In situ reversible aggregation of extracellular cellobiase in the filamentous fungus Termitomyces clypeatus

Cellobiase (E.C. 3.2.1.21), is a widely exploited industrial glycosidase with a major role in biofuel industry. Its stability and shelf life are major bottlenecks in achieving a superior formulation for industry. In the filamentous fungus Termitomyces clypeatus, the enzyme is secreted in a co-aggregated form with sucrase; the separation of this co-aggregation results in substantial loss of the enzyme??s activity. The aim of the present study was to examine the mode of aggregation of the secreted cellobiase-sucrase coaggregate and its role in the stabilization of cellobiase. Transmission electron microscopy and dynamic light scattering of purified co-aggregates revealed reversible, concentration driven self-aggregation of the extracellular enzymes to form larger entities. However, the intracellular enzyme aggregates were rigid, non-interacting, and possessed a higher percentage of disulphide bonds. Circular dichroic spectra of the two coaggregates indicated no significant difference in secondary structures. Self-association increased the stability of extracellular aggregates towards heat by 1.5 fold, SDS by 4 ?? 7 fold, and chaotropic agents, by 1.5 ?? 2 fold, than the intracellular counterpart. The K_m of extracellular aggregate varied between 0.29 and 0.45 mM as a result of spontaneous aggregation and disaggregation, whereas that of intracellular aggregate was 0.22 mM irrespective of its concentration status. In situ detection of cellobiase in native PAGE revealed two activity bands of the extracellular enzyme, which indicated a minimum of two active dissociated aggregate species, as compared to a single band for the intracellular enzyme. These studies are believed to improve the understanding of aggregation of the fungal glycosidases, which remains to be a blackbox, to increase the efficacy of these enzymes.     

Regulation of cellobiase secretion in Termitomyces clypeatus by co-aggregation with sucrase

Regulated secretory proteins are sorted via selective co-aggregation in eukaryotes. Cellobiase (C) of the filamentous fungus Termitomyces clypeatus remained co-aggregated with sucrase (S), and only one isoform of each of the enzymes was present in intra- and extracellular extracts. Kinetics of secretion of sucrase increased in vivo and in vitro in secreting (Sc) medium and decreased under non-secreting (NSc) conditions similar to those observed for cellobiase. In the Sc condition, total enzyme production and activity ratios of cellobiase and sucrase (C/S) in cell-bound, extra- and intracellular preparations increased with time and were significantly higher from those obtained in non-secretory media. It was concluded that secretion of sucrase in culture medium is under same cellular regulation as that of cellobiase, and sucrase is involved in regulating extracellular release of cellobiase through co-aggregation in the fungus. Received: 27 August 2001 / Accepted: 1 November 2001     

Secretion of cellobiase is mediated via vacuoles in Termitomyces clypeatus

The majority of cellobiase activity in Termitomyces clypeatus was localized in vacuolar fractions of the fungus under secretory and nonsecretory conditions of growth. Activities of marker proteins for subcellular organelles, e.g., vacuoles, cytosol, ER, and mitochondria, in mycelial extracts from the secreting conditions increased by approximately 20, 12, 5, and 2.5 times, respectively, as compared to those obtained from mycelium grown in nonsecreting conditions. The average size and concentration of vacuoles visualized by electron microscopy were also increased in secreting conditions in the fungus. The specific activity of cellobiase in vacuoles isolated in Ficoll-sucrose gradient, as obtained from mycelial growth in secretory medium, was more than 40 times higher in comparison to that found from nonsecretory medium. The results indicated that subcellular localization of cellobiase in vacuoles is regulated by the cellular signaling prevailing in the fungus. Mycelial extraction of intracellular proteins by hand grinding and by bead-beater from cells frozen in the presence or absence of liquid nitrogen was also compared. Maximum recovery of intracellular protein was obtained with the bead-beater under aerobic conditions in the absence of nitrogen. Highest recovery of vacuoles up to 85% was obtained by single-step ultracentrifugation of the mycelial extract of the fungus in Ficoll-sucrose gradient. The method appeared to be useful for separation of other subcellular organelles in filamentous fungi.     

Cellobiase from Termitomyces clypeatus: activity and secretion in presence of glycosylation inhibitors

In presence of the glycosylation inhibitors, 2-deoxy-d-glucose (1 mg/ml), tunicamycin (30 μg/ml), 1-deoxynojirimycin (30 μg/ml) and d-glucono-δ-lactone (1 mg/ml), total cellobiase activity, in the extracellular, intracellular and cell bound fractions, of the fungus Termitomyces clypeatus grown in 20 ml cellobiose medium (1%, w/v) increased by 50-, 1.8-, 2.4-, 1.3-fold, respectively, with respect to control medium (16.3 U). The inhibitors also stimulated secretion of 95% of the total protein in culture medium, except d-glucono-δ-lactone which released 60% of the total protein. 2-Deoxy-d-glucose (1 mg/ml) led to production of extracellular cellobiase up to 40 U/ml, whereas in absence of the inhibitors only 0.59 U/ml enzyme was detected.     

Purification and characterisation of a beta-glucosidase (cellobiase) from a mushroom Termitomyces clypeatus

A beta-glucosidase with cellobiase activity was purified to homogeneity from the culture filtrate of the mushroom Termtomyces clypeatus. The enzyme had optimum activity at pH 5.0 and temperature 65 degrees C and was stable up to 60 degrees C and within pH 2-10. Among the substrates tested, p-nitrophenyl-beta-D-glucopyranoside and cellobiose were hydrolysed best by the enzyme. Km and Vm values for these substrates were 0.5, 1.25 mM and 95, 91 mumol/min per mg, respectively. The enzyme had low activity towards gentiobiose, salicin and beta-methyl-D-glucoside. Glucose and cellobiose inhibited the beta-D-glucosidase (PNPGase) activity competitively with Ki of 1.7 and 1.9 mM, respectively. Molecular mass of the native enzyme was approximated to be 450 kDa by HPLC, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a molecular mass of 110 kDa. The high molecular weight enzyme protein was present both intracellularly and extracellularly from the very early growth phase. The enzyme had a pI of 4.5 and appeared to be a glycoprotein.     

Saccharification of xylan by an amyloglucosidase of Termitomyces clypeatus and synergism in the presence of xylanase

Summary An amyloglucosidase from a mycelial culture of the mushroom Termitomyces clypeatus hydrolysed larch wood xylan independently and synergistically with an endo-(14) xylanase of the same fungus. The glucoamylase saccharified xylan predigested with xylanase at a faster rate compared to that of xylanase acting on amylase-digested xylan. However, overall saccharification of xylan in both cases was the same. Only glucose was liberated from xylan by amylase digestion whereas xylose, xylobiose and other oligosaccharides were liberated during xylanase digestion. The synergistic response of enzyme combinations was reflected in the liberation of glucose from xylan, rather than xylose. Glucoamylase and xylanase activities on soluble and insoluble fractions of larch wood xylan with different xylose and glucose contents suggested that synergism in xylanolysis by the presence of glucoamylase was dependent on the activity of the participating xylanase on the xylan preparation. It is suggested that possibly -glucosidic linkages are present in xylan and that amyloglucosidase might be involved in xylanolysis. Correspondence to: S. Sengupta     

Increased enzyme secretion by 2-deoxy-D-glucose in presence of succinate by suppression of metabolic enzymes in Termitomyces clypeatus

Regulatory mode of secretion of proteins was detected for the industrial glycosidase, cellobiase, under secreting conditions (in presence of TCA cycle intermediates like succinate etc.) in the filamentous fungus Termitomyces clypeatus. The titers of key metabolic enzymes were investigated under secreting and non-secreting conditions of growth and compared to the corresponding production of intra and extracellular levels of cellobiase. Results were compared in presence of 2-deoxy-D-glucose, a potent glycosylation inhibitor in the secreting media. Inclusion of 2-deoxy-D-glucose in presence of succinate caused about 10 to 100 times decrease in titers of the metabolic enzymes hexokinase, fructose-1,6-bisphosphatase, isocitrate lyase and malate dehydrogenase leading to increased secretion of cellobiase by more than 100 times. The intracellular concentration of cAMP (86-fold decrease in presence of 2-deoxy-D-glucose under secreting conditions) and turnover rate of proteins also dropped significantly. In this suppressed metabolic state, a 10-fold increase in the titer of the secreted cellobiase was noticed. The results indicated elucidation of carbon catabolite repression like phenomenon in the fungus under secreting conditions which was more pronounced by 2-deoxy-D-glucose. The interdependence between secretion and regulation of metabolic enzymes will help in better understanding of the physiology of these highly adapted organisms for increasing their secretion potential of glycosidases like cellobiase with high industrial value.     

Regulation of protein secretion by mycelial culture of the mushroom Termitomyces clypeatus

Termitomyces clypeatus secreted a 24-kDa xylanase constitutively in xylan medium, but required a gluconeogenic amino acid or Krebs cycle acid for the secretion of a 56-kDa amyloglucosidase in dextrin medium. Aspartate, glutamate, succinate and fumarate all increased secretion of amyloglucosidase from 50% to >90% and enzyme production by 10-fold with little effect on xylanase production. Glutamate or succinate stimulated in vitro release of intracellular amyloglucosidase from washed mycelia in the presence of cycloheximide. Amyloglucosidase accumulated in the absence of glutamate was a high-molecular-mass protein that did not migrate in PAGE. Cellular regulation by the fungus of the secretion of amyloglucosidase is indicated.     

Evidence for an arginine-dependent route for the synthesis of NO in the model filamentous fungus Aspergillus nidulans

Nitric oxide (NO) is a signalling molecule in eukaryotic and prokaryotic organisms. NO levels transiently boost upon induction of conidiation in Aspergillus nidulans. Only one pathway for NO synthesis involving nitrate reductase has been reported in filamentous fungi so far, but this does not satisfy all the NO produced in fungal cells. Here we provide evidence for at least one additional biosynthetic pathway in A. nidulans involving l -arginine or an intermediate metabolite as a substrate. Under certain growth conditions, the addition of l -arginine to liquid media elicited a burst of NO that was not dependent on any of the urea cycle genes. The NO levels were controlled by the metabolically available arginine, which was regulated by mobilization from the vacuoles and during development. In vitro assays with protein extracts and amino acid profiling strongly suggested the existence of an arginine-dependent NO pathway analogous to the mammalian NO synthase. Addition of polyamines induced NO synthesis, and mutations in the polyamine synthesis genes puA and spdA reduced the production of NO. In conclusion, here we report an additional pathway for the synthesis of NO in A. nidulans using urea cycle intermediates.     

Regulation (alteration) of activity and conformation of sucrase by coaggregation with cellobiase in culture medium of Termitomyces clypeatus

Extracellular sucrase (S) of Termitomyces clypeatus was aggregated with cellobiase (C) in culture filtrate and coaggregates of sucrase to cellobiase with different activity ratios (S/C) were obtained during purification. Specific activity of the enzyme decreased significantly, after purification of sucrase free from cellobiase. Purified sucrase was characterized as a glycoprotein of molar mass around 55kDa as indicated by SDS-PAGE and HPGPLC. K(m) and V(max) of the purified enzyme were determined as 34.48 mM and 13.3 U/mg, respectively, at optimum temperature (45 degrees C) and pH (5.0). Substrate affinity and reaction velocity of the purified enzyme, free from cellobiase, was lowered by approximately 3.5 and 55 times, respectively, than that of the enzyme obtained from culture filtrate. The instant regain of sucrase activity up to the extent of 41% was obtained on in vitro addition of cellobiase (free from sucrase) to the enzyme in incubation mixture. Conformation of the enzyme free from cellobiase appeared to be significantly different from that of the coaggregate, as analyzed by circular dichroic and light scattering spectroscopy. It was concluded that activity and conformation of sucrase is regulated (altered) by heteroaggregation with cellobiase in the fungus.     

Stabilization and improvement of catalytic activity of a low molar mass cellobiase by cellobiase-sucrase aggregation in the culture filtrate of Termitomyces clypeatus

The extracellular cellobiase (EC 3.2.1.21) of Termitomyces clypeatus separated in two protein fractions when culture filtrate or ammonium sulfate precipitated proteins were chromatographed on BioGel P-200 column. During purification of cellobiase (CBS) from the lower molar mass (LMM) protein fraction, the enzyme behaved like a low molecular weight multimeric protein. The purified enzyme gave a single 56 kDa band in SDS-PAGE but ladderlike bands (14, 28, 42, and 56 kDa) on denaturation by reducing-SDS and urea. The protein, however, dissociated on dilution and protomeric (14 kDa) and multimeric forms (28 and 60 kDa) were eluted separately during HPGPLC. Specific activity of CBS gradually decreased as the molar mass of the enzyme was lowered in different eluted peaks. Protein present in all CBS pool fractions had the same amino acid composition and all displayed the same, single protein peak in reverse-phase HPLC and 56 kDa band in SDS-PAGE. Thus, T. clypeatus CBS was a multimeric 14 kDa protein that was optimally active as a tetramer. CBS purified from the higher molar mass fraction (HMM) as a SDS-PAGE homogeneous 110-kDa protein did not dissociate on dilution or by SDS-urea. The purified protein was a protein aggregate as CBS consistently contained 20 +/- 5% sucrase (SUC) Units in the preparation. The aggregate resolved during reverse-phase chromatography on a C(4) column, and an additional protein peak other than CBS was detected. The aggregated CBS had a higher temperature optimum and was more stable toward thermal and chemical denaturations than SUC-free CBS. Increase of stability and catalytic activity of CBS by aggregation with SUC was much higher than those by the multimerization of CBS itself. All of these observations for the first time suggested that the heterologous protein-protein aggregation, observed for a long time for fungal enzymes, might have a significant role in modulating physicochemical properties of the extracellular enzyme.     

Septum-directed secretion in the filamentous fungus Aspergillus oryzae

Although exocytosis in fungal cells takes place at hyphal tips, there also seems a line of circumstantial evidence suggesting the occurrence of exocytosis at other sites of cells, such as septa. To investigate whether exocytosis takes place at fungal septa, we monitored dynamics of EGFP‐fused α‐amylase (AmyB–EGFP), the representative secretory enzyme of the filamentous fungus Aspergillus oryzae. We found that AmyB–EGFP accumulates in Spitzenkörper at hyphal tips as well as septal periplasm between the plasma membrane and cell walls. The septal accumulation of AmyB–EGFP was a rapid process, and required microtubules but not F‐actin. Thus, this process is independent of exocytosis at hyphal tips that requires both microtubules and F‐actin. In addition, fluorescence recovery after photobleaching (FRAP) analysis of EGFP‐fused AoSnc1 revealed that secretory vesicles constitutively fuse with the septal plasma membrane. These results demonstrated that exocytosis takes place at septa in addition to hyphal tips. Analysis of two plasma membrane transporters, AoUapC and AoGap1, revealed that they preferentially accumulate at septa and the lateral plasma membrane with no clear accumulation at apical Spitzenkörper, suggesting that non‐tip directed exocytosis is important for delivery of these proteins.     

Fungal partnerships stimulate growth of Termitomyces clypeatus stalk mycelium in vitro

The symbiotic relationship between termites and Termitomyces fungi, which allows the termite to digest cellulose-rich food sources, is poorly understood. In this study, in vitro mixed symbiotic relationships between Termitomyces clypeatus and fungi isolated from individual fungus-comb communities using a culture-dependent method were analyzed. Twenty-day-old stalk cultures of three T. clypeatus isolates were co-cultured with cellulase-producing fungi on potato dextrose agar. The high cellulase-producing fungal isolate no. 18, which showed 99 % ITS sequence identity to Sordariomycetes endophyte isolate 2171 (EU687039), increased growth of T. clypeatus 18/50 by 85.7 %. The high xylanase-producing isolate no. 13, which showed 88 % ITS sequence identity to Arthrinium sacchari isolate L06 (HQ115662), stimulated T. clypeatus 18/50 growth by 58.6 %. The high cellulase- and xylanase-producing isolate no. 50, which showed 90 % ITS sequence identity to the fungal endophyte isolate 2196 (EU687056), improved T. clypeatus 18/50 growth by 45.7 %. A Gigantropanus sp. promoted the growth of T. clypeatus 18/50 and 20/50 by 45.7 and 44.1 %, respectively, and that of T. clypeatus 19/50 by 10.6 %. These results indicated the most beneficial potential partnership of T. clypeatus might involve cellulase-producing fungi isolated from the same ecological niche. The Gigantropanus sp. is a potential partner of T. clypeatus but is likely to be less common than cellulase-producing fungi isolated from fungus combs owing to the lower host specificity of the Gigantropanus sp. This study provides an interesting method to culture Termitomyces using an in vitro mixed culture method for production of Termitomyces fruiting bodies in the future.     

Development of high-molar-mass cellobiase complex by spontaneous protein-protein interaction in the culture filtrate ofTermitomyces clypeatus

The 450 kDa cellobiase fromTermitomyces clypeatus which migrates as a single band on IEF, PAGE and SDS-PAGE, was found to possess appreciable sucrase activity. The fungus produced sucrase and cellobiase constitutively in different media but with different activity ratios. The kinetics of secretion of the two enzymes was similar underin vivo andin vitro conditions. HPGPLC analysis of the culture filtrates indicated the presence of both sucrase and cellobiase in the same protein fractions of different molar mass, even in the 30-kDa protein fraction. No free sucrase or cellobiase could be detected in the culture filtrates. It was also observed that fractionation of cellobiase by (NH₄)₂SO₄ precipitation was different with different amounts of associated sucrase activity present in the culture filtrate. The (NH₄)₂SO₄-precipitated cellobiase fraction also contained cellobiases in proteins of widely varied molar mass ranges. However, none of the low-molar mass proteins other than the 450-kDa enzyme could be purified, as all low-molar-mass fractions spontaneously aggregated to the 450-kDa enzyme. Hydrophobic chromatography of the (NH₄)₂SO₄-precipitated fractions followed by HPGPLC of the eluted active fraction yielded both cellobiase-free sucrase and a very low sucrase-containing cellobiase fraction. The cellobiase fraction, homogeneous in PAGE, was also a high-molar-mass protein complex dissociating into a number of protein bands on SDS-PAGE. It was suggested that the 450-kDa cellobiase was not liberated by the fungus as a preformed enzyme complex but that the complex developed through interaction of cellobiase with sucrase underin vitro conditions and the possibility of the involvement of other proteins in the aggregation cannot be excluded.     

蚁巢伞属真菌Termitomyces clypeatus实时荧光定量PCR内参基因的筛选

蚁巢伞属真菌尖盾蚁巢伞Termitomyces clypeatus是一类与大白蚁亚科昆虫共生的野生食用真菌,因味道鲜美备受消费者喜爱。为进一步对蚁巢伞属真菌开展相关生物学和遗传学研究,本试验选取8个候选内参基因[3-磷酸甘油醛脱氢酶(GAPDH)、磷酸葡萄糖变异酶(PGM)、β微管蛋白(TUB)、β肌动蛋白(ACT)、翻译延长因子1-α (EF1)、蛋白磷酸酶2A (PP2A)、聚泛素(UBQ)和翻译延伸因子2 (EF2)],对其在T. clypeatus菌株不同生长发育时期(菌丝体、巢内萌发期及成熟子实体)的表达稳定性进行评估,通过4种软件(geNorm、NormFinder、BestKeeper以及RefFinder)进行数据分析,结果表明：在供试条件下,ACT、EF1和TUB的相对表达量处于较稳定的状态,可作为蚁巢伞属真菌功能基因转录水平分析的内参基因。     

Removal of carboxymethyl cellulase activity from the culture filtrate of Termitomyces clypeatus producing xylanase

Termitomyces clypeatus produced 450 IU xylanase ml^–1 in a medium containing starch-free wheat bran powder as the carbon source. Carboxymethyl cellulase (CMCase) activity in the culture filtrate was removed by keeping the filtrate at pH 10 for 60 min followed by a change to pH 6. Treatment of Kraft pulp (bamboo) with the filtrate at pH 7 decreased the kappa number from 10.5 to 5 with release of reducing groups equivalent to 0.15 mg glucose g^–1 pulp.     

真菌菌丝中类胡萝卜素的提取方法

建立一种从丝状真菌的菌丝体中快速提取类胡萝卜素的方法。发现类胡萝卜素在菌丝体中以色素颗粒形式存在。当饱和NaCl溶液存在于磨碎的菌丝体中,色素颗粒被破坏,类胡萝卜素可以被石油醚萃取。该方法适合根霉属（Rhizopus)、笄霉属（Choanephora)、毛霉属（Mucor)、脉孢霉属（Neurspora)、镰胞霉属（Fusarium)、布拉霉属（Blakeslea）、根霉属（Rhizopus)等丝状真菌菌丝体中类胡萝卜素的提取。     

Supramolecular organization of cytochrome c oxidase- and alternative oxidase-dependent respiratory chains in the filamentous fungus Podospora anserina

To elucidate the molecular basis of the link between respiration and longevity, we have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This established aging model is able to respire by either the standard or the alternative pathway. In the latter pathway, electrons are directly transferred from ubiquinol to the alternative oxidase and thus bypass complexes III and IV. We show that the cytochrome c oxidase pathway is organized according to the mammalian "respirasome" model (Sch?gger, H., and Pfeiffer, K. (2000) EMBO J. 19, 1777-1783). In contrast, the alternative pathway is composed of distinct supercomplexes of complexes I and III (i.e. I(2) and I(2)III(2)), which have not been described so far. Enzymatic analysis reveals distinct functional properties of complexes I and III belonging to either cytochrome c oxidase- or alternative oxidase-dependent pathways. By a gentle colorless-native PAGE, almost all of the ATP synthases from mitochondria respiring by either pathway were preserved in the dimeric state. Our data are of significance for the understanding of both respiratory pathways as well as lifespan control and aging.