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.     

Differential expression of cellulases and xylanases by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> grown on different carbon sources

The diversity of cellulases and xylanases secreted by Cellulomonas flavigena cultured on sugar cane bagasse, Solka-floc, xylan, or glucose was explored by two-dimensional gel electrophoresis. C. flavigena produced the largest variety of cellulases and xylanases on sugar cane bagasse. Multiple extracellular proteins were expressed with these growth substrates, and a limited set of them coincided in all substrates. Thirteen proteins with carboxymethyl cellulase or xylanase activity were liquid chromatography/mass spectrometry sequenced. Proteins SP4 and SP18 were identified as products of celA and celB genes, respectively, while SP20 and SP33 were isoforms of the bifunctional cellulase/xylanase Cxo recently sequenced and characterized in C. flavigena. The rest of the detected proteins were unknown enzymes with either carboxymethyl cellulase or xylanase activities. All proteins aligned with glycosyl hydrolases listed in National Center for Biotechnology Information database, mainly with cellulase and xylanase enzymes. One of these unknown enzymes, protein SP6, was cross-induced by sugar cane bagasse, Solka-floc, and xylan. The differences in the expression maps of the presently induced cultures revealed that C. flavigena produces and secretes multiple enzymes to use a wide range of lignocellulosic substrates as carbon sources. The expression of these proteins depends on the nature of the cellulosic substrate.     

Media carbon induction of extracellular cellulase activities inNeocallimastix frontalis isolate EB188

Extracellular cellulase induction in the ruminal fungusNeocallimastix frontalis isolate EB188 was followed. Glucose media-established cultures produced cellulase when switched to a variety of cellulose-containing media. High levels of cellulase and xylanase activities were present in cultures switched to sigma cell 100, solka floc, avicel, sisal fiber, and wheat straw, but not those switched to glucose, carboxymethylcellulose, or wood chips. Several assay substrates were used to show differential cellulase induction as well as-glucosidase activity. Cellulases hydrolyzed short oligosaccharides and released glucose from insoluble cellulose. Cellobiase activity was also indicated. Cellulase activity tolerated brief exposure to high temperature, was insensitive to certain metal ions, and possessed pH optima between 5.0 and 6.5.     

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     

Biochemical properties of xylanases from a thermophilic fungus,Melanocarpus albomyces, and their action on plant cell walls

Melanocarpus albomyces, a thermophilic fungus isolated from compost by enrichment culture in a liquid medium containing sugarcane bagasse, produced cellulase-free xylanase in culture medium. The fungus was unusual in that xylanase activity was inducible not only by hemicellulosic material but also by the monomeric pentosan unit of xylan but not by glucose. Concentration of bagasse-grown culture filtrate protein followed by size-exclusion and anion-exchange chromatography separated four xylanase activities. Under identical conditions of protein purification, xylanase I was absent in the xylose-grown culture filtrate. Two xylanase activities, a minor xylanase IA and a major xylanase IIIA, were purified to apparent homogeneity from bagasse-grown cultures. Both xylanases were specific forβ-1,4 xylose-rich polymer, optimally active, respectively, at pH 6.6 and 5.6, and at 65°C. The xylanases were stable between pH 5 to 10 at 50°C for 24 h. Xylanases released xylobiose, xylotriose and higher oligomers from xylans from different sources. Xylanase IA had a M_r of 38 kDa and contained 7% carbohydrate whereas xylanase IIIA had a M_r of 24 kDa and no detectable carbohydrate. The K_m for larchwood xylan (mg ml⁻¹) and V_max (μmol xylose min⁻¹ mg⁻¹ protein) of xylanase IA were 0.33 and 311, and of xylanase IIIA 1.69 and 500, respectively. Xylanases IA, II and IIIA showed no synergism in the hydrolysis of larchwood glucuronoxylan or oat spelt and sugarcane bagasse arabinoxylans. They had different reactivity on untreated and delignified bagasse. The xylanases were more reactive than cellulase on delignified bagasse. Simultaneous treatment of delignified bagasse by xylanase and cellulase released more sugar than individual enzyme treatments. By contrast, the primary cell walls of a plant, particularly from the region of elongation, were more susceptible to the action of cellulase than xylanase. The effects of xylanase and cellulase on plant cell walls were consistent with the view that hemicellulose surrounds cellulose in plant cell walls.     

Production and characterisation of lignocellulases of Panus tigrinus and their application

This study on the lignocellulases in broth cultures of the basidiomycete Panus tigrinus indicates that laccase and xylanase enzymes are constitutive and cellulase is inducible. In stationary culture at 28°C, the greatest laccase and xylanase activity was observed after growth for approximately nine days. Laccase production was dependent on the presence, and the particular brand, of malt extract in the growth medium. While production of laccase was enhanced by growth at 37°C and 42°C, xylanase was not. Raising the pH of the growth medium from pH 5.6 to pH 7.0 did not affect xylanase production, but laccase production was reduced at the higher pH. In shake culture, growth was pelleted and biomass lower than in stationary culture, and synthesis of both enzymes was strongly inhibited. Cultures of P. tigrinus decolourised Poly R-478 and the toxic triphenyl methane dye, crystal violet. It was also shown to degrade a natural lignocellulosic waste, sawdust.     

ACEI of Trichoderma reesei Is a Repressor of Cellulase and Xylanase Expression

We characterized the effect of deletion of the Trichoderma reesei (Hypocrea jecorina) ace1 gene encoding the novel cellulase regulator ACEI that was isolated based on its ability to bind to and activate in vivo in Saccharomyces cerevisiae the promoter of the main cellulase gene, cbh1. Deletion of ace1 resulted in an increase in the expression of all the main cellulase genes and two xylanase genes in sophorose- and cellulose-induced cultures, indicating that ACEI acts as a repressor of cellulase and xylanase expression. Growth of the strain with a deletion of the ace1 gene on different carbon sources was analyzed. On cellulose-based medium, on which cellulases are needed for growth, the Δace1 strain grew better than the host strain due to the increased cellulase production. On culture media containing sorbitol as the sole carbon source, the growth of the strain with a deletion of the ace1 gene was severely impaired, suggesting that ACEI regulates expression of other genes in addition to cellulase and xylanase genes. A strain with a deletion of the ace1 gene and with a deletion of the ace2 gene coding for the cellulase and xylanase activator ACEII expressed cellulases and xylanases similar to the Δace1 strain, indicating that yet another activator regulating cellulase and xylanase promoters was present.     

Effect of Carbon Source on Production of Lytic Enzymes by the Sclerotial Parasites Trichoderma atroviride and Coniothyrium minitans

Three isolates of Trichoderma atroviride and two isolates of Coniothyrium minitans known to efficiently degrade sclerotia of Sclerotinia sclerotiorum were cultured on minimal medium with sucrose, carboxymethyl cellulose (CMC), xylan, laminarin, colloidal chitin or powdered sclerotia as carbon source. The activity of endochitinase, endo‐β‐1,3‐glucanase, endoxylanase and endocellulase in culture filtrates was determined after 7 and 15 days of culture using dye‐labelled substrates. The strongest inducers of chitinase were colloidal chitin and sclerotia powder. Chitinase activity appeared to be faster induced in the isolates of T. atroviride than in the isolates of C. minitans, but the maximum level of activity present in culture filtrates of the two species was similar. With CMC and xylan as carbon source, concurrent production of the corresponding enzymes was observed for the Trichoderma isolates. The isolates of C. minitans produced cellulase on xylan but not on CMC, whereas xylanase was produced on both carbon sources. Laminarin induced the formation of glucanases in the three isolates of T. atroviride but not the isolates of C. minitans. However, in the sclerotia‐containing cultures of C. minitans glucanase activity was even higher than in the respective cultures of the Trichoderma isolates. During the 31‐day study period, the pattern of enzyme production in shake cultures containing sclerotia powder was very similar for the isolates of T. atroviride and C. minitans. Glucanase activity generally reached a peak 24 days after inoculation of the flasks, whereas the activity of chitinase, cellulase and xylanase remained fairly constant throughout the experiment.     

Effects of dietary cellulase and xylanase addition on digestion,rumen fermentation and methane emission in growing goats

The objective of this study was to evaluate the effectiveness of supplementation of cellulase and xylanase to diets of growing goats to improve nutrient digestibility, utilisation of energy and mitigation of enteric methane emissions. The experiment was conducted in a 5 × 5 Latin square design using five goats with permanent rumen fistulae and five treatments consisted of two levels of cellulase crossed over with two levels of xylanase plus unsupplemented Control. The cellulase (243 U/g) derived from Neocallimastix patriciarum was added at 0.8 and 1.6 g/kg dry matter intake (DMI) and the xylanase (31,457 U/ml) derived from Aspergillus oryzae was fed at 1.4 and 2.2 ml/kg DMI. There were no differences in apparent digestibility of organic matter, neutral detergent fibre, acid detergent fibre and rumen fermentation parameters (i.e. ammonia-nitrogen [N], volatile fatty acids) among all treatments. Dietary cellulase and xylanase addition did not influence energy and N utilisation. But compared to xylanase addition at the higher dose, at the low xylanase dose the retained N, the availability of retained N and digested N were increased (p < 0.01). Moreover, enzyme addition did not affect the enteric methane emission and community diversity of ruminal methanogens. The present results indicated that previous in vitro findings were not confirmed in ruminant trials.     

Fruiting of Agaricus bisporus

Several enzymes were assayed in extracts from mycelium-colonised compost during growth and fruiting of Agaricus bisporus (Lange) Imbach. Comparison of changes of enzyme levels in axenic and nonaxenic cultures and in cultures of non-fruiting strains indicated that they were associated directly with the fungal mycelium. Large changes were found in the amounts of laccase and cellulase which were correlated with fruit body development. Laccase concentration increased during mycelial growth and then declined rapidly at the start of fruiting. Cellulase activity could be detected throughout growth but increased at fruiting. No such changes were observed in xylanase, alkaline protease, laminarinase and acid and alkaline phosphatases. Activities of laccase and cellulase were measured in axenic cultures arrested at various stages of fruiting development. Such cultures showed that the changes in concentration of laccase and cellulase were associated with the enlargement of fruit bodies.     

Cellulase-free xylanase by thermophilic fungi: a comparison of xylanase production by two Thermomyces lanuginosus strains

Thermomyces lanuginosus strains RT9 and MH4 were studied to find favourable cultivation conditions and to compare their abilities to produce xylanolytic enzymes in three media on different substrates at 50° C or 55° C under shake-culture conditions. Both organisms produced xylanases free of cellulase at widely different levels in all cultivation conditions employed. Wheat bran, corn cobs and xylan induced xylanases in increasing order of producing with both cultures. T. lanuginosus RT9 demonstrated the highest xylanase production in all cultivation conditions but with lower soluble protein, reducing sugar, -xylosidase and debranching enzymes levels (arabinosidase, acetylxylanesterase, mannanase) when compared to T. lanuginosus MH4. The study reveals that xylanase production was highly influenced by nitrogen sources and their concentrations and by the initial pH in the cultures. The two strains may therefore be unique, when technical application is considered in terms of the quantity and quality of the xylanolytic enzymes produced.     

Activities of cellulase and other extracellular enzymes during lignin solubilization by Streptomyces viridosporus

Summary Two mutant strains of the lignin degrading bacterium Streptomyces viridosporus strain T7A with enhanced abilities to produce a soluble lignin degradation intermediate, acid-precipitable polymeric lignin (APPL) and several mutants derepressed for cellulase production were compared with the wild type to examine the roles of cellulase and selected other extracellular enzymes in lignin solubilization by S. viridosporus. The two APPL-overproducing mutants, T-81 and T-138, had higher cellulase activities than the wild type. Mutants specifically derepressed for cellulase were also isolated and were found to produce more APPL than the wild type. The results are indicative of some involvement of cellulase in the lignin solubilization process. The lignin solubilized from corn (Zea mays) lignocellulose by the mutants was slightly different chemically as compared to wild type solubilized lignin in that it had a higher coumaric acid ester content. The production of extracellular coumarate ester esterase, aromatic aldehyde oxidase, and xylanase was also examined in the mutants. Xylanase and aromatic aldehyde oxidase production did not differ significantly between the mutants and the wild type. Mutant T-81 was found to have a slightly lower activity for esterase as compared with the wild type. It was concluded that xylanase, oxidase and esterase are not the enzymes directly responsible for enhanced lignin solubilization. The results, however, do implicate cellulase in the process.Paper number 86 511 of the Idaho Agricultural Experiment Station     

Assessment of Keratinase and Other Hydrolytic Enzymes in Thermophilic Bacteria Isolated from Geothermal Areas in Patagonia Argentina

Thermophilic aerobic bacteria were isolated from two geothermal areas in Neuquén province using two different enrichment methods and a total of 30 isolates were obtained. From chicken feather enrichment cultures, strains affiliated to Bacillus cytotoxicus and Bacillus licheniformis were isolated and all of them demonstrated the capability to degrade completely chicken feather. A preliminary research on biotechnological enzymes' potential demonstrated that all the isolates displayed at least one of the extracellular hydrolytic enzymes tested. Most of the isolates showed protease, inulinase and/or pectinase activities, while cellulase and xylanase activities were less common. In light of these findings, geothermal areas of Argentina may be considered as a potential source of thermophilic bacteria able to produce many industrially relevant enzymes.     

A bifunctional cellulase–xylanase of a new Chryseobacterium strain isolated from the dung of a straw‐fed cattle

A new cellulolytic strain of Chryseobacterium genus was screened from the dung of a cattle fed with cereal straw. A putative cellulase gene (cbGH5) belonging to glycoside hydrolase family 5 subfamily 46 (GH5_46) was identified and cloned by degenerate PCR plus genome walking. The CbGH5 protein was overexpressed in Pichia pastoris, purified and characterized. It is the first bifunctional cellulase–xylanase reported in GH5_46 as well as in Chryseobacterium genus. The enzyme showed an endoglucanase activity on carboxymethylcellulose of 3237 μmol min^?1 mg^?1 at pH 9, 90 °C and a xylanase activity on birchwood xylan of 1793 μmol min^?1 mg^?1 at pH 8, 90 °C. The activity level and thermophilicity are in the front rank of all the known cellulases and xylanases. Core hydrophobicity had a positive effect on the thermophilicity of this enzyme. When similar quantity of enzymatic activity units was applied on the straws of wheat, rice, corn and oilseed rape, CbGH5 could obtain 3.5–5.0× glucose and 1.2–1.8× xylose than a mixed commercial cellulase plus xylanase of Novozymes. When applied on spent mushroom substrates made from the four straws, CbGH5 could obtain 9.2–15.7× glucose and 3.5–4.3× xylose than the mixed Novozymes cellulase+xylanase. The results suggest that CbGH5 could be a promising candidate for industrial lignocellulosic biomass conversion.     

Characterization of a New α-<Emphasis Type="SmallCaps">l</Emphasis>-Arabinofuranosidase from <Emphasis Type="Italic">Penicillium</Emphasis> sp. LYG 0704, and their Application in Lignocelluloses Degradation

A gene (arf) encoding an α-l-arabinofuranosidase (ARF) that hydrolyzes arabinose substituted on xylan was isolated from Penicillium sp. The gene was predicted to encode 339 amino acid residues showing 71–75% homology to GH family 54. E. coli expressed ARF showed optimal activity at 50°C and pH 5–6 on wheat arabinoxylan. The hydrolysis activities on oat spelt xylan by ARF and xylanase were 1.67-fold higher than that of xylanase alone. The synergistic effects of ARF and commercial enzymes (xylanase and cellulase) on popping-pretreated rice straw were 1.15–1.51-fold higher amounts of sugars released in the [ARF + xylanase + cellulase] mixture than in the mixtures [ARF + xylanase], [ARF + cellulase], and [xylanase + cellulase]. Moreover, the liberation of arabinose by ARF was enhanced 2.1–2.9-fold in a reaction with xylanase and cellulase as compared with [xylanase + cellulase] and ARF alone.     

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.     

<Emphasis Type="Italic">Xanthomonas gardneri</Emphasis> exoenzymatic activity towards plant tissue

Bacterial spot caused by Xanthomonas spp. is an important tomato and pepper disease worldwide. Recent outbreaks of bacterial spot disease in Central Brazil and Canada have been attributed to Xanthomonas gardneri, which is also recognized as group D of Xanthomonas campestris pv. vesicatoria. Carotenoid-like pigments called xanthomonadins, which are diagnostic for yellow Xanthomonas spp., were extracted from X. gardneri. It was shown that the model plant Arabidopsis thaliana, member of the Brassicaceae family, can develop disease symptoms in response to different isolates of X. gardneri. Secretion of enzymes has been shown to play an important role in pathogenicity for different pathogens, and to begin to understand the interaction of X. gardneri and A. thaliana, a biochemical analysis of secreted proteins in the presence of A. thaliana leaves was performed. Different enzymatic activities such as for cellulase, α-arabinofuranosidase, pectinase, invertase and xylanase were assayed. In the presence of leaves, cellulase activity was highest after 60 and 72 h of growth and α-arabinofuranosidase activity was detected between 12 and 72 h of growth. Pectinase, invertase and xylanase activities were not detected. Cellulase and α-arabinofuranosidase activities may be important for X. gardneri acquisition of plant nutrients through degradation of cellulose fibers and hemicellulose of the cell wall, respectively, to the invasion of the host tissue and/or may generate signal molecules that are recognized by the plant. This is the first study to address how X. gardneri responds to host plant tissue.     

High-Level Heterologous Expression of Bacillus halodurans Putative Xylanase Xyn11A (BH0899) in Kluyveromyces lactis

The putative xyn11A structural gene (BH0899) encoding a family-11 xylanase from alkaliphilic Bacillus halodurans strain C-125 was heterologously expressed in the yeast Kluyveromyces lactis CBS 1065 and secreted to a level of 156 μg/ml under selective culture conditions in shake flasks. The Xyn11A production level in shake flask cultures of K. lactis CBS 1065 was higher than that reported for other xylanase genes placed under the control of the regulated LAC4 promoter on a plasmid containing an entire sequence of pKD1 from Kluyveromyces drosophilarium. Recombinant Xyn11A was highly active over pH range from 3 to 10, with maximal activity around pH 7. The enzyme showed a specific activity of 628 U/mg-protein on birchwood xylan as substrate, but no cellulase or β-xylosidase activity.