Purification and characterization of cellobiose dehydrogenase from the plant pathogen Sclerotium (Athelia) rolfsii

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. In the presence of a suitable electron acceptor, e.g., 2,6-dichloro-indophenol (DCIP), cytochrome c, or metal ions, CDH oxidizes cellobiose to cellobionolactone. The phytopathogenic fungus Sclerotium rolfsii (teleomorph: Athelia rolfsii) strain CBS 191.62 produces remarkably high levels of CDH activity when grown on a cellulose-containing medium. Of the 7,500 U of extracellular enzyme activity formed per liter, less than 10% can be attributed to the proteolytic product cellobiose:quinone oxidoreductase. As with CDH from wood-rotting fungi, the intact, monomeric enzyme from S. rolfsii contains one heme b and one flavin adenine dinucleotide cofactor per molecule. It has a molecular size of 101 kDa, of which 15% is glycosylation, and a pI value of 4.2. The preferred substrates are cellobiose and cellooligosaccharides; additionally, beta-lactose, thiocellobiose, and xylobiose are efficiently oxidized. Cytochrome c (equine) and the azino-di-(3-ethyl-benzthiazolin-6-sulfonic acid) cation radical were the best electron acceptors, while DCIP, 1,4-benzoquinone, phenothiazine dyes such as methylene blue, phenoxazine dyes such as Meldola's blue, and ferricyanide were also excellent acceptors. In addition, electrons can be transferred to oxygen. Limited in vitro proteolysis with papain resulted in the formation of several protein fragments that are active with DCIP but not with cytochrome c. Such a flavin-containing fragment, with a mass of 75 kDa and a pI of 5.1 and lacking the heme domain, was isolated and partially characterized.     

Purification and Characterization of Cellobiose Dehydrogenase from the Plant Pathogen Sclerotium (Athelia) rolfsii

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. In the presence of a suitable electron acceptor, e.g., 2,6-dichloro-indophenol (DCIP), cytochrome c, or metal ions, CDH oxidizes cellobiose to cellobionolactone. The phytopathogenic fungus Sclerotium rolfsii (teleomorph: Athelia rolfsii) strain CBS 191.62 produces remarkably high levels of CDH activity when grown on a cellulose-containing medium. Of the 7,500 U of extracellular enzyme activity formed per liter, less than 10% can be attributed to the proteolytic product cellobiose:quinone oxidoreductase. As with CDH from wood-rotting fungi, the intact, monomeric enzyme from S. rolfsii contains one heme b and one flavin adenine dinucleotide cofactor per molecule. It has a molecular size of 101 kDa, of which 15% is glycosylation, and a pI value of 4.2. The preferred substrates are cellobiose and cellooligosaccharides; additionally, β-lactose, thiocellobiose, and xylobiose are efficiently oxidized. Cytochrome c (equine) and the azino-di-(3-ethyl-benzthiazolin-6-sulfonic acid) cation radical were the best electron acceptors, while DCIP, 1,4-benzoquinone, phenothiazine dyes such as methylene blue, phenoxazine dyes such as Meldola's blue, and ferricyanide were also excellent acceptors. In addition, electrons can be transferred to oxygen. Limited in vitro proteolysis with papain resulted in the formation of several protein fragments that are active with DCIP but not with cytochrome c. Such a flavin-containing fragment, with a mass of 75 kDa and a pI of 5.1 and lacking the heme domain, was isolated and partially characterized.     

High-throughput screening for cellobiose dehydrogenases by Prussian Blue in situ formation

Extracellular fungal flavocytochrome cellobiose dehydrogenase (CDH) is a promising enzyme for both bioelectronics and lignocellulose bioconversion. A selective high-throughput screening assay for CDH in the presence of various fungal oxidoreductases was developed. It is based on Prussian Blue (PB) in situ formation in the presence of cellobiose (<0.25 mM), ferric acetate, and ferricyanide. CDH induces PB formation via both reduction of ferricyanide to ferrocyanide reacting with an excess of Fe3? (pathway 1) and reduction of ferric ions to Fe2? reacting with the excess of ferricyanide (pathway 2). Basidiomycetous and ascomycetous CDH formed PB optimally at pH 3.5 and 4.5, respectively. In contrast to the holoenzyme CDH, its FAD-containing dehydrogenase domain lacking the cytochrome domain formed PB only via pathway 1 and was less active than the parent enzyme. The assay can be applied on active growing cultures on agar plates or on fungal culture supernatants in 96-well plates under aerobic conditions. Neither other carbohydrate oxidoreductases (pyranose dehydrogenase, FAD-dependent glucose dehydrogenase, glucose oxidase) nor laccase interfered with CDH activity in this assay. Applicability of the developed assay for the selection of new ascomycetous CDH producers as well as possibility of the controlled synthesis of new PB nanocomposites by CDH are discussed.     

Assay for cellobiose dehydrogenase in the presence of laccase

The currently used assay for cellobiose dehydrogenase (CDH), an enzyme produced by many wood degrading fungi, lacks specificity and can give false results. The presence of laccase interferes with the standard assay. We have developed an assay for CDH that is insensitive to the presence of both laccase and other phenoloxidases. The method is based on the decrease of reducing end groups in lactose determined by the DNS method. Ferricyanide is present as electron acceptor. Advantages and drawbacks of CDH assay methods are discussed     

Characterisation of cellobiose dehydrogenases from the white-rot fungi<Emphasis Type="Italic"> Trametes pubescens</Emphasis> and<Emphasis Type="Italic"> Trametes villosa</Emphasis>

Cellobiose dehydrogenase (CDH) is an extracellular haemoflavoenzyme that is produced by a number of wood-degrading and phytopathogenic fungi and it has a proposed role in the early events of lignocellulose degradation and wood colonisation. In the presence of a suitable electron acceptor, e.g. 2,6-dichloro-indophenol, cytochrome c, or metal ions, CDH oxidises cellobiose to cellobionolactone. When screening 11 different Trametes spp. for the formation of CDH activity, all the strains investigated were found to secrete significant amounts of CDH when cultivated on a cellulose-containing medium. Amongst others, Trametes pubescens and Trametes villosa were identified as excellent, not-yet-described, producer strains of this enzyme activity that has various potential applications in biotechnology. CDH from both strains was purified to apparent homogeneity and subsequently characterised. Both monomeric enzymes have a molecular mass of approximately 90 kDa (gel filtration) and a pI value of 4.2–4.4. The best substrates are cellobiose and cellooligosaccharides; additionally, lactose, thiocellobiose, and xylobiose are efficiently oxidised. Glucose and maltose are poor substrates. The preferred substrate is cellobiose with a K _m value of 0.21 mM and a k _cat value of 22 s^–1 for CDH from T. pubescens; the corresponding values for the T. villosa enzyme are 0.21 mM and 24 s^–1, respectively. Both enzymes showed very high activity with one-electron acceptors such as ferricenium, ferricyanide, or the azino-bis-(3-ethyl-benzthiazolin-6-sulfonic acid) cation radical.     

纤维二糖脱氢酶生成羟自由基和还原各种自由基的研究

利用电子顺磁共振（ＥＳＲ）技术和硫代巴比妥酸（ＴＢＡ）反应研究了纤维二糖脱氢酶（ＣＤＨ）生成·ＯＨ和还原各种自由基的能力．以纤维二糖为电子供体时,ＣＤＨ可以生成·ＯＨ．·ＯＨ生成量与ＣＤＨ、Ｆｅ３＋和Ｏ２的浓度有关．加入过氧化氢酶可使·ＯＨ的生成明显减少．ＣＤＨ可以还原自旋加合物［ＰＢＮ－ＯＨ］·、氮氧自由基和天然木素分子中的自由基．结果表明,ＣＤＨ具有生成·ＯＨ和还原各种自由基的能力．对该酶在木质纤维素降解中的作用进行了探讨     

Optimization of cellobiose dehydrogenase production by Schizophyllum commune and effect of the enzyme on kraft pulp bleaching by ligninases

The effects of various parameters on cellobiose dehydrogenase (CDH) production by Schizophyllum commune AS 5.391 were investigated. Among different carbon and nitrogen sources tested, dewaxed cotton powder and diammonium hydrogen phosphate produced the highest titers of CDH. S. commune AS 5.391 produced CDH only when grown on cellulosic substrates but the lignin-related compounds veratryl alcohol and guaiacol had no effect on CDH production. The optimum pH for CDH production was 4.5. Addition of succinate and Tween 80 to the medium significantly improved the enzyme yield. Optimized culture conditions were obtained and the highest level of CDH was 150 U/l. CDH could facilitate kraft pulp lignin degradation by ligninases. The influence of CDH on kraft pulp bleaching by ligninases was also studied.     

Production and Characterization of Recombinant Phanerochaete chrysosporium Cellobiose Dehydrogenase in the Methylotrophic Yeast Pichia pastoris

The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induction medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH. From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.     

Production and characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase in the methylotrophic yeast Pichia pastoris.

The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induction medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH. From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.     

An amperometric enzyme biosensor for real‐time measurements of cellobiohydrolase activity on insoluble cellulose

An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi‐crystalline and amorphous, can be monitored directly and in real‐time by an enzyme‐modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross‐linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH‐catalyzed reaction with cellobiose, was recorded under constant‐potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH‐biosensors showed high sensitivity (87.7 µA mM^?1 cm^?2), low detection limit (25 nM), and fast response time (t_95% ～ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH‐biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the β‐anomer of cello‐oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real‐time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 2012; 109: 3199–3204. © 2012 Wiley Periodicals, Inc.     

Properties of neutral cellobiose dehydrogenase from the ascomycete Chaetomium sp. INBI 2-26(-) and comparison with basidiomycetous cellobiose dehydrogenases

The extracellular cellobiose dehydrogenase (CDH) obtained from Chaetomium sp. INBI 2-26(-) has a molecular mass of 95 kDa and an isoelectric point of 5. This novel CDH is highly specific for the oxidation of cellobiose (K(m,app) 4.5 microM) and lactose (K(m,app) 56 microM). With 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) (cyt c(3+)) as electron acceptors, CDH was most active at pH 6. The turnover number of the enzyme for cellobiose, lactose, DCIP and cyt c(3+) was in the range of 9-14s(-1) at 20 degrees C and pH 6. The UV-visible spectrum revealed the flavohemoprotein nature of the enzyme. The cytochrome b domain of the enzyme was reduced by ascorbate, dithionite, as well as specifically by cellobiose in a wide range of pH. The apparent first order rate constants of the spontaneous re-oxidation of the reduced heme domain were estimated as 0.01 and 0.00039 s(-1) at pH 4.5 and 6.5, respectively. The half-inactivation time of CDH at pH 6 and 55 degrees C was ca. 100 min; the stability at pH 8 and, particularly, pH 4 was remarkably lower. Cellobiose stabilized the enzyme against thermal inactivation, whereas DCIP in turn sensitized the enzyme. The new enzyme revealed low affinity for crystalline cellulose, but was capable of binding onto H(3)PO(4)-swollen filter paper. The results show significant differences to already known CDHs and perspectives for several biotechnological applications, where CDH with maximal activity at neutral pH and high affinity for cellobiose and lactose night have some advantages.     

纤维二糖脱氢酶抑制酚型化合物聚合及促进木素降解的研究

裂褶菌纤维二糖脱氢酶（ＣＤＨ）可以氧化纤维二糖并还原多种物质,催化的是一双底物双产物反应,符合乒乓反应机制,在电子供体纤维二糖存在下,ＣＤＨ可以还原由豆壳过氧化物酶（ＳＨＰ）氧化多种芳香化合物所生成的产物,ＳＨＰ氧化１－羟基苯丙三唑（１－ｈｙｄｒｏｘｙｂｅｚｏｔｒｉａｚｏｌｅ,ＨＢＴ）生成的产物对ＳＨＰ有失活作用,而在纤维二糖存在下,ＣＤＨ可以还原该氧化产物从而阻止其对酶的失活作用,ＣＤＨ可以抑制     

Optimisation of cellobiose dehydrogenase production by the fungus<Emphasis Type="Italic"> Sclerotium</Emphasis> (<Emphasis Type="Italic">Athelia</Emphasis>)<Emphasis Type="Italic"> rolfsii</Emphasis>

The phytopathogenic fungus Sclerotium (Athelia) rolfsii CBS 191.62 is a very efficient producer of the hemoflavoprotein, cellobiose dehydrogenase (CDH), forming up to 225 mg l(-1) (15,000 units cytochrome c activity l(-1)) of this protein, which is of biotechnological interest for sensors, biocatalysis and bioremediation. Both cellulose as inducing substrate and the use of a rich medium containing increased concentrations of peptone from meat or suitable amino acids are important for attaining high CDH yields. CDH, containing a protease-sensitive linker region, can be cleaved by endogenous proteases into a catalytically active flavin fragment and an inactive heme domain. By using increased concentrations of peptone, or certain amino acids such as valine or leucine, or by adding exogenous protease inhibitors, this cleavage can be almost completely inhibited, so that more than 95% intact CDH is obtained under optimised culture conditions. When using non-inhibitory amino acids, e.g. glutamine or lysine, in the medium, more than 80% of the total cellobiose-oxidising activity can be attributed to the flavin fragment.     

Electrochemical oxidation of water by a cellobiose dehydrogenase from <Emphasis Type="BoldItalic">Phanerochaete chrysosporium</Emphasis>

Cellobiose dehydrogenase (CDH) is a redox protein containing two electron transfer centers; a flavin coenzyme performing a two-electron transfer reaction and an iron-heme coenzyme facilitating single-electron transfer. Purified CDH from Phanerochaete chrysosporium was immobilized on a pyrolytic graphite electrode and electron transfer from cellobiose to the electrode was generated. With cellobiose present during cyclic voltammetry, this novel enzyme/electrode system exhibited sharp, stable oxidation peaks with slower, though equivalent, reduction peaks. During cyclic voltammetry without substrate, the enzyme was rapidly oxidized during the initial scan, with no corresponding enzyme reduction during the reducing half of the cycle. After resting for several hours in aqueous buffer, the full oxidation current appeared again. These results suggest that the CDH is reduced by water splitting, albeit at a slow rate.     

Purification and characterization of cellobiose dehydrogenase from white-rot basidiomycete Trametes hirsuta

In order to save energy during the pulp making process, we tried to use white-rot basidiomycete, Trametes hirsuta, which degrades lignin efficiently. But a decrease in paper strength caused by cellulolytic activity ruled this out for practical application. Since the cellulolytic activity of the fungus must be decreased, we purified and characterized a cellobiose dehydrogenase (CDH) that was reported to damage pulp fiber. The CDH in the culture filtrate of C. hirsutus was purified by freeze-thawing and chromatographic methods. The pI of the enzyme was 4.2 and its molecular weight was 92 kDa. The optimal temperature and pH of the enzyme were 60-70 degrees C and 5.0 respectively. Since the purified CDH decreased the viscosity of pulp in the presence of Fe(III) and cellobiose, it was shown that the suppression of CDH should be an effective way to reduce cellulose damage.     

Cellobiose dehydrogenase from the ligninolytic basidiomycete Phlebia lindtneri

Cellobiose dehydrogenase (CDH), an extracellular flavocytochrome produced by several wood-degrading fungi, was detected in the culture supernatant of the selective delignifier Phlebia lindtneri maintained on a cellulose-based liquid medium. Cellobiose dehydrogenase was purified to homogeneity by a rapid procedure, using ammonium sulfate precipitation, ion-exchange chromatography, and chromatofocusing. The enzyme was recovered with a 61.2 fold increased specific activity and a yield of 47.5%. As determined by SDS-PAGE, the molecular mass of the purified enzyme was found to be 104.5 kDa and its isoelectric point was 4.0. The carbohydrate content of the purified enzymes was 22%. In this work, the cellobiose dehydrogenase gene cdh1 and its corresponding cDNA from fungi Phlebia lidnteri were isolated, cloned, and characterized. The 2319 bp full-length cDNA of cdh1 encoded a mature CDH protein containing 755 amino acids, which was preceded by a signal peptide of 17 amino acids. The deduced protein sequence of cdh1 shared significant similarity with other known fungal cellobiose dehydrogenase.     

Efficient immobilization technique for enhancement of cellobiose dehydrogenase activity on silica gel

In this study, cellobiose dehydrogenase (CDH) of Phanerochaete chrysosporium ATCC 32629 was immobilized on silica gel for the further application of CDH in the saccharification process of biomass. To prevent the loss of enzyme activity during enzyme immobilization, the pretreatment of CDH was performed by various pretreatment materials before immobilization. When pretreated enzymes were used in immobilization, the activities of immobilized CDH were higher than non-pretreated CDH even in same amounts of immobilized protein. The specific activity of pretreated immobilized CDH with lactose was about two times higher than that of non-pretreated immobilized CDH. Moreover, the pretreated immobilized CDH showed better reusability than non-pretreated immobilized CDH, with 67.3% of its original activity being retained after 9 reuses.     

Cloning, expression, and characterization of a cellobiose dehydrogenase from Thielavia terrestris induced under cellulose growth conditions

The enzyme cellobiose dehydrogenase (CDH) is of considerable interest, not only for its biotechnological applications, but also its potential biological role in lignocellulosic biomass breakdown. The enzyme catalyzes the oxidation of cellobiose and other cellodextrins, utilizing a variety of one- and two-electron acceptors, although the electron acceptor employed in nature is still unknown. In this study we show that a CDH is present in the secretome of the thermophilic ascomycete Thielavia terrestris when grown with cellulose, along with a mixture of cellulases and hemicellulases capable of breaking down lignocellulosic biomass. We report the cloning of this T. terrestris CDH gene (cbdA), its recombinant expression in Aspergillus oryzae, and purification and characterization of the T. terrestris CDH protein (TtCDH). The TtCDH shows spectral properties and enzyme activity similar to other characterized CDH enzymes. Substrate specificity was determined for a number of carbohydrate electron donors in the presence of the two-electron acceptor 2,6-dichlorophenol-indophenol. The TtCDH also shows dramatic synergy with Thermoascus aurantiacus glycoside hydrolase family 61A protein in the presence of a β-glucosidase for the cleavage of cellulose.     

Cloning and characterization of a thermostable cellobiose dehydrogenase from Sporotrichum thermophile.

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. CDH contains one heme b and one FAD per molecule and oxidizes cellobiose to cellobionolactone in the presence of cytochrome c. In this report, a thermostable CDH from the thermophilic ascomycete Sporotrichum thermophile has been purified, cloned, and characterized. The temperature optimum for this CDH reaction was 60 degrees C, and the activation energy for the reaction was 26.3 kJ/mol. The Km and kcat were temperature-dependent and increased as reaction temperature increased. These kinetic properties prove that this CDH is truly thermophilic. A 2.8-kb cDNA was isolated by screening an expression library of S. thermophile with a polyclonal antisera raised against Phanerochaete chrysosporium CDH. The cDNA encoded an 807-amino-acid protein with a predicted mass of 86,332 Da. S. thermophile CDH is organized into three domains, an N-terminal flavin domain, a middle heme domain, and a C-terminal cellulose-binding domain, which shows sequence similarity with the cellulose-binding domains of endoglucanases and cellobiohydrolases from Trichoderma reesei. Comparison with the CDH sequences of P. chrysosporium and Trametes versicolor identified Met 95 and His 143 as potential heme coordinations. EFIG, LGGPM, and VNSTH motifs in the heme domain and the XRXPXTDXPSXDGXRY motif in the flavin domain were identified as CDH-specific motifs. With regard to the amino acid composition, S. thermophile CDH has more disulfide linkages and acidic and basic amino acids compared to CDHs from P. chrysosporium and T. versicolor.     

A simple assay for measuring cellobiose dehydrogenase activity in the presence of laccase.

The commonly used assay for measuring cellobiose dehydrogenase (CDH) activity, based on the reduction of dichlorophenol-indophenol (DCIP), has been adapted to measure this enzyme activity in the presence of laccase, which is often formed concurrently with CDH by a number of fungi. Laccase interferes with the assay by rapidly reoxidizing the reduced form of DCIP and can mask CDH activity completely. It can be conveniently and completely inhibited by 4 mM fluoride in the assay, while CDH activity is only slightly affected by the addition of this inhibitor. The modified assay enables the detection of low CDH activities even in the presence of very high excesses of laccase. It should be useful for screening culture supernatants of wood-degrading fungi for CDH since the assay is rapid and uses inexpensive and nontoxic reagents. Furthermore, it might be used for the detection of other enzyme activities which are assayed by following the reduction of quinones or analogue compounds such as DCIP.