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     

Purification and characterization of a chloride-stimulated cellobiosidase from Bacteroides succinogenes S85.

A cellobiosidase with unique characteristics from the extracellular culture fluid of the anaerobic gram-negative cellulolytic rumen bacterium Bacteroides succinogenes grown on microcrystalline cellulose (Avicel) in a continuous culture system was purified to homogeneity by column chromatography. The enzyme was a glycoprotein with a molecular weight of approximately 75,000 and an isoelectric point of 6.7. When assayed at 39 degrees C and pH 6.5, the activity of the enzyme with p-nitrophenyl-beta-D-cellobioside as the substrate was stimulated by chloride, bromide, fluoride, iodide, nitrate, and nitrite, with maximum activation (approximately sevenfold) occurring at concentrations ranging from 1.0 mM (Cl-) to greater than 0.75 M (F-). The presence of chloride (0.2 M) did not affect the Km but doubled the Vmax. In the presence of chloride (0.2 M), the pH optimum of the enzyme was broadened, and the temperature optimum was increased from 39 to 45 degrees C. The enzyme released terminal cellobiose from cellotriose and cellobiose and cellotriose from longer-chain-length cellooligosaccharrides and acid-swollen cellulose, but it had no activity on cellobiose. The enzyme showed affinity for cellulose (Avicel) but did not hydrolyze it. It also had a low activity on carboxymethyl cellulose.     

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     

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.     

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.     

Characterization of the cellulolytic activity of a Bacillus isolate

A group I Bacillus strain, DLG, was isolated and characterized as being most closely related to Bacillus subtilis. When grown on any of a variety of sugars, the culture supernatant of this isolate was found to possess cellulolytic activity, as demonstrated by degradation of trinitrophenyl-carboxymethyl cellulose. Growth in medium containing cellobiose or glucose resulted in the greatest production of cellulolytic activity. The cellulolytic activity was not produced until the stationary phase of growth, and the addition of glucose or cellobiose to a culture in this phase had no apparent effect on enzyme production. Fractionation of the culture supernatant showed that the molecular weight of the enzymatic activity was less than 100,000. Maximum cellulolytic activity in assays was observed at pH 4.8 and at 58C, although maximum thermal stability of the activity. Kinetic experiments suggested that more than one enzyme was acting upon trinitrophenyl-carboxymethyl cellulose. Exocellular protein produced by this Bacillus isolate showed roughly one-fifth the cellulolytic activity displayed by Trichoderma reesei C30 on noncrystalline, cellulosic substrates. In contrast to T. reesei cellulase, the Bacillus enzymatic activity showed no ability to degrade crystalline forms of cellulose, nor was cellobiase activity detectable.     

Characterization of the cellulolytic activity of a Bacillus isolate.

A group I Bacillus strain, DLG, was isolated and characterized as being most closely related to Bacillus subtilis. When grown on any of a variety of sugars, the culture supernatant of this isolate was found to possess cellulolytic activity, as demonstrated by degradation of trinitrophenyl-carboxymethyl cellulose. Growth in medium containing cellobiose or glucose resulted in the greatest production of cellulolytic activity. The cellulolytic activity was not produced until the stationary phase of growth, and the addition of glucose or cellobiose to a culture in this phase had no apparent effect on enzyme production. Fractionation of the culture supernatant showed that the molecular weight of the enzymatic activity was less than 100,000. Maximum cellulolytic activity in assays was observed at pH 4.8 and at 58C, although maximum thermal stability of the activity. Kinetic experiments suggested that more than one enzyme was acting upon trinitrophenyl-carboxymethyl cellulose. Exocellular protein produced by this Bacillus isolate showed roughly one-fifth the cellulolytic activity displayed by Trichoderma reesei C30 on noncrystalline, cellulosic substrates. In contrast to T. reesei cellulase, the Bacillus enzymatic activity showed no ability to degrade crystalline forms of cellulose, nor was cellobiase activity detectable.     

Major characteristics of the cellulolytic system of Clostridium thermocellum coincide with those of the purified cellulosome

The properties of the cellulosome (a cellulose-binding, multiple cellulase-containing protein complex isolated from Clostridium thermocellum) have been compared with the previously reported characteristics for crude cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparations. Similar to the crude enzyme system, true cellulolytic activity was demonstrated for the purified cellulosome on the basis of extensive solubilization of microcrystalline cellulose. The cellulolytic activity of the purified cellulosome was enhanced both by calcium ions and by thiols, and was inhibited by cellobiose (the major end product of the cellulosome-mediated cellulose degradation). In addition, at low ionic strength, cellulose-adsorbed cellulosome was detached intact from the cellulose matrix. Using controlled conditions, maximum enzymatic activity was shown to correspond to suboptimal conditions of cellulosome adsorption to cellulose. The results suggest that previous data accumulated for the crude cellulase system in C. thermocellum essentially reflect the contribution of the cellulosome.     

Purification and Characterization of Cellobiose Dehydrogenases from the White Rot Fungus Trametes versicolor

The white rot fungus Trametes versicolor degrades lignocellulosic material at least in part by oxidizing the lignin via a number of secreted oxidative and peroxidative enzymes. An extracellular reductive enzyme, cellobiose dehydrogenase (CDH), oxidizes cellobiose and reduces insoluble Mn(IV)O(inf2), commonly found as dark deposits in decaying wood, to form Mn(III), a powerful lignin-oxidizing agent. CDH also reduces ortho-quinones and produces sugar acids which can promote manganese peroxidase and therefore ligninolytic activity. To better understand the role of CDH in lignin degradation, proteins exhibiting cellobiose-dependent quinone-reducing activity were isolated and purified from cultures of T. versicolor. Two distinct proteins were isolated; the proteins had apparent molecular weights of 97,000 and 81,000 and isoelectric points of 4.2 and 6.4, respectively. The larger CDH (CDH 4.2) contained both flavin and heme cofactors, whereas the smaller contained only a flavin (CDH 6.4). These CDH enzymes were rapidly reduced by cellobiose and lactose and somewhat more slowly by cellulose and certain cello-oligosaccharides. Both glycoproteins were able to reduce a very wide range of quinones and organic radical species but differed in their ability to reduce metal ion complexes. Temperature and pH optima for CDH 4.2 were affected by the reduced substrate. Although CDH 4.2 showed rather high substrate specificity among the ortho-quinones, it could also rapidly reduce a structurally very diverse collection of other species, from negatively charged triiodide ions to positively charged hexaquo ferric ions. CDH 6.4 showed a higher K(infm) and a lower V(infmax) and turnover number than did CDH 4.2 for all substrates tested. Furthermore, CDH 6.4 did not reduce the transition metals Fe(III), Cu(II), and Mn(III) at concentrations likely to be physiologically relevant, while CDH 4.2 was able to rapidly reduce even very low concentrations of these ions. The reduction of Fe(III) and Cu(II) by CDH 4.2 may be important in sustaining a Fenton's-type reaction, which produces hydroxyl radicals that can cleave both lignin and cellulose. Unlike the CDH proteins from Phanerochaete chrysosporium, CDH 4.2 and CDH 6.4 are unable to produce hydrogen peroxide.     

A cellulase assay coupled to cellobiose dehydrogenase

An assay for cellulase activity based on the oxidation of cellobiose, formed during the cellulase reaction, with ferricyanide and a cellobiose dehydrogenase derived from the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile is presented. Due to the restricted specificity of this enzyme for cellobiose and cellodextrins, glucose, which may be formed by the action of some cellulolytic components or by beta-glucosidase, does not contribute to the result. The negative interference of beta-glucosidase may be eliminated by glucono-delta-lactone inhibition. The assay, which is not influenced by cellobiose back-inhibition of the cellulase reaction, like the usual cellulase tests based on the increase in reducing power, is basically unspecific with respect to endo- or exo-acting enzymes giving rise to a total cellulase activity. With the use of an amorphous cellulose substrate (reprecipitated cellulose after dissolving in concentrated phosphoric acid), unpredictable effects due to cooperativity between endo- and exo-enzyme components were eliminated. An analytical procedure giving a linear response between activity and enzyme concentration and between activity and time of incubation has been worked out.     

Expression of cellobiose dehydrogenase from Neurospora crassa in Pichia pastoris and its purification and characterization

A gene encoding cellobiose dehydrogenase (CDH) from Neurospora crassa strain FGSC 2489 has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter. Recombinant CDH without the native signal sequence and fused with a His₆-tag (rNC-CDH1) was successfully expressed and secreted. rNC-CDH1 was produced at the level of 652 IU/L after 2 days of cultivation in the induction medium. The His₆-tagged rNC-CDH1 was purified through a one-step Ni–NTA affinity column under non-denaturing conditions. The purified rNC-CDH1 has a CDH activity of 7451 IU/L (0.89 mg protein/mL), with a specific CDH activity of 8.37 IU/mg. The purity of the enzyme was examined by SDS–PAGE, and a single band corresponding to a molecular weight of about 120 kDa was observed. Activity staining confirmed the CDH activity of the protein band. The purified rNC-CDH1 has maximum CDH activity at pH 4.5, and a rather broad temperature optimum of 25–70 °C. Kinetic analysis showed cellobiose and cellooligosaccharides are the best substrates for rNC-CDH1. The K_m value of the rNC-CDH1 for cellooligosaccharide increases with the elongation of glucosyl units. k_cat remains relatively constant when the chain length changes.     

Separation and Some Properties of Two Intracellular beta-Glucosidases of Sporotrichum (Chrysosporium) thermophile

Intracellular, inducible beta-glucosidase from the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile (ATCC 42464) was fractionated by gel chromatography or isoelectric focusing into components A and B. Enzyme A (molecular weight 440,000) had only aryl-beta-glucosidase activity, whereas enzyme B (molecular weight 40,000) hydrolyzed several beta-glucosides but had only low activity against o-nitrophenyl-beta-d-glucopyranoside (ONPG). Both enzymes had temperature optima of about 50 degrees C. The pH optimum was 5.6 for enzyme A and 6.3 for enzyme B, respectively. The K(m) (ONPG) value for enzyme A was 0.5 mM, and the corresponding values for enzyme B were 0.18 mM (ONPG) and 0.28 mM (cellobiose). Enzyme B, when tested with ONPG, showed substrate inhibition at a substrate concentration above 0.4 mM which could be released by cellobiitol and other alditols. Enzyme A was isoelectric at pH 4.48, and enzyme B was isoelectric at pH 4.64. Several inhibitors were tested for their action on the activity of enzymes A and B. Both enzymes were found to be concomitantly induced in cultures with either cellobiose or cellulose as carbon source.     

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.     

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.     

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.     

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.     

Ethanol production from cellulose by a coculture ofZymomonas mobilis and a clostridium

Zymomonas mobilis and a mesophilic cellulolytic clostridium (strain C7) were grown in coculture in a medium containing cellulose as fermentable substrate. The coculture was stable through at least ten serial transfers and produced markedly higher amounts of ethanol than monocultures of the cellulolytic clostridium. Glucose and cellobiose, derived from the breakdown of cellulose, accumulated in strain C7 monocultures, but not in cocultures. The molar ratio of ethanol to acetate was higher in cocultures than in monocultures of strain C7. The cellulolytic clostridium was relatively ethanol-tolerant, inasmuch as it grew and fermented cellulose in media containing up to 7 g of ethanol/100 ml. Cellulase (Avicelase) activity of strain C7 was inhibited by cellobiose, but not by glucose.     

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.     

Cellulase enzyme production during continuous culture growth of Sporotrichum (Chrysosporium) thermophile

Summary The cellulolytic fungus Sporotrichum (Chrysosporium) thermophile produces an extracellular cellobiose dehydrogenase during batch culture on cellulose or cellobiose. In chemostat culture at pH 5.6 on cellobiose this enzyme was produced in parallel with endo-cellulase. At pH 5.0 in continuous or fed-batch culture such a pattern was not evident. At constant growth rate in a chemostat with varying pH, activity of these enzymes was found to be poorly correlated. Thus the induction of cellobiose dehydrogenase shows a dependence on pH and cellobiose concentration which is different to that for endo-cellulase. The natural inducer of these enzymes and the role of cellobiose dehydrogenase remain to be elucidated.     

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.