Extracellular Xylanase Activity Research Articles

Effect of cultivation pH and agitation rate on growth and xylanase production by Aspergillus oryzae in spent sulphite liquor

The effects of cultivation pH and agitation rate on growth and extracellular xylanase production by Aspergillus oryzae NRRL 3485 were investigated in bioreactor cultures using spent sulphite liquor (SSL) and oats spelts xylan as respective carbon substrates. Xylanase production by this fungus was greatly affected by the culture pH, with pH 7.5 resulting in a high extracellular xylanase activity in the SSL-based medium as well as in a complex medium with xylan as carbon substrate. This effect, therefore, was not solely due to growth inhibition at the lower pH values by the acetic acid in the SSL. The xylanase activity in the SSL medium peaked at 199 U ml(-1) at pH 7.5 with a corresponding maximum specific growth rate of 0.39 h(-1). By contrast, the maximum extracellular beta-xylosidase activity pf 0.36 U ml(-1) was recorded at pH 4.0. Three low molecular weight xylanase isozymes were secreted at all pH values within the range of pH 4-8, whereas cellulase activity on both carbon substrates was negligible. Impeller tip velocities within the range of 1.56-3.12 m s(-1) had no marked effect, either on the xylanase activity, or on the maximum volumetric rate of xylanase production. These results also demonstrated that SSL constituted a suitable carbon feedstock as well as inducer for xylanase production in aerobic submerged culture by this strain of A. oryzae.

Production, optimization growth conditions and properties of the xylanase from Aspergillus carneus M34

Growth conditions, including incubation times, temperature, agitation rate and initial pH of medium, that affect xylanase production by Aspergillus carneus M34 were studied sequentially use the classical “change-one-factor-at-a-time” method. Our results showed that there was a similar trend between cellular xylanase activity and extracellular xylanase activity. The optimal conditions for xylanase production, different from their cell growth, were on the third day, 30 °C, 100 rpm and pH 4, respectively, in this test. Response surface methodology (RSM) was further introduced to optimize the cultivation conditions and to evaluate the significance of these factors. The optimal cultivation conditions predicted from canonical analysis of this model were achieved by incubation at 35.08 °C with an agitation rate of 111.9 rpm and an initial pH of 5.16. In addition, temperature was the most critical factor for xylanase production by A. carneus M34. Xylanase activity of 22.2 U/mL was verified using the predicted optimal conditions and confirmed the fitness and applicability of the model. The optimal temperature and pH of the crude xylanase activity was observed at 60 °C and acidic pH, respectively. Sustained xylanase activity in the crude extract was also detected over a broad range of pH from 3 to 10. Considering its higher specificity toward agricultural wastes, especially corn cob and coba husk, this strain can be used to develop low-cost media for the mass-production of xylanase.

Role of the Transcriptional Activator XlnR of Fusarium oxysporum in Regulation of Xylanase Genes and Virulence

Fungal infection of plants involves degradation of the host cell wall through the action of lytic enzymes secreted by the pathogen. The role of these enzymes in virulence is difficult to determine due to their functional redundancy and, therefore, remains controversial. Here, we have studied XlnR, a zinc-finger transcription factor from the vascular wilt pathogen Fusarium oxysporum that is orthologous to the major transcriptional activator of xylanase genes in Aspergillus spp. Transcription of the xlnR gene was activated by inducing carbon sources such as oat spelt xylan (OSX) and repressed by glucose. Targeted knockout of xlnR in F. oxysporum resulted in lack of transcriptional activation of structural xylanase genes, both in culture and during infection of tomato plants, as well as in dramatically reduced extracellular xylanase activity. By contrast, overexpression of xlnR under the control of the Aspergillus nidulans gpdA promoter did not significantly increase xylanase activity, suggesting that XlnR is regulated not only at the transcriptional but also at the post-translational level. The deltaxlnR mutants were still fully virulent on tomato plants. Thus, XlnR, the major transcriptional activator of xylanase genes, is not an essential virulence determinant in F. oxysporum.

Production and characterization of an enzyme complex from a new strain of Clostridium thermocellum with emphasis on its xylanase activity

A new bacterial strain (ISO II) was isolated from manure cow and identified as phylogenetically close to the thermophilic cellulolytic bacterium Clostridium thermocellum. The new strain produced extracellular xylanase, pectinase, mannanase and cellulase activities when grown in liquid culture medium containing banana stem as carbon source. The enzyme production profile after growth on banana stem showed that xylanase and cellulase activities were detected in different incubation periods. An enzyme complex containing xylanase, cellulase and mannanase activities was isolated from culture supernatant samples of strainISO II. The complex was partially purified by ultrafiltration and gel filtration chromatography on Sephacryl S-300. Zymogram analysis after SDS-PAGE presented at least 05 subunits with xylanase activity. The enzyme showed single protein and xylanase activity bands after electrophoresis under non-denaturing conditions. The hydrolysis of xylan was optimal at temperature range of 55-75oC and pH 6.0. Xylanase activity was quite stable at 65oC, retaining 80% of its original activity after 12 h incubation. The apparent Km values, using insoluble and soluble arabinoxylans as substrates, were 1.54 and 11.53 mg/mL, respectively. Xylanase was activated by dithiothreitol, L-tryptophan and L-cysteine and strongly inhibited by N-bromosuccinimide and CoCl2. The characterization of mannanase showed Km and temperature optimum of 0.846 mg/mL and 65oC, respectively and pH 8.0. By contrast to xylanase, it was less stable at 65oC with half-life of 2.5 h and inhibited by dithiothreitol and Ca2+.

INDUCTION, PROPERTIES AND APPLICATION OF XYLANASE ACTIVITY FROM SCLEROTINIA SCLEROTIORUM S2 FUNGUS

Extracellular xylanase activity was produced in a submerged culture by Sclerotinia sclerotiorum S2 fungus, on wheat bran as an inducing substrate. The enzyme was partially purified and biochemically characterized. The optimum pH and temperature for the activity were 5.5 and 65C, respectively. The xylanase was stable over a pH range of 4.0–9.0 and retained more than 75% of its activity after incubation for 24 h without substrate. The enzyme was stable at 50C and 60C for 80 min of incubation at pH 5.0, while the half-life was 20 min at 70C. The novel xylanase activity was useful as an aid in orange juice clarification. The clarification was observed with concomitant production of reducing sugars (0.350 mg/mL from juice) after 24 h of incubation of juice with the xylanase. Xylanase aided juice clarification, which resulted in a 27% decrease in insoluble materials.

Purification and characterization of two sugarcane bagasse-absorbable thermophilic xylanases from the mesophilic Cellulomonas flavigena

We report the purification and characterization of two thermophilic xylanases from the mesophilic bacteria Cellulomonas flavigena grown on sugarcane bagasse (SCB) as the only carbon source. Extracellular xylanase activity produced by C. flavigena was found both free in the culture supernatant and associated with residual SCB. To identify some of the molecules responsible for the xylanase activity in the substrate-bound fraction, residual SCB was treated with 3 M guanidine hydrochloride and then with 6 M urea. Further analysis of the eluted material led to the identification of two xylanases Xyl36 (36 kDa) and Xyl53 (53 kDa). The pI for Xyl36 was 5.0, while the pI for Xyl53 was 4.5. Xyl36 had a Km value of 1.95 mg/ml, while Xyl53 had a Km value of 0.78 mg/ml. In addition to SCB, Xyl36 and Xyl53 were also able to bind to insoluble oat spelt xylan and Avicel, as shown by substrate-binding assays. Xyl36 and Xyl53 showed optimal activity at pH 6.5, and at optimal temperature 65 and 55 degrees C, respectively. Xyl36 and Xyl53 retained 24 and 35%, respectively, of their original activity after 8 h of incubation at their optimal temperature. As far as we know, this is the first study on the thermostability properties of purified xylanases from microorganisms belonging to the genus Cellulomonas.

Xylanase production by a novel halophilic bacterium increased 20-fold by response surface methodology

Medium optimisation for a novel halophilic eubacterium, strain SX15, resulted in a 20-fold increase of extracellular xylanase activity. This facilitates the purification of xylanase produced by this strain. Prior experiments revealed that xylan concentration, source and concentration of nitrogen and salinity were important variables for xylanase production. Based on this, we adopted a fractional factorial design to determine the best combinations and approximate levels of these values. Subsequently, response surface (RS) methodology was applied to locate the optimal levels of xylan, NH 4Cl and salinity.

Enzymatic properties of a neutral endo-1,3(4)-β-xylanase Xyl II from Bacillus subtilis

A Bacillus sp. CCMI 966, characterised as Bacillus subtilis, has a duplication time of about 24 min. It produces at least two extracellular xylanases, Xyl I and Xyl II. The extracellular xylanase activity seems to be strongly correlated with the biomass growth profile. The Xyl II isoenzyme was purified by ammonium sulphate precipitation and anionic exchange chromatography, with a purification factor of 8.3. The molecular weight of the isoenzyme was estimated by SDS-PAGE revealing that Xyl II is a multimeric enzyme with a catalytic subunit of about 20 kDa. Under non-denaturing conditions, a molecular weight of about 340 kDa was obtained by native PAGE gel and of 20 kDa by gel filtration chromatography. The enzyme showed an optimum pH and temperature of 6.0 at 60 °C. Xyl II was stable at 40 °C for 180 min at pH 6.0. The specificity of Xyl II for different substrates was evaluated. Xyl II presents a higher affinity towards OSX, with a K m of 1.56 g l −1 and showed the ability to hydrolyse laminarin, with a K m of 1.02 g l −1. Xylotetraose is the main product of xylan degradation. The Xyl II ability for binding to cellulose and/or xylan was also studied.

Xylanase activity and thermostratification during the thermogenic phase of industrial composting in aerated trenches

Extracellular xylanase activity and thermostratification were monitored during the thermogenic phase of industrial composting (mainly garden waste) in aerated trenches. Xylanase activity was assayed at different temperatures and pH by incubation of clarified compost extracts with xylan. The highest xylanase activity and temperatures were measured during the summer (when waste was rich in freshly cut grass). Xylanase activity was higher in the peripheral layers of the trenches, at moderately high temperatures (48 to 68°C), than in the hottest central layers (70 to 78°C). Optimal temperature for the xylanases was between 60 and 80°C at pH 6, depending on the temperature of the sampling site. The thermostability of the xylanases from surface samples was high until 60°C, moderate at 70°C and weak at 90°C. Our results show a broad thermostratification of the compost mass. Frequent turnings of the compost stimulate xylan degradation by redistributing the substrates, the free enzymes and the microorganisms.

Xylanase activity and thermostratification during the thermogenic phase of industrial composting in aerated trenches

Extracellular xylanase activity and thermostratification were monitored during the thermogenic phase of industrial composting (mainly garden waste) in aerated trenches. Xylanase activity was assayed at different temperatures and pH by incubation of clarified compost extracts with xylan. The highest xylanase activity and temperatures were measured during the summer (when waste was rich in freshly cut grass). Xylanase activity was higher in the peripheral layers of the trenches, at moderately high temperatures (48 to 68°C), than in the hottest central layers (70 to 78°C). Optimal temperature for the xylanases was between 60 and 80°C at pH 6, depending on the temperature of the sampling site. The thermostability of the xylanases from surface samples was high until 60°C, moderate at 70°C and weak at 90°C. Our results show a broad thermostratification of the compost mass. Frequent turnings of the compost stimulate xylan degradation by redistributing the substrates, the free enzymes and the microorganisms.

Constitutive expression of a heterologous Eubacterium ruminantium xylanase gene (xynA) in Butyrivibrio fibrisolvens.

An Eubacterium ruminantium xylanase gene (xynA) was inserted into pYK4, a shuttle vector replicable in both Escherichia coli and Butyrivibrio fibrisolvens, and the resultant chimeric plasmid (pYK4XT) was electroporated into B. fibrisolvens OB156C in an attempt to obtain a more xylanolytic B. fibrisolvens. Electrotransformants were screened by the development of erythromycin resistance, followed by an activity staining and Southern hybridization. The presence of mRNA from xynA in the transformant, B. fibrisolvens NO4, was confirmed by Northern hybridization. Xylanase activity of the transformant NO4 was apparently enhanced regardless of carbon sources in the medium. When grown on glucose or cellobiose. NO4 had approximately 5-6 times higher intracellular activity than the parent OB156C on a culture volume basis as well as protein basis. The transformant showed extracellular xylanase activity much higher (between 7- and 10(4)-fold) than the parent. Transformant NO4 recorded the highest activity when grown on xylan. Most (> 90%) of the activity was extracellular. The extracellular activity was 2-fold greater in NO4. These findings indicate that the introduced xynA was expressed constitutively and the xylanase protein was exported into the culture supernatant. Growth of NO4 on glucose was similar to that of OB156C, which suggests little extra load for plasmid maintenance and foreign xylanase production in the transformant. The plasmid pYK4XT was maintained stably in the transformant for more than 100 generations.

Constitutive expression of a heterologous Eubacterium ruminantium xylanase gene ( xynA) in Butyrivibrio fibrisolvens

An Eubacterium ruminantium xylanase gene ( xynA) was inserted into pYK4, a shuttle vector replicable in both Escherichia coli and Butyrivibrio fibrisolvens, and the resultant chimeric plasmid (pYK4XT) was electroporated into B. fibrisolvens OB156C in an attempt to obtain a more xylanolytic B. fibrisolvens. Electrotransformants were screened by the development of erythromycin resistance, followed by an activity staining and Southern hybridization. The presence of mRNA from xynA in the transformant, B. fibrisolvens NO4, was confirmed by Northern hybridization. Xylanase activity of the transformant NO4 was apparently enhanced regardless of carbon sources in the medium. When grown on glucose or cellobiose, NO4 had approximately 5–6 times higher intracellular activity than the parent OB156C on a culture volume basis as well as protein basis. The transformant showed extracellular xylanase activity much higher (between 7- and 10 4-fold) than the parent. Transformant NO4 recorded the highest activity when grown on xylan. Most (>90%) of the activity was extracellular. The extracellular activity was 2-fold greater in NO4. These findings indicate that the introduced xynA was expressed constitutively and the xylanase protein was exported into the culture supernatant. Growth of NO4 on glucose was similar to that of OB156C, which suggests little extra load for plasmid maintenance and foreign xylanase production in the transformant. The plasmid pYK4XT was maintained stably in the transformant for more than 100 generations.

Purification and characterization of an alkaline xylanase from Streptomyces viridosporus T7A

Streptomyces viridosporus T7A produces a large amount of extracellular xylanase activity. We have isolated and purified an alkaline xylanase from culture supernatants of this organism. Purification was achieved by treatment of the culture supernatant with Q-Sepharose, concentration of the unbound proteins by ultrafiltration, fractionation of these proteins by Sephadex G-75 gel filtration chromatography, followed by Rotofor preparative isoelectric focusing. The xylanase has a molecular mass of 59 kDa as determined by capillary electrophoresis. The purified xylanase has a pI of 10.2–10.5, a pH optimum of 7.0–8.0, and a temperature optimum of 65–70°C. The xylanase is strongly inhibited by Hg 2+, Cu 2+, and Fe 3+ metal ions. The purified protein shows activity on birchwood and oat spelt xylans, but does not exhibit activity toward galactomannan, arabinogalactan, lichenan, or carboxymethylcellulose. This enzyme could have potential uses in biotechnology applications due to its high pH and temperature optima and unique substrate specificity.

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.

Polysaccharide hydrolase production by the rumen fungus Caecomyces communis

The anaerobic fungus Caecomyces communis was grown in a fermentor in either a discontinuous cultivation system or in a culture system with daily withdrawal and addition of fresh medium. Lowe and Orpin media were tested. The Lowe medium was best for the stimulation of enzyme production, the Orpin medium, for the stimulation of fungal growth and enzyme release. Xylanase activity was predominant among the polysaccharide hydrolases. Most of the enzymes studied were associated with cells except when the culture medium contained glucose or Ray grass hay. Enzymatic activities were constitutive, but their level was regulated by a carbon source. Cellulase production in both the cellular and extracellular fractions and the extracellular xylanase activity were stimulated by the presence of glucose. Cell-associated xylanase activity, however, was stimulated by glucose plus cellobiose. The presence of glucose enhanced enzyme release.

Cloning and characterization of a xylanase gene from corn strains of Erwinia chrysanthemi.

The gene encoding a 42-kDa endoxylanase was cloned from Erwinia chrysanthemi strain D1. Sequencing of this gene, called xynA, showed that it encoded a primary protein product of 413 amino acids with an unusual and long (31 amino acid) leader peptide that was cleaved during secretion to the bacterial periplasm. This protein is distinct from xylanases in glycohydrolase families 10 and 11 and, instead, appears to be intermediate between families 5 and 30. The xynA gene is located downstream from a gene with high homology to ATP-dependent RNA helicases and the Escherichia coli recD gene. Large amounts of the mature xylanase were produced by E. coli cells carrying a T7 expression plasmid construct and the protein was isolated from the bacterial periplasmic fraction by chromatography on a CM Bio-gel column. Marker exchange mutagenesis of the xynA gene eliminated the ability of strain D1 to produce detectable extracellular xylanase activity but did not affect virulence on corn leaves.

Optimization of extracellular xylanase production by Dictyoglomus sp. B1 in continuous culture

The thermophilic, xylanolytic, anaerobic organism, Dictyoglomus sp. B1, was cultivated in batch and continuous cultures in media containing insoluble beech-wood xylan. The extracellular xylanase activity levels obtained for the two cultivation methods were compared. Experiments were performed separately to determine the optimum substrate concentration, dilution rate, pH and temperature for xylanase production. Maximum xylanase activity was found at a substrate concentration of 1.5 g xylan/l, a dilution rate of 0.112 h−1, pH 8.0 and at 7°C. Different combinations of these optimum values were used in a 23 factorial experiment to investigate whether an increase in the xylanase production/activity could be achieved. A maximum xylanase activity of 2312 U/l was found when fermentors were operated at 73°C with a substrate concentration of 1.5 g xylan/l, pH 8.0, and a dilution rate of 0.112 h−1. Thus, the optimum xylanase activity in the factorial experiment was obtained when the conditions that gave the maximum xylanase activities in the individual experiments were combined. Optimum xylanase activity obtained in the 23 factorial experiment was 6.2 times higher than the activity found in the initial batch culture (373 U/l) and 3.0 times higher than the activity of a batch culture (783 U/l) grown at the same optimum conditions as the factorial experiment. The higher specific xylanase activity (217 U/mg protein) found in the 23 factorial experiment was 4.1 times higher than the specific activity in the initial batch culture (53 U/mg protein).

Effect of heterotrophic ruminal bacteria on xylan metabolism by the anaerobic fungus Piromyces communis

Six rumen bacteria were cocultured with the rumen fungus Piromyces communis and the effects on xylanolysis determined. The rate and extent of xylan utilization was enhanced in cocultures with Prevotella ruminicola or Succinivibrio dextrinosolvens. The positive effects of Suc. dextrinosolvens and Prev. ruminicola on xylanolysis by P. communis correlated with effective cross-feeding by the bacteria on arabinose and xylose released from xylan. Xylanolysis was not enhanced in cocultures of P. communis with Streptococcus bovis, Veillonella parvula or Ruminococcus flavefaciens. A comparison of fermentation product profiles and of extracellular enzyme activities showed that whereas saccharolytic species and Butyrivibrio fibrisolvens were dominant in cocultures, P. communis dominated in the culture with R. flavefaciens. Extracellular xylanase and β-xylosidase activities were not increased by cocultivation of P. communis with any of the heterotrophic bacteria.

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.

Two xylanases from Gaeumannomyces graminis with identical N-terminal amino acid sequence

Gaeumannomyces graminis var. avenae produces extracellular xylanase activity when grown in minimal medium containing xylan as sole carbon source. A 26·5 kDa and a 28 kDa xylanase were isolated from culture filtrates of the fungus grown on xylan. Both xylanases had a pI of 10·5 and behaved very similarly during hydrophobic interaction, cation exchange, and size exclusion chromatography. Enzyme activity of the combined xylanases was maximal at 50 °C and between pH 5·0 and 5·5. The N-terminal amino acid sequences of the two xylanases were identical and showed strong homology with xylanase from Schizophyllum commune and three other microbial xylanases. Xylanase activity with similar properties to the enzymes purified from culture fluids was detected in oat and wheat roots infected with G. graminis var. avenae but was not detected in healthy roots.