Production and Properties of Galactosidases from Corticium rolfsii

When Corticum rolfsii was grown in a medium containing bran extract under aerobic conditions, it secreted alpha-D-galactosidase and beta-D-galactosidase into the culture fluid. Pectin also stimulated the production of these enzymes, whereas galactose, glucose, and sucrose stimulated their production to a lesser degree. C. rolfsii produced greater amounts of both enzymes than Aspergillus niger. Both galactosidases in the culture medium hydrolyzed alpha- and beta-p-nitrophenyl-D-galactosides as well as lactose, stachyose, melibiose, and raffinose. Both exhibited optimal activity at pH 2 to 4 and were quite stable under acidic conditions. alpha-Galactosidase was separated from beta-galactosidase by column chromatography.     

Purification and properties of the raw-starch-digesting glucoamylases from Corticium rolfsii

Corticium rolfsii AHU 9627, isolated from a tomato stem, is one of the strongest producers of a raw-starch-digesting amylase. The amylase system secreted by C. rolfsii AHU 9627 consisted of five forms of glucoamylase (G1–G5) and a small amount of α-amylase. Among these amylases, G1, G2 and G3 were able to hydrolyze raw starch. Five forms of glucoamylase were separated from each other and purified to an electrophoretically homogeneous state. The molecular masses were: G1 78 kDa, G2 78 kDa, G3 79 kDa, G4 70 kDa, and G5 69 kDa. The isoelectric points were: G1 3.85, G2 3.90, G3 3.85, G4 4.0, and G5 4.1. These glucoamylases showed nearly identical characteristics except that G4 and G5 were unable to hydrolyze raw starch. Received: 16 December 1997 / Received last revision: 6 May 1998 / Accepted: 1 June 1998     

Cloning of Corticium rolfsii glucoamylase cDNA and its expression in Saccharomyces cerevisiae

A cDNA coding for the glucoamylase of Corticium rolfsii AHU 9627 was cloned using synthetic oligonucleotide probes that code for inner amino acid sequences of the purified enzyme. This clone (CG 15) is 1900 base pairs long and contains the entire coding region for a polypeptide of 579 residues. Comparison with amino acid sequences of other fungal glucoamylases showed homologies of 35%–56%, and most homology with that of Aspergillus niger. The expression plasmid pACG 115 was constructed by introduction of the coding region of CG 15 into a yeast expression vector pAAH 5, containing the promoter and terminator of alcohol dehydrogenase (ADH1). Saccharomyces cerevisiae AH 22, containing the recombinant plasmid pACG 115, acquired starch-saccharifying ability.     

Cloning,sequencing, and heterologous expression of a cellobiohydrolase cDNA from the basidiomycete Corticium rolfsii

From a Corticium rolfsii cDNA library, a clone homologous to other fungal cellobiohydrolase (CBH1) genes was isolated using the polymerase chain reaction. In the nucleotide sequence, one 1.6 kb long open reading frame coding for a polypeptide of 530 amino acid residues was detected which showed 64% identity with CBH1 of Phanerochaete chrysosporium. With expression of the 1.8 kb cDNA using the Aspergillus oryzae expression system, we detected microcrystalline cellulose (Avicel) hydrolyzing activity in the culture supernatant. The secreted protein, accompanied by the activity, was 89 kDa by SDS-polyacrylamide gel electrophoresis.     

Purification and Properties of Lysophospholipase Produced by Corticium centrifugum

Lysophospholipase (EC 3. 1. 1. 5) from the culture broth of Corticium centrifugum was purified 92-fold in specific activity by DEAE-Sephadex and hydroxylapatite column chromatography. The isoelectric point was at about pH 3.9, and the molecular weight was about 130,000. The optimal pH was about 3.5~5.0. The stable pH range was from 7.0 to 8.0. Lysophospholipase activity was inhibited by Fe³⁺, Hg²⁺ and Al³⁺, but stimulated by various organic solvents. Diazobenzene p-sulfonic acid, N-bromosuccinimide and diisopropyl-fluorophosphate also inhibited the activity. This enzyme did not hydrolyze mono-, di-or tripalmitin or phosphatidylcholine. Apparent Michaelis constants of lysophospholipase activity for 1-acyl-LPC, 1-palmitoyl-LPC and 1-oleoyl-LPC were 0.35, 0.16 and 0.09 mm, respectively. The effect of detergents on the enzyme activity was observed to differ with the fatty acid composition of substrate.     

Phospholipid Acyl-hydrolases from a Mold,Corticium centrifugum

The activities of phospholipids acyl-hydrolases in an enzyme preparation from a mold, Corticium centrifugum, were examined. Lecithin acyl-hydrolase had an optimal pH at 3.5. The reaction proceeded beyond the range of 50%. Sigmoidal curves observed suggested the presence of lysophospholipase in the preparation. The latter enzyme activity was found to be seven times as strong as the former at the same pH. Fractionation by DEAE-Sephadex chromatography and analysis of the reaction products demonstrated that the main component of lecithin acyl-hydrolase was phospholipase B, which hydrolyzed both of fatty acyl ester groups of lecithin. This activity was found to be present as a separate enzyme from most of lysophospholipase.     

Enhanced Cellulase Production by a Mutant of Sclerotium rolfsii

A mutant of Sclerotium rolfsii CPC 142 that secretes about two times more filter paper-degrading activity in NM-2 growth medium in submerged cultures than the parent strain was obtained by ultraviolet mutagenesis of crushed sclerotia. The production of endo-β-glucanase in the mutant was affected to a lesser extent. With the parent strain, the addition of 3% rice bran to NM-2 medium was essential for optimal formation of cellulase, including filter paper-degrading activity. However, with the mutant the addition of rice bran to NM-2 medium increased the formation of endo-β-glucanase but not filter paper-degrading or cellobiase activity. An altered control mechanism for the production of filter paper-degrading enzymes is suggested. The genome(s) controlling the cellulase complex of enzymes in the UV-8 mutant is not under coordinate control.     

Acetylcholinesterase-inhibiting steroidal alkaloid from the sponge Corticium sp

A new stigmastane-type steroidal alkaloid, 4-acetoxy-plakinamine B (1), was isolated from the Thai sponge Corticium sp. The compound was subjected to the acetylcholinesterase inhibitory activity determination to reveal a high inhibitory activity (IC(50) 3.75+/-1.69 microM). The kinetics of enzyme inhibition showed a decrease in V(max), whereas K(m) was increased, thus suggesting an unusual mixed-competitive mode of inhibition. Compound 1 is the first steroidal alkaloid bearing a stigmastane skeleton ever been reported to exhibit such good potency in the acetylcholinesterase inhibition bioassay.     

The Role of Ethylene in Growth and Endopolygalacturonase Production by Sclerotium rolfsii

Growth of and endopolygalacturonase production by Sclerotium rolfsii was better on a defined mineral medium than on a medium containing segments of tomato leaf petioles. The effect of treatment with ethylene (10μl/l) upon endopolygalacturonase activity with investigated at various stages of growth, in a mineral defined medium. Addition of ethylene to a 10 days-old culture of S. rolfsii resulted in a decrease in activity by day 14, whereas the addition of ethylene to a 4, 6 and 8 days old cultures resulted in an increase in endopolygalacturonase activity. Ethylene seems to have little or no stimulating effect upon growth of S. rolfsii when applied after 8 days. However, inhibited fungal growth, after the addition of ethylene at earlier stages of growth, was obtained due to the depletion of oxygen from sealed culture flasks. Endopolygalacturonase was extracted and purified from control cultures after 14 days of growth. Fractionation of this enzyme protein on Sephadex G-100 gel filtration columns resulted in two peaks of activity measured by the release of reducing sugars from polygalacturonic acid (PGA).     

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.     

Production and Properties of Xylan-Degrading Enzymes from Cellulomonas uda

Xylan degradation and production of β-xylanase and β-xylosidase activities were studied in cultures of Cellulomonas uda grown on purified xylan from birchwood. β-Xylanase activity was found to be associated with the cells, although in various degrees. The formation of β-xylanase activity was induced by xylotriose and repressed by xylose. β-Xylosidase activity was cell bound. Both constitutive and inducible β-xylosidase activities were suggested. β-Xylanase and β-xylosidase activities were inhibited competitively by xylose. β-Xylanase activity had a pronounced optimum pH of 5.8, whereas the optimum pH of β-xylosidase activity ranged from 5.4 to 6.1. The major products of xylan degradation by a crude preparation of β-xylanase activity, in decreasing order of amount, were xylobiose, xylotriose, xylose, and small amounts of xylotetraose. This pattern suggests that β-xylanase activity secreted by C. uda is of the endosplitting type. Supernatants of cultures grown on cellulose showed not only β-glucanase but also β-xylanase activity. The latter could be attributed to an endo-1,4-β-glucanase activity which had a low β-xylanase activity.     

Activities of glycosidases from Sclerotium rolfsii in non-aqueous media

The rates of hydrolysis by b-D-glucosidase, b-D-xylosidase and a-L-arabinofuranosidase isolated from Sclerotium rolfsii were increased 17 to 220 fold in organic solvents as compared to aqueous system with the highest rates occurring in chlorinated hydrocarbons. The molecular weight, log P, Hildebrand solubility parameter and dielectric constant of the solvents correlated with the activities of glycosidases.     

High Cellobiase and Xylanase Production by Sclerotium rolfsii UV-8 Mutant in Submerged Culture

Sclerotium rolfsii UV-8 mutant secretes high levels of cellobiase and xylanase in addition to having high cellulase production. The apparent K_m and V_max of cellobiase (grown in NM-2 + 2% corn steep liquor medium) with cellobiose as a substrate were 5.6 mM and 22.2 μmol of glucose liberated per min per ml of culture filtrate, respectively. The addition of 2% corn steep liquor to NM-2 medium increased endo-β-glucanase, cellobiase, and xylanase yields by approximately 1.5-fold.     

Production,Purification and Properties of Ribonuclease from Aspergillus niger

Aspergillus niger NRC–A–1–233 was cultivated by the shaking method. The optimal cultural conditions for ribonuclease (RNase) production were: composition of medium: sucrose, 15%; NH₄NO₃, 0.2%; KH₂PO₄, 0.1%; MgSO₄·7 aq., 0.025%; initial pH, 2.2; shaking conditions: 50 ml of medium /500 ml flask; cultivation time, 120 hr. The RNase was purified by acid clay treatment and chromatography on DEAE-cellulose and Sephadex G–75 columns. The purified RNase was homogeneous by ultracentrifuge and disc electrophoresis.

The molecular weight of the RNase was estimated to be 28,500 on SDS-polyacrylamide gel and its isoelectric point was 2.8 by Ampholine electrofocusing method. Digestion rate of RNA by the RNase was 100%. The RNase did not have an exact base specificity and produced four kinds of 3′-nucleotides from yeast RNA.     

Sclerotial formation in Corticium olivascens and other hymenomycetes

Sclerotia ofCorticium olivascens are reported and described for the first time. Examples include sclerotia collected from a stump ofPinus virginiana in Greenbelt, Maryland, and those formed in several cultures originally developed from spores produced by basidiocarps. Among outstanding characteristics of basidiocarps ofC. olivascens are the greenish or olivaceous color of the hymenial surface, constant presence of clamp-connections, development of septate cystidia, and production of nonamyloid but dextrinoid basidiospores. Cultural characteristics are described, and the negative oxidase reaction is noted.C. olivascens is a highly distinctive fungus which requires further taxonomic attention.

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Zusammenfassung Sclerotia vonCorticium olivascens sind das erste Mal mitgeteilt und beschrieben. Beispiele betrifft Sclerotia, die vom Baumstumpf vonPinus virginiana in Greenbelt, Maryland gesammelt worden sind und diejenigen, die sich in mehreren Kulturen, ursprünglich von Sporen der Basidiocarpen entwickelt haben. Unter den besonderen Kennzeichen der Basidiocarpen vonC. olivascens sind die grünliche oder olive Farbe der Hymenialoberfläche, ständiges Vorkommen der Klammerverbindungen, Entwicklung septierter Cystidien und die Produktion von nicht-amyloiden, sondern dextrinoiden Basidiosporen. Kulturkennzeichen sind beschrieben und die negative Oxidasenreaktion ist erwähnt.C. olivascens ist ein hoch distinguierter Pilz, der eine weitere, taxonomische Aufmerksamkeit verlangt.

     

Indigo degradation with purified laccases from Trametes hirsuta and Sclerotium rolfsii

The degradation of the textile dye indigo with purified laccases from the fungi Trametes hirsuta (THL1 and THL2) and Sclerotium rolfsii (SRL1) was studied. All laccases were able to oxidize indigo yielding isatin (indole-2,3-dione), which was further decomposed to anthranilic acid (2-aminobenzoic acid). Based on the oxygen consumption rate of the laccases during indigo degradation, a potential mechanism for the oxidation of indigo involving the step-wise abstraction of four electrons from indigo by the enzyme was suggested. Comparing the effect of the known redox-mediators acetosyringone, 1-hydroxybenzotriazole (HOBT) and 4-hydroxybenzenesulfonic acid (PHBS) on laccase-catalyzed degradation of indigo, we found a maximum of about 30% increase in the oxidation rate of indigo with SRL1 and acetosyringone. The particle size of indigo agglomerates after laccase treatment was influenced by the origin of the laccase preparation and by the incubation time. Diameter distributions were found to have one maximum and compared to the indigo particle size distribution of the control, for all laccases, the indigo agglomerates seemed to have shifted to smaller diameters. Bleaching of fabrics by the laccases (based on K/S values) correlated with the release of indigo degradation products.