Transglycosylation activities of exo- and endo-type cellulases from Irpex lacteus (Polyporus tulipiferae)

Two highly purified cellulases, Ex-1 [exo-type, exo-cellobiohydrolase, EC 3.2.1.91] and En-1 [endo-type, EC 3.2.1.4] obtained from Driselase, a commercial enzyme preparation from Irpex lacteus (Polyporus tulipiferae), were used in this work. Both cellulases produced 14C-cellooligosaccharides such as 14C-G2 and 14C-G3 by transglycosylation when G3, G5, or beta-PNPC was used as a donor and 14C-G1 as an acceptor. However, the transglycosylation activity of Ex-1 was far higher than that of En-1. When Ex-1 or En-1 was incubated with beta-PNPG only, no p-nitrophenol was released, but it was readily released when G3 was added to the reaction mixture. In this reaction, the optimal donor (G3) concentration for Ex-1 was 1.0 mM, and the optimal pH values of Ex-1 were at 2.7 and 3.7 for beta-PNPG and beta-PG as acceptors, respectively, these values being far lower than the ordinary optimal pH values of the cellulase (4.0-5.0).     

Purification and properties of two endo-1,4-beta-xylanases from Irpex lacteus (Polyporus tulipiferae)

Two different endo-1,4-beta-xylanases [1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8], named Xylanases I and III, were purified to homogeneity by gel filtration and ion exchange column chromatography from Driselase, a commercial enzyme preparation from Irpex lacteus (Polyporus tulipiferae). The purified enzymes were found to be homogeneous on polyacrylamide disc electrophoresis and their specific activities toward xylan were increased approximately 28.7 and 19.8 times, respectively. The activities of each enzyme were considerably inhibited by Hg2+, Ag+, and Mn2+. Their molecular weights were estimated to be approximately 38,000 and 62,000 by gel filtration and sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis, respectively. Their carbohydrate contents were 2.5% and 8.0% as glucose, and their amino acid composition patterns resembled each other, showing high contents of acidic amino acids, serine, threonine, alanine, and glycine. Both enzymes were most active at pH 6.0 but Xylanase I was more stable as to pH. Their optimum temperatures were 60 degrees C and 70 degrees C, respectively. Xylanase I split up to 34.5% of larchwood xylan whereas Xylanase III split only 18.9% of it. The products with the former were mainly xylose (X1), xylobiose (X2), and xylotriose (X3), whereas X2 and X3 were the main products with the latter. Both enzymes did not hydrolyze X2. Xylanase I produced almost equal quantities of X1 and X2 from X3, while Xylanase III did not attack this substrate. Both enzymes showed no activity toward glycans, other than xylan, such as starch, pachyman and Avicel (microcrystalline cellulose), except the almost one twentieth activity of Xylanase III toward sodium carboxymethyl cellulose (CMC).     

Purification and Characterization of a Pepstatin-insensitive Carboxyl Proteinase from Polyporus tulipiferae (Irpex lacteus)

A pepstatin-insensitive carboxyl proteinase of Polyporus tulipiferae (formerly Irpex lacteus) was purified by methods including affinity chromatography with chymostatin as the ligand.

Although the enzyme’s maximum proteolytic activity on hemoglobin was at pH 2.8, it was not affected by carboxyl proteinase inhibitors such as DAN, EPNP, and pepstatin. On the other hand, the enzyme was inhibited by chymostatin competitively and its Ki value was estimated to be 1.6×10^?5 m. The enzyme was very heat labile and was inactivated completely at pH 4.6 by heating at 45°C for 15min. Polyporus enzyme and Scytalidium lignicolum enzyme A₁ hydrolyzed the same peptide bond of Z-tetrapeptides, but their primary specificities were slightly different. Polyporus enzyme as well as Ganoderma lucidum enzyme contains histidine, and the amino acid compositions of Polyporus enzyme and other pepstatin-insensitive carboxyl proteinases resembled each other.     

Purification and properties of an endo-cellulase of avicelase type from Irpex lacteus (Polyporus tulipiferae).

A culture filtrate of Irpex lacteus (Polyporus tulipiferae) was fractionated initially by salting out with ammonium sulfate, and a cellulase [EC 3.2.1.4.] fraction with high Avicel-hydrolyzing activity (formerly called Avicelase) was extensively purified by a series of column chromatography procedures. This purified endo-cellulase showed a less random hydrolytic mechanism, and was obtained in a yield of 0.04% with respect to the starting material. Its specific activity was enhanced approximately 30 times over that of the starting material. The cellulase component showed a single peak on both ultracentrifugal and acrylamide disc electrophoretic analyses. Its molecular weight was estimated to be 56,000. It contained 12.2% carbohydrate; the major sugar constituents were glucose and mannose. Regarding the amino acid composition, the contents of aspartic acid and glycine were highest, followed by those of glutamic acid, serine, and theonine. The cellulase component was not markedly inhibited by most metal ions tested excepted for Hg2+. This purified endo-cellulase attacked a series of cellooligosaccharides, beta-cellobioside, CM-cellulose, and insoluble, cellulosic substrates. In the digests from insoluble substrates, glucose, cellobiose, cellotriose, and cellotetraose were detectable, but the amount of cellobiose was the largest by far. In constrast, cellobiose and glucose were produced in almost equal amounts from beta-cellobioside.     

Purification of an exo-beta-(1----3)-D-galactanase of Irpex lacteus (Polyporus tulipiferae) and its action on arabinogalactan-proteins

An exo-beta-(1----3)-D-galactanase from Driselase, a commercial enzyme preparation from Irpex lacteus (Polyporus tulipiferae) has been purified 166-fold. Apparent molecular weights of the purified enzyme, estimated by denaturing gel electrophoresis and gel filtration, were found to be 51,000 and 42,000, respectively. It hydrolyzed specifically oligosaccharides and polymers of (1----3)-linked beta-D-galactopyranosyl residues, and exhibited a maximal activity toward these substrates at pH 4.6. Based on the mode of the liberation of D-galactose from beta-(1----3)-D-galactan and the methyl beta-glycoside of beta-(1----3)-D-galactopentaose, the enzyme can be classified as an exo-glycanase capable of catalyzing the sequential hydrolytic release of single D-galactosyl residues from the nonreducing termini. The extent of the hydrolysis of the carbohydrate portion of acacia gum and radish arabinogalactan-proteins increased with their decreasing branching. Isolation and characterization of the major products formed from the proteoglycans indicated the action pattern of the enzyme to include the capability of bypassing the branching points. Consequently, the side chains carrying an additional D-galactosyl group at the reducing termini are released as neutral (1----6)-linked beta-D-galactooligosaccharides and their acidic derivatives having a 4-O-methyl-beta-D-glucuronosyl residue as the nonreducing end-group. The specificity and the mode of action showed the enzyme to be a useful tool for analyzing the fine structure of type II arabinogalactans and arabinogalactan-protein conjugates.     

Cellulose-splitting enzymes: VII. Starch zone electrophoresis of cellulose and other carbohydrases from Irpex lacteus

The cellulase of the culture filtrates of Irpex lacteus (Polyporus tulipiferae) was separated into nine components by starch zone electrophoresis with Veronal buffer at pH 8.7. The component which migrated toward the cathode appeared to represent the major portion of the cellulase, when carboxymethylcellulose was used as substrate. Some components showed differences in their power of lowering the viscosity and of producing reducing sugars.In addition, β-glucosidase, β-1,3- and β-1,4-xylanases, laminaranase and amylase existing in the filtrates were also resolved into a number of components with their characteristic patterns. Since all the β-glucosidase components migrated toward the anode, the major cellulase component was completely free of β-glucosidase activity.     

Crystallization and preliminary X-ray diffraction studies of aspartic proteinase from Irpex lacteus.

Crystals of ILAP (Irpex lacteus aspartic proteinase) have been obtained by the hanging drop method using ammonium sulfate as a precipitant. The crystals are monoclinic, space group P2(1) with cell dimensions a = 54.5 A, b = 79.6 A, c = 37.5 A, beta = 96.8 degrees. The crystals are quite stable to X-rays and diffract beyond 1.9 A resolution. There is one molecule in the asymmetric unit.     

Capacity of Irpex lacteus and Pleurotus ostreatus for decolorization of chemically different dyes

The rate and efficiency of decolorization of poly R-478- or Remazol Brilliant Blue R (RBBR)-containing agar plates (200 μg g⁻¹) were tested to evaluate the dye degradation activity in a total of 103 wood-rotting fungal strains. Best strains were able to completely decolorize plates within 10 days at 28 °C. Irpex lacteus and Pleurotus ostreatus were selected and used for degradation of six different groups of dyes (azo, diazo, anthraquinone-based, heterocyclic, triphenylmethane, phthalocyanine) on agar plates. Both fungi efficiently degraded dyes from all groups. Removal of RBBR, Bromophenol blue, Cu-phthalocyanine, Methyl red and Congo red was studied with I. lacteus also in liquid medium. Within 14 days, the following color reductions were attained: RBBR 93%, Bromophenol blue 100%, Cu-phthalocyanine 98%, Methyl red 56%, Congo red 58%. The ability of I. lacteus to degrade RBBR spiked into sterile soil was checked, the removal being 77% of the dye added within 6 weeks. The capacity of selected white rot fungal species to remove efficiently diverse synthetic dyes from water and soil environments is documented.     

Crystal structure of aspartic proteinase from Irpex lacteus in complex with inhibitor pepstatin

The crystal structure of Irpex lacteus aspartic proteinase (ILAP) in complex with pepstatin (a six amino acid residue peptide-like inhibitor) was determined at 1.3A resolution. ILAP is a pepsin-like enzyme, widely distributed in nature, with high milk-clotting activity relative to proteolytic activity. The overall structure was in good topological agreement with pepsin and other aspartic proteases. The structure and interaction pattern around the catalytic site were conserved, in agreement with the other aspartic proteinase/inhibitor complex structures reported previously. The high-resolution data also supported the transition state model, as proposed previously for the catalytic mechanism of aspartic proteinase. Unlike the other aspartic proteinases, ILAP was found to require hydrophobic residues either in the P(1) or P(1') site, and also in the P(4) and/or P(3) site(s) for secondary interactions. The inhibitor complex structure also revealed the substrate binding mechanism of ILAP at the P(3) and P(4) site of the substrate, where the inserted loop built up the unique hydrophobic pocket at the P(4) site.     

Purification, characterization, and mode of action of a rhamnogalacturonan hydrolase from Irpex lacteus, tolerant to an acetylated substrate

A novel rhamnogalacturonase (RGase) acting on an acetylated substrate was detected in the commercial preparation Driselase, an enzymatic mixture derived from the basidiomycete Irpex lacteus. The activity was isolated by hydrophobic interaction chromatography, gel filtration, and preparative isoelectric focusing, resulting in the isolation of five different rhamnogalacturonan hydrolases exhibiting various isoelectric points from 6.2 to 7.7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry analyses after trypsin cleavage of the five fractions revealed that the five rhamnogalacturonases have a molar mass of 55 kDa without any divergences in the identified peptides. The RGase with a pI of 7.2 exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40°C and 50°C. Its mode of action was analyzed by mass spectrometry of the oligosaccharides produced after hydrolysis of acetylated and nonacetylated rhamnogalacturonan. Oligomers esterified by an acetyl group on the reducing galacturonic acid residue or fully acetylated were detected in the hydrolysate showing that the novel enzyme is able to bind acetylated galacturonic acid in its active site.     

Characterization of a Novel Dye-Decolorizing Peroxidase (DyP)-Type Enzyme from Irpex lacteus and Its Application in Enzymatic Hydrolysis of Wheat Straw

Irpex lacteus is a white rot basidiomycete proposed for a wide spectrum of biotechnological applications which presents an interesting, but still scarcely known, enzymatic oxidative system. Among these enzymes, the production, purification, and identification of a new dye-decolorizing peroxidase (DyP)-type enzyme, as well as its physico-chemical, spectroscopic, and catalytic properties, are described in the current work. According to its N-terminal sequence and peptide mass fingerprinting analyses, I. lacteus DyP showed high homology (>95%) with the hypothetical (not isolated or characterized) protein cpop21 from an unidentified species of the family Polyporaceae. The enzyme had a low optimal pH, was very stable to acid pH and temperature, and showed improved activity and stability at high H₂O₂ concentrations compared to other peroxidases. Other attractive features of I. lacteus DyP were its high catalytic efficiency oxidizing the recalcitrant anthraquinone and azo dyes assayed (k_cat/K_m of 1.6 × 10⁶ s^-1 M^-1) and its ability to oxidize nonphenolic aromatic compounds like veratryl alcohol. In addition, the effect of this DyP during the enzymatic hydrolysis of wheat straw was checked. The results suggest that I. lacteus DyP displayed a synergistic action with cellulases during the hydrolysis of wheat straw, increasing significantly the fermentable glucose recoveries from this substrate. These data show a promising biotechnological potential for this enzyme.     

The Disintegration of Soybean by a Cellulase Preparation from Irpex lacteus Fr. and the Enzyme Relating to It

It was found that there was a fairly well correlation between the soybean disintegrating activity (SD activity) of Driselase, a cellulase preparation from Irpex lacteus, and the improvement of feed efficiency caused from its supplementation to a ration.

When the seed coat of soybean was hydrolysed by Driselase, arabinose, galactose, and other aldoses were liberated; on the other hand, some ketoses such as fructose, sucrose, raffinose and such were detected as a result of the hydrolysis of the cotyledon. On the fractionation of Driselase with column chromatography, acid proteinase was appeared to be parallel, in a certain extent, with SD activity. These suggested that Driselase partially attacked the cell walls of the cotyledon and led to the leakage of intracellular substances such as ketoses and protein.

Since it was revealed, however, that only a kind of pectin hydrolase was detected in the fraction with high SD activity, the maceration of soybean by a pectin hydrolase was thought to be chiefly concerned with SD activity.     

Gene cloning of an endoglucanase from the basidiomycete Irpex lacteus and its cDNA expression in Saccharomyces cerevisiae

A gene (cen1) coding for an endoglucanase I (En-1) was isolated from white rot fungus Irpex lacteus strain MC-2. The cen1 ORF was comprised of 399 amino acid residues and interrupted by 14 introns. The deduced amino acid sequence of the cen1 ORF revealed a multi-domain structure composed of a cellulose-binding domain, a Ser-/Thr-rich linker, and a catalytic domain from the N-terminus. It showed a significant similarity to those of other endoglucanases that belong to family 5 of glycosyl hydrolases. cen1 cDNA was inserted into a yeast expression vector, YEpFLAG-1, and introduced into Saccharomyces cerevesiae. The resulting S. cerevisiae transformant secreted a recombinant En-1 that had enzymatic properties similar to the original En-1. A strong synergistic effect for a degradation of Avicel and phosphoric acid swollen cellulose was observed when recombinant En-1 was used together with a major exo-type cellobiohydrolase I of I. lacteus MC-2.     

Polyporus phyllostachydis sp. nov. with notes on other rhizophilic species of Polyporus (Basidiomycota,Polyporaceae)

Polyporus phyllostachydis is described and illustrated as a new species. This species is characterized by its occurrence on bamboo roots, the small and centrally stipitate basidiocarps, the white pileus, usually becoming darker from the center at maturity, and the cylindrical stipe with a distinct crust. Morphological characters of the present species were compared with those of P. cryptopus and P. rhizophilus, other rhizophilic species of the genus. Polyporus cryptopus and P. rhizophilus are morphologically distinct by contextual texture, basidiospores, and hyphae, and possibly represent two distinct species. Contribution no. 205, Laboratory of Plant Parasitic Mycology, Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan     

Adsorption of Cu(II) and Pb(II) onto diethylenetriamine-bacterial cellulose

Diethylenetriamine-bacterial cellulose (EABC) was synthesized by amination with diethylenetriamine on bacterial cellulose (BC). Its adsorption properties for Cu(II) and Pb(II) were investigated. The parameters affecting the metal ions adsorption, such as contact time, solution pH, and initial metal ions concentration have been investigated. The adsorption kinetics and adsorption isotherms were further studied. The results show that the adsorption rate could be well fitted by pseudo-second-order rate model, and adsorption isotherm could be described by the Langmuir model. The regeneration of EABC was also studied. This study provides the relatively comprehensive data for the EABC application to the removal of metal ion in the wastewater.     

Gene Cloning of Cellobiohydrolase II from the White Rot Fungus Irpex lacteus MC-2 and Its Expression in Pichia pastoris

A gene (cel4) coding for a cellobiohydrolase II (Ex-4) was isolated from the white rot basidiomycete, Irpex lacteus strain MC-2. The cel4 ORF was composed of 452 amino acid residues and was interrupted by eight introns. Its deduced amino acid sequence revealed a multi domain structure composed of a cellulose-binding domain, a linker, and a catalytic domain belonging to family 6 of glycosyl hydrolases, from the N-terminus. cel4 cDNA was successfully expressed in the yeast Pichia pastoris. Recombinant Ex-4 showed endo-processive degrading activity towards cellulosic substrates, and a synergistic effect in the degradation of Avicel was observed when the enzyme acted together with either cellobiohydrolase I (Ex-1) or endoglucanase (En-1) produced by I. lacteus MC-2.     

Adsorption of Cu(II), Cd(II), and Pb(II) from aqueous single metal solutions by cellulose and mercerized cellulose chemically modified with succinic anhydride

This work describes the preparation of new chelating material from mercerized cellulose. The first part treats the chemical modification of non-mercerized cellulose (cell 1) and mercerized cellulose (cell 2) with succinic anhydride. Mass percent gains (mpg) and degree of succinylation (DS) of cell 3 (from cell 1) and cell 4 (from cell 2) were calculated. Cell 4 in relation to cell 3 exhibited an increase in mpg and in the concentration of carboxylic functions of 68.9% and 2.8 mmol/g, respectively. Cells 5 and 6 were obtained by treatment of cells 3 and 4 with bicarbonate solution to release the carboxylate functions and characterized by FTIR. The second part compares the adsorption capacity of cells 5 and 6 for Cu2+, Cd2+, and Pb2+ ions in an aqueous single metal solution. Adsorption isotherms were developed using Langmuir model. Cell 6 in relation to cell 5 exhibited an increase in Qmax for Cu2+ (30.4 mg/g), Cd2+ (86.0 mg/g) and Pb2+ (205.9 mg/g).     

Adsorption of chromium (VI) ion from aqueous solution by succinylated mercerized cellulose functionalized with quaternary ammonium groups

Succinylated mercerized cellulose (cell 1) was used to synthesize an anion exchange resin. Cell 1, containing carboxylic acid groups was reacted with triethylenetetramine to introduce amine functionality to this material to obtain cell 2. Cell 2 was reacted with methyl-iodide to quaternize the amine groups from this material to obtain cell 3. Cells 2 and 3 were characterized by mass percent gain, degree of amination and quaternization, FTIR and CHN. Cells 2 and 3 showed degrees of amination and quaternization of 2.8 and 0.9 mmol/g and nitrogen content of 6.07% and 2.13%, respectively. Cell 3 was used for Cr (VI) adsorption studies. Adsorption equilibrium time and optimum pH for Cr (VI) adsorption were found to be 300 min and 3.1, respectively. The Langmuir isotherm was used to model adsorption equilibrium data. The adsorption capacity of cell 3 was found to be 0.829 mmol/g. Kinetic studies showed that the rate of adsorption of Cr (VI) on cell 3 obeyed a pseudo-second-order kinetic model.