Heterologous expression of Phanerochaete chrysosporium cellobiose dehydrogenase in Trichoderma reesei.

BACKGROUND: Cellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDHTr) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDHPp). RESULTS: PcCDHTr was recombinantly produced with a yield of 600 U L-1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDHTr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDHTr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDHTr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-D-glucosamine additions at the six potential N-glycosylation sites of PcCDHTr, which indicates the presence of an endo-N-acetyl-ß-D-glucosaminidase in the supernatant. CONCLUSIONS: Heterologous production of PcCDHTr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDHTr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDHTr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.

Purified cellobiose dehydrogenase of Termitomyces sp. OE147 fuels cellulose degradation resulting in the release of reducing sugars.

Termitomyces sp. OE 147 is one of the active cellulose degraders in the ecosphere and produces large amount of cellobiose dehydrogenase (CDH) and ß-glucosidases when cultivated on cellulose. In order to investigate its effect on cellulose, a highly purified preparation of CDH was obtained from the culture supernatant of the fungus cultivated on cellulose. A combination of ultrafiltration, ion-exchange and gel-filtration chromatography was used to purify CDH by ∼172-fold to a high specific activity of ∼324 U/mg protein on lactose which was used for routine measurement of enzyme activity. The enzyme displayed a pH optimum of 5.0 and stability between pH 5.0 and 8.0 with maximum catalytic efficiency (kcat/Km) of 397 mM-1 s-1 on cellobiose. Incubation of microcrystalline cellulose with the purified CDH led to production of reducing sugars which was accelerated by the addition of FeCl3 during the early stages of incubation. A mass spectrometric analysis revealed fragmentation products of cellulose which were concluded to be cellodextrins, sugars, and corresponding aldonic acids suggesting that CDH can release reducing sugars in the absence of externally added lytic polysaccharide monooxygenases. Polymerized products of glucose were also detected at low intensity.

Antimicrobial and antioxidative potential of free and immobilised cellobiose dehydrogenase isolated from wood degrading fungi.

Cellobiose dehydrogenase (CDH, EC 1.1.99.18) is a glycoprotein having many biotechnological applications. In the present study, CDHs isolated from Phlebia lindtneri (PlCDH), Phanerochaete chrysosporium (PchCDH), Cerrena unicolor (CuCDH), and Pycnoporus sanguineus (PsCDH) were studied the first time for their ability to generate antioxidant and antimicrobial agents. The aim of the research was to evaluate the antioxidant and antimicrobial activity of systems composed of four CDHs and lactose or cellobiose as a reaction substrate. The free radical scavenging effect of free and immobilised enzymes was evaluated using the DPPH method. The lowest values of EC50 (10.04 ± 0.75 µg/ml) was noted for PlCDH/lactose and for PlCDH/cellobiose (12.06 ± 1.35 µg/ml). The EC50value reached 12.6 ± 1.51 µg/ml in the case of PsCDH/lactose and 15.96 ± 1.35 for PsCDH. The CDH preparations were also effectively immobilised in alginate (the immobilisation efficiency expressed as a protein yield ranged from 61.6 to 100 %). The operational stability expressed as a scavenging effect showed the possibility of using the alginate beads 4 times. Both the free and immobilised CDHs as well as the CDH/substrate were tested against Gram-negative Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Gram-positive Staphylococcus aureus ATCC 25923 bacteria. All samples, except PlCDH, were potentially effective in suppression of bacterial growth. The highest percentage of inhibition (100 %) was obtained for S. aureus bacteria using PsCDH and PchCDH with lactose as a substrate, whereas a slightly lesser effect was observed for E. coli and P. aeruginosa bacterial cells, i.e. 64.1 % and 86.5 % (PsCDH) and 94.1 % and 41.4 % (PchCDH), respectively. Furthermore, the concentrations of the reaction products (aldonic acids and hydrogen peroxide) were quantified and the surface morphology of the alginate beads was analysed using SEM visualisation.

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.

Molecular and catalytic properties of fungal extracellular cellobiose dehydrogenase produced in prokaryotic and eukaryotic expression systems.

BACKGROUND: Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by lignocellulolytic fungi. cdh gene expression is high in cellulose containing media, but relatively low CDH concentrations are found in the supernatant of fungal cultures due to strong binding to cellulose. Therefore, heterologous expression of CDH in Pichia pastoris was employed in the last 15 years, but the obtained enzymes were over glycosylated and had a reduced specific activity. RESULTS: We compare the well-established CDH expression host P. pastoris with the less frequently used hosts Escherichia coli, Aspergillus niger, and Trichoderma reesei. The study evaluates the produced quantity and protein homogeneity of Corynascus thermophilus CDH in the culture supernatants, the purification, and finally compares the enzymes in regard to cofactor loading, glycosylation, catalytic constants and thermostability. CONCLUSIONS: Whereas E. coli could only express the catalytic dehydrogenase domain of CDH, all eukaryotic hosts could express full length CDH including the cytochrome domain. The CDH produced by T. reesei was most similar to the CDH originally isolated from the fungus C. thermophilus in regard to glycosylation, cofactor loading and catalytic constants. Under the tested experimental conditions the fungal expression hosts produce CDH of superior quality and uniformity compared to P. pastoris.

Characterization of three pyranose dehydrogenase isoforms from the litter-decomposing basidiomycete Leucoagaricus meleagris (syn. Agaricus meleagris).

Multigenicity is commonly found in fungal enzyme systems, with the purpose of functional compensation upon deficiency of one of its members or leading to enzyme isoforms with new functionalities through gene diversification. Three genes of the flavin-dependent glucose-methanol-choline (GMC) oxidoreductase pyranose dehydrogenase (AmPDH) were previously identified in the litter-degrading fungus Agaricus (Leucoagaricus) meleagris, of which only AmPDH1 was successfully expressed and characterized. The aim of this work was to study the biophysical and biochemical properties of AmPDH2 and AmPDH3 and compare them with those of AmPDH1. AmPDH1, AmPDH2 and AmPDH3 showed negligible oxygen reactivity and possess a covalently tethered FAD cofactor. All three isoforms can oxidise a range of different monosaccarides and oligosaccharides including glucose, mannose, galactose and xylose, which are the main constituent sugars of cellulose and hemicelluloses, and judging from the apparent steady-state kinetics determined for these sugars, the three isoforms do not show significant differences pertaining to their reaction with sugar substrates. They oxidize glucose both at C2 and C3 and upon prolonged reaction C2 and C3 double-oxidized glucose is obtained, confirming that the A. meleagris genes pdh2 (AY753308.1) and pdh3 (DQ117577.1) indeed encode CAZy class AA3_2 pyranose dehydrogenases. While reactivity with electron donor substrates was comparable for the three AmPDH isoforms, their kinetic properties differed significantly for the model electron acceptor substrates tested, a radical (the 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] cation radical), a quinone (benzoquinone) and a complexed iron ion (the ferricenium ion). Thus, a possible explanation for this PDH multiplicity in A. meleagris could be that different isoforms react preferentially with structurally different electron acceptors in vivo.

Novel thin layer flow-cell screen-printed graphene electrode for enzymatic sensors.

A new Screen-printed electrodes (SPE) integrated in one channel flow-cell was developed. The one channel flow-cell is attached and directly changeable with electrode. In the new flow-cell the injection is done through an "in-line luer injection port" which can be less aggressive than wall-jet flow cell for a biological recognition element immobilized on the surface of the electrode. The sample volume can be easily controlled by the operator through a syringe. In this novel thin layer flow-cell screen-printed electrodes, the working electrode was modified with graphene materials, and an enhancement of electroactive area to 388% over a standard electrode was found. This new configuration was applied to study the entrapped cellobiose dehydrogenase from the ascomycete Corynascus thermophilus (CtCDH) in a photocrosslinkable PVA-based polymer. The calibration curve of lactose using optimized parameters shows a wide linear measurement ranges between 0.25 and 5mM. A good operational stability of the CtCDH-PVA-modified graphene electrode is obtained, which keeps the same initial activity during 8h and exhibits a good storage stability with a decrease of only 9% in analytical response after 3 months storage at 4◦C.

Comparative characterization of two GDP-mannose dehydrogenase genes from Saccharina japonica (Laminariales, Phaeophyceae).

BACKGROUND: Saccharina japonica is an important commercial brown seaweed, its main product is alginate, which is used in food, textile and by the cosmetic and pharmaceutical industries. GDP-mannose dehydrogenase (GMD) is the key enzyme involved in the synthesis of alginate. However, little is known about GMD in S. japonica. Here we report comparative biochemical analysis of two GMD genes in S. japonica. RESULTS: Two GMD genes from S. japonica (Sjgmd1, Sjgmd2) were cloned. The open reading frame lengths of Sjgmd1, Sjgmd2 are 963 bp and 948 bp, respectively. Alignment analysis showed that the two SjGMD sequences shared 79.38 % identity. Both proteins possess the GGxCLPKDV and GxGxVG sequence motifs characteristic of the short-chain dehydrogenase/reductase superfamily. The optimum temperatures for SjGMDs were 30 °C (SjGMD1) and 20 °C (SjGMD2), and the optimum pH values were 8.0 (SjGMD1) and 8.25 (SjGMD2). Kinetic analysis demonstrated the Km values for the substrate GDP-mannose were 289 µM (SjGMD1) and 177 µM (SjGMD2), and the Km values for the cofactor NAD(+) were 139 µM (SjGMD1) and 195 µM (SjGMD2). The metal iron Zn(2+) is a potent inhibitor of SjGMD1 and SjGMD2. Real-time PCR analysis showed that heat and desiccation treatments resulted in a significant increase in Sjgmd1 and Sjgmd2 transcript abundance, suggesting that the SjGMDs are directly involved in the acclimitisation of S. japonica to abiotic stresses. CONCLUSION: Our work identified two novel genes encoding GMD in S. japonica, comparatively characterized their structural characteristics and enzyme kinetics, and revealed the function of GMD in the stress adaptability of S. japonica. The knowledge obtained here enriched our understanding of the alginate synthesis mechanism in S. japonica, and may promote further research on functional differences between GMD genes.

Differential production of lignocellulolytic enzymes by a white rot fungus Termitomyces sp. OE147 on cellulose and lactose.

White-rot fungi are the only organisms known to degrade all basic wood polymers using different strategies of employing a variety of hydrolytic and oxidative enzymes. A comparative secretome analysis of Termitomyces sp. OE147 cultivated on cellulose and lactose was carried out by two-dimensional gel electrophoresis followed by MALDI-TOF/TOF-MS analysis to identify the enzymes coordinately expressed on cellulose. A total of 29 proteins, belonging to CAZy hydrolases (11), CAZy oxidoreductases (13) and some 'other' (5) proteins were identified. Among the CAZy hydrolases, a distinct repertoire of cellulolytic and hemicellulolytic enzymes were produced while among the CAZy oxidoreductases, cellobiose dehydrogenase and laccase were the predominant enzymes along with H2O2 dependent peroxidases. This coordinated expression indicated a unique and integrated system for degradation of not only crystalline cellulose but also other components of lignocellulolytic substrates, namely lignin and xylan. Activities of the identified proteins were confirmed by plate assays and activity measurements. Many of the enzyme activities were also correlated with reduction in the crystallinity index of cellulose. Based on the enhanced production of CDH, ß-glucosidases and several oxidoreductases, a more prominent role of these enzymes is indicated in this fungus in cellulose breakdown.

Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii.

BACKGROUND: The enzyme cellobiose dehydrogenase (CDH) can be used to oxidize lactose to lactobionic acid. As Sclerotium rolfsii is known to be a good producer of CDH, the aim of this paper was to simplify its production and secondly to systematically study its purification aiming for a high yield. Two preservation methods (freezing and freeze-drying) and the influence of several protectants were investigated. RESULTS: Production of cellobiose dehydrogenase was optimized leading to a more simplified medium composition. Purification of the enzyme was evaluated by determining breakthrough profiles on different ion exchange (IEX) and hydrophobic interaction (HIC) materials with regard to buffer composition. Highest purification with an acceptable loss during the capture step using IEX was obtained with a Q Sepharose XL medium and a 100 mM sodium acetate buffer at pH 4.5. Subsequent purification using hydrophobic interaction chromatography was done at 1.1 M ammonium sulfate concentration. Purification was moderate, yielding a specific activity of 11.9 U/mg (56% yield). However, as could be shown in a preliminary experiment, purity of the obtained enzyme solution was sufficient for its intended use to oxidize lactose to lactobionic acid. Various sugars and sugar alcohols were investigated to study their protective effect during lyophilisation and freezing at -20 °C. Glucose and lactulose could be identified to have a high lyoprotective effect while loss of enzyme activity was high (77%) when using no additives. CONCLUSION: By simplifying the cultivation medium of Sclerotium rolfsii, the costs of cellobiose dehydrogenase production could be reduced. Simultaneously, CDH production was increased by 21%. The production of lactobionic acid from lactose is possible using partially purified and unpurified enzyme. Storage at -20 °C using 50% (w/v) glycerol was considered to be most suited for preservation of the enzyme.

Heterologous production of cellobiose dehydrogenases from the basidiomycete Coprinopsis cinerea and the ascomycete Podospora anserina and their effect on saccharification of wheat straw.

Cellobiose dehydrogenases (CDHs) are extracellular glycosylated haemoflavoenzymes produced by many different wood-degrading and phytopathogenic fungi. Putative cellobiose dehydrogenase genes are recurrently discovered by genome sequencing projects in various phylogenetically distinct fungi. The genomes from the basidiomycete Coprinopsis cinerea and the ascomycete Podospora anserina were screened for candidate cdh genes, and one and three putative gene models were evidenced, respectively. Two putative cdh genes were selected and successfully expressed for the first time in Aspergillus niger. CDH activity was measured for both constructions (CDHcc and CDHpa), and both recombinant CDHs were purified to homogeneity and subsequently characterised. Kinetic constants were determined for several carbohydrates including ß-1,4-linked di- and oligosaccharides. Optimal temperature and pH were 60 °C and 5 for CDHcc and 65-70 °C and 6 for CDHpa. Both CDHs showed a broad range of pH stability between 4 and 8. The effect of both CDHs on saccharification of micronized wheat straw by an industrial Trichoderma reesei secretome was determined. The addition of each CDH systematically decreased the release of total reducing sugars, but to different extents and according to the CDH concentration. Analytical methods were carried out to quantify the release of glucose, xylose and gluconic acid. An increase of glucose and xylose was measured at a low CDHcc concentration. At moderated and high CDHcc and CDHpa concentrations, glucose was severely reduced with a concomitant increase of gluconic acid. In conclusion, these results give new insights into the physical and chemical parameters and diversity of basidiomycetous and ascomycetous CDHs. These findings also demonstrated that CDH drastically influenced the saccharification on a natural substrate, and thus, CDH origin, concentration and potential enzymatic partners should be carefully considered in future artificial secretomes for biofuel applications.

Cellulose oxidation and bleaching processes based on recombinant Myriococcum thermophilum cellobiose dehydrogenase.

Myriococcum thermophilum cellobiose dehydrogenase (MtCDH) was expressed in Pichia pastoris using the pPICZαA expression vector under the control of methanol inducible AOX promoter. The purified recombinant MtCDH with a specific activity of 3.1 Umg(-1) was characterized to obtain kinetic constants for various carbohydrate substrates. Additionally, the C1 oxidation of the reducing ends of cellobiose, cellotetraose and maltotriose by MtCDH was verified by HPLC-MS. MtCDH was employed to oxidize several different cellulose-based materials by production of hydrogen peroxide. Based on the obtained results a one-pot enzymatic scouring/bleaching process for cotton fabrics was developed using pectinases as scouring agent and MtCDH to produce H(2)O(2) for bleaching. An average increase in whiteness (Berger) ΔE of 26 and an average 95% increase in wettability were observed in all MtCDH treated fabrics. In addition, MtCDH oxidized typical colored cotton flavonoids (morin, rutin, isoquercitrin).

Heterologous expression and biochemical characterization of novel pyranose 2-oxidases from the ascomycetes Aspergillus nidulans and Aspergillus oryzae.

A gene encoding a pyranose 2-oxidase (POx; pyranose/oxygen 2-oxidoreductase; glucose 2-oxidase; EC 1.1.3.10) was identified in the genome of the ascomycete Aspergillus nidulans. Attempts to isolate POx directly from A. nidulans cultures or to homologously overexpress the native POx (under control of the constitutive gpdA promoter) in A. nidulans were unsuccessful. cDNA encoding POx was synthesized from mRNA and expressed in Escherichia coli, and the enzyme was subsequently purified and characterized. A putative pyranose 2-oxidase-encoding gene was also identified in the genome of Aspergillus oryzae. The coding sequence was synthetically produced and was also expressed in E. coli. Both purified enzymes were shown to be flavoproteins consisting of subunits of 65 kDa. The A. nidulans enzyme was biochemically similar to POx reported in literature. From all substrates, the highest catalytic efficiency was found with D-glucose. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones and 2,6-dichloroindophenol. As judged by the catalytic efficiencies (k (cat)/k(m)), some of these quinone electron acceptors are better substrates for pyranose oxidase than oxygen. The enzyme from A. oryzae was physically similar but showed lower kinetic constants compared to the enzyme from A. nidulans. Distinct differences in the stability of the two enzymes may be attributed to a deletion and an insertion in the sequence, respectively.

Catalytic properties and classification of cellobiose dehydrogenases from ascomycetes.

Putative cellobiose dehydrogenase (CDH) genes are frequently discovered in various fungi by genome sequencing projects. The expression of CDH, an extracellular flavocytochrome, is well studied in white rot basidiomycetes and is attributed to extracellular lignocellulose degradation. CDH has also been reported for plant-pathogenic or saprotrophic ascomycetes, but the molecular and catalytic properties of these enzymes are currently less investigated. This study links various ascomycetous cdh genes with the molecular and catalytic characteristics of the mature proteins and suggests a differentiation of ascomycete class II CDHs into two subclasses, namely, class IIA and class IIB, in addition to the recently introduced class III of hypothetical ascomycete CDHs. This new classification is based on sequence and biochemical data obtained from sequenced fungal genomes and a screening of 40 ascomycetes. Thirteen strains showed CDH activity when they were grown on cellulose-based media, and Chaetomium atrobrunneum, Corynascus thermophilus, Dichomera saubinetii, Hypoxylon haematostroma, Neurospora crassa, and Stachybotrys bisbyi were selected for detailed studies. In these strains, one or two cdh-encoding genes were found that stem either from class IIA and contain a C-terminal carbohydrate-binding module or from class IIB without such a module. In several strains, both genes were found. Regarding substrate specificity, class IIB CDHs show a less pronounced substrate specificity for cellobiose than class IIA enzymes. A pH-dependent pattern of the intramolecular electron transfer was also observed, and the CDHs were classified into three groups featuring acidic, intermediate, or alkaline pH optima. The pH optimum, however, does not correlate with the CDH subclasses and is most likely a species-dependent adaptation to different habitats.

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 His(6)-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 His(6)-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 745 1IU/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.

Crystallization of carbohydrate oxidase from Microdochium nivale.

Microdochium nivale carbohydrate oxidase was produced by heterologous recombinant expression in Aspergillus oryzae, purified and crystallized. The enzyme crystallizes with varying crystal morphologies depending on the crystallization conditions. Several different crystal forms were obtained using the hanging-drop vapour-diffusion method, two of which were used for diffraction measurements. Hexagon-shaped crystals (form I) diffracted to 2.66 A resolution, with unit-cell parameters a = b = 55.7, c = 610.4 A and apparent space group P6(2)22. Analysis of the data quality showed almost perfect twinning of the crystals. Attempts to solve the structure by molecular replacement did not give satisfactory results. Recently, clusters of rod-shaped crystals (form II) were grown in a solution containing PEG MME 550. These crystals belonged to the monoclinic system C2, with unit-cell parameters a = 132.9, b = 56.6, c = 86.5 A, beta = 95.7 degrees . Data sets were collected to a resolution of 2.4 A. The structure was solved by the molecular-replacement method. Model refinement is currently in progress.

Cellobiose dehydrogenase from the ligninolytic basidiomycete Ceriporiopsis subvermispora.

Cellobiose dehydrogenase (CDH), an extracellular flavocytochrome produced by several wood-degrading fungi, was detected in cultures of the selective delignifier Ceriporiopsis subvermispora when grown on a cellulose- and yeast extract-based liquid medium. CDH amounted to up to 2.5% of total extracellular protein during latter phases of the cultivation and thus suggested an important function for the fungus under the given conditions. The enzyme was purified 44-fold to apparent homogeneity. It was found to be present in two glycoforms of 98 kDa and 87 kDa with carbohydrate contents of 16 and 4%, respectively. The isoelectric point of both glycoforms is around 3.0, differing by 0.1 units, which is the most acidic value so far reported for a CDH. By using degenerated primers of known CDH sequences, one cdh gene was found in the genomic DNA, cloned, and sequenced. Alignment of the 774-amino-acid protein sequence revealed a high similarity to CDH from other white rot fungi. One notable difference was found in the longer interdomain peptide linker, which might affect the interdomain electron transfer at higher temperatures. The preferred substrate of C. subvermispora CDH is cellobiose, while glucose conversion is strongly discriminated by a 155,000-fold-lower catalytic efficiency. This is a typical feature of a basidiomycete CDH, as are the acidic pH optima for all tested electron acceptors in the range from 2.5 to 4.5.

Improving thermostability and catalytic activity of pyranose 2-oxidase from Trametes multicolor by rational and semi-rational design.

The fungal homotetrameric flavoprotein pyranose 2-oxidase (P2Ox; EC 1.1.3.10) catalyses the oxidation of various sugars at position C2, while, concomitantly, electrons are transferred to oxygen as well as to alternative electron acceptors (e.g. oxidized ferrocenes). These properties make P2Ox an interesting enzyme for various biotechnological applications. Random mutagenesis has previously been used to identify variant E542K, which shows increased thermostability. In the present study, we selected position Leu537 for saturation mutagenesis, and identified variants L537G and L537W, which are characterized by a higher stability and improved catalytic properties. We report detailed studies on both thermodynamic and kinetic stability, as well as the kinetic properties of the mutational variants E542K, E542R, L537G and L537W, and the respective double mutants (L537G/E542K, L537G/E542R, L537W/E542K and L537W/E542R). The selected substitutions at positions Leu537 and Glu542 increase the melting temperature by approximately 10 and 14 degrees C, respectively, relative to the wild-type enzyme. Although both wild-type and single mutants showed first-order inactivation kinetics, thermal unfolding and inactivation was more complex for the double mutants, showing two distinct phases, as revealed by microcalorimetry and CD spectroscopy. Structural information on the variants does not provide a definitive answer with respect to the stabilizing effects or the alteration of the unfolding process. Distinct differences, however, are observed for the P2Ox Leu537 variants at the interfaces between the subunits, which results in tighter association.

Biochemical and structural characterization of a short-chain dehydrogenase/reductase of Thermus thermophilus HB8: a hyperthermostable aldose-1-dehydrogenase with broad substrate specificity.

Thermus thermophilus HB8 is a hyperthermophilic bacterium, thriving at environmental temperature near 80 degrees C. The genomic analysis of this bacterium predicted 18 genes for proteins belonging to the short-chain dehydrogenase/reductases (SDR) superfamily, but their functions remain unknown. A SDR encoded in a gene (TTHA0369) was chosen for functional and structural characterization. Enzymatic assays revealed that the recombinant tetrameric protein has a catalytic activity as NAD(+)-dependent aldose 1-dehydroganse, which accepts various aldoses such as d-fucose, d-galactose, d-glucose, l-arabinose, cellobiose and lactose. The enzyme also oxidized non-sugar alicyclic alcohols, and was competitively inhibited by hexestrol, 1,10-phenanthroline, 2,3-benzofuran and indole. The enzyme was stable at pH 2-13 and up to 85 degrees C. We have determined the crystal structure of the enzyme-NAD(+) binary complex at 1.65A resolution. The structure provided evidence for the strict coenzyme specificity and broad substrate specificity of the enzyme. Additionally, it has unusual features, aromatic-aromatic interactions among Phe141 and Phe249 in the subunit interface and hydrogen networks around the C-terminal Asp-Gly-Gly sequence at positions 242-244. Stability analysis of the mutant D242N, F141A and F249A enzymes indicated that the two unique structural features contribute to the hyperthermostability of the enzyme. This study demonstrates that aldose 1-dehydrogenase is a member of the SDR superfamily, and provides a novel structural basis of thermostability.

Eukaryotic and bacterial gene clusters related to an alternative pathway of nonphosphorylated L-rhamnose metabolism.

The Entner-Doudoroff (ED) pathway is a classic central pathway of d-glucose metabolism in all three phylogenetic domains. On the other hand, Archaea and/or bacteria possess several modified versions of the ED pathway, in which nonphosphorylated intermediates are involved. Several fungi, including Pichia stipitis and Debaryomyces hansenii, possess an alternative pathway of L-rhamnose metabolism, which is different from the known bacterial pathway. Gene cluster related to this hypothetical pathway was identified by bioinformatic analysis using the metabolic enzymes involved in analogous sugar pathways to the ED pathway. Furthermore, the homologous gene cluster was found not only in many other fungi but also several bacteria, including Azotobacter vinelandii. Four putative metabolic genes, LRA1-4, were cloned, overexpressed in Escherichia coli, and purified. Substrate specificity and kinetic analysis revealed that nonphosphorylated intermediates related to L-rhamnose are significant active substrates for the purified LRA1-4 proteins. Furthermore, L-2-keto-3-deoxyrhamnonate was structurally identified as both reaction products of dehydration by LRA3 and aldol condensation by LRA4. These results suggested that the LRA1-4 genes encode L-rhamnose 1-dehydrogenase, L-rhamnono-gamma-lactonase, L-rhamnonate dehydratase, and L-KDR aldolase, respectively, by which L-rhamnose is converted into pyruvate and L-lactaldehyde through analogous reaction steps to the ED pathway. There was no evolutionary relationship between L-KDR aldolases from fungi and bacteria.