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.     

Electron transfer chain reaction of the extracellular flavocytochrome cellobiose dehydrogenase from the basidiomycete Phanerochaete chrysosporium

Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome containing flavin and b-type heme, and plays a key role in cellulose degradation by filamentous fungi. To investigate intermolecular electron transfer from CDH to cytochrome c, Phe166, which is located in the cytochrome domain and approaches one of propionates of heme, was mutated to Tyr, and the thermodynamic and kinetic properties of the mutant (F166Y) were compared with those of the wild-type (WT) enzyme. The mid-point potential of heme in F166Y was measured by cyclic voltammetry, and was estimated to be 25 mV lower than that of WT at pH 4.0. Although presteady-state reduction of flavin was not affected by the mutation, the rate of subsequent electron transfer from flavin to heme was halved in F166Y. When WT or F166Y was reduced with cellobiose and then mixed with cytochrome c, heme re-oxidation and cytochrome c reduction occurred synchronously, suggesting that the initial electron is transferred from reduced heme to cytochrome c. Moreover, in both enzymes the observed rate of the initial phase of cytochrome c reduction was concentration dependent, whereas the second phase of cytochrome c reduction was dependent on the rate of electron transfer from flavin to heme, but not on the cytochrome c concentration. In addition, the electron transfer rate from flavin to heme was identical to the steady-state reduction rate of cytochrome c in both WT and F166Y. These results clearly indicate that the first and second electrons of two-electron-reduced CDH are both transferred via heme, and that the redox reaction of CDH involves an electron-transfer chain mechanism in cytochrome c reduction.     

Activation of bacterial lytic polysaccharide monooxygenases with cellobiose dehydrogenase

Lytic polysaccharide monooxygenases (LPMOs) represent a recent addition to the carbohydrate‐active enzymes and are classified as auxiliary activity (AA) families 9, 10, 11, and 13. LPMOs are crucial for effective degradation of recalcitrant polysaccharides like cellulose or chitin. These enzymes are copper‐dependent and utilize a redox mechanism to cleave glycosidic bonds that is dependent on molecular oxygen and an external electron donor. The electrons can be provided by various sources, such as chemical compounds (e.g., ascorbate) or by enzymes (e.g., cellobiose dehydrogenases, CDHs, from fungi). Here, we demonstrate that a fungal CDH from Myriococcum thermophilum (MtCDH), can act as an electron donor for bacterial family AA10 LPMOs. We show that employing an enzyme as electron donor is advantageous since this enables a kinetically controlled supply of electrons to the LPMO. The rate of chitin oxidation by CBP21 was equal to that of cosubstrate (lactose) oxidation by MtCDH, verifying the usage of two electrons in the LPMO catalytic mechanism. Furthermore, since lactose oxidation correlates directly with the rate of LPMO catalysis, a method for indirect determination of LPMO activity is implicated. Finally, the one electron reduction of the CBP21 active site copper by MtCDH was determined to be substantially faster than chitin oxidation by the LPMO. Overall, MtCDH seems to be a universal electron donor for both bacterial and fungal LPMOs, indicating that their electron transfer mechanisms are similar.     

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.     

Direct electrochemistry and electrocatalytic activity of catalase incorporated onto multiwall carbon nanotubes-modified glassy carbon electrode

The direct voltammetry and electrocatalytic properties of catalase, which was adsorbed on the surface of multiwall carbon nanotubes (MWCNTs), was investigated. A pair of well-defined and nearly reversible cyclic voltammetry peaks for Fe(III)/Fe(II) redox couple of catalase adsorbed on the surface of MWCNTs at approximately -0.05 V versus reference electrode in pH 6.5 buffer solution, indicating the direct electron transfer between catalase and electrode. The surface coverage of catalase immobilized on MWCNTs glassy carbon electrode was approximately 2.4x10(-10) molcm-2. The transfer coefficient (alpha) was calculated to be 0.4, and the heterogeneous electron transfer rate constant was 80 s-1 in pH 7, indicating great facilitation of the electron transfer between catalase and MWCNTs adsorbed on the electrode surface. The formal potential of catalase Fe(III)/Fe(II) couple in MWCNTs film had a linear relationship with pH values between 2 and 11 with a slope of 58 mV/pH, showing that the electron transfer is accompanied by single proton transportation. Catalase adsorbed on MWCNTs exhibits a remarkable electrocatalytic activity toward the reduction of oxygen and hydrogen peroxide. The value for calculated Michaelis-Menten constant (1.70 mM) was high, indicating the potential applicability of the films as a new type of reagentless biosensor based on the direct electrochemistry of the catalase enzyme.     

Kinetics and reactivity of the flavin and heme cofactors of cellobiose dehydrogenase from Phanerochaete chrysosporium

The flavin cofactor within cellobiose dehydrogenase (CDH) was found to be responsible for the reduction of all electron acceptors tested. This includes cytochrome c, the reduction of which has been reported to be by the reduced heme of CDH. The heme group was shown to affect the reactivity and activation energy with respect to individual electron acceptors, but the heme group was not involved in the direct transfer of electrons to substrate. A complicated interaction was found to exist between the flavin and heme of cellobiose dehydrogenase. The addition of electron acceptors was shown to increase the rate of flavin reduction and the electron transfer rate between the flavin and heme. All electron acceptors tested appeared to be reduced by the flavin domain. The addition of ferric iron eliminated the flavin radical present in reduced CDH, as detected by low temperature ESR spectroscopy, while it increased the flavin radical ESR signal in the independent flavin domain, more commonly referred to as cellobiose:quinone oxidoreductase (CBQR). Conversely, no radical was detected with either CDH or CBQR upon the addition of methyl-1,4-benzoquinone. Similar reaction rates and activation energies were determined for methyl-1,4-benzoquinone with both CDH and CBQR, whereas the rate of iron reduction by CDH was five times higher than by CBQR, and its activation energy was 38 kJ/mol lower than that of CBQR. Oxygen, which may be reduced by either one or two electrons, was found to behave like a two-electron acceptor. Superoxide production was found only upon the inclusion of iron. Additionally, information is presented indicating that the site of substrate reduction may be in the cleft between the flavin and heme domains.     

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.     

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.     

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.     

Voltammetric discrimination of mandelic acid enantiomers

We report a novel electrochemical biosensor for direct discrimination of d- and l-mandelic acid (d- and l-MA) in aqueous medium. The glassy carbon electrode (GCE) surface was modified with reduced graphene oxide (rGO) and γ-globulin (GLOB). Electrochemical characterization of the modified electrodes was investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The modified electrode surfaces were also characterized by scanning electron microscopy. Electrochemical response of the prepared electrode (GCE/rGO/GLOB) for discrimination of d- and l-MA enantiomers was investigated by cyclic voltammetry and was compared with bare GCE in the concentration range of 2 to 10 mM. Whereas the bare GCE showed no electrochemical response for the MA enantiomers, the GCE/rGO/GLOB electrode exhibited direct and selective discrimination with different oxidation potential values of 1.47 and 1.71 V and weak reduction peaks at potential values of −1.37 and −1.48 V, respectively. In addition, electrochemical performance of the modified electrode was investigated in mixed solution of d- and l-MA. The results show that the produced electrode can be used as electrochemical chiral biosensor for MA.     

Electrochemical investigation of cellobiose dehydrogenase from new fungal sources on Au electrodes

Following previous electrochemical investigations of cellobiose dehydrogenase (CDH), the present investigation reports on the initial screening of the electrochemistry of three new CDHs, two from the white rot basidiomycetes Trametes villosa and Phanerochaete sordida and one from the soft rot ascomycete Myriococcum thermophilum, for their ability to directly exchange electrons with 10 different alkanethiol-modified Au electrodes. Direct electron transfer (DET) between the enzymes and some of the modified Au electrodes was shown, both, in the presence and in the absence of cellobiose. However, the length and the head functionality of the alkanethiols drastically influenced the efficiency of the DET reaction and also influenced the effect of pH on the biocatalytic/redox currents, suggesting the importance of structural/sequence differences between these CDH enzymes. In this respect, the white rot CDHs exhibit excellent biocatalytic and redox currents, whereas for the soft rot CDH the DET communication is much less efficient. Cyclic voltammograms indicate that the heme domain of the CDHs is the part of the enzymes that most readily exchanges electrons with the electrode. However, for P. sordida CDH on 11-mercaptoundecanol or dithiopropionic acid-modified Au electrodes, a second voltammetric wave was noticed suggesting that for some orientations of the enzyme, DET communication with the FAD cofactor can also be obtained.     

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     

Bubble-free oxygenation of a bi-enzymatic system: effect on biocatalyst stability

The effect of bubble-free oxygenation on the stability of a bi-enzymatic system with redox mediator regeneration for the conversion of lactose to lactobionic acid was investigated in a miniaturized reactor with bubbleless oxygenation. Earlier investigations of this biocatalytic oxidation have shown that the dispersive addition of oxygen can cause significant enzyme inactivation. In the process studied, the enzyme cellobiose dehydrogenase (CDH) oxidizes lactose at the C-1 position of the reducing sugar moiety to lactobionolactone, which spontaneously hydrolyzes to lactobionic acid. 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt was used as electron acceptor for CDH and was continuously regenerated (reoxidized) by laccase, a blue multi-copper oxidase. Oxygen served as the terminal electron acceptor of the reaction and was fully reduced to water by laccase. The overall mass transfer coefficient of the miniaturized reactor was determined at 30 and 45 degrees C; conversions were conducted both in the reaction-limited and diffusion-limited regime to study catalyst inactivation. The bubbleless oxygenation was successful in avoiding gas/liquid interface inactivation. It was also shown that the oxidized redox mediator plays a key role in the inactivation mechanism of the biocatalysts unobserved during previous studies.     

Electrochemistry and electrocatalysis of myoglobin immobilized in sulfonated graphene oxide and Nafion films

In this study, sulfonated graphene oxide (SGO) was synthesized and characterized by Fourier transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). It was used to make Mb–SGO–Nafion composite films by coating myoglobin (Mb) on the glassy carbon electrodes (GCE). Positions of the Soret absorption bands suggested that Mb retained its native conformation in the films. Mb–SGO–Nafion film modified electrode showed a pair of well-defined and nearly reversible cyclic voltammetry peaks at around −0.39 V versus saturated calomel electrode (SCE) in pH 7.0 buffers, characteristic of heme Fe(III)/Fe(II) redox couples. Electrochemical parameters such as electron transfer rate constant (k_s) and formal potential (E^o′) were estimated by fitting the data of square-wave voltammetry with nonlinear regression analysis. Experimental data demonstrated that the electron transfer between Mb and electrode was greatly facilitated and showed good electrocatalytic properties toward various substrates, such as H₂O₂ and NaNO₂, with significant lowering of reduction overpotential.     

Myoglobin immobilization on electrodeposited nanometer-scale nickel oxide particles and direct voltammetry

Prosperity of information on the reactions of redox-active sites in proteins can be attained by voltammetric studies in which the protein sample is located on a suitable surface. This work reports the presentation of myoglobin/nickel oxide nanoparticles/glassy carbon (Mb/NiO NPs/GC) electrode, ready by electrochemical deposition of the NiO NPs on glassy carbon electrode and myoglobin immobilization on their surfaces by the potential cycling method. Images of electrodeposited NiO NPs on the surface of glassy carbon electrode were obtained by scanning electron microscopy (SEM) and atomic force microscopy (AFM). A pair of well-defined redox peaks for Mb(Fe(III)-Fe(II)) was obtained at the prepared electrode by direct electron transfer between the protein and nanoparticles. Electrochemical parameters of immobilized myoglobin such as formal potential (E(0')), charge transfer coefficient (alpha) and apparent heterogeneous electron transfer rate constant (k(s)) were estimated by cyclic voltammetry and nonlinear regression analysis. Biocatalytic activity was exemplified at the prepared electrode for reduction of hydrogen peroxide.     

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.     

Direct electrochemistry of glucose oxidase and electrochemical biosensing of glucose on quantum dots/carbon nanotubes electrodes

Because of their unique chemical, physical and electronic properties, Quantum dots (QDs) and carbon nanotubes (CNTs) are now extremely attractive and important nanomaterials in bioanalytical applications. In this work, CdTe QDs with the size of about 3 nm were prepared and a novel electrochemical biosensing platform of glucose based on CdTe/CNTs electrode was explored. This CdTe/CNTs electrode was prepared by first mixing CdTe QDs, CNTs, Nafion, and glucose oxidase (GOD) in appropriate amounts and then modifying this mixture on the glass carbon electrode (GC). Transmission electron microscopy (TEM) was used to observe the dispersion of CdTe QDs on carbon nanotubes and cyclic voltammetry (CV) was used to investigate the electrochemical behavior of the CdTe/CNTs electrode. A pair of well-defined quasi-reversible redox peaks of glucose oxidase were obtained at the CdTe/CNTs based enzyme electrode by direct electron transfer between the protein and the electrode. The immobilized glucose oxidase could retain bioactivity and catalyze the reduction of dissolved oxygen. Due to the synergy between the CdTe QDs and CNTs, this novel biosensing platform based on QDs/CNTs electrode responded even more sensitively than that based on GC electrode modified by CdTe QDs or CNTs alone. The inexpensive, reliable and sensitive sensing platform based on QDs/CNTs electrode provides wide potential applications in clinical, environmental, and food analysis.     

Direct electrochemistry of glucose oxidase and biosensing for glucose based on boron-doped carbon nanotubes modified electrode

Due to their unique physicochemical properties, doped carbon nanotubes are now extremely attractive and important nanomaterials in bioanalytical applications. In this work, selecting glucose oxidase (GOD) as a model enzyme, we investigated the direct electrochemistry of GOD based on the B-doped carbon nanotubes/glassy carbon (BCNTs/GC) electrode with cyclic voltammetry. A pair of well-defined, quasi-reversible redox peaks of the immobilized GOD was observed at the BCNTs based enzyme electrode in 0.1M phosphate buffer solution (pH 6.98) by direct electron transfer between the protein and the electrode. As a new platform in glucose analysis, the new glucose biosensor based on the BCNTs/GC electrode has a sensitivity of 111.57 microA mM(-1)cm(-2), a linear range from 0.05 to 0.3mM and a detection limit of 0.01mM (S/N=3). Furthermore, the BCNTs modified electrode exhibits good stability and excellent anti-interferent ability to the commonly co-existed uric acid and ascorbic acid. These indicate that boron-doped carbon nanotubes are the good candidate material for the direct electrochemistry of the redox-active enzyme and the construction of the related enzyme biosensors.     

Kinetic studies of the electron exchange reaction between the octaheme cytochrome c3 (Mr 26000) and the hydrogenase from Desulfovibrio desulfuricans Norway.

The octaheme cytochrome c3 (Mr 26000) from Desulfovibrio desulfuricans Norway was studied using cyclic voltammetry at the pyrolytic graphite electrode. The kinetics of reduction of the octaheme cytochrome c3 (Mr 26000) from D. desulfuricans Norway by the Ni-Fe-Se hydrogenase purified from the same organism was investigated by an electrochemical method. From cyclic voltammetry experiments a value of 8.108M-1S-1 was obtained for the second order homogenous rate constant of the electron transfer between the two proteins. Results are compared with similar experiments performed on the electron exchange between the tetrahemic cytochrome c3 (Mr 13000) and hydrogenase.