase activity at 400 nkat (nanokatal) ml⁻¹ after 168-h shake culture at 4°C. In addition, very little endoglucanase, β-mannanase, β-xylosidase, β-glucosidase, α-l-arabinofuranosidase, and no filter paper cellulase activities were detected. Among 12 carbon sources tested, maximum xylanase activity was induced by xylan, followed by lignocelluloses such as steamed wheat straw and alkali-treated bagasse. The level of enzyme activity produced on other carbon sources appeared to be constitutive. Among the complex organic nitrogen sources tested, the xylanase activity was most enhanced by yeast extract, followed by soymeal, Pharmamedia (cotton seed protein), and Alburex (potato protein). A batch culture at 10°C in a 5-l fermenter (3.5-1 working volume) using the optimized medium gave 385 nkat at 111 h of cultivation. The crude xylanase showed optimal activity at pH 5.0–5.5 and good stability at pH 4–9 (21 h at 4°C). Although the enzyme was maximally active at 45°–50°C, it appeared very thermolabile, showing a half-life of 78 min at 35°C. At 40°–50°C, it lost 71%–95% activity within 5 min. This is the first report on the production as well as on the properties of thermolabile xylanase produced by an Antarctic yeast. Received: December 10, 1999 / Accepted: March 23, 2000     

Detection and assay of xylanolytic enzymes in a Cellulomonas isolate

Summary The accuracy of xylanase assay was tested and improved. The assay was used to monitor xylanase production by a Cellulomonas isolate and to demonstrate that this activity is distinct from the organism's -xylosidase activity.     

Influence of vanillin on the production of cellulolytic and xylanolytic enzymes from a wood-rotting fungus,Coriolus versicolor

To examine the influence of a phenolic compound on the production of cellulolytic and xylanolytic enzymes of a woodrotting fungusCoriolus versicolor, a two-dimensional map of enzyme activity was constructed with various concentrations of cellobiose and vanillin. The productions of CMCase, xylanase, β-glucosidase, and β-xylosidase increased with higher cellobiose concentration and were markedly enhanced by addition of vanillin. Higher ratio of vanillin/cellobiose activated the production of these enzymes. Only acetyl esterase, which is not actively produced at the ligninolytic stage ofC. versicolor, was inhibited by the monolignol vanillin. As the presence of vanillin is considered to approximate conditions of wood decay more closely than its absence, the present result demonstrates that addition of vanillin, a phenolic compound, enhanced the production of cellulolytic and xylanolytic enzymes for wood cell wall degradation.