Catalytic hydrolysis of cellobiose using different acid-functionalised Fe3O4 magnetic nanoparticles.

The present study demonstrated the preparation of three different acid-functionalised magnetic nanoparticles (MNPs) and evaluation for their catalytic efficacy in hydrolysis of cellobiose. Initially, iron oxide (Fe3O4)MNPs were synthesised, which further modified by applying silica coating (Fe3O4-MNPs@Si) and functionalised with alkylsulfonic acid (Fe3O4-MNPs@Si@AS), butylcarboxylic acid (Fe3O4-MNPs@Si@BCOOH) and sulphonic acid (Fe3O4-MNPs@Si@SO3H) groups. The Fourier transform infrared analysis confirmed the presence of above-mentioned acid functional groups on MNPs. Similarly, X-ray diffraction pattern and energy dispersive X-ray spectroscopy analysis confirmed the crystalline nature and elemental composition of MNPs, respectively. TEM micrographs showed the synthesis of spherical and polydispersed nanoparticles having diameter size in the range of 20-80 nm. Cellobiose hydrolysis was used as a model reaction to evaluate the catalytic efficacy of acid-functionalised nanoparticles. A maximum 74.8% cellobiose conversion was reported in case of Fe3O4-MNPs@Si@SO3H in first cycle of hydrolysis. Moreover, thus used acid-functionalised MNPs were magnetically separated and reused. In second cycle of hydrolysis, Fe3O4-MNPs@Si@SO3H showed 49.8% cellobiose conversion followed by Fe3O4-MNPs@Si@AS (45%) and Fe3O4-MNPs@Si@BCOOH (18.3%). However, similar pattern was reported in case of third cycle of hydrolysis. The proposed approach is considered as rapid and convenient. Moreover, reuse of acid-functionalised MNPs makes the process economically viable.

Transformation of cellobiose during the interaction of cellobiose dehydrogenase and ß-glucosidase of Cerrena unicolor.

This work investigated the regulatory role of the interaction between cellobiose dehydrogenase (CDH) and ß-glucosidase (ß-GLU) in the conversion of cellobiose into cellobionolactone or glucose in vitro. To study the regulation, the two enzymes were isolated from the culture medium of the fungus Cerrena unicolor grown on a medium with microcrystalline cellulose. The enzymes were obtained in an electrophoretically homogeneous state. Their properties were studied. Both enzymes had acidic pH optima and were more stable in the acidic pH range. CDH was moderately thermostable, while ß-GLU had a low thermostability. Both enzymes efficiently catalyzed the transformation of cellobiose. A mixture of CDH and ß-GLU transformed cellobiose to glucose or cellobionolactone in the presence of various concentrations of laccase and hydroquinone. Formation of glucose and cellobionolactone in vitro during the competition between CDH and ß-GLU for cellobiose depended on the availability of quinones, formed as a result of the interaction of laccase and hydroquinone, for CDH. At low laccase and hydroquinone concentrations, the formation of glucose was found to predominate over that of cellobionolactone. The possible physiological role of the enzymes' interaction is discussed.

Metabolic characterization of anaerobic fungi provides a path forward for bioprocessing of crude lignocellulose.

The conversion of lignocellulose-rich biomass to bio-based chemicals and higher order fuels remains a grand challenge, as single-microbe approaches often cannot drive both deconstruction and chemical production steps. In contrast, consortia based bioprocessing leverages the strengths of different microbes to distribute metabolic loads and achieve process synergy, product diversity, and bolster yields. Here, we describe a biphasic fermentation scheme that combines the lignocellulolytic action of anaerobic fungi isolated from large herbivores with domesticated microbes for bioproduction. When grown in batch culture, anaerobic fungi release excess sugars from both cellulose and crude biomass due to a wealth of highly expressed carbohydrate active enzymes (CAZymes), converting as much as 49% of cellulose to free glucose. This sugar-rich hydrolysate readily supports growth of Saccharomyces cerevisiae, which can be engineered to produce a range of value-added chemicals. Further, construction of metabolic pathways from transcriptomic data reveals that anaerobic fungi do not catabolize all sugars that their enzymes hydrolyze from biomass, leaving other carbohydrates such as galactose, arabinose, and mannose available as nutritional links to other microbes in their consortium. Although basal expression of CAZymes in anaerobic fungi is high, it is drastically amplified by cellobiose breakout products encountered during biomass hydrolysis. Overall, these results suggest that anaerobic fungi provide a nutritional benefit to the rumen microbiome, which can be harnessed to design synthetic microbial communities that compartmentalize biomass degradation and bioproduct formation.

Creating Oxidase-Peroxidase Fusion Enzymes as a Toolbox for Cascade Reactions.

A set of bifunctional oxidase-peroxidases has been prepared by fusing four distinct oxidases to a peroxidase. Although such fusion enzymes have not been observed in nature, they could be expressed and purified in good yields. Characterization revealed that the artificial enzymes retained the capability to bind the two required cofactors and were catalytically active as oxidase and peroxidase. Peroxidase fusions of alditol oxidase and chitooligosaccharide oxidase could be used for the selective detection of xylitol and cellobiose with a detection limit in the low-micromolar range. The peroxidase fusions of eugenol oxidase and 5-hydroxymethylfurfural oxidase could be used for dioxygen-driven, one-pot, two-step cascade reactions to convert vanillyl alcohol into divanillin and eugenol into lignin oligomers. The designed oxidase-peroxidase fusions represent attractive biocatalysts that allow efficient biocatalytic cascade oxidations that only require molecular oxygen as an oxidant.

Whole-cell biosensor of cellobiose and application to wood decay detection.

Fungal biodegradation of wood is one of the main threats regarding its use as a material. So far, the detection of this decaying process is empirically assessed by loss of mass, when the fungal attack is advanced and woody structure already damaged. Being able to detect fungal attack on wood in earlier steps is thus of special interest for the wood economy. In this aim, we designed here a new diagnostic tool for wood degradation detection based on the bacterial whole-cell biosensor technology. It was designed in diverting the soil bacteria Streptomyces CebR sensor system devoted to cellobiose detection, a cellulolytic degradation by-product emitted by lignolytic fungi since the onset of wood decaying process. The conserved regulation scheme of the CebR system among Streptomyces allowed constructing a molecular tool easily transferable in different strains or species and enabling the screen for optimal host strains for cellobiose detection. Assays are performed in microplates using one-day culture lysates. Diagnostic is performed within one hour by a spectrophotometric measuring of the cathecol deshydrogenase activity. The selected biosensor was able to detect specifically cellobiose at concentrations similar to those measured in decaying wood and in a spruce leachate attacked by a lignolytic fungus, indicating a high potential of applicability to detect ongoing wood decay process.

Application of diffusion ordered-1H-nuclear magnetic resonance spectroscopy to quantify sucrose in beverages.

This work focuses on a quantitative analysis of sucrose using diffusion ordered-quantitative (1)H-nuclear magnetic resonance spectroscopy (DOSY-qNMR), where an analyte can be isolated from interference based on its characteristic diffusion coefficient (D) in gradient magnetic fields. The D value of sucrose in deuterium oxide at 30°C was 4.9 × 10(-10)m(2)/s at field gradient pulse from 5.0 × 10(-2) to 3.0 × 10(-1)T/m, separated from other carbohydrates (glucose and fructose). Good linearity (r(2)=0.9999) was obtained between sucrose (0.5-20.0 g/L) and the resonance area of target glucopyranosyl-α-C1 proton normalised to that of cellobiose C1 proton (100.0 g/L, as an internal standard) in 1D sliced DOSY spectrum. The DOSY-qNMR method was successfully applied to quantify sucrose in orange juice (36.1 ± 0.5 g/L), pineapple juice (53.5 ± 1.1g/L) and a sports drink (24.7 ± 0.6g/L), in good agreement with the results obtained by an F-kit method.

Development and evaluation of methods to infer biosynthesis and substrate consumption in cultures of cellulolytic microorganisms.

Concentrations of biosynthate (microbial biomass plus extracellular proteins) and residual substrate were inferred using elemental analysis for batch cultures of Clostridium thermocellum. Inferring residual substrate based on elemental analysis for a cellulose (Avicel)-grown culture shows similar results to residual substrate determined by quantitative saccharification using acid hydrolysis. Inference based on elemental analysis is also compared to different on-line measurements: base addition, CO2 production, and Near Infra Red optical density (OD850 ). Of these three on-line techniques, NIR OD850 has the best correlation with residual substrate concentration and is the most practical to use. Both biosynthate and residual substrate concentration demonstrate typical sigmoidal trends that can easily be fitted with a five-parameter Richards curve. The sigmoidal character of the inferred concentrations and on-line data, especially the CO2 production rate, suggest that there is a maximum in cell-specific rates of growth and substrate utilization during batch fermentations of crystalline cellulose, which is not observed during grown on cellobiose. Using a sigmoidal fit curve, the instantaneous specific growth rate was determined. While soluble substrate grown cultures show a constant growth rate, cultures grown on solid substrate do not. Features of various approaches are compared, with some more appropriate for rapid general indication of metabolic activity and some more appropriate for quantitative physiological studies.

A simplified filter paper assay method of cellulase enzymes based on HPLC analysis.

A simplified filter paper assay (FPA) method of cellulase enzymes was proposed based on high-performance liquid chromatography (HPLC) measurement. The method was according to the sum of glucose and cellobiose concentrations measured by HPLC that was able to be correlated with filter paper units (FPU) of the cellulase enzymes assayed by the traditional FPA method, regardless of the differences in the sources, activities, and components of the cellulases. This simple and quick assay method for the cellulase enzymes provided another parameter of the ratio of glucose to cellobiose (G/C ratio) representing the capacity of cellulase enzymes degrading cellulose into fermentable monomeric sugars.

Aislamiento y evaluación de la actividad enzimática de hongos descomponedores de madera (Quindío, Colombia) / Isolation of wood-decaying fungi and evaluation of their enzymatic activity (Quindío, Colombia)

Se realizó la colecta de hongos (rdas.) en troncos caídos con diferentes estados de descomposición en un bosque subandino (la reserva natural La Montaña del Ocaso) y se evaluó su actividad ligninolítica. Se cultivaron en Agar extracto de malta y se realizaron pruebas semicuantitativas de actividad lacasa utilizando como inductor enzimático el ácido 2,2’azino-bis-[3-etilbenzotiazolin-6-sulfónico] y el 2,6-diclorofenolindofenol para la celobiosa deshidrogenasa (CDH). Se seleccionaron los hongos con mayor actividad enzimática de troncos con diferente grado de descomposición: Cookeina sulcipes (de estado 1), un hongo de la familia Corticiaceae (de estado 2), Xylaria polymorpha (de estado 3) y Earliella sp. (de estado 4). La fermentación se realizó a 28°C durante 11 días, a 150r.p.m., con mediciones diarias para biomasa, glucosa, actividad lacasa, actividad CDH y proteínas. Los hongos de los troncos con estados de descomposición 1 a 3 presentaron mayor actividad lacasa, a medida que aumentaba el estado de descomposición. Hubo un aumento en la actividad CDH a medida que se incrementó el estado de descomposición de los troncos. Hubo una relación positiva entre la producción de las 2 enzimas. Earliella sp. fue el hongo con mayor producción de biomasa (1.140,19g/l), actividad lacasa (157Ul−1) y CDH (43,50Ul−1). Este trabajo es el primer reporte de actividad lacasa y CDH en C. sulcipes y Earliella sp. Además, sienta las bases para la utilización de estos hongos nativos en aplicaciones biotecnológicas y se adentra en el conocimiento de su función dentro del proceso de descomposición de la madera en bosques(AU)

White rotfungi(AscomycotaandBasidiomycota)werecollectedonfallentrunkswithdifferentdecaystages, inasubandeanforest(LaMontan˜a delOcasonaturereserve),anditwasevaluatedtheirligninoliticactivity.Theywereculturedonmaltextractagar.Thenitwasperformedsemiquantitativetestsforlaccaseand cellobiosedehydrogenase(CDH)activityusingABTSandDCPIPasenzymaticinducers.Basedontheresults ofthesetests,thefungiwithhigheractivitiesfromtrunkswithdifferentdecaystageswereselected:Cookeina sulcipes (for stage1),afungusfromthefamilyCorticiaceae(forstage2), Xylariapolymorpha (forstage 3)and Earliella sp. (forstage4).Afermentationwasperformedat28 1C, during11days,inarotatoryshaker at150rpm.Biomass,glucose,proteinsandenzymeactivitiesmeasurementswereperformeddaily.The fungithatwereinthetrunkswithdecaystatesfrom1to3,showedhigherlaccaseactivityasthestateof decayincreased.AhigherDCHactivitywasalsoassociatedwithahigher.Also,therewasapositiverelationship betweenbothenzymes’activities.Erliellawasthefunguswhichpresentedthehighestbiomass production(1140,19g/l),laccaseactivity(157UL 1) andCDHactivity(43,50UL 1). Thisworkisthefirst reportoflaccaseandCDHactivityfor Cookeina sulcipes and Earliella sp. Moreover,itgivesbasisforthe useofthesenativefungiinbiotechnologicalapplicationsandtheacknowledgmentoftheirfunctioninthe wooddecayprocessinnativeforest

Determination of glucose and cellobiose dissolved in the ionic liquid 1-ethyl-3-methylimidazolium acetate using Fourier transform infrared spectroscopy.

The conversion of biogenic carbohydrate feedstock to chemicals or energy equivalents is a promising approach to solve the problem of limited fossil fuel reserves. Some concepts to accomplish these transformations are based on ionic liquids (ILs) due to their ability to dissolve biopolymers, such as cellulose, and even complex biopolymer mixtures, such as wood. However, concerning control of such conversions, a reliable tool for process analytics is required. In this paper we demonstrate the applicability of Fourier transform infrared (FT-IR) spectroscopy to perform quantitative concentration measurements of glucose and cellobiose as two examples of carbohydrates dissolved in the room-temperature ionic liquid [EMIM][OAc] (1-ethyl-3-methylimidazolium acetate). For this purpose, binary mixtures in the range 0-20 wt% have been studied. A previously developed method for the data analysis, which was based on the Beer-Lambert relation, has been universalized by employing empirical correlations between the measured quantity (i.e., extinction) and the carbohydrate concentration. In the entire spectral range under investigation (500-4000 cm(-1)) numerous individual wave-numbers have been identified, allowing quantitative measurements with high accuracy and precision.

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.

Synergistic activity of Paenibacillus sp. BP-23 cellobiohydrolase Cel48C in association with the contiguous endoglucanase Cel9B and with endo- or exo-acting glucanases from Thermobifida fusca.

Cellobiohydrolase Cel48C from Paenibacillus sp. BP-23, an enzyme displaying limited activity on most cellulosic substrates, was assayed for activity in the presence of other bacterial endo- or exocellulases. Significant enhanced activity was observed when Cel48C was incubated in the presence of Paenibacillus sp. BP-23 endoglucanase Cel9B or Thermobifida fusca cellulases Cel6A and Cel6B, indicating that Cel48C acts synergistically with them. Maximum synergism rates on bacterial microcrystalline cellulose or filter paper were obtained with a mixture of Paenibacillus cellulases Cel9B and Cel48C, accompanied by T. fusca exocellulase Cel6B. Synergism was also observed in cell extracts from recombinant clone E. coli pUCel9-Cel48 expressing the two contiguous Paenibacillus cellulases Cel9B and Cel48C. The enhanced cellulolytic activity displayed by the cellulase mixtures assayed could be used as an efficient tool for biotechnological applications like pulp and paper manufacturing.

Synergistic cellulose hydrolysis can be described in terms of fractal-like kinetics.

A fractal-like kinetics model was used to describe the synergistic hydrolysis of bacterial cellulose by Trichoderma reesei cellulases. The synergistic action of intact cellobiohydrolase Cel7A and endoglucanase Cel5A at low enzyme-to-substrate ratios showed an apparent substrate inhibition consistent with a case where two-dimensional (2-D) surface diffusion of the cellobiohydrolase is rate-limiting. The action of Cel7A core and Cel5A was instead consistent with a three-dimensional (3-D) diffusion-based mode of action. The synergistic action of intact Cel7A was far superior to that of the core at a high enzyme-to-substrate ratio, but this effect was gradually reduced at lower enzyme-to-substrate ratios. The apparent fractal kinetics exponent h obtained by nonlinear fit of hydrolysis data to the fractal-like kinetics analogue of a first-order reaction was a useful empirical parameter for assessing the rate retardation and its dependence on the reaction conditions.

Cellulase production of Trichoderma reesei Rut C 30 using steam-pretreated spruce. Hydrolytic potential of cellulases on different substrates.

Various techniques are available for the conversion of lignocellulosics to fuel ethanol. During the last decade processes based on enzymatic hydrolysis of cellulose have been investigated more extensively, showing good yield on both hardwood and softwood. The cellulase production of a filamentous fungi, Trichoderma reesei Rut C 30, was examined on carbon sources obtained after steam pretreatment of spruce. These materials were washed fibrous steam-pretreated spruce (SPS), and hemicellulose hydrolysate. The hemicellulose hydrolysate contained, besides water-soluble carbohydrates, lignin and sugar degradation products, which were formed during the pretreatment and proved to be inhibitory to microorganisms. Experiments were performed in a 4-L laboratory fermentor. The hydrolytic capacity of the produced enzyme solutions was compared with two commercially available enzyme preparations, Celluclast and Iogen Cellulase, on SPS, washed SPS, and Solka Floc cellulose powder. There was no significant difference among the different enzymes produced by T. reesei Rut C 30. However, the conversion of cellulose using these enzymes was higher than that obtained with Iogen or Celluclast cellulases using steam-pretreated spruce as substrate.

Thin gold film-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry of biomolecules.

A new thin gold film-assisted (TGFA) laser desorption/ionization (LDI) technique has been combined with Fourier transform ion cyclotron resonance (FT-ICR) high-resolution mass analysis. A thermally labile organic sample is coated onto a thin gold film deposited on a glass plate and desorbed and ionized by Nd:YAG laser irradiation. The wavelength for maximum light absorption may be tuned by varying the thickness of the metal film; e.g., a 10-nm-thick gold film absorbs maximally near the Nd:YAG fundamental wavelength (1064 nm). A key advantage of the method is the stability of the gold film, which facilitates deposition of samples from any of a variety of solvent systems. Coupled with recently introduced quadrupolar excitation and collisional cooling for ion axialization, TGFA-LDI FT-ICR mass spectra provide high sensitivity (e.g., 100-fmol single-shot detection limit and mass resolving power, m/delta m approximately 113,000, for (M+K)+ ions from gramicidin S at m/z 1180).

Nucleotide sequence of the celG gene of Clostridium thermocellum and characterization of its product, endoglucanase CelG.

The nucleotide sequence of the celG gene of Clostridium thermocellum, encoding endoglucanase CelG, was determined. The open reading frame extended over 1,698 bp and encoded a 566-amino-acid polypeptide (molecular weight of 63,128) similar to the C. thermocellum endoglucanase CelB (51.5% identical residues). The N terminus displayed a typical signal peptide, followed by a catalytic domain. The C terminus, which was separated from the catalytic domain by a 25-amino-acid segment rich in Pro, Thr, and Ser, contained two conserved stretches of 22 amino acids closely similar to those previously described in other cellulases from the same organism. Expression of the gene in Escherichia coli was increased by fusing the fragment coding for the catalytic domain in frame with the start of the lacZ' gene present in the vector. A low- and a high-M(r) form of the protein were purified. The two forms displayed identical enzymatic properties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that both forms consist of a major polypeptide of M(r) 50,000 and two minor polypeptides of M(r)s 49,000 and 48,000, resulting from heterogeneous proteolytic cleavage at the C terminus. An antiserum raised against the forms purified from E. coli reacted with an immunoreactive polypeptide of M(r) 66,000, which was associated with the extracellular cellulolytic complex of C. thermocellum known as the cellulosome.

Purification and properties of the Clostridium thermocellum bglB gene product expressed in Escherichia coli.

The Clostridium thermocellum beta-glucosidase B was purified to homogeneity in its recombinant form from Escherichia coli. The purification protocol included ion exchange, hydrophobic interaction and hydroxyapatite chromatography. The polypeptide was found to have a molecular mass of 84,000 daltons and a pI of 4.4. There was a differential effect of temperature on the aryl-beta-glucosidase and cellobiase activities of the purified protein. The cellobiase activity had an optimum of 45 degrees C, and aryl-beta-glucosidase 60 degrees C. Both activities had an optimum pH of 5.6, although the aryl-beta-glucosidase had a secondary peak at 7.0. Both activities were stimulated by divalent cations and DTT, but inhibited by thiol reagents. The enzyme was found to have a broad substrate specificity. Using cellobiose as substrate and a temperature of 45 degrees C, the Km and Vmax values were 1.6 mM and 5.5 U mg-1 respectively. The aryl-beta-glucosidase when assayed against pNP glucopyranoside and a temperature of 60 degrees C had Km and Vmax of 2.9 mM and 1.1 U mg-1 respectively. The enzyme was very stable at 45 degrees C, but rapidly inactivated at 60 degrees C.

Intestinal lactase (beta-galactosidase) and other disaccharidase activities of suckling and adult common brushtail possums, Trichosurus vulpecula (Marsupialia:Phalangeridae).

Small-intestinal disaccharidase activities of eight suckling T. vulpecula, aged from 34 to 150 days, and of two adult animals were investigated. Intestinal maltase, isomaltase and sucrase activities increased with age, whereas lactase activities decreased. Trehalase activities were relatively high in all animals and showed no obvious age-related changes. Three separate beta-galactosidase activities, one neutral and two acid, acted on lactose. The neutral beta-galactosidase activity appeared to be due to a brush border enzyme similar to that of eutherian mammals, whereas the acid beta-galactosidases were soluble and probably of lysosomal origin. One of these, acid beta-galactosidase-1, had similar properties to the sole intestinal beta-galactosidase of macropodid marsupials, whereas the other, acid beta-galactosidase-2, has not previously been described. Galactosyl oligosaccharides isolated from macropodid milk were readily hydrolysed by both acid beta-galactosidases but not by the neutral beta-galactosidase. The total intestinal lactase activity in animals aged up to 125 days was due mainly to acid beta-galactosidase-1, whereas in older animals it was due mostly to the neutral beta-galactosidase; this suggests that late in lactation the young T. vulpecula change from a macropodid mode of digestion of galactosyl oligosaccharides to a eutherian mechanism for the digestion of lactose. These findings may have implications for the hand-rearing of orphaned T. vulpecula.