Effect of pH on production of xylanase by Trichoderma reesei on xylan- and cellulose-based media

Trichoderma reesei VTT-D-86271 (Rut C-30) was cultivatedon media based on cellulose and xylan as the main carbon source in fermentors with different pH minimum controls. Production of xylanase was favoured by a rather high pH minimum control between 6.0 and 7.0 on both cellulose- and xylan-based media. Although xylanase was produced efficiently on cellulose as well as on xylan as the carbon source, significant production of cellulose was observed only on the cellulose-based medium and best production was at lower pH (4.0 minimum). Production of xylanase at pH 7.0 was shown to be dependent on the nature of the xylan in the cultivation medium but was independent of other organic components. Best production of xylanase was observed on insoluble, unsubstituted beech xylan at pH 7.0. Similar results were obtained in laboratory and pilot (200-l) fermentors. Downstream processing of the xylanase-rich, low-cellulose culture filtrate presented no technical problems despite apparent autolysis of the fungus at the high pH. Enzyme produced in the 200-l pilot fermentor was shown to be suitable for use in enzyme-aided bleaching of kraft pulp. Due to the high xylanase/cellulase ratio of enzyme activities in the culture filtrate, pretreatment for removal of cellulase activity prior to pulp bleaching was unnecessary. Correspondence to: M. J. Bailey     

A third xylanase from Trichoderma reesei PC-3-7

A third xylanase (Xyn III) from Trichoderma reesei PC-3–7 was purified to electrophoretic homogeneity by gel filtration and ion-exchange chromatographies. The enzyme had a molecular mass of 32 kDa, and its isoelectric point was 9.1. The pH optimum of Xyn III was 6.0, similar to that of Xyn II, another basic xylanase of  T. reesei. The purified Xyn III showed high activity with birchwood xylan but no activity with cellulose and aryl glycoside. The hydrolysis of birchwood xylan by Xyn III produced mainly xylobiose, xylotriose and other xylooligosaccharides. The amino acid sequences of the N-terminus and internal peptides of Xyn III exhibited high homology with the family F xylanases, showing that they were distinct from those of Xyn I and Xyn II of  T. reesei, which belong to family G. These results reveal that Xyn III is a new specific endoxylanase, differing from Xyn I and Xyn II in  T. reesei. It is noteworthy that this novel xylanase was induced only by cellulosic substrates and l-sorbose but not by xylan and its derivarives. Furthermore,  T. reesei PC-3-7 produced Xyn III in quantity when grown on Avicel or lactose as a carbon source, while  T. reesei QM9414 produced little or no Xyn III. Received: 7 November 1997 / Received last revision: 2 February 1988 / Accepted: 23 February 1998     

The role of two Trichoderma reesei xylanases in the bleaching of pine kraft pulp

Summary The two major xylanases of Trichoderma reesei with different pI values and pH optima were compared for increasing the bleachability of pine kraft pulp. The efficiencies of the two enzymes acting on pulp substrate were very similar in hydrolysis yield, extraction kappa number or final brightness value. Only slight synergism between the two enzymes was observed in both hydrolysis and bleaching tests. The pH optimum of the pI 5.5 xylanase was similar in pulp treatment and in the hydrolysis of isolated substrates, and the bleaching result also correlated well with the hydrolysis of pulp xylan. By contrast, the pI 9.0 xylanase acted differently on pulp than on isolated xylans at different pH values and the pH optimum on pulp was increased. The bleachability of pulp by the pI 9.0 xylanase was improved more than expected at pH 7.0, although the hydrolysis of pulp xylan was substantially decreased. A similar phenomenon was also observed when the hydrolysis was performed in water instead of buffer. It thus appears that the degree of hydrolysis needed to obtain improved bleachability with pI 9.0 xylanase can be minimized by proper adjustment of the hydrolysis conditions. Correspondence to: J. Buchert     

Production of mycelial protein and hydrolytic enzymes from paper mill sludges by cellulolytic fungi

Summary Characterization of lignocellulosic wastes from three paper mills in New York State indicated that a kraft mill sludge contained substantial quantities of utilizable cellulose and hemicellulose. This residue was tested as a carbon source for seven cellulolytic fungi.Trichoderma reesei DAOM 167654 accumulated a product of over 22% crude protein, and caused a conversion of sludge to protein of almost 15% in 3 days growth in shake flasks.T. reesei also produced the highest levels of cellulase, whileT. longibrachiatum produced more xylanase (35 units/ml) than other fungi examined.     

Production and characterization of xylanase from Bacillus thermoalkalophilus grown on agricultural wastes

Summary Bacillus thermoalkalophilus isolated from termite-infested mound soils of the semi-arid zones of India had the ability to produce good amounts of xylanase(s) from cheap agricultural wastes. Of the two hemicellulosic substrates tested, bagasse was found to be the better inducer for xylanase production. Alkali treatment of bagasse and rice husk had varied effects on enzyme production. The enzyme preparation had activity optima at 60° C and 70° C and a half-life of 60 min at 65° C. The enzyme was stable for 24 h over a pH range of 4.0–6.0, while maximum activity was observed at pH 6.0–7.0. Enzyme production and activity were inhibited by the end-product of xylan hydrolysis, xylose. Offprint requests to: Ajit Varma     

Influence of pH on the production of xylanases by Trichoderma reesei Rut C-30

Trichoderma reesei Rut C-30 was cultivated in bioreactors at different pH on a medium with lactose as the main carbon source. Compared to an earlier study, in which T. reesei Rut C-30 was cultivated using polysaccharides (cellulose or xylan) as the main carbon sources, we now report a slightly lower pH value for maximal xylanase levels. The highest xylanase activity (IU/ml) on the lactose-based medium was observed at pH 6.0 compared to pH 7.0 on the polysaccharide-based media. When the pattern of different xylanases was analyzed by isoelectric focusing and activity zymogram, we observed that a low pH (4.0) favoured the production of xylanase I, whilst a high pH (6.0) favoured the production of xylanase III. Xylanase II was clearly produced at both pH values. The results at pH 4 and 6 correlate with the pH activity profiles of xylanase I, II and III. Hence, the different T. reesei xylanases were produced according to which enzyme is most active in that particular environment.     

Production of Polysaccharidases in Different Carbon Sources by Leucoagaricus gongylophorus Möller (Singer), the Symbiotic Fungus of the Leaf-Cutting Ant Atta sexdens Linnaeus

Leucoagaricus gongylophorus, the fungus cultured by the leaf-cutting ant Atta sexdens, produces polysaccharidases that degrade leaf components by generating nutrients believed to be essential for ant nutrition. We evaluated pectinase, amylase, xylanase, and cellulase production by L. gongylophorus in laboratory cultures and found that polysaccharidases are produced during fungal growth on pectin, starch, cellulose, xylan, or glucose but not cellulase, whose production is inhibited during fungal growth on xylan. Pectin was the carbon source that best stimulated the production of enzymes, which showed that pectinase had the highest production activity of all of the carbon sources tested, indicating that the presence of pectin and the production of pectinase are key features for symbiotic nutrition on plant material. During growth on starch and cellulose, polysaccharidase production level was intermediate, although during growth on xylan and glucose, enzyme production was very low. We propose a possible profile of polysaccharide degradation inside the nest, where the fungus is cultured on the foliar substrate.     

Production of alpha-amylase and xylanase by an alkalophilic strain of Penicillium griseoroseum RR-99

An alkalophilic strain of Penicillium sp. RR 99 was isolated that was found to synthesise extra-cellular alpha-amylase and xylanase, when cultivated in presence of starch and xylan respectively. The strain showed maximum alpha-amylolytic activity on 4th day and maximum xylanolytic activity on 6th day of cultivation. The ability of the strain to hydrolyse starchy and hemicellulosic wastes made the strain competent not only for the commercial production of these enzymes but also for successful utilization of wastes.     

Addendum to Issue 1 - ENZITEC 2012Simultaneous biosynthesis of biomass-degrading enzymes using co-cultivation of Aspergillus niger and Trichoderma reesei

Abstract

The biotransformation of lignocellulosic materials into biofuels and chemicals requires the simultaneous action of multiple enzymes. Since the cost of producing an efficient enzyme system maybe high, mixed cultures of microorganisms maybe an alternative to increase enzymatic production and consequently reduce costs. This study investigated the effects of different inoculum ratios and inoculation delays on the biosynthesis of cellulases and xylanases during co-cultivation of Aspergillus niger and Trichoderma reesei under solid-state fermentation (SSF). While the monoculture of T. reesei was more efficient for CMCase production than the co-cultivation of A. niger and T. reesei, a significant increase in β-glucosidase and xylanase production was achieved by co-cultivation of both species. The maximum CMCase activity of 153.91 IU/g was obtained with T. reesei after 48 h of cultivation, while the highest β-glucosidase activity of 119.71 IU/g (after 120 h) was obtained by co-cultivation of A. niger and T. reesei with a 3:1 inoculum ratio (A. niger: T. reesei). The greatest xylanase activity observed was 589.39 IU/g after 72 h of mixed culturing of A. niger and T. Reesei with a 1:1 inoculum ratio. This is the first study where the effects of inoculum ratio and inoculation delay in mixed culture of T. reesei and A. niger under SSF have been systematically assessed, and it indicates co-cultivation as a feasible alternative to increase enzymatic production.     

The use of canola meal as a substrate for xylanase production by Trichoderma reesei

Summary Tests made utilizing canola meal as a substrate for the production of xylanase indicate that Trichoderma reesei produced this enzyme in similar or better yields from canola meal than from Solka-floc, xylan or glucose. The maximum xylanase activity obtained from canola meal was 210 IU/ml in 9–12 days. The enzyme system produced using canola meal also contained a higher proportion of acetyl-xylan esterase, cellulase, and xylosidase activities. This system was more than or equally efficient as that produced using Solka-floc in hydrolysing canola meal, corn cobs, corn and wheat brans, straw, and larchwood xylan to fermentable sugars. Offprint requests to: Z. Duvnjak     

Ultrastructural localization of cellular compartments involved in secretion of the low molecular weight,alkaline xylanase by Trichoderma reesei

The intracellular location of the low-molecular weight, alkaline xylanase (XYN II) of Trichoderma reesei RUT C-30 was investigated during growth on xylan, using immunoelectron microscopy. A monoclonal antibody, produced against XYN II, was used for this purpose. The enzyme was found at the endoplasmic reticulum and in electron dense 0.2 to 0.8 m vesicles, as well as in the vacuole, at the plasma membrane and in the fungal cell-wall. No staining occured in the cytoplasm, the mitochondria and the nucleus. No Golgi-like structures could be seen. Addition of the carboxylic ionophore monensin blocked xylanase as well as total protein secretion. The results are discussed with respect to XYN II being secreted by T. reesei via a pathway involving the endoplasmic reticulum and secretory vesicles and/or the vacuole.     

Cellulose and xylan degrading enzymes of Fusarium avenaceum

It was found that crude preparation obtained from the culture medium of Fusarium avenaceum degraded cellulose and xylan. After chromatography on CM-Sepharose CL-6B of this preparation six fraction were obtained. The eluted fractions II and V showed xylanase activity, fraction IV — cellulase activity and fraction III — xylanase and cellulase activity. The end products of xylan hydrolysis by all xylanase fractions (II, III, V) were xylobiose, xylose, xylotriose and xylotetrose. The end products of cellulose hydrolysis by fractions III and IV was cellobiose, glucose and cellotriose. The data from gel filtration on Sephacryl S-200 indicated a molecular weight of more than 250,000 for both cellulase IV and xylanase V. After gel filtration in the presence of urea disaggregation of those high molecular xylanase and cellulase particles was observed. Xylanase II in difference from the other fractions contained higher amount of sugar. Digestion of fraction II with cellulase-hemicellulase preparation from Phoma hibernica decreased the content of sugar from 17% to 8%, but did not change its enzymatic properties. Cellulase IV as well as xylanase V were inactivated by N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and tetranitromethane, hence it is suggested that tryptophan and tyrosine are the essential for the activity of these enzymes.     

Mixed culture solid substrate fermentation for cellulolytic enzyme production

Summary Cellulolytic and hemicellulolytic enzymes were produced on extracted sweet sorghum silage by mixed culture solid substrate fermentation with Trichoderma reesei LM-1 (a Peruvian mutant) and Aspergillus niger ATCC 10864. Optimal cellulose and xylanase levels of 4 IU/g dry weight (DW) and 180 IU/g DW, respectively, were achieved in 120 h-fermentation when T. reesei, inoculated at 0 h, was followed by the inoculation of A. niger at 48 h.     

Increased Production of Xylanase by Expression of a Truncated Version of the xyn11A Gene from Nonomuraea flexuosa in Trichoderma reesei

We have previously shown that the Nonomuraea flexuosa Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the N. flexuosa xyn11A gene and the truncated gene was expressed in Trichoderma reesei. The xylanase sequence was fused to the T. reesei mannanase I (Man5A) signal sequence or 3′ to a T. reesei carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 (cel7A, cbh1) promoter. Single-copy isogenic transformants in which the expression cassette replaced the cel7A gene were cultivated and analyzed. The transformants expressing the truncated N. flexuosa xyn11A produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length N. flexuosa xyn11A. The transformant expressing the cel6A CBD-truncated N. flexuosa xyn11A produced about 1.9 g liter⁻¹ of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length N. flexuosa xyn11A with an exact fusion to the cel7A terminator was expressed. We suggest that the T. reesei folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length N. flexuosa xyn11A is efficiently translated.     

Production of cellulases and xylanase by Trichoderma reesei F-522 on pretreated wheat straw

Autohydrolyzed and ethanol-alkali pulped wheat straw was examined as a candidate feedstock for both cellulase and xylanase production and enzymatic hydrolysis. Submerged cultures of Trichoderma reesei F-522 grown on hydrothermally modified straw provided culture supernatants of the highest enzymatic activities, whereas the maximal efficiency of enzymatic hydrolysis was recorded in straw treated with ethanol-NaOH mixture. Some culture conditions were optimized to improve the growth and cellulase production by T. reesei on autohydrolyzed wheat straw.     

Production of Xylanase of Bacillus coagulans and its bleaching potential

Summary The production of xylanase from Bacillus coagulans has been studied with respect to the environmental parameters, the carbon source and the concentration of carbon source at the shake flask level. Among the various carbon sources used, wheat straw powder favoured higher enzyme production. Xylan isolated from wheat straw gave higher enzyme production as compared to the birchwood xylan. Maximum enzyme activity of 165 IU/ml was obtained with 2% wheat straw xylan in a shake flask study. Improvement of xylanase production was achieved by increasing the wheat straw powder concentration up to 3%. Enzyme has optimum activity at a temperature of 55 °C and pH of 7. The concentrated crude enzyme was found to reduce the kappa number of enzyme-treated eucalyptus pulp by␣5.45% with a marginal increase in the CED viscosity of the enzyme treated pulp as compared to the non-enzymatically treated pulp.     

Diversity in Production of Xylan-Degrading Enzymes Among Species Belonging to the Trichoderma Section Longibrachiatum

Xylan is an important part of plant biomass and represents a renewable raw material for biorefineries. Contrary to cellulose, the structure of hemicellulose is quite complex. Therefore, the biodegradation of xylan needs the cooperation of many enzymes. For industrial production of xylanase multienzyme complexes (cocktails) and selected monocomponent xylanases, different Trichoderma reesei mutants and recombinants are used. T. reesei QM 6a (wild-type parent of best existing mutants) was selected as a starting material in the 1960s when the modern in-depth analytical methods were not yet in use. Therefore, screening of fungi genetically close to T. reesei in biodegradation of xylan may have a scientific value. Fifteen different strains from Trichoderma section Longibrachiatum have been tested for extracellular xylan-degrading enzyme production on three carbon sources (wheat straw, corn fiber, and eucalyptus wood) in shake flask cultivation. The enzyme activities were evaluated by traditional colorimetric enzyme assays and by HPLC and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Degradation of xylan was studied on four different xylan-rich model substrates. T. reesei CPK 155, Trichoderma parareesei TUB F-2535, and Trichoderma gracile TUB F-2543 isolates were equally good or better in degradation of the wheat arabinoxylan (WAX) and corn fiber alcohol insoluble solids as hydolysis substrates than the well-known T. reesei QM 6a and RUT C30 strains. Though Trichoderma saturnisporum ATCC 18903 gave relatively low volumetric enzyme activities by traditional colorimetric assays, it could release quite large amount of hydrolysis products (mono- and oligosaccharides) from WAX. Therefore, these fungi may be potential candidates for further experiments. Enzyme production on wheat straw and corn fiber carbon sources was more effective than on eucalyptus wood.     

Production of xylanase by Aspergilli using alternative carbon sources: application of the crude extract on cellulose pulp biobleaching

The ability of xylanolytic enzymes produced by Aspergillus fumigatus RP04 and Aspergillus niveus RP05 to promote the biobleaching of cellulose pulp was investigated. Both fungi grew for 4–5 days in liquid medium at 40°C, under static conditions. Xylanase production was tested using different carbon sources, including some types of xylans. A. fumigatus produced high levels of xylanase on agricultural residues (corncob or wheat bran), whereas A. niveus produced more xylanase on birchwood xylan. The optimum temperature of the xylanases from A. fumigatus and A. niveus was around 60–70°C. The enzymes were stable for 30 min at 60°C, maintaining 95–98% of the initial activity. After 1 h at this temperature, the xylanase from A. niveus still retained 85% of initial activity, while the xylanase from A. fumigatus was only 40% active. The pH optimum of the xylanases was acidic (4.5–5.5). The pH stability for the xylanase from A. fumigatus was higher at pH 6.0–8.0, while the enzyme from A. niveus was more stable at pH 4.5–6.5. Crude enzymatic extracts were used to clarify cellulose pulp and the best result was obtained with the A. niveus preparation, showing kappa efficiency around 39.6% as compared to only 11.7% for that of A. fumigatus.     

Ferulic Acid Esterase Activity from Schizophyllum commune

Schizophyllum commune produced an esterase which released ferulic acid from starch-free wheat bran and from a soluble ferulic acid-sugar ester that was isolated from wheat bran. The preferred growth substrate for the production of ferulic acid esterase was cellulose. Growth on xylan-containing substrates (oat spelt xylan and starch-free wheat bran) resulted in activity levels that were significantly lower than those observed in cultures grown on cellulose. Similar observations were made for endoglucanase, p-nitrophenyllactopyranosidase, xylanase, and acetyl xylan esterase. Of the enzymes studied, only arabinofuranosidase was produced at maximum levels during growth on xylan-containing materials. Ferulic acid esterase that had been partially purified by DEAE chromatography released significant amounts of ferulic acid from wheat bran only in the presence of a xylanase-rich fraction, indicating that the esterase may not be able to readily attack high-molecular-weight substrates. The esterase acted efficiently, without xylanase addition, on a soluble sugar-ferulic acid substrate.     

Application of thermostable xylanase of Dictyoglomus sp. in enzymatic treatment of kraft pulps

Enzymatic treatment of pine and birch kraft pulps with a xylanase preparation from a thermophilic anaerobic bacterium Dictyoglomus sp. strain B1 was studied in order to improved pulp bleachability. Maximal solubilization of pulp xylan was obtained at 90°C and pH 6.0–7.0. The enzyme was also active in the alkaline pH range; at pH 9.0 xylan hydrolysis was decreased by only 18% from the maximum at pH 7.0. The positive effect of xylanase pretreatment at 80°C and pH 6.0 or 8.0 on bleachability of pine kraft pulp was demonstrated. The brightness was increased by two ISO units in one-stage peroxide delignification, which corresponds well to values obtained with other enzymes at lower temperatures and pH values. Thus, the Dictyoglomus xylanase is well suited for pulp treatments at elevated temperatures in neutral and alkaline conditions.Correspondence to: M. Rättö