The N-terminal cellulose-binding domain of EGXA increases thermal stability of xylanase and changes its specific activities on different substrates

A full-length EGXA enzyme from a mollusk, Ampullaria crossean, was cloned into pFastBac vector and then heterogeneously expressed in insect Tn5 cells. Its natural N-terminal signal peptide worked well in the insect Tn5 cells. The recombinant EGXA was a 63 kDa protein and had active endo-beta-1,4-glucanase (EC 3.2.1.4) and endo-beta-1,4-xylanase (EC 3.2.1.8). The specific activity of endo-beta-1,4-xylanase was higher than in the EGX, which was purified from the stomach tissues of Ampullaria crossen. The N-terminal cellulose-binding domain of EGXA made it bind to cellulose and xylan more efficiently. This cellulose-binding domain also increased the thermal stability of this recombinant enzyme and decreased the recombinant EGXA's specific activities on p-nitrophenyl-beta-D-cellobioside and sodium carboxymethyl cellulose.     

The endo-beta-1,4-xylanase xyn11A is required for virulence in Botrytis cinerea

Phytopathogenic fungi can degrade xylan, an abundant hemicellulose in plant cell walls, by the coordinate action of a group of extracellular enzymes. Among these, endo-beta-1,4-xylanases carry out the initial breakdown by cleaving internal bonds in the polymer backbone. We have isolated and characterized a gene, xyn11A, coding for an endo-beta-1,4-xylanase belonging to family 11 of glycosyl hydrolases. xyn11A was shown to be induced by xylan and repressed by glucose and to be expressed in planta. The disruption of xyn11A caused only a moderate decrease, about 30%, in the level of extracellular endo-beta-1-4-xylanase activity and in the growth rate, with beechwood xylan as the only carbon source. However, deletion of the gene had a more pronounced effect on virulence, delaying the appearance of secondary lesions and reducing the average lesion size by more than 70%. Reintroducing the wild-type gene into the mutant strains reversed this phenotype back to wild type.     

A monovalent anion affected multi-functional cellulase EGX from the mollusca,Ampullaria crossean

A cellulose hydrolytic enzyme was isolated from the stomach juice of Ampullaria crossean, a kind of herbivorous mollusca. The enzyme was purified 45.3-fold to homogenety by ammonium sulfate precipitation, DEAE-Sephadex A-50 column, Bio-gel P-100 gel filtration column, and phenyl-Sepharose CL-4B column chromatography. The enzyme was designated as cellulase EGX. The purified enzyme is a multi-functional enzyme with the activities of exo-beta-1,4-glucanase (14.84 U/mg for p-nitrophenyl beta-D-cellobioside), endo-beta-1,4-glucanase (40.3 U/mg for carboxymethyl cellulose), and endo-beta-1,4-xylanase (196 U/mg for soluble xylan from birchwood). The monovalent anions such as F(-), Cl(-), Br(-), I(-), and NO(3)(-) are essential for its exo-beta-1,4-glucanase activity but have no effect on the activity for xylan, while I(-) higher than 5mM would inhibit the exo-beta-1,4-glucanase activity. The monovalent anions Cl(-) and Br(-) activate its endo-beta-1,4-glucanase activity. Binding of Cl(-) enhances the thermostability of EGX, but does not affect its fluorescence emission spectrum. The molecular mass of EGX is 41.5 kDa, as determined by SDS-PAGE. The pI value is about pH 7.35. The xylan hydrolytic activity of EGX reaches to the maximum between pH 4.8 and 6.0 and the pNPC hydrolytic activity reaches the maximum between pH 4.8 and 5.6, while that for CMC hydrolytic activity is between pH 4.4 and 4.8. Preliminary results showed that the enzyme was secreted by the mollusca itself.     

Molecular phylogeny of three oxymonad genera: Pyrsonympha,Dinenympha and Oxymonas

Oxymonads are a morphologically well-characterized and highly diverse lineage of protists. They are, however, under sampled at a molecular level. It has recently been demonstrated that a genus of oxymonads, Pyrsonympha, is phylogenetically related to the excavate taxon Trimastix. Here, we addressed issues of internal oxymonad evolution. Pyrsonympha and Dinenympha are shown, by fluorescent in situ hybridization and phylogenetic evidence, to be separate genera and not morphotypes of the same organism. We demonstrated that three genera of oxymonads, Dinenympha, Pyrsonympha, and Oxymonas are each monophyletic and together form a clade which excludes other known eukaryotes. We have presented a taxonomic scheme of oxymonads taking into account their sisterhood with Trimastix and speculated on morphological evolution of oxymonads, particularly of their attachment apparatuses. Our biogeographical analysis with Japanese and Canadian Pyrsonympha and Dinenympha suggests that these genera diverged before the separation of termites that inhabit Eastern Asia and Western North America.     

Diet and carbohydrate digestion in the yellow-spotted longicorn beetle Psacothea hilaris

Larval and adult Psacothea hilaris feed on mulberry wood and leaves, respectively. High levels of endogenous activity against the major dietary carbohydrates, cellulose, hemicellulose, starch and soluble sugars were secreted in the gut of larvae and adults. Activity against pectin was also high and multiple polygalacturonase (EC 3.2.1.15) components were secreted in the gut of larvae. One glycanase component, beta-EG1, which was primarily an endo-beta-1,4-glucanase (EC 3.2.1.4) and another, beta-EG2, which was mostly an endo-beta-1,4-xylanase (EC 3.2.1.8), were also secreted, while at least four additional components hydrolysed laminarin, lichenin and crystalline cellulose. The beta-glycosidase component beta-GD1 was associated with most of the beta-mannosidase (EC 3.2.1.25) and beta-xylosidase (EC 3.2.1.37) activity secreted in the gut of larvae, while another, beta-GD2, was a beta-glucosidase (EC 3.2.1.21), the activity of which was directed against cellobiose and other beta-linked disaccharides, and a beta-fucosidase (EC 3.2.1.38). A beta-galactosidase (EC 3.2.1.23), which did not hydrolyse lactose, was also secreted, as were distinct beta-N-acetylhexosaminidase (EC 3.2.1.52), trehalase (EC 3.2.1.28), alpha-L-arabinosidase (EC 3.2.1.55), alpha-galactosidase (EC 3.2.1.22) and a minimum of four alpha-glucosidase (EC 3.2.1.20) components, one of which was also likely to be associated with a peak of alpha-mannosidase (EC 3.2.1.24) activity. The alpha-glucosidase components varied in their specificity for alpha-linked disaccharides, but none was active against sucrose, which was hydrolysed by a beta-fructofuranosidase (EC 3.2.1.26) component. Overall average levels of activity in larvae were twice those of adults, but the secretion of individual carbohydrases in both was not regulated in response to the relative abundance of particular carbohydrate components in their respective diets.     

Patterns of functional enzyme activity in fungus farming ambrosia beetles

ABSTRACT: INTRODUCTION: In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles, wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. RESULTS: We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-beta-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-beta-1,4-xylanase activity was exclusively detected in larvae. CONCLUSION: Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles do not degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray-parenchyma cells in the wood xylem. Furthermore, the detection of xylanolytic enzymes exclusively in larvae and not in adults indicates that larvae (pre-) digest plant cell wall structures exclusively in larvae (which feed on fungus colonized wood) and not in adults (which feed only on fungi). This implies that in X. saxesenii and likely also in many other ambrosia beetles, adults and larvae do not compete for the same food within their nests - in contrast, larvae increase colony fitness by facilitating enzymatic wood degradation and fungus cultivation.     

Effect of Temperature and Termite Starvation on Phagocytosis by Protozoan Symbionts of the Eastern Subterranean Termite Reticulitermes flavipes Kollar

Abstract Many termite species rely on intestinal protozoan symbionts to digest their cellulosic foods. We examined cellulose acquisition by the symbionts of the Eastern subterranean termite Reticulitermes flavipes Kollar (Isoptera; Rhinotermitidae) by following their phagocytosis of red paper fed to the termite host. The effects of termite host starvation and environmental temperature on feeding activity were studied in the zooflagellates Trichonympha agilis Leidy (Trichonymphidae), Pyrsonympha vertens Leidy, Dinenympha fimbriata Kirby, and D. gracilis Leidy (Pyrsonymphidae), which are among the largest residents in R. flavipes' hindguts. Protozoans in termites starved for 24 h ingested red paper significantly sooner than protozoans in termites with continuous access to food. Trichonympha, Pyrsonympha, and Dinenympha all ingested red paper particles at approximately the same rate. Red paper appeared significantly sooner in protozoans in termites maintained at 32°C than in those maintained at 22°C or 26°C. At 32°C, numbers of Trichonympha per gut remained constant over 96 h. Pyrsonympha and Dinenympha cells were absent or significantly reduced in number by 72 h at that temperature. These results provide insight into the environmental factors that shape the termite–protozoan symbiosis. They may aid in the development of protozoicides used to control pest termites. Received: 1 August 1997; Accepted: 26 November 1997     

Distribution and properties of endo-beta-1,4-glucanase from a lower termite, Coptotermes formosanus (Shiraki)

A multi-enzyme distribution of endo-beta-1,4-glucanase activity was found in the digestive system of a worker caste of the lower termite Coptotermes formosanus (Shiraki) by zymogram analysis. Its distribution analysis demonstrated that about 80% of this activity was localized in salivary glands from where only one component (EG-E) was secreted into the digestive tract. EG-E was isolated by a combination of chromatographic and electrophoretic techniques. Its molecular mass, optimal pH and temperature, isoelectric point, and Km were 48 kDa, 6.0, 50 degrees C, 4.2, and 3.8 (mg/ml on carboxymethylcellulose), respectively. EG-E hydrolyzed cellooligosaccharides with a degree of polymerization of 4 and larger, and had low activity on crystalline cellulose. Main reaction products from low molecular weight cellulose were cellobiose and cellotriose. The N-terminal amino acid sequence of EG-E has similarity with fungal endo-beta-1,4-glucanases and cellobiohydrolases of the glycosyl hydrolase family 7 rather than the other insect endo-beta-1,4-glucanases of family 9.     

Thermostable xylanolytic enzymes from Rhodothermus marinus grown on xylan

The thermophilic eubacterium Rhodothermus marinus was cultivated in a fermentor and studied with respect to activities of induced xylanolytic enzymes. Growth in the fermentor on xylan occurred with a maximum specific growth rate of 0.43 h^–1 for a batch culture. The final cell concentration was 4 g cell dry weight (CDW)/l for cells grown on xylan compared to 2 g CDW/l for cells grown without xylan in the cultivation medium. At least two xylanolytic enzymes, endo-1,4--xylanase and xylan 1,4--xylosidase, were secreted into the culture medium when cells were cultivated on xylan. Of the three cellulolytic enzymes tested for activity, -glucosidase activity was in the range of the xylanolytic enzyme activities whereas cellulose-1,4--cellobiosidase and cellulase activities were hardly detectable. The expression of endo-1,4--xylanase activities during cultivation indicates the existance of more than one xylanase in R. marinus. This is also observed in fractions from gel filtration. The xylanolytic enzymes are heat-stable. At 90°C and at pH 7.0 the half-life of the endo-1,4--xylanase was about 14 h and that of xylan 1,4--xylosidase was 45 min. Correspondence to: L. Dahlberg     

Cellulolytic environment in the midgut of the wood-feeding higher termite Nasutitermes takasagoensis

Unlike lower termites, xylophagous higher termites thrive on wood without the aid of symbiotic protists. In the higher termite Nasutitermes takasagoensis, both endogenous endo-β-1,4-glucanase and β-glucosidase genes are expressed in the midgut, which is believed to be the main site of cellulose digestion. To further explore the detailed cellulolytic system in the midgut of N. takasagoensis, we performed immunohistochemistry and digital light microscopy to determine distributions of cellulolytic enzymes in the salivary glands and the midgut as well as the total cellulolytic activity in the midgut. Although cellulolytic enzymes were uniformly produced in the midgut epithelium, the concentration of endo-β-1,4-glucanase activity and luminal volume in the midgut were comparable to those of the wood-feeding lower termite Coptotermes formosanus, which digests cellulose with the aid of hindgut protists. However, the size of ingested wood particles was considerably larger in N. takasagoensis than that in C. formosanus. Nevertheless, it is possible that the cellulolytic system in the midgut of N. takasagoensis hydrolyzes highly crystalline cellulose to a certain extent. The glucose produced did not accumulate in the midgut lumen. Therefore, the present study suggests that the midgut of the higher termite provides the necessary conditions for cellulolysis.     

Rice straw-decomposing fungi and their cellulolytic and xylanolytic enzymes

Filamentous fungi colonizing rice straw were collected from 11 different sites in Korea and were identified based on characterization of their morphology and molecular properties. The fungi were divided into 25 species belonging to 16 genera, including 14 ascomycetes, one zygomycete, and one basidiomycete. Fungal cellulolytic and xylanolytic enzymes were assessed through a two-step process, wherein highly active cellulase- and/or hemicellulaseproducing fungi were selected in a first screening step followed by a second step to isolate the best enzymeproducer. Twenty-five fungal species were first screened for the production of total cellulase (TC), endo-beta-1,4 glucanase (EG), and endo-beta-1,4 xylanase (XYL) using solid-state fermentation with rice straw as substrate. From this screening, six species, namely, Aspergillus niger KUC5183, A. ochraceus KUC5204, A. versicolor KUC5201, Mucor circinelloides KUC6014, Trichoderma harzianum 1 KUC5182, and an unknown basidiomycete species, KUC8721, were selected. These six species were then incubated in liquid Mandels' media containing cellulose, glucose, rice straw, or xylan as the sole carbon source and the activities of six different enzymes were measured. Enzyme production was highly influenced by media conditions and in some cases significantly increased. Through this screening process, Trichoderma harzianum 1 KUC5182 was selected as the best enzyme producer. Rice straw and xylan were good carbon sources for the screening of cellulolytic and xylanolytic enzymes.     

Xylan-degrading enzymes of the yeast Cryptococcus albidus. Identification and cellular localization

During growth on wood beta-1,4-xylans the yeast Cryptococcus albidus produced at least two enzymes which convert the polysaccharide to xylose catabolized by the cells. The enzyme almost completely secreted into culture fluid was identified as an endo-1,4-beta-xylanase. The function of the extracellular beta-xylanase is to hydrolyze xylan to oligosaccharides, mainly to xylobiose and xylotriose, which enter the cell where they are split by the second identified enzyme, a cell-bound beta-xylosidae (xylobiase). Aryl beta-xylosidase activity detected in the culture fluid was snown to be due to low affinity of beta-xylanase for p-nitrophenyl beta-D-xylopyranoside. This property of beta-xylanase was preserved after purification of the enzyme by chromatography on DEAE-cellulose, CM-Sephadex and Biogel A 1.5 m or Biogel P 100. Purified beta-xylanase exhibited certain microheterogeneity after polyacrylamide gel electrophoresis. Both extracellular beta-xylanase and intracellular beta-xylosidase were produced in much lower amounts by the cells grown on glucose than by the cells grown on xylan. This suggested that they are not produced constitutively. The investigated strain was not able to grow on cellulose and the crude and purified beta-xylanase were unable to hydrolyze cellulose or its soluble derivatives.     

Simultaneous production of endo-beta-1,4-xylanase and branched xylooligosaccharides by Thermomyces lanuginosus

When grown on beech-wood glucuronoxylan, two strains of the thermophilic fungus Thermomyces lanuginosius, IMI 84400 and IMI 96213, secreted endo-beta-1,4-xylanase of glycoside hydrolase family 11 and simultaneously accumulated an acidic pentasaccharide in the medium. The aldopentaouronic acid was purified and its structure was established by a combination of NMR spectroscopy and enzyme digestion with glycosidases as MeGlcA(3)Xyl(4). Both strains showed limited growth on wheat arabinoxylan as a carbon source. An essential part of the polysaccharide was not utilized, and it was converted to a series of arabinoxylooligosaccharides differing in the degree of polymerization. The structure of the shorter arabinoxylooligosaccharides remaining in the wheat arabinoxylan-spent medium was established using mass spectrometry and digestion with glycosidases. Xylose and linear beta-1,4-xylooligosaccharides generated extracellularly during growth on either hardwood or cereal xylan were efficiently taken up by the cells and metabolized intracellularly. The data suggest that due to a lack of extracellular beta-xylosidase, alpha-glucuronidase, and alpha-l-arabinofuranosidase, the widely used T. lanuginosus strains might become efficient producers of branched xylooligosaccharides from both types of xylans.     

Cellulose digestion in termites and cockroaches: What role do symbionts play?

• 1.1. Termites and cockroaches are excellent models for studying the role of symbionts in cellulose digestion in insects: they eat cellulose in a variety of forms and may or may not have symbionts.
• 2.2. The wood-eating cockroach, Panesthia cribrata, can be maintained indefinitely, free of microorganisms, on a diet of crystalline cellulose. Under these conditions the RQ is 1, indicating that the cockroach is surviving on glucose produced by endogenous cellulase.
• 3.3. The in vitro rate at which glucose is produced from crystalline cellulose by gut extracts from P. cribrata and Nasutitermes walkeri is comparable to the in vivo production of CO₂ in these insects, clearly indicating that the rate of glucose production from crystalline cellulose is sufficient for their needs.
• 4.4. In all termites and cockroaches examined, cellulase activity was found in the salivary glands and predominantly in the foregut and midgut. These regions are the normal sites of secretion of digestive enzymes and are either devoid of microorganisms (salivary glands) or have very low numbers.
• 5.5. Endogeneous cellulases from termites and cockroaches consist of multiple endo-β-1,4-glucanase (EC 3.2.1.4) and β-1,4-glucosidase (EC 3.2.1.21) components. There is no evidence that an exo-β-1,4-glucanase (cellobiohydrolase) (EC 3.2.1.91) is involved in, or needed for, the production of glucose from crystalline cellulose in termites or cockroaches as the endo-β-1,4-glucanase components are active against both crystalline cellulose and carboxymethylcellulose.
• 6.6. There is no evidence that bacteria are involved in cellulose digestion in termites and cockroaches. The cellulase associated with the fungus garden of M. michaelseni is distinct from that in the midgut; there is little indication that the fungal enzymes are acquired or needed. Lower termites such as Coptotermes lacteus have Protozoa in their hindgut which produce a cellulase(s) quite distinct from that in the foregut and midgut.

     

A role of xylanase,alpha-L-arabinofuranosidase,and xylosidase in xylan degradation

Renewable natural resources such as xylans are abundant in many agricultural wastes. Penicillium sp. AHT-1 is a strong producer of xylanolytic enzymes. The sequential activities of its xylanase, alpha-L-arabinofuranosidase, and beta-xylosidase on model hemicellulose oat-spelt xylan was investigated. Optimum production of the enzymes was found in culture containing oat-spelt xylan at 30 degrees C and initial pH 7.0 after 6 days. The enzymes were partially purified by ammonium sulphate fractionation and anion-exchange chromatography on DEAE-Toyopearl 650 S. The apparent molecular mass was 21 kDa, and the protein displayed an "endo" mode of action. The xylanase exhibited glycotansferase activity. It synthesized higher oligosaccharides from the initial substrates, and xylotriose was the shortest unit of substrate transglycosylated. Xylanolytic enzymes (enzyme mixture) produced by this Penicillium sp. interacted cooperatively and sequentially in the hydrolysis of oat-spelt xylan in the following order: alpha-L-arabinofuranosidase --> xylanase --> beta-xylosidase. All three enzymes exhibited optimal activity under the same conditions (temperature, pH, cultivation), indicating that they alone are sufficient to completely depolymerize the test xylan. Results indicate that the xylanolytic enzyme mixture of Penicillium sp. AHT-1 could be useful for bioconversion of xylan-rich plant wastes to value-added products.     

Synthesis and regulation of D-xylanase from Cellulomonas flavigena wild type and a mutant

The xylanolytic system from Cellulomonas flavigena was enhanced by adding cellulose to the growth medium. The Solka floc:xylan (60:40 w/w) mixture induced xylanase synthesis by more than 3-fold over that induced by growing C. flavigena, wild type and its mutant PN-120 on pure xylan. The hydrolysis pattern of sugar cane bagasse and xylan indicated the presence of debranching endo-;-xylanase activity.     

Xylanase B and an arabinofuranosidase from Pseudomonas fluorescens subsp. cellulosa contain identical cellulose-binding domains and are encoded by adjacent genes.

The complete nucleotide sequence of the Pseudomonas fluorescens subsp. cellulosa xynB gene, encoding an endo-beta-1,4-xylanase (xylanase B; XYLB) has been determined. The structural gene consists of an open reading frame (ORF) of 1775 bp coding for a protein of Mr 61,000. A second ORF (xynC) of 1712 bp, which starts 148 bp downstream of xynB, encodes a protein, designated xylanase C (XYLC), of Mr 59,000. XYLB hydrolyses oat spelt xylan to xylobiose and xylose, whereas XYLC releases only arabinose from the same substrate. Thus XYLB is a typical xylanase and XYLC is an arabinofuranosidase. Both enzymes bind to crystalline cellulose (Avicel), but not to xylan. The nucleotide sequences between residues 114 and 931 of xynB and xynC were identical, as were amino acid residues 39-311 of XYLB and XYLC. This conserved sequence is reiterated elsewhere in the P. fluorescens subsp. cellulosa genome. Truncated derivatives of XYLB and XYLC, in which the conserved sequence had been deleted, retained catalytic activity, but did not exhibit cellulose binding. A hybrid gene in which the 5' end of xynC, encoding residues 1-110 of XYLC, was fused to the Escherichia coli pho A' gene (encodes mature alkaline phosphatase) directed the synthesis of a fusion protein which exhibited alkaline phosphatase activity and bound to cellulose.     

Bacillus pumilus WL-11木聚糖酶A的纯化、鉴定及其底物降解方式

对一株BacilluspumilusWL_11木聚糖酶的纯化、酶学性质及其底物降解模式进行了研究。经过硫酸铵盐析、CM_Sephadex及SephadexG_75层析分离纯化,获得一种纯化的WL_11木聚糖酶A ,其分子量为2 6 0kD ,pI值9 5 ,以燕麦木聚糖为底物时的表观Km 值为16 6mg mL ,Vmax值为12 6 3μmol (min·mg)。木聚糖酶A的pH稳定范围为6 0至10 4 ,最适作用pH范围则在7 2至8 0之间,是耐碱性木聚糖酶;最适作用温度为4 5℃～5 5℃,在37℃、4 5℃以下时该酶热稳定性均较好;5 0℃保温时,该酶活力的半衰期大约为2h ,在超过5 0℃的环境下,该酶的热稳定较差,5 5℃和6 0℃时的酶活半衰期分别为35min和15min。WL_11木聚糖酶A对来源于燕麦、桦木和榉木的可溶性木聚糖的酶解结果发现,木聚糖酶A对几种不同来源的木聚糖的降解过程并不一致。采用HPLC法分析上述底物的降解产物生成过程发现木聚糖酶A为内切型木聚糖酶,不同底物的降解产物中都无单糖的积累,且三糖的积累量都较高;与禾本科的燕麦木聚糖底物降解不同的是,木聚糖酶A对硬木木聚糖降解形成的五糖的继续降解能力较强。采用TLC法分析了WL_11粗木聚糖酶降解燕麦木聚糖的过程,结果表明燕麦木聚糖能够被WL_11粗木聚糖酶降解生成系列木寡糖,未检出木糖,这说明WL_11主要合成内切型木聚     

Production and Properties of Extracellular Endoxylanase from Neurospora crassa

Neurospora crassa 870 produced 14 and 0.025 U of extracellular xylanase (1,4-beta-d-xylan xylanohydrolase; EC 3.2.1.8) and beta-xylosidase (1,4-beta-xylan xylohydrolase; EC 3.2.1.37) per ml, respectively, in 4 days when commercial xylan was used as a carbon source. The effects of pH and carbon sources on xylanase production by N. crassa are discussed. Two xylanases (I and II) were purified and had pI values of 4.8 and 4.5 and molecular weights of 33,000 and 30,000. The maximum degree of hydrolysis of xylan by the extracellular culture broth was 66% in 4 h. The end products of xylan hydrolysis by xylanase I and II showed the presence of xylose, xylobiose, xylotriose, xylotetraose, xylopentose, and arabinose, indicating that they are endoxylanases capable of hydrolyzing 1,3-alpha-l-arabinofuranosyl branch points. Both xylanases showed activity toward carboxymethyl cellulose but no activity toward para-nitrophenyl-beta-d-xyloside or laminarin. Xylanase I showed appreciable activity toward para-nitrophenyl-beta-d-glucoside, whereas xylanase II was inactive.