The extracellular constitutive production of chitin deacetylase in Metarhizium anisopliae: possible edge to entomopathogenic fungi in the biological control of insect pests

The possible contribution of extracellular constitutively produced chitin deacetylase by Metarhizium anisopliae in the process of insect pathogenesis has been evaluated. Chitin deacetylase converts chitin, a beta-1,4-linked N-acetylglucosamine polymer, into its deacetylated form chitosan, a glucosamine polymer. When grown in a yeast extract-peptone medium, M. anisopliae constitutively produced the enzymes protease, lipase, and two chitin-metabolizing enzymes, viz. chitin deacetylase (CDA) and chitosanase. Chitinase activity was induced in chitin-containing medium. Staining of 7.5% native polyacrylamide gels at pH 8.9 revealed CDA activity in three bands. SDS-PAGE showed that the apparent molecular masses of the three isoforms were 70, 37, and 26 kDa, respectively. Solubilized melanin (10microg) inhibited chitinase activity, whereas CDA was unaffected. Following germination of M. anisopliae conidia on isolated Helicoverpa armigera, cuticle revealed the presence of chitosan by staining with 3-methyl-2-benzothiazoline hydrazone. Blue patches of chitosan were observed on cuticle, indicating conversion of chitin to chitosan. Hydrolysis of chitin with constitutively produced enzymes of M. anisopliae suggested that CDA along with chitosanase contributed significantly to chitin hydrolysis. Thus, chitin deacetylase was important in initiating pathogenesis of M. anisopliae softening the insect cuticle to aid mycelial penetration. Evaluation of CDA and chitinase activities in other isolates of Metarhizium showed that those strains had low chitinase activity but high CDA activity. Chemical assays of M. anisopliae cell wall composition revealed the presence of chitosan. CDA may have a dual role in modifying the insect cuticular chitin for easy penetration as well as for altering its own cell walls for defense from insect chitinase.     

Vitellin of the sweet potato whitefly,Bemisia tabaci: Biochemical characterization and titer changes in the adult

SDS-PAGE of the sweet potato whitefly (Bemisia tabaci) egg extract showed one major band (approximately 190 kDa) and two minor bands (approximately 75 kDa and 67 kDa). A distinct 190 kDa band was also present in male extract. On SDS gels the vitellin band of the greenhouse whitefly (Trialeurodes vaporarium) was larger, about 220 kDa. The native molecular mass of sweet potato whitefly vitellin was estimated to be 375 kDa using 4–20% native pore-limiting gel electrophoresis. Its isoelectric point was estimated to be 7.3 using isoelectric focusing. Two-dimensional gel electrophoresis and densitometry were used to estimate vitellin subunit composition; the data suggest that the sweet potato whitefly vitellin is likely to be a 380 kDa native molecule formed by two 190 kDa subunits. The two minor bands (75 kDa and 67 kDa) may be breakdown products of the native vitellin. This conclusion was supported by a Western blot of an SDS-PAGE gel of partially degraded female and egg extracts, which showed that polyclonal antiserum raised against the 190 kDa polypeptide recognized the 75 kDa and 67 kDa bands. Seven hybridoma cell lines secreting monoclonal antibodies against the 190 kDa band were screened, and one of them (S1A2G9H2) was mass produced. The antibody recognized the 190 kDa band in a Western blot. All the screened monoclonal antibodies were female and egg-specific by ELISA and/or Western blot, suggesting that the 190 kDa band in male extract was not a vitellin. A sensitive ELISA was established that could detect as little as 1/40 of an egg equivalent of vitellin using the monoclonal antibody from S1A2G9H2. Profiles of female sweet potato whitefly reproductive activities (egg laying, amount of vitellin in the female, and total vitellin produced by a female) within 2 days after eclosion were determined. Arch. Insect Biochem. Physiol. 34:223–237, 1997. © 1997 Wiley-Liss, Inc.     

Chitin deacetylase activity of the rust Uromyces viciae-fabae is controlled by fungal morphogenesis

Abstract The broad bean rust fungus Uromyces viciae-fabae exhibits chitin only on surfaces of those infection structures which in nature are formed on the plant cuticle, but not on those differentiated in the intercellular space of the host leaf. Chitin deacetylase, an enzyme which converts chitin to chitosan, has been studied during in vitro differentiation of rust infection structures. Radiometrie and gel electrophoretic analyses of crude extracts and extracellular washing fluids have shown that chitin deacetylase activity massively increases when the fungus starts to penetrate through the stomata, and that formation of the enzyme is strictly differentiation-specifically controlled. The extracellular portion of chitin deacetylase activity was about 53% in 24-h-old differentiated infection structures. Five isoforms with apparent molecular masses of 48.1, 30.7, 25.2, 15.2 and 12.7 kDa were detectable after substrate SDS-PAGE. The enzyme is temperature-sensitive and has a pH optimum of 5.5-6.0.     

Partial characterization ofPseudomonas fluorescens subsp.cellulosa endoglucanase activity produced inEscherichia coli

Summary The recombinant plasmid, pPFC4, which carriesPseudomonas fluorescens subsp.cellulosa chromosomal DNA was previously isolated on the basis of its ability to direct the expression of endoglucanase inEscherichia coli. In the present study, some physical and chemical properties of this activity were characterized. The major portion (78.4%) of the endoglucanase activity is found in the periplasmic space ofE. coli. This plasmid-encoded endoglucanase has a pH optimum of approximately 6.0 and a temperature optimum of approximately 50°C. With carboxymethylcellulose-zymograms, after polyacrylamide gel electrophoresis, periplasmic extracts fromE. coli carrying pPFC4 show six distinct bands with endoglucanase activity. The molecular mass of the major endoglucanase band is approximately 29 kDa while the remaining bands with endoglucanase activity range from 48 to 100 kDa. Although the basis of this heterogeneity is not known, the DNA insert of pPFC4 that encodes endoglucanase activity is not large enough to contain six separate genes; hence, the observed array of endoglucanases may result from post-translational modification of one or two primary gene products.     

Identification and Characterization of a Chitin-binding Protein Purified from Coelomic Fluid of the Lugworm Arenicola marina Defining a Novel Protein Sequence Family

We have isolated a novel type of lectin named Arenicola marina lectin-1 (AML-1) from the lugworm A. marina. The lectin was purified from the coelomic fluid by affinity chromatography on a GlcNAc-derivatized column and eluted with GlcNAc. On SDS-PAGE, AML-1 showed an apparent molecular mass of 27 and 31 kDa in the reduced state. The N-terminal amino acid sequences were identical in these two bands. In the unreduced state, a complex band pattern was observed with bands from 35 kDa to more than 200 kDa. Two different full-length clones encoding polypeptides of 241 and 243 amino acids, respectively, were isolated from a coelomocyte cDNA library. The two clones, designated AML-1a and AML-1b, were 92% identical at the protein level and represent a novel type of protein sequence family. Purified AML-1 induced agglutination of rabbit erythrocytes, which could be inhibited by N-acetylated saccharides. Recombinant AML-1b showed the same band pattern as the native protein, whereas recombinant AML-1a in the reduced state lacked a 27 kDa band. AML-1b bound GlcNAc-derivatized columns and chitin, whereas AML-1a did not bind to these matrices. Immunohistochemical analysis revealed that AML-1 is expressed by coelomocytes in the nephridium and in round cells in the epidermis and in eggs. Moreover, AML-1 expression was up-regulated in response to a parasitic infection. We conclude that AML-1 purified from coelomic fluid is encoded by AML-1b and represents a novel type of protein family that binds acetylated components.     

Identification and localization of calcium-dependent protease II inNeurospora crassa andUromyces appendiculatus

Summary The existence of Ca²⁺-dependent protease II in crude extracts ofNeurospora crassa andUromyces appendiculatus was demonstrated by immunoblotting using specific antibodies. In both extracts two immunoreacting bands were observed. The molecular mass of the major band inN. crassa corresponded to 37 kDa, while that inU. appendiculatus was 43 kDa, similar to that previously reported forAllomyces arbuscula. Immunofluorescence of the enzyme was predominantly localized in the apical regions of germlings and growing hyphae, suggesting a functional role for the enzyme in hyphal growth.     

Pea saponins in the pea-Fusarium solani interaction

Saponin-like compounds isolated fromPisum sativum were tested for antifungal activity, effect on pea tissue, and effect on chitin and chitosan synthesis inFusarium solani. Growth ofFusarium solani f. sp.phaseoli and f. sp.pisi macroconidia was inhibited by saponins at concentrations of 150 and 300 μg/ml, respectively. Pod endocarp tissue treated with saponins showed temporary reduction in cell viability (esterase activity); however, there was no significant reduction in resistance toF. solani f. sp.phaseoli, normally incompatible on peas. Macroconidia germinated in the presence of saponin showed decreased incorporation ofN-[³H]acetylglucosamine into chitin and chitosan at concentrations as low as 32 μg/ml. Thus, a reduction in chitin and chitosan synthesis may be associated with inhibition of fungal growth. Saponins may contribute to the disease resistance of peas     

Urate-mediated regulation of urate oxidase inChlamydomonas reinhardtii

Summary Expression of uncase (urate oxidase) fromChlamydomonas reinhardtii has been investigated by using specific polyclonal antibodies. By Western blot analyses performed under nondenaturing conditions, a 124 kDa protein band corresponding to active uricase was detected in protein extracts from cells cultured with urate or nitrogen-starved cells. This protein band was absent in cells cultured with ammonium. Besides the 124 kDa band, the antibodies also reacted with a massive protein band, with an apparent molecular mass of 500 kDa, that was detected in all nutritional conditions assayed. In vitro, inactive uricase from cells grown with ammonium was activated by incubation in presence of urate. The appearance of uricase activity in vitro coincided with a decrease of the 500 kDa protein and an increase of the 124 kDa band corresponding to the active enzyme. We suggest that a posttranslational regulation of uricase synthesis takes place inC. reinhardtii, and that urate may be responsible for the assembly or maturation of inactive precursors to form the active uricase.     

Cytosolic and membrane-bound chitinases of two mucoraceous fungi: a comparative study.

Chitinases isolated from membrane and cytosolic fractions of two mucoraceous fungi, Choanephora cucurbitarum and Phascolomyces articulosus, were investigated. The membrane-bound chitinase was isolated by Bio-Gel P-100 and DEAE Bio-Gel A chromatographic techniques. On SDS-PAGE the chitinase from both fungi migrated as a single band of M(r) 66 kDa. The cytosolic chitinase from the mycelial extracts of these fungi was separated by heat treatment, ammonium sulphate precipitation, and by affinity chromatography with regenerated chitin. SDS-PAGE showed two bands for each fungus with M(r) of 69.5 and 55 kDa in C. cucurbitarum and M(r) 69.5 and 53 kDa in Ph. articulosus. Chitinases, membrane bound or cytosolic, hydrolyzed regenerated chitin, colloidal chitin, glycol chitin, N,N'-diacetylchitobiose, and N,N',N"-triacetylchitotriose. Heavy metals, inhibitors, and N-acetylglucosamine inhibited chitinase activity, whereas trypsin and an acid protease enhanced its activity. Chitinase preparations showed lysozyme activity that was inhibited by histamine but not by N-acetylglucosamine. There was no N-acetylglucosamanidase activity, but beta-1,3 glucanase activity was found in cytosolic preparations only. Despite slight differences in their molecular mass, both the membrane-bound and cytosolic chitinases showed similarities in substrate utilization, response to inhibitors, and activation by trypsin and acid protease; pH and temperature optima also were similar.     

Seed protein polymorphism in Phlox pilosa (Polemoniaceae)

A survey of seed protein profiles inP. pilosa revealed three bands in addition to the typical complement in northern Illinois and Indiana populations. One of the novel proteins has the same (fast) migration velocity as a protein in the standard complement ofP. glaberrima. Both of these proteins hybridize with one of the standardP. pilosa proteins to produce two additional bands. The fast protein varies in frequency between populations and is most prevalent in areas where there is the greatest potential for hybridization withP. glaberrima. These data suggest that the fast band ofP. pilosa may have been introduced fromP. glaberrima despite strong incompatibility barriers, and increased in frequency through selective accumulation.     

Clostridium chauvoei: Immunological characterization of Antigenic Preparations

Clostridium chauvoei is an anaerobic and sporulated bacterium which produces blackleg in cattle, sheep and other ungulates. Elsewhere we have studied the immunogenic power of the following antigenic preparations in mice: cellular extract (CE), a suspension of flagella (F), formolinized cells (FC) and heated cells (ΘC), and the immunogenic power of the CE in guinea pigs. The aim of this work was to characterize these immunogenic preparations by SDS-PAGE and immunoblotting. When SDS-PAGE was carried out F showed four protein bands of 86, 59, 51 and 47 kDa; CE showed 10 major bands of 86, 75, 62, 55, 47, 41, 30, 28, 25 and 16 kDa; FC six major bands of 86, 75, 62, 53, 46 and 16 kDa; and ΘC 11 bands, the major ones having 80, 41 and 16 kDa. All four preparations share the flagellar protein band of 47 kDa except ΘC. The formol-treatment reduces the number of the protein bands when it is compared with those of CE. The heat-treatment also reduces the number of the bands compared with CE and FC. When immunoblotting is analysed it can be seen that CE induces the antibodies that react with the 47 kDa protein band, the same ones are induced by FC but not by ΘC. On the other hand, CE induces antibodies that react with the 86 kDa protein band, which is believed to be a polymeric form of a flagellin monomer. According to these results and those obtained in the protection tests, we can conclude that: (1) flagella are in part, responsible for the immunogenicity of the strain; (2) the buffer used to obtain the CE extracts flagellar and somatic antigens; (3) the formol-treatment reduces the protein bands compared with CE, but retains their immunogenic power; and (4) the heat-treatment reduces immunogenic power which is seen by its minor protein bands number and by its lower immunoprotective power.     

Relationship between the acid phosphatases of the Kurloff body and the major 30–35 kDa glycoproteins of the Kurloff cell

This study deals with the acid phosphatase (AcPase) of the Kurloff body (KB), a large (10 μm diameter) periodic acid-Schiff-positive lysosomal inclusion body present in Kurloff cells (KC). KC AcPase were extracted, with similar yields, either with non-ionic detergent solution or after Dounce homogenization in low ionic strength buffer suggesting that they mainly correspond to hydrosoluble AcPase. After DEAE-cellulose chromatography of such crude Dounce-extracts, 97% of KC AcPase activity was recovered in the unbound glycoprotein fraction (peak I). The main protein content consisted of, as testified by SDS-PAGE analysis, major KC glycoproteins of 30–35 kDa. Thus, KC AcPase, and particularly sialoAcPase, may be assumed to correspond to these N-glycosylproteins among which the presence of α(2,6) sialoglycoproteins was previously established. Following electrophoresis on a 4–15% gradient native polyacrylamide gel or isoelectric focusing, the two size populations (200 kDa and 500 kDa) and up to 20 isoforms of KC AcPase were respectively detected by zymography in peak I. After Clostridium-derived sialidase digestion of peak I, the highly active bands observed at pH 3.5 – 5.2 disappeared. These zymographic patterns were similar to those obtained with crude extracts. After Concanavalin A (ConA)-Sepharose chromatography of peak I, the single ConA-bound glucosamine-labelled peak, eluted at 200 methyl-α-d-mannopyranose, contained the AcPase activity while the ConA-unbound peak was devoid of any acid phosphatase activity. After SDS-PAGE analysis, the ConA-bound fraction appeared to correspond only to a single broad protein band in the 30–35 kDa zone. The Sambucus nigra agglutinin-reactivity of the latter band confirmed the α(2,6) sialylation pattern of KC AcPase. Moreover, their strong binding to ConA-Sepharose suggests that they could only correspond to the hybrid type of the N-glycosylproteins, since they could not correspond to the oligomannosidic type considering the absence of Galanthus nivalis agglutinin-positive structures.     

Purification and characterisation of a gentisate 1,2-dioxygenase fromRalstonia solanacearum GMI 1000

The gene encoding gentisate 1,2-dioxygenase from a soil-borne Gram-negative bacterium,Ralstonia solanacearum GMI 1000, was cloned and overexpressed inEscherichia coli. The resulting product incorporated a (His) 6 tag was purified to homogeneity from the harvested cell extracts by affinity chromatography. SDS-PAGE showed that the polypeptide exhibited an approximate molecular mass of 38 kDa. The optimal temperature and pH for gentisate cleavage catalysed by the enzyme were 30 °C and 8.0, respectively. TheK _m of the enzyme was determined to be 56 μM. ThepI is 4.6–4.8. Moreover, site-directed mutagenesis revealed that His105, His 107, and His 146 are the crucial residues involved in the catalytic activity of gentisate 1,2-dioxygenase fromRalstonia solanacearum GMI 1000.     

Changes of soluble proteins in leaf and thylakoid exposed in high saline condition of a mangrove taxa Bruguiera gymnorrhiza

One-year-old seedlings of Bruguiera gymnorrhiza (L) Savingay were exposed to 500 mM NaCl for 6d under hydroponic culture condition to characterize the changes in leaf and thylakoid protein profiles in response to short-term salt exposures. Significant changes in leaf dry mass, chlorophylls and soluble leaf proteins were observed in short term of salt exposures, as it happens under tidal situations in nature. Chlorophyll a/b ratio showed decrease of light harvesting efficiency in salt treatment. Total soluble proteins in leaves were extracted from control and NaCl-treated plants at 2d intervals and were analyzed by SDS-PAGE. Intensity of several protein bands of different molecular mass of leaf protein profile ranging from 10 to 86 kDa (10, 16, 23, 33, 37, 42, 44, 50 and 86 kDa) were decreased due to high salt treatment. Out of these, 16, 23 and 33 kDa protein bands decreased dramatically from 1–3 fold but recovered in 7d growth, except the 33 kDa band. SDSPAGE profile of thylakoid protein revealed that both number and the intensity of several protein bands got altered by salt concentration. However, 33 kDa protein band of thylakoid reappeared in recovery that might not be of the same characteristics with same molecular mass as shown in total leaf protein profile. The numbers of major bands found in SDS-PAGE were reduced when analyzed in urea-SDS-PAGE to minimize protein aggregations by high salt. It was noted that 47 kDa disappeared while some proteins of apparent molecular mass like 23 kDa, 33 kDa, 37 kDa and 50 kDa degraded to minor bands. Partial restoration of protein bands occurred when the salt-treated plants were brought back to initial growth condition. These results clearly demonstrate that short term high salt concentration could cause major alterations to photosynthetic apparatus of a true non salt-secreting tree mangrove Bruguiera gymnorrhiza and adapted against fluctuation of salinity by altering leaf protein pool relatively more than the thylakoid proteins.Key words: Bruguiera gymnorrhiza, Mangrove, Polypeptides, Salt shock, Sodium chloride, Thylakoid     

Immunoanalysis of dehydrins in Araucaria angustifolia embryos

The aim of this study was to describe the dehydrin content of mature Araucaria angustifolia embryos, a species of endangered and economically important conifers, native to southern Brazil, northeastern Argentina, and eastern Paraguay. The A. angustifolia seeds have been categorized as recalcitrant. Dehydrins were studied by western blot analysis and in situ immunolocalization microscopy using antibodies raised against the K segment, a highly conserved lysine-rich 15-amino acid sequence extensively used to recognize proteins immunologically related to the dehydrin family. Western blot analysis of the heat-stable protein fraction, as estimated by 15 % SDS-PAGE, revealed three main bands of approximately 20-, 26-, and 29-kDa; when 17.5 % SDS-PAGE was used, each band resolved into two other bands. Two thermosensitive dehydrin bands of around 16 and 35 kDa were common to the axis and cotyledons, and another thermosensitive band, with molecular mass of approximately 10 kDa, was present in the cotyledons only. Following alkaline phosphatase (AP) treatment, a gel mobility shift was detected for each one of the four main bands that can be due to phosphorylation. Dehydrins were detected in all axis and cotyledon tissues using in situ immunolocalization microscopy. At the subcellular level, dehydrins were immunolocalized in the nuclei, protein bodies, and microbodies. In the nucleus, dehydrins were found to be associated with chromatin. We concluded that the gel mobility shift for the four main bands (probably due to phosphorylation), the presence of thermosensitive bands, and the specific localizations in nuclei and protein bodies provide key starting points to understand the function of dehydrins in the embryo cells of this species.     

Characterization and cloning of chitin deacetylases from Rhizopus circinans

Chitin deacetylase catalyzes hydrolysis of the acetamido groups of N-acetylglucosamine of chitin in fungal cell walls. Here a chitin deacetylase secreted by Rhizopus circinans was purified to homogeneity and partially characterized. The enzyme exhibits an apparent molecular weight of approximately 75kDa. At 37 degrees C it shows optimal activity at pH 5.5-6. Its pH stability and thermal stability are good. Mn(2+) and Mg(2+) slightly enhance the activity of the enzyme and Cu(2+) strongly inhibits it. An R. circinans cDNA library was constructed and screened with a homologous probe synthesized by RT-PCR or with synthetic primers derived from the N-terminal amino-acid sequence of the native purified chitin deacetylase. Three chitin deacetylase cDNAs (RC, D2, and I3/2) were isolated from the cDNA library and sequenced. These cDNAs exhibit features characteristic of chitin deacetylase sequences: the presence of a polysaccharide deacetylase domain, a metal-binding triad, the conserved catalytic residues, and high homology with various chitin deacetylase genes. The cDNAs were cloned in a Pichia pastoris expression system and produced as polyhistidine-tagged proteins. Only one recombinant enzyme (called RC) was active under the tested conditions. It was purified to homogeneity in a single step and further characterized. The protein showed an apparent molecular mass of approximately 75kDa and, like the native enzyme, showed optimal activity at pH 5.5-6 at 37 degrees C. It was strongly inhibited by Cu(2+). The isolation of several chitin deacetylase cDNAs from the same microorganism is discussed.     

Cellulosome-like entities inBacteroides cellulosolvens

Cellulosome-like complexes were identified in the broth and sonic extracts of cellobiose-and cellulose-grown cells ofBacteroides cellulosolvens. The extracellular fractions contained three to four major polypeptides and several minor polypeptide bands that were localized in two major gel filtration peaks indicating average molecular weights of about 700 kDa and >10 MDa. A relatively large molecular weight component (M_r 230 kDa) was found to contain carbohydrate, but no apparent enzymatic activity of its own could be detected. The cell sonicate displayed a more complicated polypeptide profile, and glycosylated polypeptides were larger (ca. 310 and 290 kDa) than that of the extracellular fraction. The 230-kDa extracellular component interacted strongly with the GSI isolectin fromGriffonia simplicifolia, exhibited immunochemical cross-reactivity with the S1 subunit of the cellulosome fromClostridium thermocellum, and displayed anomalous pH- and salt-dependent migratory behavior in SDS-PAGE. Taken together, this evidence strongly suggests a structural similarity between the glycoconjugates of these two distinct cellulolytic bacteria. A major 84-kDa polypeptide was identified as a xylanase, and a 50-kDa polypeptide displayed endoglucanase activity. Additional biochemical and cytochemical evidence indicated that cellulosome-like cellulolytic complexes are associated with the cell surface in this bacterium.     

Extracellular proteinase and chitinase produced by a culture ofStreptomyces kurssanovii

Extracellular enzymes—a chitinase and a proteinase with molecular weights 22 and 32 kDa, respectively—were isolated fromStreptomyces kurssanovii cells. After purification on modified regenerated chitin, the enzymes were virtually homogeneous according to denaturing PAGE. Both enzymes were found to degrade chitosan.     

Chitinolytic activities of <Emphasis Type="Italic">Clostridium</Emphasis> sp. JM2 isolated from stool of human administered per orally by chitosan

The novel chitinolytic bacterium Clostridium beijerinckii strain JM2 was isolated from the stool of healthy volunteers supplied daily per orally with 3 g of chitosan. The bacterium grown on colloidal chitin produced a complete array of chitinolytic enzymes. Significant activities of endochitinase, exochitinase and chitosanase were excreted into the medium (301, 282 and 268 nkat/μg protein, respectively). The high cellular activity of N-acetyl-β-glucosaminidase (NAGase) and chitosanase were detected (732.4 and 154 nkat/μg protein, respectively). NAGase activity represented the main activity associated with the cellular fraction. The activities of both enzymes tested increased from 20 to 50 °C; the optimum reaction temperature estimated being 50 °C. Endochitinase as well as NAGase showed an activity in the pH interval of 4.0–8.0; the optimum pH values were 6.5 and 6.0, respectively. The extracellular endochitinase complex consisted of six isoenzymes with molar mass of 32–76 kDa; in the cellular fraction five bands with molar mass of 45–86 kDa were detected. Exochitinase activity was demonstrated in the form of three bands (with molar mass of 30–57 kDa), NAGase activity displayed one band of 45 kDa.     

Fts insertional mutant of Salmonella typhimurium

Abstract Cellulolytic and xylanolytic polysaccharidase enzyme activities from the multi-enzyme complex of Postia placenta MAD 698 were dissociated. Decayed wood extracts were fractionated by methyl-hydrophobic interaction chromatography and analyzed for reducing sugars and by viscosimetry and Western blot. Fractionated endoglucanases had molecular masses of 35 and 40 kDa on SDS-PAGE. Western blots probed with anti-endoglucanase and anti-xylanase antibodies revealed two endoglucanase bands at 35 and 40 kDa, but no commonality with the major xylanase band at 36 kDa. Anti-endoglucanase antibody inhibited endoglucanase enzyme activity.