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Cellobiose dehydrogenase

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Patent Title
Authors
Roland Ludwig, Dietmar Haltrich, Wolfgang Harreither, Lo Gorton
Table of Contents
EXAMPLES
Example 1
Materials
Example 2
Enzyme Assays
Example 3
Enzyme Kinetics
Example 4
Protein Characterisation
Example 5
Screening For Suitable Cellobiose Dehydrogenases
Example 6
CDH Production And Purification From Fungal Sources
Example 7
Obtaining Nucleotide And Protein Sequences Of New CDHs
Example 8
Generation Of
Example 9
Production Of Recombinant CDH
Example 10
Electrochemical Equipment
Example 11
Preparation Of Enzyme Modified Graphite Electrodes
Example 12
Preparation Of Enzyme-Modified Screen Printed Electrodes
Example 13
PH Profiles Of CDH Immobilised On Electrodes
Example 14
Heterogeneous Enzyme Kinetics On Electrodes
Example 15
CDH Of
Example 16
CDH From
Example 17
Example 18
Example 19
Example 20
Comparative Example 21
Example 22
Sequences
EXAMPLES
Example 1
Materials
Chemicals used in buffers and fermentation media were commercial products and at least of analytical grade if not otherwise stated. Peptone from meat and microcrystalline cellulose were from VWR International (Vienna, Austria), alpha-cellulose from Sigma-Aldrich (Vienna, Austria). Substrates for kinetic studies were lactose, glucose, 2,6-dichloroindophenol (DCIP) and cytochrome c from horse heart(cyt c) from Sigma-Aldrich in the highest grade of purity available. Buffers were prepared using water purified and deionised (18 MΩ) with a Milli-Q system (Millipore, Bedford, Mass., USA), fermentation media contained reversed osmosis water (0.1 MΩ).
Example 2
Enzyme Assays
Enzymatic activity of cellobiose dehydrogenase was detected by two assays. The DCIP assay, measuring the activity of the flavin domain was performed by measuring the time-dependent reduction of 300 μM DCIP in 50 mM citrate-phosphate buffer at the indicated pH (3.0-8.0), containing 30 mM lactose at 520 nm and 30° C. The absorption coefficient for DCIP is pH dependent but differs at 520 nm only about 3% within pH 3.0 to 8.0 and was determined to be 6.8 mM−1 cm−1 (Karapetyan et al., 2005 Journal of Biotechnology 121: 34-48). Alternatively, enzymatic activity was determined by the reduction of cytochrome c at 30° C. and 550 nm (cyt c, c550=19.6 mM−1 cm−1, Canevascini et al., 1991, European Journal of Biochemistry 198: 43-52) in an assay containing 20 μM cyt c and 30 mM lactose, which specifically detects the activity of the whole enzyme (flavin and haem domain). The cyt c assay gives thereby also a measure of the efficiency of the intramolecular electron transfer (IET) between both domains as an indication of the enzyme's response on electrodes in a pH range of 3.0 to 8.0 (50 mM sodium citrate-phosphate buffer). For the detection of activity with glucose the above mentioned assays were used, but lactose was exchanged for 100 mM glucose. One unit of enzymatic activity was defined as the amount of enzyme that oxidises 1 μmol of lactose per min under the assay conditions. Lactose was chosen instead of the natural substrate cellobiose, as it shows no substrate inhibition with CDH. The reaction stoichiometry with carbohydrates is 1 for the two-electron acceptor DCIP, but 2 for the one-electron acceptor cyt c.
Example 3
Enzyme Kinetics
Carbohydrate stock solutions used for measuring activity and kinetic constants with the DCIP and cyt c assays were prepared in the appropriate buffer several hours before the experiment to allow mutarotation to reach equilibrium. pH profiles were determined using 50 mM citrate-phosphate buffer (3.0-8.0). To ensure an assay temperature of 30° C. the cuvettes were incubated in a thermostated chamber for at least 20 min. After the measurement, the pH was again checked in the cuvettes. Kinetic constants were calculated by fitting the observed data to the Henri-Michaelis-Menten equation or to the adapted model for substrate inhibition using nonlinear least-squares regression and the program SigmaPlot (Systat Software, San Jose, Calif., USA).
Example 4
Protein Characterisation
The protein concentration was determined by the dye-staining method of Bradford using a pre-fabricated assay from Bio-Rad Laboratories Hercules, Calif., USA) and bovine serum albumin as standard according to the manufacturers recommendations. For spectral characterisation apparently homogeneous CDH (in the oxidised state) was diluted to an absorption of −1 at 280 nm and the spectrum from 260 to 700 nm taken with an Hitachi U3000 spectrophotometer (Tokyo, Japan). After reduction with lactose (final concentration 1 mM) the reduced spectrum was taken. For electrophoretic characterisation SDS-PAGE was carried out on a Hoefer SE 260 Mighty Small II vertical electrophoresis unit. Gels (10.5×10 cm; 10% T, 2.7% C) were cast and run according to the manufacturers' modifications of the Laemmli system. Isoelectric focusing in the range of pH 2.5 to 6.5 was performed on a Multiphor II system using precast, dry gels rehydrated with Ampholytes (GE Healthcare Biosiences, Vienna, Austria). Protein bands on the SDS-PAGE were stained with silver, bands on the IEF gel with Coomassie blue R-250, according to the instructions.
Example 5
Screening for Suitable Cellobiose Dehydrogenases
Fungal strains (Chaetomium atrobrunneum CBS 238.71, Corynascus thermophilus CBS 405.69, Hypoxylon haematostroma CBS 255.63, Myriococcum thermophilum CBS 208.89, Neurospora crassa DSMZ 2968 and Stachybotrys bisbyi DSMZ 63042) were obtained from the Centraalbureau voor Schimmelcultures (CBS, Utrecht, The Netherlands) and Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ, Braunschweig, Germany) in freeze dried or actively growing form on agar slants and were periodically subcultured on potato dextrose agar (PDA) plates. Freshly inoculated agar plates were grown at 25 or 30° C., depending on the published growth temperatures of the cultures until reaching a diameter of 5 cm and then used to inoculate shaking flasks. The medium used for submersed cultures contained (per liter): 20 g of alpha-cellulose, 5 g of peptone from meat and 0.3 ml of a trace element solution. The trace element solution contained (per liter): 1 g of ZnSC4.7H2O, 0.3 g of MnCl2.4H2O, 3 g of H3BO3, 2 g of CoCl2.6H2O, 0.1 g of CuSC4.5H2O, 0.2 g of NiCl2.6H2O, 4 ml of H2SO4 (Sachslehner et al., 1997, Applied Biochemistry and Biotechnology 6365: 189-201). For the cultivation in shaking flasks, 1 L Erlenmeyer flasks were filled with 0.3 L of medium. After sterilisation the flasks were inoculated with 3 cm2 of finely cut mycelium from PDA plates and incubated in a rotary shaker (110 rpm, eccentricity=1.25 cm) at 25 or 30° C. Samples were taken regularly and the production of CDH was monitored.
Example 6
CDH Production and Purification from Fungal Sources
CDH production was performed in up to 16 parallel shaking flask cultures per strain using identical conditions as in the screening procedure. Cultures were harvested on the day exhibiting maximum cyt c activity. The culture supernatant was separated from residual cellulose and fungal biomass by centrifugation (20 min, 6000×g) and concentrated and diafiltrated using a polyethersulfone hollow fibre cross-flow module with a 10 kDa cut-off (Microza UF module SLP-1053, Pall Corporation) until a conductivity of 2 mS cm−1 was reached. The concentrated enzyme preparation was applied to a DEAE Sepharose column (chromatography equipment from GE Healthcare Biosciences) mounted on an AKTA Explorer system and equilibrated with 50 mM sodium acetate buffer, pH 5.5. The column was eluted with a linear salt gradient (0 to 0.5 M NaCl in the same buffer) in 10 column volumes (CV). Fractions with a high specific CDH activity were pooled, saturated ammonium sulphate solution was slowly added at 4° C. to 20% final saturation and applied to a PHE-Source column equilibrated with 100 mM sodium acetate buffer, pH 5.5 containing (NH4)2SO4 (20% saturation) and 0.2 M NaCl. The column was eluted with a linear gradient (0 to 100% of 20 mM sodium acetate buffer, pH 5.5) in 10 CV. The purest CDH fractions were pooled, desalted with 20 mM sodium acetate buffer, pH 5.5, concentrated and frozen at −70° C. for further use.
Example 7
Obtaining Nucleotide and Protein Sequences of New CDHs
Mycelium for nucleic acid isolations was harvested from cellulose induced growing cultures after 5 days. The mycelium was frozen in liquid nitrogen and homogenized using mortar and pestle. Portions of 100 mg mycelium were used for DNA extraction (Liu et al., 2000, Journal of Clinical Microbiology, 38: 471). Total RNA was isolated using TriFast (Peqlab, Erlangen, Germany). cDNA synthesis was performed with the First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania) and the anchor primer (5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTT-3′). Degenerated primer on the basis of known ascomycete CDH sequences were used to amplify fragments of genomic DNA encoding for CDH. For the amplification of the adjacent upstream region the DNA Walking SpeedUp Premix Kit (Seegene, Seoul, Korea) was used. For the amplification of the 3′ region cDNA was used as a template. To obtain full-length cDNA clones encoding the CDH proteins a nested PCR with two specific forward primer upstream of the putative start codon and two reverse primer, one specific for a sequence shortly downstream of the stop codon and the universal primer (5′-GTACTAGTCGACGCGTGGCC-3′) complementary to the anchor primer, was done. Names in the following primer table are abbreviated as follows: Chaetomium atrobrunneum, CA; Corynascus thermophilus, CT; Hypoxylon haematostroma, HH; Neurospora crassa, NC; Stachybotrys bisbyi, SB. forward primer reverse primer 5′-HH-1 atgcctctcttgtttggaccg Universal 5′-HH-2 tcaactctcatacttggcttgg 3′-HH-1 TACATCCAGCTTACCGGCACTG 5′-CA-1 TAGAGTCGAGGCGAACCAG UNIVERSAL 5′-CA-2 TTGCTGCTGTGCTCCTATGC 3′-CA-1 ttccttccctccatcaactcc 5′-SB-1 tcttgctacgcacttcggtattg Universal 5′-SB-2 TGTGTACCCTGTTTACTCACC 3′-SB-1 GTACCCATTAAGTACACTGCCAG 5′-CT-1 TCTTATAAGCCTTTGGCTCC Universal 5′-CT-2 TTGGCTCCGTTGGAACAATG 3′-CT-1 TTCCCCCTTCGAATTCGGTC 5′-NC-1 cgcaccaaccgtgtgaagtg Universal 5′-NC-2 TACAAGATGAGGACCACCTCG 3′-NC-1 AGCTACCTATCACCCTCTGTC The obtained PCR products were then fully sequenced to obtain the complete nucleic acid sequence of the respective cdh gene.
Example 8
Generation of
For enhanced production of recombinant Myriococcum thermophilum CDH (Zamocky et al., 2008,) in Pichia pastoris the gene (gene bank accession code EF 492052, GI:164597963) was codon optimised (FIG. 1) for expression in P. pastoris and synthesized by GenScript (Piscataway, N.J., USA). The gene shows a maximum similarity with CDH from Thielavia heterothallica (74% identity) and only 63% identity with the gene from Humicola insolens. On the protein level, the similarity is highest to Thielavia heterothallica CDH (93% identity, 97% positives, 0% gaps) and quite low for Humicola insolens CDH (61% identity, 71% positives, 2% gaps). The synthetic M. thermophilum CDH gene was mutated by a two-step site-directed mutagenesis protocol using PCR and Dpn/digestion. The yeast vector pPICZ A carrying the synthetic CDH gene was used as template for mutagenic PCR. For the replacement of Asp160 with Lys the primers 5′-TCCAAGCTTTTAAAGATCCAGGTAAC-3′ (Mt CDH-D181K-fw) and 5′-AAAAGCTTGGACCCAACCAAG-3′ (Mt CDH-D181Krv) were used. For the double mutant D547S/E550S primers 5′-GTCTTCTATTCTTTTTACTCTGCTTGGGATG-3′ (Mt CDH-D547S/E550S-fw) and 5′-ATAGAAGACCACATCAGGG-3′ (Mt CDH-D547S/E550S-rv) were used. The mutation sites are indicated by bold letters in the mutagenic forward primers. PCR was performed under the following conditions: 98° C. for 30 s, then 32 cycles of 98° C. for 10 s; 62° C. for 20 s; 72° C. for 2 min, with a 10 min final extension at 72° C. The 50 μl reaction mix contained Phusion HF Buffer (New England Biolabs, Ipswich, Mass., USA), 0.1 μg of plasmid DNA, 1 unit of Phusion DNA polymerase (New England Biolabs), 10 μM of each dNTP and 5 pmol of each primer. PCR reactions were separated by agarose gel electrophoresis and bands at 6 kB purified using the Wizard SV Gel and PCRCleanUp System (Promega, Madison, Wis., USA). The purified PCR fragment was digested with DpnI (Fermentas, Vilnius, Lithuania) to remove methylated DNA. 10 μl of this reaction was used to transform chemically competent NEB-5-Alpha E. coli cells (New England Biolabs) according to the manufacturer. For each mutation 3 colonies were checked by sequencing for the presence of the correct mutation. The purified plasmid of a positive clone was linearized with Sad and used to transform competent X-33 P. pastoris cells. Colonies growing on YPD zeocin agar plates (100 mg/L) were checked by PCR for the integration of the construct. Two positive clones of each mutation were further cultivated under induced condition and analysed for CDH production. The clones with the highest yield were selected for fermentation.
Example 9
Production of Recombinant CDH
An overnight pre-culture of a Pichia pastoris transformant (selected from a YPD plate with 100 mg/L Zeocin) was inoculated into 0.3 L of production stage medium in a Infors HT multifermenter (Bottmingen, Switzerland). The production stage medium contained per liter: 26.7 ml of H3PO4 (85%); 0.93 g of CaSO4.2H2O; 14.9 g of MgSO4.7H2O; 18.2 g of K2SO4; 4.13 g of KOH; 4% (v/v) glycerol; 1.45 ml of PTM1 trace element solution for P. pastoris according to the Invitrogen manual 053002 Ver. B (Carlsbad, Calif., USA). The PTM1 trace element solution contains per liter: 6 g of CuSO4.5H2O, 0.08 g of NaI, 3 g of MnSO4.H2O, 0.2 g of NaMoO4.2H2O, 0.02 g of H3BO3, 0.5 g of CoCl2, 20 g of ZnCl2, FeSO47H2O, 0.2 g of biotin, 5 ml of sulfuric acid. A glycerol feed was performed with an addition of 9 gL−1h−1 until the wet cell weight exceeded 150 g per liter. As soon as the residual glycerol was used up (determined by monitoring the increase of the dissolved oxygen tension), a methanol feed (100% methanol containing 12 ml PTM1 trace element solution per liter) with an average addition of 3 gL−1h−1 was started and continued for 72 h at 30° C. and 20% oxygen tension. The culture supernatant was separated from residual biomass by centrifugation (20 min, 6000×g) and concentrated and purified by hydrophobic interaction chromatography. To that purpose, saturated ammonium sulphate solution was slowly added to the clear culture supernatant at 4° C. to 20% final saturation. After a second centrifugation step (30 min, 30,000×g) the solution was applied to a PHE-Source column (GE Healthcare Biosciences) equilibrated with 100 mM sodium acetate buffer, pH 5.5 containing (NH4)2SO4 (20% saturation) and 0.2 M NaCl. The column was eluted with a linear gradient (0 to 100% of 20 mM sodium acetate buffer, pH 5.5) in 10 CV. The purest CDH fractions were pooled, desalted with 20 mM sodium acetate buffer, pH 5.5, concentrated and stored for further use.
Example 10
Electrochemical Equipment
A three electrode flow through amperometric wall jet cell was used (Appelqvist et al, Anal. Chim. Acta, 169 (1985) 237-47.) and contained the working electrode (graphite electrode modified with CDH), a reference electrode (Ag|AgCl in 0.1 M KCl) and a counter electrode made of a platinum wire, connected to a potentiostat (Zata Elektronik, Hoor, Sweden). The enzyme modified electrode was pressfitted into a Teflon holder and inserted into the wall jet cell and kept at a constant distance (ca. 1 mm) from the inlet nozzle. The response currents were recorded on a strip chart recorder (Kipp & Zonen, Delft, The Netherlands). The electrochemical cell was connected on-line to a single line flow injection (FI) system, in which the carrier flow was maintained at a constant flow rate of 0.5 ml min−1 by a peristaltic pump (Gilson, Villier-le-Bel, France). The injector was an electrically controlled six-port valve (Rheodyne, Cotati, Calif., USA), and the injection loop volume was 50 μl. For the screen-printed electrodes a special methacrylate wall jet flow for flow injection analysis (FIA) from propSense (Oviedo, Spain) was used. The electrochemical cell consists of a carbon working electrode (4 mm diameter), a carbon counter electrode and silver reference electrode connected to a potentiostat (Zäta Elektronic). The response currents were recorded on a strip chart recorder (Kipp & Zonen). The electrochemical cell was connected on-line to a single flow injection (FI) system, in which the carrier flow was maintained at a constant flow rate of 0.5 ml min−1 by a peristaltic pump (Gilson). For injection an electronically controlled six-port valve (Rheodyne) and a injection loop (50 μl) was used.
Example 11
Preparation of Enzyme Modified Graphite Electrodes
CDH was immobilised through simple chemo-physical adsorption onto the surface of solid spectroscopic graphite electrodes (diameter=3.05 mm, Ringsdorff Spektralkohlestäbe, SGL Carbon Sigri Greatlakes Carbon Group Ringsdorff-Werke GmbH, Bonn Germany). The electrode was cut and polished on wet emery paper (Tufbak, Durite, P400) and afterwards carefully rinsed with Milli-Q water and dried. Then 5 μl of enzyme solution was spread onto the entire active surface of the electrode (0.0731 cm2). The electrode was dried at room temperature and then stored overnight at 4° C. Before use, the electrode was thoroughly rinsed with Milli-Q water in order to remove any weakly adsorbed enzyme and plugged into in the wall jet cell already containing buffer. Then, the required potential was applied until a stable background current was obtained before any substrate was injected into the flow system.
Example 12
Preparation of Enzyme-Modified Screen Printed Electrodes
Five μl of enzyme solution was placed on the carbon-based electrode (DropSens, Oviedo, Spain) so that the whole area was entirely coated with solution. The immobilisation was allowed to proceed overnight at 4° C. Before use the electrodes were thoroughly rinsed with water. Cross-linking of the biocomponent was carried out by chemical modification with glutaraldehyde where 1 μl of an aqueous 1% glutaraldehyde solution was applied on the enzyme layer at 37° C. for 10-15 min. After rinsing the electrodes were allowed to dry at room temperature. The optimum for the applied potential was determined with a 10 mM lactose solution. The potential was varied stepwise from −250 to +600 mV vs. Ag|AgCl in 0.1 M KCl and +300 mV chosen for further experiments.
Example 13
pH Profiles of CDH Immobilised on Electrodes
The activity versus pH-profile for direct electron transfer (DET) of the adsorbed enzyme was determined electrochemically using a flow injection system. The substrate was lactose with a concentration of 5 mM. As enzyme assays should proceed under saturating substrate conditions so that slight variations in the absolute concentration have no influence on the reaction rate an amount at least 10 times the KM-value should be present. The following buffers were used in the experiments: 50 mM sodium citrate buffer (pH 3.0-6.5), 50 mM sodium phosphate buffer (pH 6.0-9.0). The buffers were degassed before use to prevent micro bubbles in the flow system.
Example 14
Heterogeneous Enzyme Kinetics on Electrodes
The kinetic parameters KM (Michaelis-Menten constant) and vmax (maximum volumetric activity), in this case equal to Imax (maximum response in current), were determined for a number of substrates in the DET mode (the electron acceptor being the graphite electrode). All kinetic parameters were calculated by nonlinear least-square regression, fitting the observed data to the Henri-Michaelis-Menten equation. These calculations were done after correcting the substrate concentration values using the dispersion factor of the flow system used including the wall jet cell by dividing the steady state current registered for a 50 mM ferrocyanide solution with that of the peak current for the injected sample having an equal concentration of ferrocyanide and using an applied potential of 400 mV (Ruzicka and Hansen, Flow Injection Analysis, 2nd ed., Wiley, New York 1988). In our case, for a 1 mm distance between electrode and inlet nozzle and 0.5 ml min−1 flow rate, the dispersion factor D was equal to 1.18 (FIG. 5).
Example 15
CDH of
A cellobiose dehydrogenase with high glucose turnover rates and activity under physiological pH conditions was obtained from liquid cultures of Stachybotris bisbyi. The culture was grown and screened as described. The maximum activity under the chosen conditions was 154 U/L (cyt c assay, pH 6.0, 24th day). For enzyme production and purification the outlined procedures were applied and resulted in a CDH preparation with a specific activity of 7.9 U/mg (DCIP assay, pH 6.0), an apparent molecular weight of 100 kDa as determined by SDS-PAGE and an isoelectric point of 4.5. The calculated molecular weight of the obtained protein sequence is 86.212 kDa and fits well to the native CDH when considering a glycosylation of 14% of S. bisbyi CDH, a value which lies within the observed range (2-15%, Zámocký et al., 2006, Current Protein and Peptide Science, 7: 255-280). The calculated isoelectric point is 6.37. The spectrum of Stachybotris bisbyi CDH is typical and shows the haem alpha-, beta- and gamma-bands of the reduced enzyme at 562, 533 and 430 nm. In the oxidised enzyme the gamma-band has its absorption maximum at 420 nm with a shoulder at 450 nm, which disappears after reduction with lactose and corresponds to the absorption peak of the FAD cofactor. Kinetic characterisation with the cyt c assay resulted in a pH profile with an activity maximum at pH 5.5 and 60% relative activity at pH 7.4 (FIG. 2.e). The specific activity at pH 7.4 was 0.58 U/mg using the cyt c assay and glucose as substrate. The pH optimum of the flavin domain was obtained with the DCIP assay and shows a similar trend indicating that substrate oxidation by the enzyme is efficient at pH 7.4. Kinetic constants for glucose (obtained with the cyt c assay at pH 5.5) are a KM of 950 mM and a kcat of 14.1 s−2 for glucose, which shows in comparison to currently known enzymes a far better suitability of this enzyme for the proposed application. To test the electrochemical behaviour of Stachybotris bisbyi CDH on electrodes, the purified enzyme preparation was immobilised by adsorption on a spectroscopic graphite electrode surface. Using a flow cell and subsequent injections of 50 mM glucose, DET currents were determined to determine the pH optimum, the current at the pH optimum and the current at pH 7.4. The optimum pH under the chosen conditions is 5.0 and 27% of the maximum current was obtained at pH 7.4 in 10 mM phosphate buffered saline (PBS) containing 100 mM NaCl. The KM value of the heterogenised enzyme at optimum pH on the electrode surface was determined to be 131 mM and Imax=65 nA. The currents obtained in glucose measurements should therefore follow a nearly linear relationship for concentrations approx. five-fold below the KM value. The DET current density obtained at pH 7.4 was 237 nA/cm2 and the linear range for glucose detection at pH 7.4 with the chosen setup within 3-15 mM.
Example 16
CDH from
Myriococcum thermophilum was found to oxidise many carbohydrates, glucose being one of them (Harreither et al., 2007, Electroanalysis 19: 172-180). From a pH profile measured with 5 mM cellobiose or lactose (FIG. 3A Harreither et al. 2007) a DET current at pH 7.5 can be seen with approx. 17% and 20%, respectively, of the value of peak maximum at pH 5. One could speculate that glucose could also be detected by this method, therefore a comparative measurement was performed using the same experimental conditions, enzyme and electrode preparation procedures (50 mM sodium citrate buffer, pH 4.0, 4.5, 5.0, 5.5, 6.5, 7.5; potential 400 mV vs. Ag|AgCl in 0.1 M KCl), exchanging the originally used 5 mM cellobiose or 5 mM lactose for 5 mM glucose. The results are given in FIG. 2.i and show a strong decrease of the detected current already at pH 6.5. At pH 7.5 no signal could be detected and therefore no value calculated as the response was within the electronic noise of the measurement (2 nA). The reason for this behaviour lies in the higher KM value of M. thermophilum CDH for glucose (KM=240 mM) than for cellobiose (KM=0.027 mM) or lactose (KM=0.055 mM, all data from Harreither et al. 2007) which reduces the obtained current at pH 7.5 below the limit of detection.
Example 17
Example 18
Example 19
Example 20
Comparative Example 21
Example 22
Sequences
>M.thermophilum (SEQ ID NO: 1) mrtssrligalaaallpsalagnnvpntftdpdsgitfntwgldedspqt qggftfgvalpsdalttdasefigylkcarndesgwcgislggpmtnsll itawphedtvytslrfatgyampdvyegdaeitqvsssvnsthfslifrc knclqwshggssggastsggvlvlgwvqafddpgnptcpeqitlqqhdng mgiwgaqlntdaaspsytdwaaqatktvtgdcegptetsvvgvpvptgvs fdyivvgggaggipaadklseagksvlliekgfastantggtlgpewleg hdltrfdvpglcnqiwvdskgiacedtdqmagcvlgggtavnaglwfkpy sldwdylfpdgwkyndvqpainralsripgtdapstdgkryyqegfevls kglaaggwtsvtannapdkknrtfahapfmfaggerngplgtyfqtakkr nnfdvwlntsvkrviregghitgvevepfrdggyegivpvtkvtgrvils agtfgsakillrsgigpedqlevvaasekdgptmignsswinlpvgynld dhlntdtvishpdvvfydfyeawddpiesdknsylesrtgilaqaapnig pmfweeivgadgivrqlqwtarvegslgapnghtmtmsqylgrgatsrgr mtitpslttivsdvpylkdpndkeaviqgiinlqnalqnvanltwlfpns titpreyvesmvvspsnrrsnhwmgtnklgtddgrkggsavvdldtrvyg tdnlfvidasifpgvpttnptsyivvaaehassrilalpdlepvpkygqc ggrewtgsfvcadgstceyqnewysqcl >H.haematostroma (SEQ ID NO: 3) mgrlgslaklllavglnvqqcfgqngpptpytdsetgitfatwsggngla pwggltfgvalpenalttdateligylkcgsngtttdawcglsfggpmtn slllmawphedeiltsfrfasgytrpdlytgdakltqisstidkdhftli frcqnclawnqdgasgsastsagslilgwasalraptnagcpaeinfnfh nngqmiwgatldesaanpsysewaakatatvtgdcggatpttttttttsv ptatgipvptgtydyivvgagaggipladklseagksvlliekgppssgr wggtlkpewlkdtnltrfdvpglcnqiwvnsagvactdtdqmagcvlggg tavnaglwwkpynldwdynfprgwksrdmaaatrrvfsripgtdnpsmdg krylqqgfeilagglkaagwtevtandapnkknhtyshspfmfsggergg pmgtylvsasrrknfhlwtgtavkrvvrtgghitglevepfvnggytgvv nvtsitgrvvlsagafgsakillrsgigpedqleivksstdgptmisdss witlpvgynledhtntdtvvthpdvvfydfyeaghpnvtdkdlylnsrag ilaqaapnigpmfweeikgrdgvvrqlqwtarvegsagtpngyamtmsqy lgrgaksrgrmtitkalttvvstvpylqdkndveaviqgiknlqaalsnv knltwayppsnttvedfvnnmlvsytnrrsnhwigtnklgtddgrsrggs avvdlntkvygtdnlfvvdagifpghittnptsyiviaaeraserildlp paraqprfaqcggrtwtgsfqcaapytcqyrnerysqcr >C.attrobruneum (SEQ ID NO: 5) mrpssrfvgalaaaasflpsalaqnnaavtftdpdtgivfnswglangap qtqggftfgvalpsdalttdatefigylecasadnqgwcgvsmggpmtns llitawphednvytslrfatgyampdvysgdatitqisssinathfklif rcqnclqwthdgasggastsagvlvlgwvqafpspgnptcpdqitleqhn ngmgiwgavmdsnvanpsytewaaqatktveaecdgpsetdivgvpvptg ttfdyivvgggaggiptadklseagksvlliekgiastaehggtlgpewl egndltrfdvpglcnqiwvdskgiacedtdqmagcvlgggtavnaglwfk pysldwdylfpsgwkyrdiqaaigrvfsripgtdapstdgkryyqqgfdv lagglsaggwnkvtansspdkknrtfsnapfmfsggerggplatyltsak krsnfnlwlntsvkrviregghvtgvevepfrtggyqgivnvtaysgrvv lsagtfgsakillrggigpadqlevvkaskidgptmisnaswiplpvgyn lddhlntdtvithpdvafydfyeawntpieadknsylssrtgilaqaapn igpmmweeikgadgivrqlqwtarvegsfdtpngqamtisqylgrgater grmtitpslttvvedvpylkdpndkeaviqgivnlqnalknvagltwtyp nssitpreyvdnmvvspsnrranhwmgtakigtddgrlaggsavvdlntk vygtdnlfvvdasifpgtpttnpsayivtaaehasqrilglaapkpvgkw gqcggrqwtgsfqcvsgtkcevvnewysqcl >C.thermophilum (SEQ ID NO: 7) mkllsrvgatalaatlslkqcaaqmtegtytheatgitfktwtpsdgstf tfglalpgdaltndateyigllrcqitdpsspgycgishgqsgqmtqall lvawasedvvytsfryatgytlpelytgdakltqiassysgdsfevlfrc encfswdqngatgsvstsngalvlgyaasksgltgatcpdtaefgfhnng fgqwgavlegatsdsyeewaqlatitppttcdgngpgdkvcvpapedtyd ylvvgagaggitvadklseaghkvlliekgppstglwngtmkpewlegtd ltrfdvpglcnqiwydsagiactdtdqmagcvlgggtavnaglwwkphpa dwddnfphgwkssdladatervfsripgtwhpsqdgklyrqegfevisqg lanagwrevdanqepseknrtyshsvfmfsggerggplatylasaaqrsn fnlwvntsvrrairtgprvsgvelecladggfngtvnlkegggvifsaga fgsaklllrsgigpedqleivasskdgetfiskndwiklpvghnlidhln tdliithpdvvfydfyaawdnpitedkeaylnsrsgilaqaapnigplmw eevtpsdgitrqfqwtcrvegdssktnsthamtlsqylgrgvvsrgrmgi tsgltttvaehpylhndgdleaviqgiqnvvdalsqvpdlewvlpppntt veeyvnslivspanrranhwmgtakmglddgrsggsavvdlntkvygtdn lfvvdasifpgmstgnpsamivivaeqaaqrilslry >S.bisbyi (SEQ ID NO: 9) mlfklsnwllalalfvgnvvaqlegptpytdpdtgivfqswvnpagtlkf gytypanaatvaatefigflecqgagwcsvslggsmlnkplvvaypsgde vlaslkwatgyanpepyggnhklsqisssvtsagfrvvyrcegclawnyq gieggsptngasmpigwaysassvlngdcvdntvliqhdtfgnygfvpde sslrteyndwtelptrvvrgdcggstttssvpsstappqgtgipvptgas ydyivvgsgaggipiadklteagkkvlliekgppssgrydgklkptwleg tnltrfdvpglcnqiwvdsagiacrdtdqmagcvlgggtavnaglwwkpn pidwdynfpsgwkssemigatnrvfsriggttvpsqdgktyyqqgfnvls sglkaagwtsvslnnapaqrnrtygagpfmfsggerggplatylatakkr gnfdlwtntqvkrvirqgghvtgvevenyngdgykgtvkvtpvsgrvvls agtfgsaklllrsgigpkdqlaivknstdgptmaserdwinlpvgynled htntdivishpdvvhydfyeawtasiesdktaylgkrsgilaqaapnigp lffdevrgadnivrsiqytarvegnsvvpngkamvisqylgrgavsrgrm tisqglntivstapylsnvndleaviksleniansltskvknlkiewpas gtsirdhytnmpldpatrranhwigtnkigtkngrltggdsvvdlntkvy gtdnlfvvdasifpgmvttnpsayiviaaehaaskilslptakaaakyeq cggleyngnfqcasgltctwlndyywqct >N.crassa (SEQ ID NO: 11) MRTTSAFLSGLAAVASLLSPAFAQTAPKTFTHPDTGIVFNTWSASDSQTK GGFTVGMALPSNALTTDATEFIGYLECSSAKNGANSGWCGVSLRGAMTNN LLITAWPSDGEVYTNLMFATGYAMPKNYAGDAKITQIASSVNATHFTLVF RCQNCLSWDQDGVTGGISTSNKGAQLGWVQAFPSPGNPTCPTQITLSQHD NGMGQWGAAFDSNIANPSYTAWAAKATKTVTGTCSGPVTTSIAATPVPTG VSFDYIVVGGGAGGIPVADKLSESGKSVLLIEKGFASTGEHGGTLKPEWL NNTSLTRFDVPGLCNQIWKDSDGIACSDTDQMAGCVLGGGTAINAGLWYK PYTKDWDYLFPSGWKGSDIAGATSRALSRIPGTTTPSQDGKRYLQQGFEV LANGLKASGWKEVDSLKDSEQKNRTFSHTSYMYINGERGGPLATYLVSAK KRSNFKLWLNTAVKRVIREGGHITGVEVEAFRNGGYSGIIPVTNTTGRVV LSAGTFGSAKILLRSGIGPKDQLEVVKASADGPTMVSNSSWIDLPVGHNL VDHTNTDTVIQHNNVTFYDFYKAWDNPNTTDMNLYLNGRSGIFAQAAPNI GPLFWEEITGADGIVRQLHWTARVEGSFETPDGYAMTMSQYLGRGATSRG RMTLSPTLNTVVSDLPYLKDPNDKAAVVQGIVNLQKALANVKGLTWAYPS ANQTAADFVDKQPVTYQSRRSNHWMGTNKMGTDDGRSGGTAVVDTNTRVY GTDNLYVVDASIFPGVPTTNPTAYIVVAAEHAAAKILAQPANEAVPKWGW CGGPTYTGSQTCQAPYKCEKQNDWYWQCV >M.thermophilum (SEQ ID NO: 2) atgaggacctcctctcgtttaatcggagcccttgcggcggcacttttgcc gtctgcccttgcccagaacaatgtcccgaatacttttaccgaccctgact cgggcatcaccttcaacacgtggggtctcgacgaggattctccccagact cagggcggtttcaccttcggcgttgccctgccctctgatgccctcacaac cgacgcctcggaatttatcggttacttgaaatgcgcaaggaatgatgaga gcggttggtgtggcatttcccttggcgggcctatgaccaactcgctcctc atcacagcctggccgcacgaggacacggtctacaccagtcttcggttcgc gaccggttacgccatgccggatgtctacgagggggacgccgagattaccc aggtctcttcctctgttaattcgacgcacttcagtctcatcttcaggtgc aagaactgcctgcaatggagccacggcggctcctccggcggcgcctctac ctcgggcggcgtgttggtactcggctgggttcaggcattcgacgatcccg gcaatccaacctgccccgagcagatcacactccagcagcacgacaacggc atgggtatctggggtgcccagctcaacacggatgctgccagcccgtccta cactgactgggccgcccaggctaccaagaccgtcaccggtgactgcgagg gccccaccgagacttctgtcgtcggcgtccccgttccgacgggtgtctcg ttcgattatattgttgtcggcggcggcgccgggggcatccccgcagctga caagctcagcgaggccggcaagagtgtgttgctcatcgagaagggctttg cttcgaccgcaaacaccggaggtactctcggccctgaatggcttgagggc catgatctgacccgcttcgacgtgccgggtctgtgcaaccagatctgggt cgattccaaggggatcgcttgcgaggataccgaccagatggctggctgtg ttctcggcggcggcaccgccgtgaatgctggcctgtggttcaagccctac tcgctcgactgggactacctcttccccgatggttggaagtacaatgacgt ccagcctgccatcaaccgcgccctctcgcgcatcccaggcaccgacgccc cttctaccgacggaaagcgctactaccaggagggttttgaggtcctctcc aagggcctggccgccggcggctggacctcagtcacggccaataatgcgcc cgacaagaagaaccgcaccttcgcccatgctcccttcatgtttgccggcg gcgagcgcaatggccctctgggtacctacttccagactgccaagaagcgc aacaatttcgatgtctggctcaacacgtcggtcaagcgcgtcatccgtga gggtggccacatcaccggcgtcgaggtcgagccgttccgtgacggtggtt acgagggcattgtccccgtcaccaaggttaccggccgcgttatcctgtct gccggcaccttcggcagtgcaaagattctgttaaggagcggtattggccc ggaagatcagctagaagttgtcgcggcctccgagaaggacggccctacca tgatcggcaactcgtcctggatcaacctgcctgtcgggtacaacctcgat gaccatctcaacaccgacacagtcatctcccaccccgatgtcgtgttcta cgacttttacgaggcgtgggatgatcccatcgagtctgacaagaatagct atctcgaatcgcgtacgggcatcctcgcccaagccgctcccaacattggc cctatgttctgggaagagatcgtgggcgcggacggcatcgttcgccagct ccagtggactgcccgtgtcgagggtagcctgggcgctcccaacggccaca ctatgaccatgtcgcagtaccttggccgtggtgccacctcacgcggccgc atgaccatcaccccgtctctgacgactatcgtctcagacgtgccttacct caaagaccccaacgacaaggaggctgtcatccaaggcatcatcaacctgc agaacgcccttcagaacgtcgccaacctgacttggctcttccccaactct accattacgccgcgcgaatacgttgagagcatggtcgtctccccgagcaa ccggcggtccaaccactggatgggcaccaacaagctcggtaccgacgacg ggcggaagggtggctccgctgtcgtcgacctcgacaccagggtctacggt actgacaacctcttcgtcatcgacgcctccatcttccccggcgtgcccac cacgaatcctacttcgtacatcgtggtagcggcagagcacgcttcgtccc gcatcctcgccctgcccgacctcgagcccgtccccaagtacggccagtgt ggcggtcgcgaatggaccggtagcttcgtctgcgccgatggttccacgtg cgagtaccagaatgagtggtactcgcagtgcttgtga >H.haematostroma (SEQ ID NO: 4) atgggtcgcctaggctctctcgcgaagttgcttctcgcagtcggcttgaa tgttcagcaatgcttcgggcaaaacggacccccgaccccctacactgata gtgagaccggtatcactttcgccacctggtccggcggaaacggcttagca ccctggggcggcttgactttcggtgttgcgttacctgaaaatgccctgac caccgacgctaccgagctgattggatacctgaaatgcggttccaatggca caaccacagatgcgtggtgtggtctgtcgtttgggggcccgatgactaac agcctccttctcatggcctggccgcacgaagacgagatcttgacatcatt ccgttttgccagtggatataccagaccagacctatacaccggcgatgcca aattaacgcagatatcatccaccatcgataaagatcactttactctaatt ttcaggtgccagaactgtctagcgtggaaccaagacggcgcgtctggttc cgcttcaactagtgccggctccttgatattaggctgggccagtgcgcttc gggccccgacgaatgcaggctgtccggctgaaatcaacttcaacttccac aacaatggccagatgatatggggcgctacattagacgagagcgccgcaaa cccatcatattcggaatgggctgccaaagccaccgctacggttaccggtg actgcggcggtgcaacccctacgaccactactaccaccaccacgtccgtc cctaccgccacaggtatcccagtgccaactggcacctacgactatattgt agttggtgcgggtgctggcggaatacctttggccgacaagctgagcgagg ctggaaagagtgtgttactgatcgaaaaggggccgccatcatcgggacga tggggtggcaccctcaagccagagtggttgaaggacaccaacttgacacg gtttgacgtccctggcctgtgcaatcagatctgggtcaactctgcaggcg tcgcttgtactgacacagaccaaatggccggttgcgttcttggtggtggt acagctgtcaacgctggcctatggtggaagccctacaacctcgactggga ttataacttcccacgcggatggaagtccagggatatggccgctgcaacca ggagagtcttctctcgcattcccggtacagataatccctcaatggatggc aagcggtatttacagcaaggcttcgaaatcctcgctggtggcttgaaagc cgctggatggaccgaggttaccgcgaatgacgcacccaataagaagaacc acacctactcacactcgccgttcatgttctccggcggcgaacggggtggc ccaatgggcacctacctggtatcggccagtagacgtaagaatttccatct atggacgggaacagcagtgaagagggttgttcgcacaggcggccatatca ccggtctggaggtcgagcccttcgtaaacggcggttataccggtgttgtc aacgtcacctcgattactggtcgggtcgtcttgtctgctggtgcgttcgg gtcggctaagatattactgaggagcggcatcggacctgaggatcagttgg agattgtcaagtcatcaaccgatggcccgaccatgatttccgattcttct tggattacgctacccgtcggttataatctagaggatcacacaaacaccga cacggtcgttacgcatcctgacgtcgtattttacgacttctacgaggctg gacatcctaatgttaccgacaaggacttgtatctcaactcacgggccgga atccttgctcaagcagcgcctaatatcggcccaatgttctgggaagagat taagggtagggacggcgtcgttagacagctccagtggacagccagagttg aaggaagtgccggtacaccgaatgggtacgccatgacaatgagccaatac cttggacgaggcgctaagtcgaggggccgaatgactatcacgaaggcgtt gacgaccgtcgtttctacagtaccttacctacaggataagaacgacgtgg aagcagtcatccagggaatcaagaaccttcaagcagcactttcgaacgtg aagaatctcacatgggcctacccaccatctaatacgacggtggaggactt tgttaacaacatgctggtttcatacactaataggcgttccaaccactgga ttgggaccaacaagctcggaaccgatgatggccgatcgcgcggaggttca gctgtcgtggacctcaacactaaggtatacggcaccgacaacctgttcgt cgttgacgcaggaatattccccggtcatattaccacgaacccgacttcgt atatcgtgatcgccgctgagcgcgcttctgagaggatcctcgaccttccc ccggctagagcacaaccgcgcttcgcgcagtgcggcgggcgaacgtggac gggtagcttccagtgtgcagcgccgtacacttgtcagtacaggaatgagc ggtattcccagtgccggtaa >C.attrobruneum (SEQ ID NO: 6) atgaggccctcctctcggtttgttggtgccctggcggcggcggcgtcgtt cctgccgtctgcccttgcccagaacaatgctgcagtcaccttcactgacc cggacaccggcatcgtcttcaactcctggggtcttgccaatggagcacca cagactcagggaggcttcacctttggtgtcgctctgccctctgatgcgct cacgaccgatgctaccgagttcattggttatttggaatgtgcctccgcgg acaaccagggctggtgcggtgtctcgatgggcggccccatgaccaactcg cttcttatcaccgcctggccgcacgaggacaacgtctacacctccctccg gtttgcaacaggatacgccatgccggatgtctactcgggagacgccacca tcacgcagatctcgtcgagcatcaacgcgacccacttcaagctcatcttc aggtgccagaactgcctgcaatggacccacgacggcgcttccggtggcgc ctccacgtctgccggtgttctggtcctcggctgggtccaggctttccctt cccctggcaacccgacgtgcccggaccagatcacgctcgagcagcacaac aacggcatgggcatctggggtgcggtgatggactccaacgtcgccaaccc gtcctacacagagtgggccgcgcaggccaccaagacggtcgaggccgagt gcgacggcccgagtgagacggatattgtcggcgtgcccgtgccgaccggc accaccttcgactacatcgtcgtgggcggcggtgccggcggtatccccac tgccgacaagctcagcgaggccggcaagagtgtgctgctgattgagaagg gcatcgcctcgactgctgagcacggcggcactctcggacccgagtggctc gagggcaacgacctgacgcggttcgacgtgcccggtctttgcaaccagat ctgggttgactccaagggcatcgcctgcgaggacaccgaccagatggccg gttgcgtcctcggcggcggcacggccgtcaacgccggcctctggttcaag ccctactcgctcgactgggactacctcttcccaagcggctggaagtaccg cgacatccaggccgccatcggcagggtgttctcgcgcatcccgggcactg acgcgccctcgaccgacggcaagcgctactaccagcagggcttcgacgtg ctcgcgggcggcctgagtgccggcggctggaacaaggtcacggccaactc gtctccagacaagaagaaccgcaccttctcgaacgcgcctttcatgttct cgggcggcgagcgcggcgggcccctggccacttatctcaccagcgccaag aagcgcagcaacttcaacctgtggctcaacacgtcggtcaagcgcgtcat ccgtgagggcggccacgtcacaggtgtcgaggtcgagcctttccggacgg gcgggtaccagggtatcgtgaacgttaccgccgtttcgggccgtgtcgtc ctgtcggctggtaccttcggcagtgccaagattctgctcagaggcggtat tggcccagcggatcagctcgaggttgtcaaggcgtcgaagatcgacgggc cgaccatgatcagcaatgcgtcttggattcctctgcctgttgggtacaac ctggatgaccatctcaacactgacactgtcattacccaccccgacgttgc cttctacgacttctacgaggcatggaacacgcccattgaggcggacaaga acagctacctgagcagccgcactggtatcctcgctcaggccgcgcccaac attggcccaatgatgtgggaggaaatcaagggtgccgacggtatcgtccg ccagctgcaatggaccgcccgtgtcgagggtagctttgacacgcctaacg ggcaggcgatgaccatctcgcagtacctcggccgcggcgcgacctcgcgc ggccgtatgaccatcaccccttcgctgacgaccgtcgtctcggacgtgcc gtacctcaaggacccgaacgataaggaggccgtcatccagggcatcgtca acctgcagaacgccctcaaaaacgtcgccggcctgacctggacctacccc aactcgagcatcacaccgcgcgaatacgtcgataatatggtagtctcccc tagcaaccggcgcgcaaaccactggatgggcacggccaaaatcggcaccg acgacggccgcctggccggcggctccgccgtcgtggacttgaacaccaag gtctacggcaccgacaacctctttgtcgtggacgcgtccatcttccccgg cacgcccaccaccaatccctcggcgtacatcgtcacggctgcggagcatg cttcgcagaggatcttggggttggctgcgccgaagccggttgggaaatgg ggccagtgtggcgggcggcagtggacagggagcttccagtgcgtgagtgg gacaaagtgtgaggtggtgaatgagtggtactcgcagtgcttgtag >C.thermophilum (SEQ ID NO: 8) atgaagcttctcagccgcgttggggccaccgccctagcggcgacgttgtc cctgaaacaatgtgcagctcagatgaccgaagggacgtacacccatgagg ctaccggtatcacgttcaagacatggactccttccgacggctcgactttc actttcggcttggccctccctggggacgcgctgacaaatgatgccaccga gtacattggtctcctgcgttgccaaatcaccgatccctcttcgcccggct actgtggcatctcccacggccagtccggccagatgacgcaggcgctgctg ctggtcgcttgggccagcgaggatgtcgtctacacgtcgttccgctacgc caccggctacacactccccgagctctacacgggcgacgccaagctgaccc agatcgcctcctcggtcagcggggacagcttcgaggtgctgttccgctgc gagaactgcttctcctgggaccagaacggcgccacgggcagtgtctcgac cagcaacggcgccctggttctcggctacgctgcctcgaagagtggtttga cgggcgccacgtgcccggacacggccgagtttggcttccacaacaatggt ttcggacagtggggtgcagtgctcgagggtgcgacctcggactcgtatga ggagtgggctcagctggccactatcacgcccccgaccacctgcgatggca acggccctggcgacaaggtgtgcgttccggctcccgaagacacgtatgat tacatcgttgtcggcgccggcgccggcggcatcacggtcgccgacaagct cagcgaggccggccacaaggtcctccttatcgagaagggtcctccgtcga ccggcctgtggaacgggaccatgaagcccgagtggctcgagggtaccgac ctcacccgcttcgacgtccccggtctgtgcaaccagatctgggtcgactc tgccggcattgcctgcaccgataccgaccagatggcgggctgcgttctcg gcggtggcaccgctgtcaatgctggtctgtggtggaagccccaccccgct gactgggacgacaacttccctcatggctggaagtcgagcgatctcgcgga tgcgaccgagcgtgtcttcagccgcattcccggcacgtggcacccgtcgc aggatggcaaactgtaccgccaggagggcttcgaggtcatcagccagggc ctggccaacgccggctggagggaagtcgacgccaaccaggagcccagcga gaagaaccgcacgtattcccacagtgtgttcatgttctcgggcggtgagc gcggcggccccctggcgacgtacctcgcctcggctgcccagcgcagcaac ttcaacttgtgggtcaacacttcggtccggagggccatccgcaccggccc cagggtcagtggcgtcgaactcgagtgccttgcggacggcggcttcaacg gtactgtcaacctgaaggagggtggtggtgtcatcttttcggctggcgct ttcggctcggccaagctgctccttcgcagcggcatcggtcctgaggacca gctcgagattgtggcgagctccaaggacggcgagaccttcatttccaaga atgattggatcaagctccccgtcggccataacctgatcgatcatctcaac accgacctcattattactcacccggatgtcgttttctatgacttctacgc ggcttgggacaatcccatcaccgaggacaaggaggcctacctgaactcgc ggtccggcattctcgcccaagcggcgcccaacatcggccctctgatgtgg gaggaagtcacgccatccgacggcatcacccgccagttccagtggacatg ccgtgttgagggcgacagctccaagaccaactcgacccacgccatgaccc tcagccagtatctcggccgtggcgtcgtctcgcgcggccggatgggcatc acttccgggctgaccacgacggtggccgagcacccgtacctgcacaacga cggcgacctggaggcggtgatccagggtatccagaacgtggtggacgcgc tcagccaggtgcccgacctcgagtgggtgctcccgccgcccaacacgacg gtggaggaatacgtcaacagcctgatcgtgtctccggctaaccgccgggc caaccactggatgggcacggccaagatgggcctcgatgacggccgctcgg gcggctccgcggtcgtcgacctcaacacaaaggtgtatggcaccgacaac ctgtttgtcgtcgacgcctccatcttccctggcatgtcgacgggcaaccc gtcggctatgatcgtcatcgtggccgagcaggcggcccagcgcatcctgt ccctgcggtattag >S.bisbyi (SEQ ID NO: 10) atgctgttcaagctctcaaattggttgctagcgcttgcgctctttgttgg caatgtcgttgctcaactcgaggggcctaccccgtacacggatccagata ccggcattgtctttcagtcctgggtcaatccagcagggaccctgaagttt ggttacacttaccccgcaaatgctgctacggttgccgccacggaatttat cggtttcctggaatgccaaggggctggatggtgtagcgtctcactcggtg gctccatgcttaacaagccgcttgttgttgcctaccctagtggcgatgaa gtcctcgcttctttgaagtgggccacaggctacgcgaatccagagcctta cggcggcaatcacaagctgtcccagatcagctcgtccgtcacctctgctg gcttcagggtcgtctatcgatgtgagggatgtctcgcctggaactaccag ggaattgagggagggagccccaccaatggtgcgtccatgcctatcggttg ggcttacagcgcaagttctgtactcaacggggattgtgtggataacactg ttctcattcaacatgacacctttggcaattatggcttcgtacctgatgaa tcatctcttcgcacggagtacaatgactggacggagcttccgaccagggt tgtcaggggagactgcggcggttccacaactacctcttcggtgccctcct caacggcgcctcctcaaggtactggcataccggttcctactggcgcaagc tatgactacatagttgttggctcgggtgctggaggtattcccattgcgga taagcttaccgaggctggcaaaaaggttttgttgattgagaagggaccac cctcttctggtcgctacgatggaaagctaaagccgacgtggcttgaggga actaatctcacccgattcgatgtgcctggcctctgcaaccaaatatgggt cgactccgctggcattgcatgccgtgataccgatcagatggctggttgtg ttcttggcggtggtactgctgtcaatgcaggtctatggtggaagcctaac cctattgattgggactataatttcccttcaggctggaagtcaagcgagat gataggcgcgacaaaccgtgtcttttcacgtattggtggtactactgttc cttcgcaggacggaaagacctactatcagcaaggtttcaacgttctttcc agcggtctcaaggctgcgggctggacatctgttagcctgaataacgcccc tgcgcaaaggaaccgcacctatggtgctggccctttcatgttctctggtg gagagcgaggtggacctttggccacctacctggccactgccaagaagaga ggaaacttcgacctctggacgaatacccaagttaagcgtgtaattcgaca gggaggtcatgttactggagtggaggtcgaaaactataacggtgatgggt acaagggcactgtcaaggtgactcctgtatctgggcgagttgtcctatct gctggtacctttggcagtgctaagcttttgctccgaagcggtatcggtcc caaggatcaactagctattgtcaagaactcgactgatggccctactatgg cttccgagagggactggattaatcttcccgttggctacaacttggaggac catactaacaccgacattgtcatctcccatccagatgtggtccattacga cttctatgaggcttggacagcgtcaatcgagtctgacaagactgcttatt tgggcaagcgttctggcatcctcgctcaagccgcccccaacatcgggcct ctcttctttgacgaagttcgcggtgctgacaacattgtccgctcaattca gtacactgctcgtgtggagggcaacagtgtggtccctaatggcaaggcca tggtgatcagccagtaccttggtcgtggcgctgtttccaggggtcgaatg accatctctcaaggtctcaatacgattgtttccaccgctccatacctctc aaacgtcaatgatctcgaggccgtcattaagagccttgagaacatagcga acagcttgacgtcaaaggttaaaaacctcaagattgaatggcctgcctct ggtacatccattcgcgatcacgtcacgaatatgcctctcgacccggccac ccgccgagcgaatcattggattggcactaacaagatcggaaccaagaatg gtcgactgacaggtggtgattccgtcgttgatttgaacactaaggtctat ggtacagacaatctgtttgtggtcgatgcttctattttccctggcatggt tacgaccaacccctcggcctacattgtaattgccgctgagcatgctgcat cgaagattctgagcctacctactgctaaggctgccgcgaagtacgaacaa tgtggtggccttgaatataatggtaactttcagtgtgcgtctggattaac ctgcacttggttaaacgactactactggcagtgtacttaa >N.crassa (SEQ ID NO: 12) atgaggaccacctcggcctttctcagcggcctggcggcggtggcttcatt gctgtcgcccgccttcgcccaaaccgctcccaagaccttcactcatcctg ataccggcattgtcttcaacacatggagtgcttccgattcccagaccaaa ggtggcttcactgttggtatggctctgccgtcaaatgctcttactaccga cgcgactgaattcatcggttatctggaatgctcctccgccaagaatggtg ccaatagcggttggtgcggtgtttctctcagaggcgccatgaccaacaat ctactcattaccgcctggccttctgacggagaagtctacaccaatctcat gttcgccacgggttacgccatgcccaagaactacgctggtgacgccaaga tcacccagatcgcgtccagcgtgaacgctacccacttcacccttgtcttt aggtgccagaactgtttgtcatgggaccaagacggtgtcaccggcggcat ttctaccagcaataagggggcccagctcggttgggtccaggcgttcccct ctcccggcaacccgacttgccctacccagatcactctcagtcagcatgac aacggtatgggccagtggggagctgcctttgacagcaacattgccaatcc ctcttatactgcatgggctgccaaggccaccaagaccgttaccggtactt gcagtggtccagtcacgaccagtattgccgccactcctgttcccactggc gtttcttttgactacattgtcgttggtggtggtgccggtggtattcccgt cgctgacaagctcagcgagtccggtaagagcgtgctgctcatcgagaagg gtttcgcttccactggtgagcatggtggtactctgaagcccgagtggctg aataatacatcccttactcgcttcgatgttcccggtctttgcaaccagat ctggaaagactcggatggcattgcctgctccgataccgatcagatggccg gctgcgtgctcggcggtggtaccgccatcaacgccggtctctggtacaag ccctacaccaaggactgggactacctcttcccctctggctggaagggcag cgatatcgccggtgctaccagcagagccctctcccgcattccgggtacca ccactccttctcaggatggaaagcgctaccttcagcagggtttcgaggtt cttgccaacggcctcaaggcgagcggctggaaggaggtcgattccctcaa ggacagcgagcagaagaaccgcactttctcccacacctcatacatgtaca tcaatggcgagcgtggcggtcctctagcgacttacctcgtcagcgccaag aagcgcagcaacttcaagctgtggctcaacaccgctgtcaagcgcgtcat ccgtgagggcggccacattaccggtgtggaggttgaggccttccgcaacg gcggctactccggaatcatccccgtcaccaacaccaccggccgcgtcgtt ctttccgccggcaccttcggcagcgccaagatccttctccgttccggcat tggccccaaggaccagctcgaggtggtcaaggcctccgccgacggcccta ccatggtcagcaactcgtcctggattgacctccccgtcggccacaacctg gttgaccacaccaacaccgacaccgtcatccagcacaacaacgtgacctt ctacgacttttacaaggcttgggacaaccccaacacgaccgacatgaacc tgtacctcaatgggcgctccggcatcttcgcccaggccgcgcccaacatt ggccccttgttctgggaggagatcacgggcgccgacggcatcgtccgtca gctgcactggaccgcccgcgtcgagggcagcttcgagacccccgacggct acgccatgaccatgagccagtaccttggccgtggcgccacctcgcgcggc cgcatgaccctcagccctaccctcaacaccgtcgtgtctgacctcccgta cctcaaggaccccaacgacaaggccgctgtcgttcagggtatcgtcaacc tccagaaggctctcgccaacgtcaagggtctcacctgggcttaccctagc gccaaccagacggctgctgattttgttgacaagcaacccgtaacctacca atcccgccgctccaaccactggatgggcaccaacaagatgggcaccgacg acggccgcagcggcggcaccgcagtcgtcgacaccaacacgcgcgtctat ggcaccgacaacctgtacgtggtggacgcctcgattttccccggtgtgcc gaccaccaaccctaccgcctacattgtcgtcgccgctgagcatgccgcgg ccaaaatcctggcgcaacccgccaacgaggccgttcccaagtggggctgg tgcggcgggccgacgtatactggcagccagacgtgccaggcgccatataa gtgcgagaagcagaatgattggtattggcagtgtgtgtag
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