CN114981438A

CN114981438A - Process for producing glucuronic acid

Info

Publication number: CN114981438A
Application number: CN202180009959.XA
Authority: CN
Inventors: 藤井昂洸; 宅见高史; 荒木俊雄; 本田通济
Original assignee: Ikeda Food Research Co Ltd
Current assignee: Ikeda Food Research Co Ltd
Priority date: 2020-01-23
Filing date: 2021-01-14
Publication date: 2022-08-30
Also published as: WO2021149587A1; JPWO2021149587A1

Abstract

The present invention provides a simple and low-cost method for producing glucuronic acid or glucuronic acid derivatives with reduced environmental load compared with conventional methods. The method for producing glucuronic acid comprises the following steps: a flavin-bound glucose dehydrogenase having glucose-6-dehydrogenase activity is allowed to act on glucose in the presence of a mediator to produce glucuronic acid.

Description

Process for producing glucuronic acid

Technical Field

The present invention relates to a method for producing glucuronic acid using a flavin-bound glucose dehydrogenase, a method for producing a glucuronic acid derivative, and a catalyst for producing glucuronic acid or a glucuronic acid derivative.

Background

Glucuronic acid (chemical formula: C) ₆ H ₁₀ O ₇ ) Is a representative uronic acid derived from glucose. Glucuronic acid has a detoxifying effect of binding harmful substances in the body and discharging them into urine. Currently, glucuronic acid and glucuronolactone, which is a lactone of glucuronic acid, are used in pharmaceutical products and quasi-pharmaceutical products in japan.

As a conventional method for producing glucuronic acid, the following methods have been reported: enzymes that oxidize the hydroxymethyl group at the 6-position of glucose, for example, galactose oxidase (patent document 1), aldehyde dehydrogenase (patent document 2), and alcohol dehydrogenase (patent document 3) are used. However, these enzymes have the following problems: the enzyme has a low specific activity and low oxidation specificity for hydroxymethyl groups of glucose, and produces glucose oxides such as gluconic acid in addition to glucuronic acid.

As a method for producing glucuronic acid using an enzyme that uses a saccharide other than glucose as a substrate, the following methods have been reported: a method for producing glucuronic acid by allowing a paenibacillus bacterium or a glycosidic bond hydrolase derived from the bacterium to act on α -1, 4-polyglucuronic acid produced by oxidizing starch (patent document 4); a method for producing glucuronic acid using oxidized trehalose as a substrate and an enzyme for hydrolyzing the oxidized trehalose (patent document 5); a method for producing glucuronic acid using an inositol oxygenase using inositol as a substrate (patent document 6). However, these production methods have problems such as complicated production of a substrate and high price of a substrate.

Further, as a method for producing glucuronic acid from glucose by a fermentation method, a production method using pseudomonas saccharoketogenes Rh47-3 strain has been reported (patent document 7), but the fermentation method has the following problems compared with an enzymatic method: the impurity substance in the culture solution increases, and the purity of glucuronic acid as a target product decreases, and high purification is required.

Further, as a method for producing glucuronic acid without using an enzyme, a method is known in which starch is converted into oxidized starch with nitric acid and then hydrolyzed with sulfuric acid to produce glucuronic acid (non-patent document 1), but this method has a problem that a large amount of a reagent having a large environmental load, such as nitric acid or sulfuric acid, has to be used.

It is known that glucuronidation, which is a bonding of glucuronic acid to another compound via a glycosidic bond, increases the water solubility of the resulting glucuronide compared to other compounds, thereby enhancing physiological effects. In order to utilize this property, a method of glucuronidation of a compound has been studied, and as an enzymatic method, for example, a method of glucuronidation by transferring glucuronic acid to an arbitrary substrate using UDP-glucuronyltransferase present in a living body has been reported (non-patent document 2). Further, as a chemical synthesis method, for example, a method of glucuronidation by reacting an object having a hydroxyl group to be glucuronidated with 2,3, 4-tri-o-acetyl- α -D-methyl glucuronide as a glucuronic acid donor and trimethylsilyl trifluoromethanesulfonate as a lewis acid catalyst (non-patent document 3) has been reported. However, these methods have problems that the reagents used in the reaction are expensive, the yield of the target glucuronide is low, and purification for the purpose of removing by-products after the reaction is required.

On the other hand, flavin-bound glucose dehydrogenase (flavin-bound GDH, EC 1.1.5.9) is an enzyme that catalyzes a reaction of dehydrogenating (oxidizing) the hydroxyl group at the 1-position of glucose using flavin as a coenzyme. Flavin-binding GDH derived from Aspergillus (Aspergillus) is not affected by dissolved oxygen, has low action on maltose and galactose, and has high substrate specificity for glucose, and therefore, is used for measurement of blood glucose concentration.

Documents of the prior art

Patent document

Patent document 1 International publication No. 2003/072742

Patent document 2 International publication No. 2013/183610

Patent document 3, Japanese patent application laid-open No. 5-68541

Patent document 4 Japanese patent laid-open publication No. 2009-165415

Patent document 5 Japanese laid-open patent publication No. 2002-153294

Patent document 6 specification of U.S. Pat. No. 7923231

Patent document 7 International publication No. 2008/139844

Patent document 8 International publication No. 2006/101239

Non-patent document

Non-patent document 1 journal of Japan chemistry, vol.82, No. 11, p.1536-1539

Non-patent document 2, chemical and biological 1999, Vol.37, No. 4, p.242-247

Non-patent document 3 journal of Japan scientific and technical society, 2016 (Vol. 21, No. 2), p.149-155

Disclosure of Invention

The present invention addresses the problem of providing a method for producing glucuronic acid or a glucuronic acid derivative, which is simpler and less expensive than conventional methods, and which has a reduced environmental load.

The present inventors have conducted intensive studies on a method for directly producing glucuronic acid from glucose, which is an inexpensive raw material, and as a result, have found that an enzyme having specificity for oxidation of the hydroxymethyl group at the 6-position of glucose is present in flavin-bound GDH, which is an enzyme for oxidizing glucose to glucono-1, 5-lactone, and that when this enzyme having glucose-6-dehydrogenase activity is allowed to act on glucose, the hydroxymethyl group at the 6-position of glucose is oxidized to specifically produce glucuronic acid. Further, it was found that when the enzyme is allowed to act on a glucose derivative such as glucoside, the hydroxymethyl group at the 6-position of the glucose skeleton is oxidized to specifically produce a glucuronic acid derivative.

That is, the present invention relates to the following [1] to [11 ].

[1] A method for producing glucuronic acid, comprising the steps of: a flavin-bound glucose dehydrogenase having glucose-6-dehydrogenase activity is allowed to act on glucose in the presence of a mediator to produce glucuronic acid.

[2] A method for producing a glucuronic acid derivative, comprising the steps of: a glucuronic acid derivative is produced by allowing a flavin-bound glucose dehydrogenase having glucose-6-dehydrogenase activity to act on a glucose derivative in the presence of a mediator.

[3] The process according to [2], wherein the glucose derivative is an amino sugar or an N-acetyl compound thereof, a glucoside, or a glucose analog.

[4] The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of [1] to [3], wherein the flavin-bound glucose dehydrogenase is any one of the following proteins (i) to (iii):

(i) protein having amino acid sequence represented by SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38

(ii) A protein having glucose-6-dehydrogenase activity which has an amino acid sequence in which 1 to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38

(iii) A protein having glucose-6-dehydrogenase activity, which has an amino acid sequence having 80% or more sequence homology with the amino acid sequence represented by SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38.

[5] The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of [1] to [4], wherein the flavin-bound glucose dehydrogenase has the following properties (1) to (8):

(1) the function is as follows: catalyzing dehydrogenation (oxidation) reaction of hydroxymethyl at 6-position of glucose by using flavin as coenzyme

(2) Solubility: water solubility

(3) pH stability: is stable at a pH of at least 5.5 to 8.7

(4) Thermal stability: is stable at 35 deg.C or more

(5) Substrate specificity: the action on maltose, xylose and galactose is less than 2.0% when the action on glucose is 100%

(6) Km value (relative to glucose): 30mM or more

(7) Molecular weight: 64 to 66kDa (calculated from the amino acid sequence after removal of the signal)

(8) Glucose oxidase activity: and cannot be detected.

[6] The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of [1] to [5], wherein the flavin-bound glucose dehydrogenase is derived from a microorganism belonging to the genus anthrax (Colletotrichum), genus Pleurospora (Glomerella), genus Ascophyllum (Diaporthe), genus Husky (Khuskia), genus Acremonium (Acremonium), genus Lasiosphaeria, genus Fusarium (Fusarium), or genus Phyemonopsis.

[7] The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of [1] to [6], wherein a recombinant microorganism into which a gene encoding a flavin-bound glucose dehydrogenase is introduced is used as the flavin-bound glucose dehydrogenase.

[8] The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to [7], wherein the gene encoding the flavin-bound glucose dehydrogenase is a gene composed of any one of the following DNAs (a) to (e):

(a) DNA having the base sequence represented by SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37

(b) A DNA encoding a protein having glucose-6-dehydrogenase activity which has a base sequence in which 1 to several bases are deleted, substituted or added from the base sequence represented by SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 and which has been modified by addition of at least one nucleotide

(c) A DNA encoding a protein having glucose-6-dehydrogenase activity and having a nucleotide sequence having 80% or more sequence homology to the nucleotide sequence represented by SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37

(d) A DNA which hybridizes under stringent conditions to a DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 and which encodes a protein having glucose-6-dehydrogenase activity

(e) DNA encoding the protein of the following (i), (ii) or (iii)

[9] The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of [1] to [8], wherein an oxidase is further caused to act.

[10] A flavin-bound glucose dehydrogenase having glucose-6-dehydrogenase activity, which is any one of the following proteins (i) to (iii):

[11] A catalyst for producing glucuronic acid or a glucuronic acid derivative, comprising a flavin-bound glucose dehydrogenase protein having glucose-6-dehydrogenase activity.

According to the present invention, glucuronic acid can be specifically and directly produced from glucose which is an inexpensive raw material, and therefore, the production cost is significantly reduced compared to conventional methods, which are simple and convenient. Further, since the reaction is carried out at normal temperature and pressure without using a strong acid such as nitric acid or sulfuric acid, the risk during production can be avoided, and the environmental burden can be suppressed.

In addition, since the glucuronic acid derivative can be directly produced specifically from the glucose derivative, the production cost can be reduced compared with the conventional glucuronidation method, because of simplicity. In addition, an effect of improving the water solubility of the glucose derivative by oxidation of the hydroxymethyl group can be expected.

Drawings

FIG. 1 shows the thermostability of CpGDH, FGDH and CsGDH.

FIG. 2 shows the pH stability of CpGDH, FGDH and CsGDH.

FIG. 3 shows the results of TLC analysis of glucose oxide.

FIG. 4 shows the results of HPLC analysis of glucose oxide.

FIG. 5 shows the results of TLC analysis of the substrate oxide.

FIG. 6 shows the results of TLC analysis of glucose oxide.

FIG. 7 shows the thermostability of CglGDH, CoGDH, CtoGDH, CgoGDH, GsGDH and DhGDH.

FIG. 8 shows the pH stability of CglGDH, CoGDH, CgoGDH, GsGDH and DhGDH.

FIG. 9 shows the results of TLC analysis of glucose oxide.

FIG. 10-1 shows the thermostability of Ko37GDH, AsGDH, Ko38GDH, LhGDH, DsGDH and CtaGDH.

FIG. 10-2 shows the thermostability of FlaGDH, PcGDH, Fla _ A.oGDH and Pc _ A.oGDH.

FIG. 11-1 shows the pH stability of Ko37GDH, AsGDH, Ko38GDH, LhGDH, DsGDH and CtaGDH.

FIG. 11-2 shows the pH stability of FlaGDH, PcGDH, Fla _ A.oGDH, and Pc _ A.oGDH.

FIG. 12 shows the production of piceid ¹ H-NMR analysis and ¹³ C-NMR analysis results.

FIG. 13 shows the oxidation of picea asperata neoside ¹ H-NMR analysis and ¹³ C-NMR analysis results.

Detailed Description

In the present specification, a flavin-bound glucose dehydrogenase having glucose-6-dehydrogenase activity (hereinafter, sometimes referred to as "flavin-bound GDH") refers to an enzyme that catalyzes a reaction of dehydrogenating (oxidizing) a hydroxymethyl group at the 6-position of glucose using flavin as a coenzyme. Since the flavin-bound GDH of the present invention selectively acts on the 6-position of glucose to specifically oxidize the hydroxymethyl group at the 6-position of glucose to a carboxyl group, glucuronic acid is specifically produced when the enzyme is allowed to act on glucose. When the enzyme is allowed to act on a glucose derivative such as glucoside, the hydroxymethyl group at the 6-position of the glucose skeleton is specifically oxidized, and thus a glucuronic acid derivative is specifically produced from the glucose derivative.

The glucose-6-dehydrogenase activity can be confirmed by allowing glucose-6-dehydrogenase to act on glucose, analyzing the reaction product by thin layer chromatography or HPLC, and comparing the analyzed product with a standard glucuronic acid product in which the 6-position of glucose is oxidized.

The flavin-bound GDH of the present invention has substantially no glucose-1-dehydrogenase activity and does not substantially produce gluconic acid from glucose as a substrate. Here, it essentially means that the reaction product was analyzed by thin layer chromatography or HPLC, and as a result, the formation of gluconic acid was not confirmed.

The flavin-bound GDH of the present invention is not particularly limited as long as it is an enzyme having glucose-6-dehydrogenase activity, and is preferably any one of the following proteins (i) to (iii).

(iii) A protein having glucose-6-dehydrogenase activity, which has an amino acid sequence having 80% or more sequence homology with the amino acid sequence represented by SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38

The number of deletions, substitutions, or insertions of 1 to several amino acid residues in the amino acid sequence in which 1 to several amino acid residues are deleted, substituted, or inserted in the amino acid sequence represented by SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38 is not limited as long as it shows an enzyme activity equivalent to that of a protein having the amino acid sequence represented by SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38, and is preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 8.

In the present specification, the sequence homology with the amino acid sequence represented by seq id No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 is 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and further preferably 99% or more. The percent homology of such sequences can be calculated using published or commercially available software with algorithms for comparing reference sequences as query sequences. For example, BLAST, FASTA, GENETYX (GENETYX corporation), or the like can be used.

The amino acid sequences shown by SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 are each based in turn on Colletotrichum plura MAFF305790, Fuscus _ F5126, Colletotrichum sp, Colletotrichum gloeosporioides, Colletotrichum orbiculare (Colletotrichum orbiculare), Colletotrichum anthracis (Colletotrichum tobermanii), Colletotrichum roseum (Colletotrichum trichothecoides) MAFF240289, Pectinatum minor (glotrichum roseum sp.) 057037, Stem Helianthus nigricans (aphylvaninthianth), Sphaerotheca nigrella (Kskyronyensis), Sphaeria rosea, Fusarium rosea (Fusarium oxysporium), Fusarium oxysporium (Fusarium oxysporium), Fusarium oxysporum (Fusarium oxysporum), Fusarium oxysporum, Fusarium species (Fusarium oxysporum) and Fusarium species (Fusarium oxysporum) in this species. Further, SEQ ID Nos. 36 and 38 are sequences obtained by replacing the sequences of the secretion signals estimated as SEQ ID Nos. 32 and 34 with the signal sequence of GDH derived from Aspergillus oryzae (Aspergillus oryzae).

The amino acid sequence represented by the sequence No. 4 or 6 is the same as the known amino acid sequence represented by the sequence No. 2 described in Japanese patent No. 6455714 and the sequence No. 1 described in Japanese patent No. 5435180, and the amino acid sequences represented by the sequence Nos. 8, 10, 12, 18, 20, 22, 24, 26, 28, 30, 32 and 34 are amino acid sequences registered in a known database, but there is no report that a protein having the amino acid sequence has glucose-6-dehydrogenase activity.

The flavin-bound GDH of the present invention preferably has the following properties (1) to (8). Examples of flavin include flavin adenine dinucleotide (F A D) and flavin mononucleotide (F M N), and F A D is preferable.

(2) Solubility: water solubility

(3) pH stability: is stable at a pH of at least 5.5 to 8.6

The enzyme has a residual enzyme activity of 80% or more after 1 hour of treatment at 30 ℃ as long as the enzyme has a pH of at least 5.5 to 8.6. The pH stability is preferably 4.3 to 9.3, 5.5 to 8.7, 3.2 to 9.3, 5.5 to 9.3, 4.4 to 9.3, 4.0 to 9.6, 4.4 to 9.3, 5.0 to 9.3, 3.3 to 9.6, 5.5 to 8.6, 4.3 to 9.6, 5.0 to 9.6, 4.0 to 8.6, 4.9 to 8.7, 3.3 to 9.9, 4.4 to 9.8, 4.0 to 8.8, 4.4 to 9.8, and 4.0 to 9.6.

Even if the pH is the same, the residual activity may vary depending on the type of the buffer.

(4) Thermal stability: is stable at 35 deg.C or more

The enzyme has a residual enzyme activity of 80% or more after treatment in 100mM potassium phosphate buffer (pH6.0, 7.0 or 8.0) or 100mM Tris-HCl buffer (pH8.0) for 60 minutes, as long as the enzyme is at least 35 ℃. Preferably below 40 ℃, below 45 ℃ or below 50 ℃.

The enzyme has low action on maltose, xylose and galactose, and has high substrate specificity on glucose. The activity on 50mM maltose, D-xylose, and D-galactose is 2.0% or less, preferably 0.3% or less, 0.9% or less, or 0.2% or less, assuming that the activity on 50mM D-glucose is 100%.

(6) Km value (relative to glucose): 30mM or more

The Km value to D-glucose is preferably 150 to 300mM, 50 to 120mM, or 30 to 80 mM.

The molecular weight of the present enzyme was calculated from the amino acid sequence by predicting the signal sequence of the secretion signal sequence of the enzyme by the signal sequence prediction site (SignalP-5.0, http:// www.cbs.dtu.dk/services/SignalP /) excluding the predicted signal portion.

(8) Glucose oxidase activity: can not detect out

As described above, the flavin-bound GDH of the present invention specifically acts on the 6-position of glucose and therefore has a low effect on xylose. Therefore, the enzyme can be used for the measurement of glucose and is useful as an enzyme for biosensors for the measurement of glucose.

Examples of microorganisms derived from the flavin-bound GDH of the present invention include

The flavin-bound GDH of the present invention may be any of an enzyme derived from the above-mentioned microorganism (wild strain, mutant strain), a recombinant enzyme obtained by a genetic engineering method using a gene encoding the flavin-bound GDH of the present invention, and a synthetic enzyme obtained by chemical synthesis. Preferably a recombinase.

The gene encoding the flavin-bound GDH of the present invention is preferably a gene comprising any one of the following DNAs (a) to (e).

(d) A DNA which hybridizes under stringent conditions to a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 and which encodes a protein having glucose-6-dehydrogenase activity

(e) DNA encoding the protein of the following (i), (ii) or (iii)

1 to 10 bases, more preferably 1 to 5 bases, further preferably 1 to 3 bases, further preferably 1 or 2 bases in the base sequence in which 1 to several bases are deleted, substituted or added in the base sequence represented by SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37. The deletion of a base means the absence or disappearance of a base, the substitution of a base means the substitution of a base with another base, and the addition of a base means an additional base. "appended" includes the addition of bases to one or both ends of a sequence, as well as the insertion of other bases between bases in the sequence.

In the present specification, the sequence homology of the base sequence is preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and further preferably 99% or more.

Sequence homology of base sequences can be determined using the algorithm BLAST (Pro.Natl.Acad.Sci.USA,1993,90:5873-5877) based on Karlin and Altschul. Based on the algorithm BLAST, programs called BLASTN and BLASTX were developed (j.mol.biol.,1990,215, p.403-410). In addition, the homology analysis (Search homology) program of genetic information processing software Genetyx may be used. Specific methods for these analysis methods are known (see www.ncbi.nlm.nih.gov).

In the present specification, stringent conditions are conditions under which base sequences having high homology hybridize to each other and base sequences having lower homology do not hybridize to each other. The "stringent conditions" may be appropriately changed depending on the degree of homology required. The more stringent the conditions, the more only the more homologous sequences hybridize. For example, the stringent conditions include those described in molecular cloning protocols (second edition, J.Sambrook et al, 1989). That is, there may be mentioned conditions under which a solution containing 6 XSSC (1 XSSC composition: 0.15M sodium chloride, 0.015M sodium citrate, pH7.0), 0.5% SDS, 5 XDenhardt's and 100mg/mL herring sperm DNA is hybridized together with a probe at a constant temperature of 65 ℃ for 8 to 16 hours.

Production of a flavin-bound GDH using a gene encoding a flavin-bound GDH of the present invention, for example, an expression vector containing a gene encoding a flavin-bound GDH of the present invention can be introduced into a host cell such as a microorganism and the obtained transformant can be cultured to produce a flavin-bound GDH of the present invention. In the transformant, the gene encoding the flavin-bound GDH of the present invention may be maintained in the form of a vector, or the gene may be maintained in the genome.

The gene encoding the flavin-bound GDH of the present invention can be prepared in an isolated state by any method used in the art, such as a PCR method, with reference to the gene sequence information disclosed in the present specification.

The type of vector is not particularly limited, and examples thereof include vectors generally used for protein production, such as plasmids, cosmids, phages, viruses, YACs, and BACs. Among these, plasmid vectors are preferable, and commercially available plasmid vectors for protein expression, for example, pET, pBIC, and the like can be suitably used. Procedures for introducing genes into plasmid vectors are well known in the art.

Examples of host microorganisms to be transformed for expressing the flavin-bound GDH of the present invention include bacteria, yeasts and filamentous bacteria belonging to the genus Escherichia (Escherichia), Rhodococcus (Rhodococcus), Streptomyces (Streptomyces), Bacillus (Bacillus), Brevibacillus (Brevibacillus), Staphylococcus (Staphylococcus), Enterococcus (Enterococcus), Listeria (Listeria), Yeast (Saccharomyces), Pichia (Pichia), Schizosaccharomyces (Shizosaccharomyces), Kluyveromyces (Kluyveromyces), Aspergillus (Aspergillus), Penicillium (Penicillium), Trichoderma (Trichoderma).

The medium and culture conditions used for culturing the transformant can be appropriately selected by those skilled in the art according to the type of the transformant.

For example, the culture can be carried out under aerobic conditions such as aeration, agitation, shaking, etc. using a medium containing a carbon source assimilable by the microorganism, an inorganic nitrogen source, an organic nitrogen source, inorganic salts, and other necessary organic micronutrient sources. The medium may be any of a synthetic medium, a natural medium, a semisynthetic medium, or may be a commercially available medium. The culture medium is preferably a liquid culture medium.

The pH of the medium is preferably in the range of, for example, pH5 to pH9, and the pH can be adjusted during the culture in consideration of productivity. For example, the culture temperature is preferably 10 to 40 ℃ and the culture time is in the range of 2 to 14 days.

After the culture, the culture supernatant may be obtained by a separation operation such as centrifugation, and then used. Or obtaining microbial cells, disrupting the microbial cells by any method, and obtaining a supernatant from the disrupted solution. The culture may be a culture solution, a microbial cell, or a treated product thereof (freeze-dried cell, acetone-dried cell, or the like). Further, the immobilized enzyme or immobilized microorganism may be immobilized by any method.

The flavin-bound GDH of the present invention produced by the transformant can be purified by a known purification method. For example, the purified enzyme can be obtained by combining purification operations such as ultrafiltration, salting out, solvent precipitation, heat treatment, dialysis, ion exchange chromatography, hydrophobic chromatography, gel filtration, affinity chromatography, and the like.

The method for producing glucuronic acid of the present invention comprises a step of reacting the flavin-bound GDH of the present invention with glucose in the presence of a mediator to produce glucuronic acid. In this step, the form of the flavin-bound GDH is not particularly limited, and may be a crude enzyme, a purified enzyme, or a microorganism containing the flavin-bound GDH. The microorganism containing a flavin-bound GDH is preferably a recombinant microorganism into which a gene encoding a flavin-bound GDH has been introduced. The microorganism containing the flavin-bound GDH may be either alive or dead, and may contain a treated product of the microbial cell as described above.

The formation of glucuronic acid is generally carried out in an aqueous medium. Examples of the aqueous medium include water, buffer, monohydric alcohol, and dihydric alcohol.

The glucose as a substrate is usually D-glucose. In this step, the substrate concentration is preferably about 10mM to 2M.

The mediator used in this step may be a chemical substance having excellent electron transfer ability. Mediators are also known as electron transporters, electron acceptors, redox mediators.

Examples of the mediator include osmium-based compounds (e.g., 2, 2' -bipyridine osmium (ll) complex), quinone-based compounds (e.g., benzoquinone, 1, 4-naphthoquinone, vitamin K3 (menadione)), phenol-based compounds (e.g., tert-butylhydroquinone, hydroquinone, 4-aminophenol, butylhydroxyanisole, eugenol, catechol, guaiacol, pyrogallol, vanillin, and n-propyl gallate), phenazine-based compounds (e.g., phenazine methosulfate, 1-methoxy-5-methylphenazin

Methyl sulfate, methylene blue), ferricyanide (e.g., potassium ferricyanide), flavonoids (quercetin dihydrate, hesperidin), and the like. Among them, tert-butylhydroquinone and 1-methoxy-5-methylphenazines are preferable

Methyl sulfate salt.

The amount of the mediator to be used may be appropriately set depending on the type of mediator, and is usually preferably about 0.5mM to 50 mM.

The conditions for allowing the flavin-bound GDH of the present invention to act on glucose are not particularly limited as long as they are conditions under which the flavin-bound GDH is not inactivated. The reaction is carried out at normal temperature and normal pressure. In addition, since the reaction is carried out under neutral to alkaline conditions, a strong acid is not required to be used in the reaction process.

The reaction temperature is usually 10 to 60 ℃ and preferably 20 to 40 ℃ and the reaction time is usually 30 minutes to 72 hours, preferably 1 hour to 48 hours, and more preferably 3 hours to 24 hours.

The reaction pH is preferably from pH5.0 to pH 9.0.

The amount of the flavin-bound GDH of the present invention to be used is preferably about 1U/mL to 50U/mL in terms of final concentration.

In this step, it is preferable to further act an oxidase from the viewpoint of carrying out the reoxidation of the mediator. Examples of the oxidase include phenol oxidases such as laccase (EC 1.10.3.2) and peroxidases (EC 1.11.1.7).

Further, from the viewpoint of removing active oxygen in the reaction system, catalase (EC 1.11.1.6) is preferably used.

The amount of these enzymes to be used is preferably about 0.25U/mL to 500U/mL in terms of final concentration.

Glucuronic acid is specifically produced from glucose as a substrate in this manner. On the other hand, gluconic acid is not substantially produced. The term "substantial" means that the formation of gluconic acid is not observed even in a method such as a gluconic acid measurement kit, thin layer chromatography, or HPLC when a reaction product is analyzed. Therefore, according to this step, the load of purification of glucuronic acid can be reduced. When glucuronic acid is separated and purified, a known separation and purification method can be used.

The produced glucuronic acid can be used as glucuronic acid or in the state of glucuronolactone, which is a molecular lactone thereof, in pharmaceuticals, quasi-pharmaceuticals, foods and the like.

The method for producing a glucuronic acid derivative of the present invention comprises a step of allowing the flavin-bound GDH of the present invention to act on a glucose derivative in the presence of a mediator to produce a glucuronic acid derivative. The flavin-bound GDH is the same as the flavin-bound GDH described above.

The formation of glucuronic acid derivatives is also generally carried out in an aqueous medium such as water, a buffer, a monohydric alcohol, a dihydric alcohol, or the like.

In the present specification, the glucose derivative is selected from glucose analogs in which hydroxyl groups constituting glucose are substituted with hydrogen, such as aminosugars and N-acetylates thereof, glucosides, and polyalcohols. The glucose is preferably D-glucose.

As the amino sugar and its N-acetyl compound, glucosamine, N-acetylglucosamine and the like can be mentioned.

Glucoside is a generic name of a glycoside in which a hemiacetal hydroxyl group of glucose is ether-bonded to another compound, or a glycoside in which glucose is thioether-bonded to a saturated hydrocarbon such as octane via a sulfur atom.

Examples of the other compounds include aglycone, monosaccharides, disaccharides, and trisaccharides or more polysaccharides. Examples of the aglycone include a non-sugar portion, and saturated hydrocarbons such as alcohol, phenol, phenylpropanoid, and octane.

The glucoside is preferably cellobiose, arbutin, spruce neo-glucoside, methyl glucoside, or octyl thioglucoside.

Examples of the glucose analogs include 1, 5-anhydro-D-glucitol and 2-deoxy-D-glucose.

In this step, the substrate concentration is preferably about 10mM to 1000 mM.

The mediator used in this step is the same as the above-described mediator.

The amount of mediator used is preferably about 0.5mM to 50 mM.

The conditions for allowing the flavin-bound GDH of the present invention to act on a glucose derivative are not particularly limited as long as the flavin-bound GDH is not inactivated. This step also allows the reaction to be carried out at normal temperature and pressure, and also allows the reaction to be carried out under neutral to alkaline conditions.

The reaction pH is preferably from pH5.0 to pH 9.0.

The amount of the flavin-bound GDH of the present invention to be used is preferably 1U/mL to 50U/mL in terms of final concentration.

In this step, it is also preferable to further act an oxidase from the viewpoint of reoxidation of the mediator. Examples of the oxidase include phenol oxidases such as laccase (EC 1.10.3.2) and peroxidases (EC 1.11.1.7).

In addition, from the viewpoint of removing active oxygen, catalase (EC 1.11.1.6) is preferably used.

In this manner, the hydroxymethyl group at the 6-position of the glucose skeleton of the glucose derivative as a substrate is oxidized to a carboxyl group, and a glucuronic acid derivative is specifically produced. The yield of glucuronide was approximately 100% as a result of thin layer chromatography analysis.

The produced glucuronic acid derivative can be used as a standard substance for glucuronic acid conjugates produced in vivo, a raw material for pharmaceuticals, foods or cosmetics, a surfactant, and the like.

The catalyst for producing glucuronic acid or glucuronic acid derivatives of the present invention is an enzyme catalyst for producing glucuronic acid or glucuronic acid derivatives from glucose or glucose derivatives, which comprises a flavin-bound glucose dehydrogenase having glucose-6-dehydrogenase activity.

The catalyst for producing glucuronic acid or glucuronic acid derivatives may contain, in addition to the flavin-bound GDH of the present invention, for example, excipients, suspending agents, buffers, stabilizers, preservatives, physiological saline solutions, and the like.

Examples

The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

(glucose dehydrogenase (GDH) Activity assay)

100mM potassium phosphate buffer (pH6.0)1.00mL, 1M D-glucose solution 1.00mL, 3mM 2, 6-dichlorophenolindolol (hereinafter referred to as "DCIP") 0.14mL, and 3mM 1-methoxy-5-methylphenazin

0.20mL of methyl sulfate (hereinafter referred to as "1-m-PMS") was mixed with 0.61mL of ultrapure water, and the mixture was incubated at 37 ℃ for 10 minutes, followed by addition of 0.05mL of an enzyme solution to start the reaction.

The decrease (. DELTA.A 600) per 1 minute in absorbance at 600nm with the progress of the enzyme reaction was measured 5 minutes from the start of the reaction, and the enzyme activity was calculated from the linear portion according to the following equation. At this time, the amount of enzyme that reduced 1. mu. mol of DCIP in 1 minute at 37 ℃ and pH6.0 was defined as 1U for the enzyme activity.

Glucose Dehydrogenase (GDH) activity (U/mL) ═ (- (Δ a600- Δ a600blank) × 3.0 × dilution ratio of enzyme)/(10.8 × 1.0 × 0.05)

In the formula, 3.0 represents the liquid amount (mL) of the reaction reagent + enzyme solution, 10.8 represents the molar absorption coefficient of DCIP at pH6.0, 1.0 represents the optical path length (cm) of the cuvette, 0.05 represents the liquid amount (mL) of the enzyme solution, and Δ A600blank represents the decrease per 1 minute of the absorbance at 600nm when the reaction was started by adding the enzyme-diluted solution instead of the enzyme solution.

[ example 1]

(obtaining of flavin-bound glucose dehydrogenase CpGDH)

As a result of searching for GDH-producing bacteria, GDH activity was confirmed in the culture supernatant of Colletotrichum plurivorum MAFF 305790.

(1) Cultivation of bacteria

A liquid medium composed of 2% (w/v) dextrin (Fuji film and Wako Junyaku Co.), 1% (w/v) polypeptone (Fuji film and Wako Junyaku Co.), 0.5% (w/v) potassium dihydrogen phosphate (Nacalai Tesque Co.), 0.05% (w/v) magnesium sulfate heptahydrate (Nacalai Tesque Co.), and water was prepared, 10mL of which was put in a crude tube and autoclaved at 121 ℃ for 20 minutes. The GDH-producing strain was inoculated onto a cooled liquid medium, cultured with shaking at 25 ℃ for 72 hours, and then wet cells were collected using a bleaching cloth.

(2) Isolation of Total RNA

200mg of the wet cells obtained in (1) were frozen at-80 ℃ and 100. mu.g of total RNA was extracted using ISOGENII (NIPPON GENE Co.).

(3) Preparation of cDNA library

Preparing a cDNA library from the RNA obtained in (2) by a reverse transcription reaction using a reverse transcriptase and an oligo dT primer having a linker sequence. The reaction reagent was prepared using a SMARTER RACE cDNA Amplification kit (Takara Bio Inc.), and the reaction conditions were as described in the specification.

(4) Cloning of GDH Gene

PCR was performed using the cDNA library obtained in (3) as a template and a primer set for GDH gene acquisition. As a result, a PCR product which is considered to be an internal sequence of the GDH gene was confirmed. The primer set is designed to obtain various GDH genes based on a plurality of GDH sequences already elucidated by the present inventors. The PCR product was purified to determine the nucleotide sequence.

Based on the determined base sequence, primers for elucidating the upstream and downstream sequences of the GDH gene were designed. The full length of the GDH gene (hereinafter referred to as "CpGDH") derived from the Colletotrichum pluravorum MAFF305790GDH strain was elucidated by the 5 'RACE method and the 3' RACE method using these primers.

The sequence obtained by optimizing the sequence of the CpGDH gene to the codon frequency of Aspergillus oryzae is shown in SEQ ID NO. 1. Further, the amino acid sequence predicted from the gene sequence is shown in SEQ ID NO. 2.

(5) Preparation of plasmid vector for expression containing CpGDH Gene

A plasmid vector was prepared using the modified promoter of the amylase system from Aspergillus oryzae described in publicly known document 1 (heterologous gene expression system of Aspergillus, Peak cost, chemistry and biology, 38, 12, 831-. First, using the cDNA library obtained in (3) as a template, a PCR product containing the CpGDH gene was obtained. Next, using the PCR product as a template, a CpGDH gene for vector insertion was prepared.

Finally, the prepared CpGDH gene was ligated to the downstream of the promoter to prepare a plasmid vector capable of expressing the gene. The prepared plasmid vector for expression was introduced into Escherichia coli JM109 and transformed. The obtained transformant was cultured, and the plasmid vector was extracted from the cells of the collected strain using the illustra plasmidPrep Midi Flow Kit (GE Healthcare Co.). The nucleotide sequence of the CpGDH gene was confirmed by sequence analysis of the inserted sequence in the plasmid vector.

(6) Obtaining of transformant

Using the plasmid vector extracted in (5), a recombinant microorganism (Aspergillus oryzae) producing CpGDH was prepared according to the methods described in publicly known document 2(biosci. Biotech. biochem.,61(8), 1367. sup.,. sup.1369, 1997) and publicly known document 3 (Aspergillus sakei. gene manipulation technique, Schizama strain, 494. sup.,. was prepared). The resulting recombinant strain was purified using Czapek-Dox solid medium.

Aspergillus oryzae strain NS4 was used as the host. This strain was grown by brewing trials in 1997 (in 9 years), as described in publicly known document 2, and it is now possible to obtain a strain distributed by the Alcoholic liquor integration research institute of independent administrative scientists.

(7) Confirmation of CpGDH from recombinant molds

A liquid medium composed of 2% (w/v) dextrin (Fuji film and Wako Junyaku Co.), 1% (w/v) polypeptone (Fuji film and Wako Junyaku Co.), 0.5% (w/v) potassium dihydrogen phosphate (Nacalai Tesque Co.), 0.05% (w/v) magnesium sulfate heptahydrate (Nacalai Tesque Co.), and water was prepared, 10mL of which was put in a crude tube (22 mm. times.200 mm), and autoclaved at 121 ℃ for 20 minutes. The transformant obtained in (6) was planted on a cooled liquid medium and cultured with shaking at 30 ℃ for 120 hours. After the completion of the culture, the supernatant was collected by centrifugation, and the CpGDH activity of the present invention was confirmed by measuring the GDH activity by the GDH activity measurement method described above.

(8) Purification of CpGDH

150mL of the liquid medium described in (7) was placed in a 500mL sakaguchi flask and autoclaved at 121 ℃ for 20 minutes. The transformant obtained in (6) was planted on a cooled liquid medium and cultured with shaking at 30 ℃ for 72 hours. After completion of the culture, the culture solution was filtered through a filter cloth, and the collected filtrate was centrifuged to collect the supernatant, which was further filtered through a membrane filter (10 μm, ADVANTEC) to collect the culture supernatant.

The recovered culture supernatant was purified by removing contaminating proteins using a TOYOPEARL DEAE-650S (Tosoh Co.). The purified sample was concentrated with an ultrafiltration membrane with a cut-off of 10000 and then replaced with water to produce purified CpGDH. This purified CpGDH was subjected to SDS-polyacrylamide electrophoresis, and as a result, a single band was confirmed.

(obtaining of FGDH and CsGDH)

A gene sequence 1872bp of SEQ ID NO. 1 of Japanese patent No. 6455714 and a gene sequence 1908bp of SEQ ID NO. 2 of Japanese patent No. 5435180 were synthesized, and expressed and purified in Aspergillus oryzae NS4 strain in the same manner as in example 1. The purified enzyme of sequence No. 1 from Japanese patent No. 6455714 was designated as FGDH, and the purified enzyme of sequence No. 2 from Japanese patent No. 5435180 was designated as CsGDH. The gene sequence and amino acid sequence of FGDH are shown in SEQ ID Nos. 3 and 4, and the gene sequence and amino acid sequence of CsGDH are shown in SEQ ID Nos. 5 and 6.

[ example 2]

(investigation of enzymatic chemistry of each GDH)

The properties of CpGDH, FGDH and CsGDH obtained in example 1 were examined.

(1) Determination of absorption spectra

For each GDH obtained in example 1, the absorption spectrum at 600nm at 300-fold (K.sub.300) before and after addition of D-glucose was measured using a microplate reader (SpectraMax Plus384, Molecular Devices). As a result, the absorption maximum peaks observed at around the wavelengths 360-380nm and 450-460nm disappeared by the addition of D-glucose, and it was thus confirmed that all GDH were flavin-bound proteins.

(2) Determination of Glucose Oxidase (GOD) Activity

0.2mL of 1M potassium phosphate buffer (pH7.0), 2.0mL of 1M D-glucose, 0.2mL of 25mM 4-aminoantipyrine, 0.2mL of 420mM phenol, 0.2mL of 1mg/mL peroxidase (Fuji film and Wako pure chemical industries, Ltd., from horseradish) and 0.2mL of ultrapure water were mixed, and 0.1mL of the mixture was put into a 96-well plate and incubated at 25 ℃ for 5 minutes. 0.1mL of each GDH obtained in example 1 was added to start the reaction. 5 minutes after the start of the reaction, the change in absorbance at 500nm with the progress of the enzyme reaction was measured with the above microplate reader to examine the GOD activity. The reaction was started by adding water or GOD (Nacalai Tesque Co.) derived from Aspergillus niger (Aspergillus niger) instead of GDH as a control. As a result, although a change in absorbance at 500nm was observed in the control to which GOD derived from Aspergillus niger was added, no change in absorbance was observed in GDH of the present invention as in the control to which water was added. Therefore, it was confirmed that any GDH was a dehydrogenase not utilizing oxygen as an electron acceptor.

(3) Substrate specificity

According to the GDH activity assay described above, the substrate was measured for each GDH activity relative to each substrate using D-glucose, maltose, D-xylose or D-galactose at a final concentration of 50 mM. The results are shown in Table 1.

[ Table 1]

Substrate specificity

When the activity relative to D-glucose is defined as 100%, the activity of any GDH relative to maltose, D-xylose or D-galactose is 0.3%, 0.4% or 0.2% or less, and 2.0% or less in all cases.

(4) Km value relative to D-glucose

For each GDH, the activity was measured by changing the concentration of D-glucose as a substrate according to the above-mentioned activity measurement method, and the Michaes-Woolf curve was used to determine the Michael constant (Km). The activity measurement was carried out at 25 ℃. The results are shown in Table 2.

[ Table 2]

Km value

	Km value (mM)
		CpGDH	196
FGDH	52
		CsGDH	50

As a result, CpGDH was 196mM, CsGDH was 50mM, and FGDH was 52 mM. Since the Km value is likely to vary depending on the measurement method and the calculated curve, it is considered that the Km of CpGDH is 150 mM-300 mM, the Km of FGDH is 30 mM-80 mM, and the Km of CsGDH is 30 mM-80 mM.

(5) Thermal stability

Each GDH was prepared at 6U/mL and treated in 100mM potassium phosphate buffer (pH8.0) at each temperature for 60 minutes, and as a result, the temperature range in which the residual enzyme activity becomes 80% or more assuming that the enzyme activity before treatment is 100% was 40 ℃ or less in CpGDH, 40 ℃ or less in FGDH, and 45 ℃ or less in CsGDH (see FIG. 1).

(6) Range of stable pH

The stable pH of each GDH was investigated. Each GDH was prepared to 6U/mL, and the residual activity of each GDH was measured after treatment at 30 ℃ for 1 hour at each pH by adding buffers to the final concentration of 100mM sodium citrate phosphate buffer (pH2.2-7.0), 100mM potassium acetate buffer (pH3.0-6.0), 100mM potassium phosphate buffer (pH6.0-8.0), 100mM Tris-HCl buffer (pH7.0-9.0) and 100mM glycine-NaOH buffer (pH 9.0-10.0). As a result, the pH region in which the residual enzyme activity value of each GDH becomes 80% or more, assuming that the enzyme activity value before treatment is 100%, is pH4.3 to 9.3 in CpGDH, pH5.5 to 8.7 in FGDH, and pH3.2 to 9.3 in CsGDH (see FIG. 2). As a result, it was found that the GDH of the present invention is stable at a pH of at least 5.5 to 8.7. Even at the same pH, the residual activity may vary depending on the type of buffer.

[ example 3]

(analysis of glucose oxide obtained in the Presence of 1-m-PMS)

A reaction system using purified CpGDH and 1-m-PMS as mediators was constructed to obtain glucose oxide. The preparation method and various analysis methods are described below.

(1) Preparation of glucose oxide

0.04mL of 1M potassium phosphate buffer (pH7.0), 0.01mL of 2M D-glucose solution, 0.02mL of 0.2M 1-M-PMS solution, 0.02mL of 10000U/mL of catalase (Fuji film & Wako pure chemical industries, from bovine liver), 0.02mL of 90U/mL of CpGDH, FGDH or CsGDH, and 0.23mL of ultrapure water were mixed, and after mixing the mixture in reverse at room temperature, the air in the reaction vessel was replaced and further mixed in reverse for several hours to obtain a reaction product containing glucose oxide.

(2) Thin layer chromatography of glucose oxide

The reaction product obtained in (1) was sampled and analyzed by thin layer chromatography (hereinafter referred to as "TLC"). A sample of 0.001mL was dropped onto a Silica gel plate (Merck Millipore, Silica gel 60F 254), dried, and developed with acetonitrile/ultrapure water (70: 30) for 10 minutes. When the silica gel plate was dried and sprayed with concentrated sulfuric acid/ethanol (5: 95), and then heated, the CpGDH, FGDH, and CsGDH reaction products were all observed to have spots at the same positions as the glucuronic acid standard, and no spots were observed at the same positions as the D-glucose standard and the gluconic acid standard. On the other hand, as a comparative example, GDH which is generally known, i.e., GDH from Aspergillus terreus (patent document 8) was purified and subjected to the same test, and as a result, no spot was detected at the same position as the glucuronic acid standard and the glucose standard, and a spot was detected at the same position as the gluconic acid standard. From this, it was confirmed that general GDH oxidizes the 1-position of glucose to produce gluconic acid, whereas CpGDH, FGDH and CsGDH oxidize the 6-position of glucose to produce glucuronic acid.

(3) Glucose Oxidation Studies under various conditions

(1) Under the conditions described above, the reaction product containing glucose oxide was prepared under the conditions in which the buffer solution used was changed to 1M sodium phosphate buffer solution (pH7.0 or 8.0) or 1M potassium phosphate buffer solution (pH8.0), the amount of D-glucose added was changed to 2 times the amount, or the amount of 1-M-PMS added was changed to 0.0025mL or 0.005mL, and TLC analysis was performed, whereby the reaction products CpGDH, FGDH, and CsGDH were observed in the same positions as those of the glucuronic acid standard and no spots were observed in the same positions as those of the D-glucose standard and the gluconic acid standard under any of the conditions.

(4) High performance liquid chromatography analysis of glucose oxide

To remove 1-m-PMS from the CpGDH reaction product obtained in (1), 2 to 20mg of powdered activated carbon (Futamura CHEMICAL Co., Ltd., Tai activated carbon SG) which had been crushed and washed with ultrapure water was added, and the mixture was allowed to stand at room temperature for 5 minutes. By this treatment, 1-m-PMS was adsorbed on activated carbon, and the supernatant was collected by centrifugation at 4 ℃ and 8000 Xg for 1 minute, whereby a CpGDH reaction product from which 1-m-PMS was removed was obtained. This supernatant was fluorescently labeled with a GlyScope ABEE labeling kit (J-CHEMICAL Co., Ltd.), and HPLC analysis was performed using a sugar analysis column Honenpak C18(J-CHEMICAL Co., Ltd.), whereby no peak of D-glucose as a substrate was observed and a peak was observed at the same position as that of a glucuronic acid standard (see FIG. 4). The fluorescence labeling and HPLC analysis were carried out according to the protocol described in the kit, and SIL-10A series (Shimadzu corporation) was used as the HPLC analysis device, and a fluorescence detector RF-10AXL (excitation wavelength/fluorescence wavelength: 305/360nm, Shimadzu corporation) was used as the detector.

(5) Analysis of glucose oxide Using Nuclear Magnetic Resonance (NMR) apparatus

The CpGDH reaction product from (4) from which 1-m-PMS was removed was pre-frozen at-40 ℃ and FREEZE-dried using a FREEZE-DRYER (EYELA, FREEZE DRYER FD-1). The freeze-dried sample was analyzed by a nuclear magnetic resonance apparatus, and as a result, a peak indicating that the hydroxymethyl group at the 6-position of glucose was replaced with a carboxyl group was obtained, and it was confirmed that glucuronic acid was selectively produced from glucose.

(6) Analysis Using gluconic acid assay kit

To confirm whether or not gluconic acid was contained in the CpGDH reaction product obtained in (4) from which 1-m-PMS was removed, activity measurement was performed using F-kit D-gluconic acid/gluconolactone (JK. INTERNATIONAL Co., Ltd.). The evaluation was performed according to the protocol described in the specification, and as a result, an accurate gluconic acid concentration could be measured in the gluconic acid standard used as a control, but no gluconic acid was detected in the CpGDH reaction product.

(7) Analysis Using glucuronic acid assay kit

In order to measure the concentration of glucuronic acid contained in the CpGDH reaction product obtained in (4) from which 1-m-PMS was removed, analysis was performed using a glucuronic acid measurement kit (Megazyme, K-URONIC). Evaluation was carried out according to the protocol described in the specification, and as a result, it was confirmed that glucuronic acid was produced.

[ example 4]

(analysis of glucose oxide obtained in the Co-presence of t-butylhydroquinone)

A reaction system using purified CpGDH and t-butylhydroquinone (hereinafter referred to as TBHQ) as mediators was constructed to obtain glucose oxide. The preparation and analysis thereof are described below.

(1) Production of glucose oxide using TBHQ

0.02mL of 1M sodium phosphate buffer (pH8.0), 0.01mL of 1M D-glucose solution, 0.002mL of 1M TBHQ (Tokyo chemical industry Co., Ltd.), 0.005mL of 10U/mL laccase (Sigma-Aldrich Co., Ltd., from Aspergillus), 0.04mL of 90U/mL CpGDH0.04mL of ultrapure water and 0.123mL of ultrapure water were mixed together, and the mixture was inverted and mixed at room temperature in a light-shielded state to obtain a reaction product containing glucose oxide. In the above composition, a reaction product containing glucose oxide was obtained in the same manner as in the case where the laccase concentration was changed to 1/5.

(2) TLC analysis of glucose oxides

As a result of TLC analysis in the same manner as in (2) in example 3, in the CpGDH reaction product using TBHQ as a mediator, a spot was also observed at the same position as the glucuronic acid standard, and no substance was detected at the same position as the glucose standard and the gluconic acid standard.

(3) Glucose Oxidation study with peroxidase and Catalase addition

A reaction solution was prepared by mixing 0.02mL of 1M sodium phosphate buffer (pH8.0), 0.01mL of 1M D-glucose solution, 0.002mL of 1M TBHQ (Tokyo chemical industry Co., Ltd.), 0.01mL of 10000U/mL of catalase (Fuji film and Wako pure chemical industries, Ltd., from bovine liver), 0.005mL of 100U/mL of horseradish peroxidase (Fuji film and Wako pure chemical industries, Ltd., from horseradish), 0.04mL of 90U/mL of CpGDH0.04mL and 0.113mL of ultrapure water, and the reaction product was reacted, and the TLC analysis of the reaction product was carried out, whereby spots were confirmed at the same positions as those of the glucuronic acid standard product in the same manner as in (1) and no substance was detected at the same positions as those of the glucose standard product and the gluconic acid standard product.

(4) Analysis Using gluconic acid assay kit

The analysis was performed using the D-gluconic acid/gluconolactone measurement kit (F-kit) in the same manner as in (6) of example 3, and it was found that no gluconic acid was produced from CpGDH in the reaction system using TBHQ as a mediator.

(5) Analysis Using glucuronic acid assay kit

As a result of analysis using a glucuronic acid measurement kit (K-URONIC) in the same manner as in (7) of example 3, it was confirmed that glucuronic acid was produced in the same amount as glucose used in the reaction system using TBHQ as a mediator.

[ example 5]

(analysis of glucose oxide obtained in the Co-presence of butyl hydroxyanisole)

And constructing a reaction system of the purified CpGDH and the butyl hydroxyanisole as a mediator to obtain the glucose oxide. The preparation and analysis thereof are described below.

(1) Preparation of glucose oxide Using butylhydroxyanisole

0.02mL of 1M sodium phosphate buffer (pH8.0), 0.01mL of 1M D-glucose solution, 0.1M butylhydroxyanisole (Fuji film & Wako Junyaku Co., Ltd.), 0.02mL of 10000U/mL catalase (Fuji film & Wako Junyaku Co., Ltd.), 0.01mL of 10U/mL laccase (Sigma-Aldrich Co., Ltd., from Aspergillus), 0.005mL of 90U/mL CpGDH0.04mL of ultrapure water, and 0.095mL of ultrapure water were mixed together, and the mixture was inverted and mixed in a room-temperature light-shielded state to obtain a reaction product containing glucose oxide.

(2) TLC analysis of glucose oxides

As a result of TLC analysis in the same manner as in (2) of example 3, in the CpGDH reaction product prepared using butylhydroxyanisole as a mediator, a spot was observed at the same position as the glucuronic acid standard, and no substance was detected at the same position as the glucose standard and the gluconic acid standard.

[ example 6]

(analysis of various substrate oxides)

Oxides such as glycosides were obtained using purified CpGDH. The preparation method and various analysis methods are described below.

(1) Preparation of substrate oxides

To a reaction solution composed of potassium phosphate buffer (pH8.0) at a final concentration of 100mM, D- (+) -cellobiose as a substrate at 13 to 100mM, α -arbutin, β -arbutin, piceid, N-acetyl-D-glucosamine, 1-m-PMS as a mediator at 10mM, and catalase (Fuji film and Wako pure chemical industries, from bovine liver) at 500U/mL as an active oxygen scavenger was added CpGDH so that the final concentration became 14U/mL, and the mixture was mixed by inversion at room temperature. Then, the air in the reaction vessel was replaced, and the mixture was further mixed in reverse at room temperature for several hours, thereby obtaining a substrate oxide containing a glycoside oxide and the like.

(2) TLC analysis of substrate oxides

As a result of TLC analysis in the same manner as in (2) in example 3, a new spot was detected at a position different from that before the reaction (see fig. 5). This indicates that CpGDH oxidizes the glucose residue in the glycoside.

[ example 7]

(obtaining of CglGDH, CoGDH, CtoGDH, CgoGDH, GsGDH and DhGDH)

Separately expressed in Aspergillus oryzae strain NS4 and purified in the same manner as in example 1 using Colletotrichum gloeosporioides (Colletotrichum gloeosporioides), Colletotrichum (Colletotrichum orbiculare), fungus Colletotrichum (Colletotrichum tofieldiae), Colletotrichum floribunda (Colletotrichum collectiae) MAFF240289, Pleurotus minor sp.RD 057037 or Helianthus annuus (Diaporterherbia helionthi) in place of Colletotrichum purivorum MAFF 305790. The purified enzyme from Colletotrichum gloeosporioides (Colletotrichum gloeosporioides) was designated cglGDH, the purified enzyme from Colletotrichum (Colletotrichum orbiculare) was designated CoGDH, the purified enzyme from Colletotrichum (Colletotrichum toroidium) was designated CtoGDH, the purified enzyme from Colletotrichum (Colletotrichum nodetiae) MAFF240289 was designated CgGDH, the purified enzyme from Microthecium minor strain RD057037 was designated GsGDH, and the purified enzyme from Helianthus annuus Leptotomus (Diaporter heliophili) was designated DhGDH. The gene sequences are shown in SEQ ID Nos. 7, 9, 11, 13, 15 and 17, and the amino acid sequences are shown in SEQ ID Nos. 8, 10, 12, 14, 16 and 18.

(investigation of enzyme chemistry)

(1) Determination of glucose-6-dehydrogenase Activity

Glucose oxides were prepared using CglGDH, CoGDH, CtoGDH, cgodh, GsGDH and DhGDH under the conditions described in example 4, and TLC analysis was performed, and as a result, spots were observed in the same positions as those of the standard glucuronic acid product, and no substance was detected in the same positions as those of the standard glucose product and the standard gluconic acid product (see fig. 6).

(2) Substrate specificity

The activity of each GDH with respect to each substrate was measured in the same manner as in example 2. The results are shown in Table 3.

[ Table 3]

Substrate specificity

When the activity against D-glucose is assumed to be 100%, the activity against maltose or D-xylose of either GDH is 0.2% or 0.9% or less, and both are 2.0% or less.

(3) Km value relative to D-glucose

The michaelis constant (Km) was determined in the same manner as in example 2. The results are shown in Table 4.

[ Table 4]

Km value

	Km value (mM)
		Cg1GDH	573
COGDH	441
		CtoGDH	541
CgoGDH	307
		GsGDH	425
DhGDH	86

Since the Km value is liable to vary depending on the measurement method and the calculated curve, it is considered that the Km of CglGDH is 400 mM-900 mM, the Km of CoGDH is 250 mM-650 mM, the Km of CtoGDH is 400 mM-900 mM, the Km of CgoGDH is 200 mM-450 mM, the Km of GsGDH is 250 mM-650 mM, and the Km of DhGDH is 60 mM-140 mM.

(4) Thermal stability

Each GDH was prepared at 6U/mL by the same method as in example 2, and treated in 100mM potassium phosphate buffer (pH8.0) for 60 minutes at each temperature, and as a result, the temperature range in which the residual enzyme activity becomes 80% or more assuming that the enzyme activity before treatment is 100% was 40 ℃ or less among CglGDH, CtoGDH, CgoGDH and GsGDH, 45 ℃ or less among CoGDH and 50 ℃ or less among DhGDH (see FIG. 7).

(5) Stable pH range

As a result of examining the stable pH of each GDH in the same manner as in example 2, the pH range in which the residual enzyme activity value of each GDH becomes 80% or more assuming that the enzyme activity value before treatment is 100% was pH5.5 to 9.3 in CglGDH, pH4.4 to 9.3 in CoGDH, pH4.0 to 9.6 in CgoGDH, pH4.4 to 9.3 in CgoGDH, pH5.0 to 9.3 in GgGDH, and pH3.3 to 9.6 in DhGDH (see FIG. 8). As a result, it was found that the GDH of the present invention is stable at a pH of at least 5.5 to 9.3.

[ example 8]

(Ko37GDH, AsGDH, Ko38GDH, LhGDH, DsGDH, CtaGDH, FlaGDH, PcGDH, Fla _ A.oGDH and Pc _ A.oGDH)

Instead of Colletotrichum purivorum MAFF305790, the published genomic data of Blastomyces nigrescens (Khuskokia oryzae), Acremonium (Acremonium strictum), Lasiosphaeris hirsute, Sphaerotheca intervariensis (Diaporthaceae sp.), anthrax (Colletotrichum tanaceti), Fusarium lambertianum (Fusarium lankshirae), and Phyemonopsis curvata gave sequence information with high amino acid sequence homology to CpGDH, which was expressed and purified in Aspergillus oryzae strain NS4 in the same manner as in example 1 on the basis of the codon frequency optimized to Aspergillus oryzae, respectively. Separately, the sequences obtained by substituting the signal sequence of GDH derived from Fusarium langsithiae and Pheialemopsis curvata with the signal sequence of GDH derived from Aspergillus oryzae were expressed and purified in the same manner as in example 1 in Aspergillus oryzae strain NS 4.

The purified enzymes from nigrospora melanosporum (Khuskia oryzae) were designated as Ko37GDH and Ko38GDH, the purified enzyme from Acremonium cladosporium (Acremonium strictum) was designated as AsGDH, the purified enzyme from lasiosphaeria hirsute was designated as LhGDH, the purified enzyme from Diaporthaceae sp was designated as DsGDH, the purified enzyme from anthrax (Colletotrichum tanaceti) was designated as CtaGDH, the purified enzyme from Fusarium langerii (Fusarium langeri) was designated as FlaGDH, the purified enzyme from philus flaveri (Fusarium lanuge) was designated as PcGDH, the purified enzyme from Fusarium solani (Fusarium lanuge) was substituted for the signal sequence from the putative aspergillus oryzae was designated as Fla _ Pc, and the purified enzyme from Fusarium lanuginose was designated as humigdh _ a.

The gene sequences of Ko37GDH, AsGDH, Ko38GDH, LhGDH, DsGDH, CtaGDH, FlaGDH, PcGDH, flaa _ a.ogdh, and Pc _ a.ogdh optimized to the codon frequency of aspergillus oryzae are set forth in seq id nos 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37, and the amino acid sequences are set forth in

seq id nos

20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.

(investigation of enzyme chemistry)

(1) Determination of glucose-6-dehydrogenase Activity

Glucose oxides were prepared using Ko37GDH, AsGDH, Ko38GDH, LhGDH, DsGDH, CtaGDH, FlaGDH, PcGDH, Fla _ a.ogdh, and Pc _ a.ogdh under the conditions described in example 4, and TLC analysis was performed, and as a result, spots were observed in the same positions as those of the glucuronic acid standard, and no substance was detected in the same positions as those of the glucuronic acid standard and the gluconic acid standard (see fig. 9).

(2) Substrate specificity

The activity of each GDH with respect to each substrate was measured in the same manner as in example 2. The results are shown in Table 5.

[ Table 5]

Substrate specificity

When the activity against D-glucose is defined as 100%, the activity against maltose or D-xylose of either GDH is 0.5% or 0.2% or less, and 2.0% or less in each case.

(3) Km value relative to D-glucose

The michaelis constant (Km) was determined in the same manner as in example 2. The results are shown in Table 6.

[ Table 6]

Km value

	Km value (mM)
		KO37GDH	40
AsGDH	42
		Ko38GDH	1,033
DsGDH	215
		CtaGDH	167
FlaGDH	1,119
		PcGDH	Cannot measure
F1a_A.oGDH	870
		Pc_A.oGDH	Cannot measure

The Km value is likely to vary depending on the measurement method and the calculated curve, and therefore, it is considered that the Km of Ko37GDH is 25 mM-60 mM, the Km of AsGDH is 25 mM-60 mM, the Km of Ko38GDH is 700 mM-1500 mM, the Km of DsGDH is 150 mM-300 mM, the Km of CtaGDH is 110 mM-240 mM, the Km of F1aGDH is 900 mM-1600 mM, and the Km of Fla _ A.oGDH is 600 mM-1200 mM.

(4) Thermal stability

Each GDH was prepared to 6U/mL by the same method as in example 2, and treated in 100mM buffers shown in Table 7 for 60 minutes at each temperature, and as a result, the temperature range in which the residual enzyme activity becomes 80% or more assuming that the enzyme activity before treatment is 100% was 35 ℃ or less in Ko37GDH, 40 ℃ or less in DsGDH, 45 ℃ or less in Ko38GDH, LhGDH, FlaGDH, and Fla _ A.oGDH, and 50 ℃ or less in AsGDH, CtaGDH, PcGDH, and Pc _ A.oGDH (see FIG. 10).

[ Table 7]

Enzyme name	Buffer solution used
		Ko37GDH	Potassium phosphate buffer (pH7.0)
AsGDH	Potassium phosphate buffer (pH7.0)
		Ko38GDH	Potassium phosphate buffer (pH7.0)
LhGDH	Potassium phosphate buffer (pH6.0)
		DsGDH	Potassium phosphate buffer (pH7.0)
CtaGDH	Tris-HCl buffer (pH8.0)
		FlaGDH	Tris-HCl buffer (pH8.0)
PcGDH	Potassium phosphate buffer (pH7.0)
		Fla_A.o	Tris-HCl buffer (pH8.0)
Pc_A.o	Potassium phosphate buffer (pH7.0)

(5) Stable pH range

The pH range in which the residual enzyme activity value of each GDH becomes 80% or more when the enzyme activity value before treatment is set to 100% was examined as the stable pH of each GDH in the same manner as in example 2, and as a result, the pH range in which the residual enzyme activity value of each GDH becomes 80% or more was pH5.5 to 8.6 in Ko37GDH, pH4.3 to 9.6 in AsGDH, pH5.0 to 9.6 in Ko38GDH, pH4.0 to 8.6 in LhGDH, pH4.9 to 8.7 in DsGDH, pH3.3 to 9.9 in CtaGDH, pH4.4 to 9.8 in Fla GDH, pH4.0 to 8 in PcGDH, pH4.4 to 9.8 in Fla _ A.oGDH, and pH4.0 to 9.6 in Pc _ A.oGDH (see FIG. 11).

[ example 9]

(analysis of spruce neoside oxide Using Nuclear magnetic resonance apparatus (NMR))

Oxides of picroside were obtained using purified CpGDH. The preparation method and ¹ H-NMR analysis and ¹³ C-NMR analysis results.

(1) Preparation of substrate oxides

To a reaction solution comprising 20mM potassium phosphate buffer (pH7.0) at the final concentration, 50mM spruce neo-glycoside powder as a substrate, 10mM TBHQ as a mediator, 200U/mL catalase (Fuji film & Wako pure chemical industries, from bovine liver) as an active oxygen scavenger, and 2U/mL laccase (Sigma-Aldrich industries, from Aspergillus) was added CpGDH at the final concentration of 14U/mL, and the mixture was stirred while aeration was carried out at room temperature with 1N NaOH so that the pH in the reaction system became 6.5 to 7.0, to obtain spruce neo-glycoside oxide.

(2) Process for preparing spruce neoside oxide ¹ H-NMR analysis and ¹³ C-NMR analysis

The spruce neoside oxide obtained in (1) was purified by using a C18 column, and after removing impurities, its structure was identified by nmr analysis in the same manner as in example 3. On the other hand, as a comparative example, the same analysis was performed for piceid.

According to ¹ H-NMR、 ¹³ As a result of C-NMR, it was confirmed that the hydroxymethyl group at C6 position of the glucose residue of piceid was a carboxyl group, and that the other groups were common (see FIGS. 12 and 13). This indicates that CpGDH specifically oxidizes glucose residues in piceid.

[ example 10]

(1) Mass production of glucuronic acid

CpGDH was added to a reaction solution comprising a sodium phosphate buffer (pH7.0) at a final concentration of 20mM, 2M glucose as a substrate, guaiacol as a mediator at a final concentration of 10mM, and laccase (Sigma-Aldrich, from Aspergillus) at a final concentration of 2.1U/mL, and the reaction system was aerated and stirred at room temperature while adding 1N NaOH so that the pH in the reaction system became 6.5 to 7.0, to obtain glucuronic acid.

(2) TLC analysis of glucuronic acid

As a result of TLC analysis in the same manner as in (2) in example 3, a deep spot was observed at the same position as the standard product glucuronic acid, and a glucose spot was not observed. This indicates that glucose as a substrate can be completely converted into glucuronic acid under the conditions of (1).

[ example 11]

(Mass production of spruce neoside oxide)

Oxides of picroside were obtained in large quantities using purified CpGDH. The preparation method thereof is described below.

(1) Preparation of spruce neoside oxide (adding polyethylene glycol and ethanol)

In a reaction solution comprising sodium phosphate buffer (pH7.0) at a final concentration of 20mM, spruce neoside powder corresponding to 200mM as a substrate, TBHQ as a mediator at a final concentration of 10mM, catalase (Fuji film and Wako pure chemical industries, from bovine liver) at a final concentration of 500U/mL as an active oxygen scavenger, polyethylene glycol (molecular weight: 200, Nacalai Tesque, Inc.) at a final concentration of 10% (v/v), ethanol at a final concentration of 10% (v/v), laccase (Sigma-Aldrich, from Aspergillus) at a final concentration of 20U/mL, CpGDH was added, and it was confirmed that almost all spruce neoside as a substrate was oxidized after aeration at room temperature and stirring for 165 hours at one year by adding 1N NaOH so that the pH in the reaction system became 6.5 to 7.0.

(2) Analysis of Picea neoglycoside oxide

TLC analysis was performed on the spruce neoside oxide prepared under the condition of (1), and as a result, spots were confirmed at the same positions as the spruce neoside oxide subjected to NMR analysis in example 9. In addition, no substance was detected at the same position as the spruce neoside standard analyzed in example 9. This confirmed that under the condition (1), all of the spruce neoside as a substrate became spruce neoside oxide.

(3) Preparation of spruce neoside oxide (adding ethanol)

To a reaction solution comprising sodium phosphate buffer (pH7.0) at a final concentration of 20mM, spruce neo-glycoside powder corresponding to 50mM as a substrate, TBHQ as a mediator at a final concentration of 10mM, catalase (Fuji film and Wako pure chemical industries, Ltd., from bovine liver) at a final concentration of 500U/mL as an active oxygen scavenger, ethanol at a final concentration of 10% (v/v), and laccase (Sigma-Aldrich Co., Ltd., from Aspergillus) at a final concentration of 20U/mL was added CpGDH, and while aeration was conducted at room temperature and stirring was carried out with 1N NaOH so that the pH in the reaction system became 6.5 to 7.0, spruce neo-glycoside oxide was obtained. Also, spruce neoside oxide can be obtained similarly under the condition that the final concentration of ethanol is changed to 20% or 30%.

(4) Analysis of spruce neoside oxide

TLC analysis was performed on the spruce neoside oxide prepared under the condition of (3), and as a result, spots were confirmed at the same positions as the spruce neoside oxide subjected to NMR analysis in example 9. In addition, no substance was detected at the same position as the spruce neoside standard analyzed in example 9. This confirmed that all of the spruce neoside as a substrate became spruce neoside oxide under the condition (3).

(5) Preparation of spruce neoside oxide (alkaline condition)

To a reaction solution composed of glycine-NaOH buffer (pH 10.0) having a final concentration of 20mM, 50 mM-equivalent piceid powder as a substrate, and 10 mM-equivalent TBHQ as a mediator, a culture supernatant containing CtaGDH was added so that the final concentration became 20U/mL, and the mixture was aerated and stirred at room temperature for 1 hour to obtain piceid oxide.

(6) Analysis of spruce neoside oxide

TLC analysis was performed on the spruce neoside oxide prepared under the condition of (5), and as a result, spots were confirmed at the same positions as the spruce neoside oxide subjected to NMR analysis in example 9. In addition, no substance was detected at the same position as the spruce neo-glycoside analyzed in example 9. In the condition described in (5), it was confirmed by TLC analysis that spruce neo-glycoside as a substrate remained even in the same reaction time under the conditions in which the glycine-NaOH buffer (pH 10) was changed to a sodium phosphate buffer (pH7.0) and 3.2mU/mL of laccase (Sigma-Aldrich, from Aspergillus) was added. From this, it was confirmed that under the alkaline condition of (5), all of the spruce neoside as the substrate rapidly became spruce neoside oxide as compared with the neutral condition.

(7) Preparation of spruce neoside oxide (mediator natural oxidation)

CtaGDH was added to a reaction solution comprising glycine-NaOH buffer (pH 10.0) at a final concentration of 20mM, 50mM of spruce neoside powder as a substrate, and TBHQ as a mediator at a final concentration of 10mM, and the mixture was stirred at room temperature under aeration for 1 hour to obtain spruce neoside oxide.

(8) Analysis of spruce neoside oxide

TLC analysis of the spruce neoside oxide prepared under the condition of (7) showed that spots were identified in the same positions as the spruce neoside oxide analyzed by NMR in example 9. In addition, no substance was detected at the same position as the spruce neo-glycoside analyzed in example 9. This confirmed that under the condition (7), all of the spruce neoside as a substrate rapidly became spruce neoside oxide. In addition, even if no laccase was added, the spruce neoside as the substrate was all converted to spruce neoside oxide, and thus it was confirmed that the mediator was rapidly autoxidized under alkaline conditions.

[ example 12]

(analysis of arbutin oxide)

Arbutin oxides were obtained using purified CpGDH and analyzed for glucuronic acid residues.

(1) Preparation of arbutin oxide

A reaction solution composed of a sodium phosphate buffer (pH7.0) having a final concentration of 20mM, 50mM arbutin as a substrate, 10mM TBHQ as a mediator, 500U/mL catalase (Fuji film and Wako pure chemical industries, Ltd., from bovine liver) as an active oxygen scavenger, and 30mU/mL laccase (Sigma-Aldrich Ltd., from Aspergillus) was aerated at room temperature with CtaGDH added so that the final concentration became 20U/mL, and 1N NaOH was added so that the pH in the reaction system became 6.5 to 7.0, and the mixture was stirred for 48 to 72 hours to obtain arbutin oxide.

(2) Sugar cleavage of arbutin oxides

After adding trifluoroacetic acid to the arbutin oxide prepared under the condition of (1) so that the final concentration becomes 4M and heating at 100 ℃ for 3 hours, isopropanol was added to the treatment solution so that the final concentration becomes 80% (v/v), and the mixture was allowed to stand at room temperature for 24 hours to dry and solidify. The dried solidified sample was dissolved in ultrapure water for analysis of the cleaved sugar moieties.

(3) Analysis of sugar residues cleaved from arbutin oxides

TLC analysis of the sugar residue derived from arbutin oxide prepared under the condition of (2) was performed in the same manner as in (2) of example 3, and as a result, a spot was observed at the same position as the glucuronic acid standard, and no substance was detected at the same position as the glucose standard and the gluconic acid standard. From this, it was confirmed that the use of CpGDH in arbutin also converted the glucose residue into glucuronic acid.

[ example 13]

(solubility of Picea neoglycoside oxide)

The solubility of spruce neoside oxide in water was confirmed.

(1) Solubility of spruce neoside oxide in water

The spruce neoside powder was dissolved in water so that the final concentration became 2mM, and as a result, the powder remained without being completely dissolved. On the other hand, the spruce neoside oxide prepared in (1) of example 11 was purified by a C18 column, and the solubility of the obtained purified spruce neoside oxide in water was confirmed, and as a result, the added spruce neoside oxide was completely dissolved even under the condition that the final concentration was about 200 mM. From this, it was confirmed that the conversion of the glucose residue of picroside into a glucuronic acid residue improves the solubility in water by at least 100 times.

Sequence listing

<110> Seika Kaisha

<120> Process for producing glucuronic acid

<130> IS0007

<150> JP2020-009502

<151> 2020-01-23

<160> 38

<170> PatentIn version 3.5

<210> 1

<211> 1908

<212> DNA

<213> Colletotrichum_plurivorum

<400> 1

atgacgctct ttcgccagtc caagtcctgg cccgggctcg cctccgcagc cctgctcgcc 60

gtgagctccg tcgccgacgc ctatgtcatc ccgcgccaga tcaactcgtc cgagctgctg 120

acgagctacg actatgtcat cgtcggaggc ggcaccgccg gtctgaccgt ggctgaccgc 180

ctgacggagg accccgagac caaggtcctg gtcctcgagg ccgccgactg gggcaacatg 240

gacaacaacc tcatggccta cgttgccggc agaaccggtg ccttcaccga catcctgtgg 300

cccggcctcc agtccgtccc ccagcccggc ctgaacggaa ggaccggcaa cgtcttcatc 360

gccaagcagg tcggcggtgg atcctcggtt aacgccatga tgaacatgcg tggctccgcc 420

gaggactacg accgctgggc ctccctgttc ggctccgcgg cccagcaggg taccgccgac 480

tggagctggg atggcatcct gcccttcttc aagaagggtc tccacttcac cgagcctccc 540

cccgagctca ccgacaactt ccccagcgtc aagacagacg cctcctactg gggcgactcc 600

tccgacatct acgccggctg gcctcggttc tactacccgg gcgtgaaccc cctggtcgag 660

gccttcaagg aggtcgaggg agtcgagttc cctccggaca gcggtgccgg acagcctggt 720

gtcttctggt tccccgccct catggacccc cggaccgtca cccgctccta cgccggcacc 780

ggccactacc tcaacgtcaa cgccacccgc cccaactacc acctcctcgt caacacccag 840

gcccgcaagc tcctgcttaa cgacgagctc gtcgccaccg gcgtcgagtt ccccgtcggc 900

aacgccctgg tcaccgtcag cgccaagaag gaggtcatcg tctctgccgg cgccatccac 960

accccccagc tgctccagct gagtggtatc ggccccaaga agctcctcga ggccgccggc 1020

attgacgtcc gcgtcgacct gcccggtgtc ggccagaact tccaggacca cagcagtctg 1080

tccgccgtga acatcactct ccacaagctc gcgtcgatcc accccaaccc gaacgacctc 1140

gtcgaaggaa acgagttcaa ggcctgggcc gacgaggtct gggcggccaa caagaccggc 1200

ccttactcgc tccccttcac caacctggcc ggctggctcc ccttcaccgt cgtctccgac 1260

aaggccgagg agctcgccgc caagctcgag gcgcaggacc ccgccagcct cctccaggag 1320

ggcgccgacc ccaccgtcgt cgccggcttc gcggcgcaga tgaagatcct cgcgtcccag 1380

atgcgctcca aggacaccgc cttcacccgc taccagctcg tccccgccca gggcgcccag 1440

ggccccgtcg ccatgcagtc cttctcccgc ggcaccgtca acatcaacgc cgaggacccc 1500

tggggctccg agcccgtcat cgactaccgc gtgctcagca accccgtcga ggccgacttc 1560

ttcgtcgagt cgatccgctt cctgcgccgc tacaacttcg agacttctct cgctaaggag 1620

ttcgatcccg tcgagtacgc gcccggcccc gaggtcgtct ccgacgagga cctcaaggcg 1680

tacatcgccg acaccctgtc gcccacggac taccaccccg tcggcacctc gtccatgctg 1740

ccgctcgagc tcggcggcgt cgtcgaccag accctccgcg tctacggcgt caagaacctc 1800

cgcgtcgtgg acgctagcgt catgcccatg gtccccagcg ccaacacctg ccagcccacc 1860

tacgccctgg ccgagaaggc cgccgagatc atcaagcaga gcatctaa 1908

<210> 2

<211> 635

<212> PRT

<213> Colletotrichum_plurivorum

<400> 2

Met Thr Leu Phe Arg Gln Ser Lys Ser Trp Pro Gly Leu Ala Ser Ala

1 5 10 15

Ala Leu Leu Ala Val Ser Ser Val Ala Asp Ala Tyr Val Ile Pro Arg

20 25 30

Gln Ile Asn Ser Ser Glu Leu Leu Thr Ser Tyr Asp Tyr Val Ile Val

35 40 45

Gly Gly Gly Thr Ala Gly Leu Thr Val Ala Asp Arg Leu Thr Glu Asp

50 55 60

Pro Glu Thr Lys Val Leu Val Leu Glu Ala Ala Asp Trp Gly Asn Met

65 70 75 80

Asp Asn Asn Leu Met Ala Tyr Val Ala Gly Arg Thr Gly Ala Phe Thr

85 90 95

Asp Ile Leu Trp Pro Gly Leu Gln Ser Val Pro Gln Pro Gly Leu Asn

100 105 110

Gly Arg Thr Gly Asn Val Phe Ile Ala Lys Gln Val Gly Gly Gly Ser

115 120 125

Ser Val Asn Ala Met Met Asn Met Arg Gly Ser Ala Glu Asp Tyr Asp

130 135 140

Arg Trp Ala Ser Leu Phe Gly Ser Ala Ala Gln Gln Gly Thr Ala Asp

145 150 155 160

Trp Ser Trp Asp Gly Ile Leu Pro Phe Phe Lys Lys Gly Leu His Phe

165 170 175

Thr Glu Pro Pro Pro Glu Leu Thr Asp Asn Phe Pro Ser Val Lys Thr

180 185 190

Asp Ala Ser Tyr Trp Gly Asp Ser Ser Asp Ile Tyr Ala Gly Trp Pro

195 200 205

Arg Phe Tyr Tyr Pro Gly Val Asn Pro Leu Val Glu Ala Phe Lys Glu

210 215 220

Val Glu Gly Val Glu Phe Pro Pro Asp Ser Gly Ala Gly Gln Pro Gly

225 230 235 240

Val Phe Trp Phe Pro Ala Leu Met Asp Pro Arg Thr Val Thr Arg Ser

245 250 255

Tyr Ala Gly Thr Gly His Tyr Leu Asn Val Asn Ala Thr Arg Pro Asn

260 265 270

Tyr His Leu Leu Val Asn Thr Gln Ala Arg Lys Leu Leu Leu Asn Asp

275 280 285

Glu Leu Val Ala Thr Gly Val Glu Phe Pro Val Gly Asn Ala Leu Val

290 295 300

Thr Val Ser Ala Lys Lys Glu Val Ile Val Ser Ala Gly Ala Ile His

305 310 315 320

Thr Pro Gln Leu Leu Gln Leu Ser Gly Ile Gly Pro Lys Lys Leu Leu

325 330 335

Glu Ala Ala Gly Ile Asp Val Arg Val Asp Leu Pro Gly Val Gly Gln

340 345 350

Asn Phe Gln Asp His Ser Ser Leu Ser Ala Val Asn Ile Thr Leu His

355 360 365

Lys Leu Ala Ser Ile His Pro Asn Pro Asn Asp Leu Val Glu Gly Asn

370 375 380

Glu Phe Lys Ala Trp Ala Asp Glu Val Trp Ala Ala Asn Lys Thr Gly

385 390 395 400

Pro Tyr Ser Leu Pro Phe Thr Asn Leu Ala Gly Trp Leu Pro Phe Thr

405 410 415

Val Val Ser Asp Lys Ala Glu Glu Leu Ala Ala Lys Leu Glu Ala Gln

420 425 430

Asp Pro Ala Ser Leu Leu Gln Glu Gly Ala Asp Pro Thr Val Val Ala

435 440 445

Gly Phe Ala Ala Gln Met Lys Ile Leu Ala Ser Gln Met Arg Ser Lys

450 455 460

Asp Thr Ala Phe Thr Arg Tyr Gln Leu Val Pro Ala Gln Gly Ala Gln

465 470 475 480

Gly Pro Val Ala Met Gln Ser Phe Ser Arg Gly Thr Val Asn Ile Asn

485 490 495

Ala Glu Asp Pro Trp Gly Ser Glu Pro Val Ile Asp Tyr Arg Val Leu

500 505 510

Ser Asn Pro Val Glu Ala Asp Phe Phe Val Glu Ser Ile Arg Phe Leu

515 520 525

Arg Arg Tyr Asn Phe Glu Thr Ser Leu Ala Lys Glu Phe Asp Pro Val

530 535 540

Glu Tyr Ala Pro Gly Pro Glu Val Val Ser Asp Glu Asp Leu Lys Ala

545 550 555 560

Tyr Ile Ala Asp Thr Leu Ser Pro Thr Asp Tyr His Pro Val Gly Thr

565 570 575

Ser Ser Met Leu Pro Leu Glu Leu Gly Gly Val Val Asp Gln Thr Leu

580 585 590

Arg Val Tyr Gly Val Lys Asn Leu Arg Val Val Asp Ala Ser Val Met

595 600 605

Pro Met Val Pro Ser Ala Asn Thr Cys Gln Pro Thr Tyr Ala Leu Ala

610 615 620

Glu Lys Ala Ala Glu Ile Ile Lys Gln Ser Ile

625 630 635

<210> 3

<211> 1872

<212> DNA

<213> Fungus_F5126

<400> 3

atgtcgttct ctcgctcgtc tctgctggtg acgctgacag ccgcgtcttc tgcgctcggc 60

tttgcgatcc cacgctccgc gtaccaggct cgccaagtca gcgatgcctc ggagctgctg 120

ccagcctacg actatgtgat cgtgggagga ggtactgcag gtcttacggt ggctgaccgc 180

ctaacggaag acccggatac aacggtcctc gttctcgagt ccggtgtttt cgctcccgac 240

gaggacgtcc tgcccatcac cggcggtggc acccgcagac agcctcgcat ctccttccag 300

tcggtgcccc agaagggcct tggcggtaag aacttcaccg tcaccatggg ccacatggtt 360

ggcggcagtt ccggtgtcaa tgcaatgatg accgtgagag gctctgctga ggattacgat 420

cgctggggcc agctgttcgg cgaggatagc cagggctgga gctgggacgg cttgttgccc 480

tacttcaaga agggcttgac tatgactccc ccgtctgccg agttggccaa gcgtttcaac 540

atcaagaccg acacgtctta ctggggcaag gactcgccca tccgagtcag cttcccgagc 600

ttccagtacc cgggccttga cccccttctc agcgccttcg aggagctccc tggcattgag 660

atggttgccg acagcggtgc tggtggtgcc ggtgtctact ggttccctac tttcatggat 720

gaggtcaacg tggagcggtc gtacgctggc aacgtgcaca agaagctcga ccgatccaac 780

taccacatcc ttgccaagac accagtgcgc cgcgtccttc ttgacgagaa cactgcagtc 840

agtggcgtgg agttctacac caacgacacc aacgttgcta ccattcaggc cactaaggag 900

gttctcatgg ctgccggcgc catccacact cccaagctgc tgcagctcag tggcatcggc 960

cccaagaatg tcctcgaggc tgctggcatc gacaccatcg tcgacttgcc cggtgtcggc 1020

caaaacttcc aggaccactc taatatcggc tctgctatct cgctcccagg tctcgcccag 1080

attcacccca acgccaacga tctcaccaac gatgctgcct tcaagaagct tgcggatgag 1140

ctctgggtcg ccaaccgcac cggtcccaag tcaattgcct tcggcaacgt cgccggctgg 1200

cttccgttca cagccatctc gcccgaacgc tttgatgagc tggccactga actggagaac 1260

caggaccacg ctgcctactt gccggctaat gtgcacccaa ccgttgcaaa gggctacgcc 1320

gctcagatga aaggtctggc tgctgcgatg agaagcaagg acacagtctt cgcacgctac 1380

cacgttgatg ccaccagagg tgcaacggcc cctatcctca accagccctt cagccgcgga 1440

tccgtcaaca tcgaccccaa ggatcccttc aacgcatacc ctatcgtcga ctaccgctcg 1500

ctcagcaacc ccgtcgagac cagtgtcgtt gtcgagatga tcaagtggta ccgccgttac 1560

aactttgaga cgtctctggc ctcgctgaac cccaaggaga cggctcccgg cgccgctgtt 1620

gtcaccgacg agcagctcgc tgcctgggtg cccaccgcgc tcaaccccac cgactaccac 1680

cccgccggca ccgccgccct gagccccctt gagctgggtg gcgttgtcga tcagaccctg 1740

cgcgtttatg gcgtgcagaa gttgcgtgtc attgatgcca gtgtcatccc tgtgctccct 1800

ggcgccaaca catgccagcc tacttatgct attgctgaga aggcggctga tatcatcaag 1860

agccaggtct aa 1872

<210> 4

<211> 623

<212> PRT

<213> Fungus_F5126

<400> 4

Met Ser Phe Ser Arg Ser Ser Leu Leu Val Thr Leu Thr Ala Ala Ser

1 5 10 15

Ser Ala Leu Gly Phe Ala Ile Pro Arg Ser Ala Tyr Gln Ala Arg Gln

20 25 30

Val Ser Asp Ala Ser Glu Leu Leu Pro Ala Tyr Asp Tyr Val Ile Val

35 40 45

Gly Gly Gly Thr Ala Gly Leu Thr Val Ala Asp Arg Leu Thr Glu Asp

50 55 60

Pro Asp Thr Thr Val Leu Val Leu Glu Ser Gly Val Phe Ala Pro Asp

65 70 75 80

Glu Asp Val Leu Pro Ile Thr Gly Gly Gly Thr Arg Arg Gln Pro Arg

85 90 95

Ile Ser Phe Gln Ser Val Pro Gln Lys Gly Leu Gly Gly Lys Asn Phe

100 105 110

Thr Val Thr Met Gly His Met Val Gly Gly Ser Ser Gly Val Asn Ala

115 120 125

Met Met Thr Val Arg Gly Ser Ala Glu Asp Tyr Asp Arg Trp Gly Gln

130 135 140

Leu Phe Gly Glu Asp Ser Gln Gly Trp Ser Trp Asp Gly Leu Leu Pro

145 150 155 160

Tyr Phe Lys Lys Gly Leu Thr Met Thr Pro Pro Ser Ala Glu Leu Ala

165 170 175

Lys Arg Phe Asn Ile Lys Thr Asp Thr Ser Tyr Trp Gly Lys Asp Ser

180 185 190

Pro Ile Arg Val Ser Phe Pro Ser Phe Gln Tyr Pro Gly Leu Asp Pro

195 200 205

Leu Leu Ser Ala Phe Glu Glu Leu Pro Gly Ile Glu Met Val Ala Asp

210 215 220

Ser Gly Ala Gly Gly Ala Gly Val Tyr Trp Phe Pro Thr Phe Met Asp

225 230 235 240

Glu Val Asn Val Glu Arg Ser Tyr Ala Gly Asn Val His Lys Lys Leu

245 250 255

Asp Arg Ser Asn Tyr His Ile Leu Ala Lys Thr Pro Val Arg Arg Val

260 265 270

Leu Leu Asp Glu Asn Thr Ala Val Ser Gly Val Glu Phe Tyr Thr Asn

275 280 285

Asp Thr Asn Val Ala Thr Ile Gln Ala Thr Lys Glu Val Leu Met Ala

290 295 300

Ala Gly Ala Ile His Thr Pro Lys Leu Leu Gln Leu Ser Gly Ile Gly

305 310 315 320

Pro Lys Asn Val Leu Glu Ala Ala Gly Ile Asp Thr Ile Val Asp Leu

325 330 335

Pro Gly Val Gly Gln Asn Phe Gln Asp His Ser Asn Ile Gly Ser Ala

340 345 350

Ile Ser Leu Pro Gly Leu Ala Gln Ile His Pro Asn Ala Asn Asp Leu

355 360 365

Thr Asn Asp Ala Ala Phe Lys Lys Leu Ala Asp Glu Leu Trp Val Ala

370 375 380

Asn Arg Thr Gly Pro Lys Ser Ile Ala Phe Gly Asn Val Ala Gly Trp

385 390 395 400

Leu Pro Phe Thr Ala Ile Ser Pro Glu Arg Phe Asp Glu Leu Ala Thr

405 410 415

Glu Leu Glu Asn Gln Asp His Ala Ala Tyr Leu Pro Ala Asn Val His

420 425 430

Pro Thr Val Ala Lys Gly Tyr Ala Ala Gln Met Lys Gly Leu Ala Ala

435 440 445

Ala Met Arg Ser Lys Asp Thr Val Phe Ala Arg Tyr His Val Asp Ala

450 455 460

Thr Arg Gly Ala Thr Ala Pro Ile Leu Asn Gln Pro Phe Ser Arg Gly

465 470 475 480

Ser Val Asn Ile Asp Pro Lys Asp Pro Phe Asn Ala Tyr Pro Ile Val

485 490 495

Asp Tyr Arg Ser Leu Ser Asn Pro Val Glu Thr Ser Val Val Val Glu

500 505 510

Met Ile Lys Trp Tyr Arg Arg Tyr Asn Phe Glu Thr Ser Leu Ala Ser

515 520 525

Leu Asn Pro Lys Glu Thr Ala Pro Gly Ala Ala Val Val Thr Asp Glu

530 535 540

Gln Leu Ala Ala Trp Val Pro Thr Ala Leu Asn Pro Thr Asp Tyr His

545 550 555 560

Pro Ala Gly Thr Ala Ala Leu Ser Pro Leu Glu Leu Gly Gly Val Val

565 570 575

Asp Gln Thr Leu Arg Val Tyr Gly Val Gln Lys Leu Arg Val Ile Asp

580 585 590

Ala Ser Val Ile Pro Val Leu Pro Gly Ala Asn Thr Cys Gln Pro Thr

595 600 605

Tyr Ala Ile Ala Glu Lys Ala Ala Asp Ile Ile Lys Ser Gln Val

610 615 620

<210> 5

<211> 1908

<212> DNA

<213> Anthrax species

<400> 5

atgcccttgt ttcgccagtc caagtcccag cctcggtggc cgggcgtcgc atccgcggtc 60

tttctcgcag cgagctctgt cgccaatgct tacgccattc cgcgtgacat caagccatct 120

gagctgctgc agagctatga ttatgtcatt gttggaggtg gaacggcagg cctgactgtc 180

gcagaccgtc tcacggagga ccctaacacg acagtcttgg tccttgaagc cggcggctgg 240

agcaacatga ctgacaacct gatggcctac gtcgcgggca gatccggcag gattctgtgg 300

cccggcctcc agtctgtgcc gcaagagcac ttgaatggaa gaaccaacac cgtctccgtt 360

gccaggcaag ttggaggcgg ctccgccata aacgccatga tcaccatgcg tggctctgca 420

gaggactatg accgctgggc gaccctgttc ggacccgagg ctcagcgggg cactgctgac 480

tggagctggg atggtatcct gccgttcttc aagaaggctc tccacttcac tgagccccct 540

cctgagctta ccgacaactt tgatatcaag tatgacgcct cctactgggg cgactcttcc 600

gagctctacg ccggttggcc ccggttctac tacccaggag tgactcccct cttggaagca 660

ttcaaggaga tcgagggcgt tgaattccct cccgacagtg gtgccggcca gccaggtgtt 720

tactggttcc ccgccttcat ggacccccgt actgtcactc gctcctacgc cgccactggt 780

cactatctca acgttaacgc gacccgccaa aactaccacc tgttgattaa cagccaggct 840

cgcaagctga tcttggacga caacctcacc gccactggag ttgagttccc cctggcaaac 900

aacaccctat tcactgtcaa cgcaaggaag gaggtcattc tctctgctgg taccgttcac 960

actcctcagc ttctgcagct gagcggtgtc ggtcccaaga agcttcttga ggaggcgggc 1020

attgacgtgc gtgttgacct tcccggtgtt ggccagaact tccaggacca tagcagtctc 1080

tccacagtga acatcactct ctccaagatt acatcgattc accccaaccc caaggacctg 1140

gtcgatggaa acgacttcaa gacctgggcc gacgaggttt ggcaagctaa caagactggc 1200

ccttactcca tctcatggac caacttggct ggctggctcc ctttcaccgt catttcggac 1260

aaggctgacg agcttgccac caagctggag caacaagact tcgccagcct gctgcccgct 1320

ggcaccgacg ccacagtggt cgccggtttt gaggcgcaga tgaagctcct ggccgcccag 1380

atgcgctcca aaaacacggc cttcacccgt taccagctta tcgcggagca cggcgtccag 1440

ggccccgtgg ggttgcaatc cttcagccgc ggcaccatca acatcaacac caccaacccg 1500

tggaacacgg agccggtgat cgactaccgc gtcctcagta accccctcga ggccgactac 1560

ttcgtcgagt caatcaagtt ccttcgccgc tacaacttcg agacctccct ggcctccaag 1620

tttgagccgg tcgagtacgt ccctggcccc gacgtcacct ctgatgagga cctgaaggcc 1680

tacatcgccc gtgctttgtc cccctccgac taccaccccg tgggtacagc gtccatgctg 1740

cctctgaact tgggtggtgt cgttgaccag accctgcgcg tgtacggagt caagaacctg 1800

agagtcgttg acgccagtgt catgcccatg gtccccggtg ccaatacttg ccagcctacg 1860

tacgctcttg ccgagaaggc ttcggaaatc atcaagcaag gcatctaa 1908

<210> 6

<211> 635

<212> PRT

<213> Anthrax species

<400> 6

Met Pro Leu Phe Arg Gln Ser Lys Ser Gln Pro Arg Trp Pro Gly Val

1 5 10 15

Ala Ser Ala Val Phe Leu Ala Ala Ser Ser Val Ala Asn Ala Tyr Ala

20 25 30

Ile Pro Arg Asp Ile Lys Pro Ser Glu Leu Leu Gln Ser Tyr Asp Tyr

35 40 45

Val Ile Val Gly Gly Gly Thr Ala Gly Leu Thr Val Ala Asp Arg Leu

50 55 60

Thr Glu Asp Pro Asn Thr Thr Val Leu Val Leu Glu Ala Gly Gly Trp

65 70 75 80

Ser Asn Met Thr Asp Asn Leu Met Ala Tyr Val Ala Gly Arg Ser Gly

85 90 95

Arg Ile Leu Trp Pro Gly Leu Gln Ser Val Pro Gln Glu His Leu Asn

100 105 110

Gly Arg Thr Asn Thr Val Ser Val Ala Arg Gln Val Gly Gly Gly Ser

115 120 125

Ala Ile Asn Ala Met Ile Thr Met Arg Gly Ser Ala Glu Asp Tyr Asp

130 135 140

Arg Trp Ala Thr Leu Phe Gly Pro Glu Ala Gln Arg Gly Thr Ala Asp

145 150 155 160

Trp Ser Trp Asp Gly Ile Leu Pro Phe Phe Lys Lys Ala Leu His Phe

165 170 175

Thr Glu Pro Pro Pro Glu Leu Thr Asp Asn Phe Asp Ile Lys Tyr Asp

180 185 190

Ala Ser Tyr Trp Gly Asp Ser Ser Glu Leu Tyr Ala Gly Trp Pro Arg

195 200 205

Phe Tyr Tyr Pro Gly Val Thr Pro Leu Leu Glu Ala Phe Lys Glu Ile

210 215 220

Glu Gly Val Glu Phe Pro Pro Asp Ser Gly Ala Gly Gln Pro Gly Val

225 230 235 240

Tyr Trp Phe Pro Ala Phe Met Asp Pro Arg Thr Val Thr Arg Ser Tyr

245 250 255

Ala Ala Thr Gly His Tyr Leu Asn Val Asn Ala Thr Arg Gln Asn Tyr

260 265 270

His Leu Leu Ile Asn Ser Gln Ala Arg Lys Leu Ile Leu Asp Asp Asn

275 280 285

Leu Thr Ala Thr Gly Val Glu Phe Pro Leu Ala Asn Asn Thr Leu Phe

290 295 300

Thr Val Asn Ala Arg Lys Glu Val Ile Leu Ser Ala Gly Thr Val His

305 310 315 320

Thr Pro Gln Leu Leu Gln Leu Ser Gly Val Gly Pro Lys Lys Leu Leu

325 330 335

Glu Glu Ala Gly Ile Asp Val Arg Val Asp Leu Pro Gly Val Gly Gln

340 345 350

Asn Phe Gln Asp His Ser Ser Leu Ser Thr Val Asn Ile Thr Leu Ser

355 360 365

Lys Ile Thr Ser Ile His Pro Asn Pro Lys Asp Leu Val Asp Gly Asn

370 375 380

Asp Phe Lys Thr Trp Ala Asp Glu Val Trp Gln Ala Asn Lys Thr Gly

385 390 395 400

Pro Tyr Ser Ile Ser Trp Thr Asn Leu Ala Gly Trp Leu Pro Phe Thr

405 410 415

Val Ile Ser Asp Lys Ala Asp Glu Leu Ala Thr Lys Leu Glu Gln Gln

420 425 430

Asp Phe Ala Ser Leu Leu Pro Ala Gly Thr Asp Ala Thr Val Val Ala

435 440 445

Gly Phe Glu Ala Gln Met Lys Leu Leu Ala Ala Gln Met Arg Ser Lys

450 455 460

Asn Thr Ala Phe Thr Arg Tyr Gln Leu Ile Ala Glu His Gly Val Gln

465 470 475 480

Gly Pro Val Gly Leu Gln Ser Phe Ser Arg Gly Thr Ile Asn Ile Asn

485 490 495

Thr Thr Asn Pro Trp Asn Thr Glu Pro Val Ile Asp Tyr Arg Val Leu

500 505 510

Ser Asn Pro Leu Glu Ala Asp Tyr Phe Val Glu Ser Ile Lys Phe Leu

515 520 525

Arg Arg Tyr Asn Phe Glu Thr Ser Leu Ala Ser Lys Phe Glu Pro Val

530 535 540

Glu Tyr Val Pro Gly Pro Asp Val Thr Ser Asp Glu Asp Leu Lys Ala

545 550 555 560

Tyr Ile Ala Arg Ala Leu Ser Pro Ser Asp Tyr His Pro Val Gly Thr

565 570 575

Ala Ser Met Leu Pro Leu Asn Leu Gly Gly Val Val Asp Gln Thr Leu

580 585 590

Arg Val Tyr Gly Val Lys Asn Leu Arg Val Val Asp Ala Ser Val Met

595 600 605

Pro Met Val Pro Gly Ala Asn Thr Cys Gln Pro Thr Tyr Ala Leu Ala

610 615 620

Glu Lys Ala Ser Glu Ile Ile Lys Gln Gly Ile

625 630 635

<210> 7

<211> 1923

<212> DNA

<213> colletotrichum gloeosporioides

<400> 7

atgccgttcc ttcgccagtc caagtcccag cccctgtggc cccggactgc gttcacagct 60

cttctcgcag tgagctccgt cgccaacgcc ttggccgtcc ctcgccagat caagtcatct 120

cagctgctgc aaagctacga ctatgtcgtt gtcggaggtg gaacggccgg cctgaccctg 180

gcagaccgtt tgactgagga ccctgaaacg agtgtcttgg tcctcgaggc cggcaactgg 240

ggcaatatgg aggagaatct tgcggcttac ttcaccgagt cgcgatatgc caacctcatg 300

agctccctct ggcccggcgt ggagtctgtg ccgcagccca acttgaacgg aagagttggc 360

accgtctgga ttgccaagca agtcggaggc ggctcttccg tgaacgccat gatgaacatg 420

cgaggttccg ccgaggacta cgaccgctgg gcaaccctat tcggagccga agctcagcag 480

ggagtcgctg actggagctg ggatggcatg ttgaagtcct tcaagaaggg cctccacttt 540

accgagccca ctcccgagct gaccgacaac ttcgatagct tcaagtatga tccctcctac 600

tggggtgatt cttcggagat ctatgctggc tggcctcgct tctactaccc cggcgtaaag 660

cccctggtgg aagcgttcaa ggagatcgac ggcgttgagt tccctcccga cagtggtgcc 720

ggacagcccg gcgtcttctg gttccccacg ctgatggacc cccggactgt cacccgctcc 780

tacgctggta ccggtcacta cctgagcgtc aacgctaccc gccccaacta ccaccttctg 840

ctcgacaccc aggctcgcaa gctggtagtt gatgatgacc tcactgccac tggcgtcgtc 900

ttcccttccg gaaacaacac cctcgtcacc gtcaacgcaa agaaggaggt cattctctct 960

gctggtaccg ttcacactcc tcacctcttg cagctgagcg gtatcggccc caagaaggtt 1020

ctcgaggctg gaggcatcga tgtccgcgtc gatcttcctg gtgttggcca gaacttccag 1080

gaccacagca gtttgtctgc tatgaacatt acgctatcta agctcgcatc gattcacccg 1140

aacccgagtg acctagtgga tggcaacgag ttcaaggact gggctgaaga agtgtgggca 1200

gccaacaaga ccggccccta ctctctcaca ttcaccaacc tggccggctg gctccccttc 1260

accgtcgtgt ctggaaaggc cgacgagctc gccaccaagt tggaacaaca agacttcgct 1320

agcctgctcc ctgccgatgc cgactccacc gtcgtcgctg gtttcgcggc gcagatgaag 1380

atcctggccg cccagatgcg ctccaagaac accgccttca cccgcatgca gctcgtccct 1440

gaccacggct cccagggccc cgtcgcgatg cagtctttca gccgcggcac catcaacatc 1500

aacacgaccg acccgtggaa caccgagccc gtgatcgact accgcgccct cagcaacccc 1560

gtcgaggccg acttcttcgt cgaggcgatc aagttcctcc gccgctacaa cttcgagacc 1620

tcgctggcca ccgagtacga gcctgtcgag tacgcccctg gcccggatgt cacttccgac 1680

gaggacctca aggcctacat cgccggtgct ctgtcgccta ccgactacca ccccgtgggc 1740

acttcgtcta tgctgccgct ggagttgggc ggtgtcgtcg accagaccct tcgcgtttac 1800

ggagtcaaga acctcagagt cgttgacgcc agtgtcatgc ccatggttcc cagtgccaac 1860

acttgccagc ccacttatgc cctggccgag aaggccgcgg aaatcattaa gcaaggtatc 1920

taa 1923

<210> 8

<211> 640

<212> PRT

<213> colletotrichum gloeosporioides

<400> 8

Met Pro Phe Leu Arg Gln Ser Lys Ser Gln Pro Leu Trp Pro Arg Thr

1 5 10 15

Ala Phe Thr Ala Leu Leu Ala Val Ser Ser Val Ala Asn Ala Leu Ala

20 25 30

Val Pro Arg Gln Ile Lys Ser Ser Gln Leu Leu Gln Ser Tyr Asp Tyr

35 40 45

Val Val Val Gly Gly Gly Thr Ala Gly Leu Thr Leu Ala Asp Arg Leu

50 55 60

Thr Glu Asp Pro Glu Thr Ser Val Leu Val Leu Glu Ala Gly Asn Trp

65 70 75 80

Gly Asn Met Glu Glu Asn Leu Ala Ala Tyr Phe Thr Glu Ser Arg Tyr

85 90 95

Ala Asn Leu Met Ser Ser Leu Trp Pro Gly Val Glu Ser Val Pro Gln

100 105 110

Pro Asn Leu Asn Gly Arg Val Gly Thr Val Trp Ile Ala Lys Gln Val

115 120 125

Gly Gly Gly Ser Ser Val Asn Ala Met Met Asn Met Arg Gly Ser Ala

130 135 140

Glu Asp Tyr Asp Arg Trp Ala Thr Leu Phe Gly Ala Glu Ala Gln Gln

145 150 155 160

Gly Val Ala Asp Trp Ser Trp Asp Gly Met Leu Lys Ser Phe Lys Lys

165 170 175

Gly Leu His Phe Thr Glu Pro Thr Pro Glu Leu Thr Asp Asn Phe Asp

180 185 190

Ser Phe Lys Tyr Asp Pro Ser Tyr Trp Gly Asp Ser Ser Glu Ile Tyr

195 200 205

Ala Gly Trp Pro Arg Phe Tyr Tyr Pro Gly Val Lys Pro Leu Val Glu

210 215 220

Ala Phe Lys Glu Ile Asp Gly Val Glu Phe Pro Pro Asp Ser Gly Ala

225 230 235 240

Gly Gln Pro Gly Val Phe Trp Phe Pro Thr Leu Met Asp Pro Arg Thr

245 250 255

Val Thr Arg Ser Tyr Ala Gly Thr Gly His Tyr Leu Ser Val Asn Ala

260 265 270

Thr Arg Pro Asn Tyr His Leu Leu Leu Asp Thr Gln Ala Arg Lys Leu

275 280 285

Val Val Asp Asp Asp Leu Thr Ala Thr Gly Val Val Phe Pro Ser Gly

290 295 300

Asn Asn Thr Leu Val Thr Val Asn Ala Lys Lys Glu Val Ile Leu Ser

305 310 315 320

Ala Gly Thr Val His Thr Pro His Leu Leu Gln Leu Ser Gly Ile Gly

325 330 335

Pro Lys Lys Val Leu Glu Ala Gly Gly Ile Asp Val Arg Val Asp Leu

340 345 350

Pro Gly Val Gly Gln Asn Phe Gln Asp His Ser Ser Leu Ser Ala Met

355 360 365

Asn Ile Thr Leu Ser Lys Leu Ala Ser Ile His Pro Asn Pro Ser Asp

370 375 380

Leu Val Asp Gly Asn Glu Phe Lys Asp Trp Ala Glu Glu Val Trp Ala

385 390 395 400

Ala Asn Lys Thr Gly Pro Tyr Ser Leu Thr Phe Thr Asn Leu Ala Gly

405 410 415

Trp Leu Pro Phe Thr Val Val Ser Gly Lys Ala Asp Glu Leu Ala Thr

420 425 430

Lys Leu Glu Gln Gln Asp Phe Ala Ser Leu Leu Pro Ala Asp Ala Asp

435 440 445

Ser Thr Val Val Ala Gly Phe Ala Ala Gln Met Lys Ile Leu Ala Ala

450 455 460

Gln Met Arg Ser Lys Asn Thr Ala Phe Thr Arg Met Gln Leu Val Pro

465 470 475 480

Asp His Gly Ser Gln Gly Pro Val Ala Met Gln Ser Phe Ser Arg Gly

485 490 495

Thr Ile Asn Ile Asn Thr Thr Asp Pro Trp Asn Thr Glu Pro Val Ile

500 505 510

Asp Tyr Arg Ala Leu Ser Asn Pro Val Glu Ala Asp Phe Phe Val Glu

515 520 525

Ala Ile Lys Phe Leu Arg Arg Tyr Asn Phe Glu Thr Ser Leu Ala Thr

530 535 540

Glu Tyr Glu Pro Val Glu Tyr Ala Pro Gly Pro Asp Val Thr Ser Asp

545 550 555 560

Glu Asp Leu Lys Ala Tyr Ile Ala Gly Ala Leu Ser Pro Thr Asp Tyr

565 570 575

His Pro Val Gly Thr Ser Ser Met Leu Pro Leu Glu Leu Gly Gly Val

580 585 590

Val Asp Gln Thr Leu Arg Val Tyr Gly Val Lys Asn Leu Arg Val Val

595 600 605

Asp Ala Ser Val Met Pro Met Val Pro Ser Ala Asn Thr Cys Gln Pro

610 615 620

Thr Tyr Ala Leu Ala Glu Lys Ala Ala Glu Ile Ile Lys Gln Gly Ile

625 630 635 640

<210> 9

<211> 1917

<212> DNA

<213> colletotrichum anthracnose colletotrichum

<400> 9

atgtcgttct ttcgccagtc caagtctcag cctcgctggc aacgcctcgc tctcgcggcc 60

ctcgccgcgt gttccgtcgt cgacgcctac gttatcccgc gtcagatcaa gccgtctgag 120

cttctcgaca gctacgacta tgtcatcgtc ggaggcggta ccgccggtct caccgtcgcc 180

gaccgtctga ccgaagaccc cgagaccaag gtcttggttc tcgaggccgg tgactttggc 240

aacatggaca acaacctcct ggtgtacgtc atgggcaggt ctggtggctt caccgacccc 300

ctctggcccg gtctccagtc cgtgccgcag cccggtctga acggaagacc cggcaccgtc 360

ttcgtcgcca agcaggtcgg aggtggttcc ggcgtcaacg ccatgatgaa catgcgtggc 420

tcggccgagg actacgaccg ctgggctacc ctgttcggag ccgacgccca gcagggcacc 480

gccgactgga gctgggacgg catcctgcca ttcttcaaga agggtcttca cttcaccgag 540

cctcccaagg agctgacgga caacttcccc agcatcaaga ccgatgcgtc ctactggggc 600

gactcgtccg agatctacgc cggatggcct cgcttctact accccggagt caaccctctg 660

gtggaggcct tcaaggagat cgagggagtc gagttccctc ccgacagtgg tgccggacag 720

cccggtgtct tctggttccc tacgctcatg gatccccgtt ccaccacgcg ttcgtacgcc 780

gccactggtc actacctcaa cgtcaacgcc acccgctcca actaccacct cttggtcaac 840

accctggctc gcaagctggt gctgaacgac gagctgaccg ccaccggggt cgagttcccc 900

ctcggaaaca acactcttgt taccgtcaac gccaagaagg aggtcattct ctctgccggt 960

gccgtccaca cccctcacct cctgcagctc agcggcatcg gacccaagaa gcttctcgag 1020

gctggcaaca ttgacgtccg cgtcgacctg cccggcgtcg gccagaactt ccaggaccac 1080

agcagtctgt ctgccatgaa catcacgctc tccaagctcg cctcgatcca ccccaacccc 1140

aacgacctcg tcgatggaaa cgagttcaag acctgggctg aggaggtctg ggccgccaac 1200

aagaccggcc cctactccct cgccttcacc aacctcgccg gctggctccc cttcaccgcc 1260

gtctccgaca gggccgagga gctcgccgcc aagctcgaga cccaggactt tgccagcctc 1320

ctccccgccg gcgccgacgt caccgtcgtc gccggcttcg aggcgcagat gaagatcctc 1380

gcctcccagc tgcgctccaa gaacaccgcc ttcacccgcc tccagctcgt ccccgagcac 1440

ggcgcccagg gccccgtggc catgcagtcc ttcagccgcg gcaccatcaa catcaacacc 1500

acggacccct ggaacacgga acccgtcatc gactaccgcg tcctgagcaa ccccgtcgag 1560

gccgacttct tcgtcgagag catcaagttc ctccgccgct acaacttcga gacctccctg 1620

gccgccgagt acgaccccgt cgagtacgcc cccggcgccg acgtcacctc cgacgccgac 1680

ctcaaggagt acatcgccaa caccctctcg cccaccgact accaccccgt cggcaccgcg 1740

tccatgctgc cgctggagct gggcggcgtc gtcgaccaga ccctccgcgt gtacggcgtc 1800

aagaacctca gggtcgtcga cgcgagcgtc atgcccatgg ttccaagtgc caacacgtgc 1860

cagcccacct acgctctggc cgagaaggct gcggagatta tcaagcaggg agtttaa 1917

<210> 10

<211> 638

<212> PRT

<213> colletotrichum anthracnose colletotrichum

<400> 10

Met Ser Phe Phe Arg Gln Ser Lys Ser Gln Pro Arg Trp Gln Arg Leu

1 5 10 15

Ala Leu Ala Ala Leu Ala Ala Cys Ser Val Val Asp Ala Tyr Val Ile

20 25 30

Pro Arg Gln Ile Lys Pro Ser Glu Leu Leu Asp Ser Tyr Asp Tyr Val

35 40 45

Ile Val Gly Gly Gly Thr Ala Gly Leu Thr Val Ala Asp Arg Leu Thr

50 55 60

Glu Asp Pro Glu Thr Lys Val Leu Val Leu Glu Ala Gly Asp Phe Gly

65 70 75 80

Asn Met Asp Asn Asn Leu Leu Val Tyr Val Met Gly Arg Ser Gly Gly

85 90 95

Phe Thr Asp Pro Leu Trp Pro Gly Leu Gln Ser Val Pro Gln Pro Gly

100 105 110

Leu Asn Gly Arg Pro Gly Thr Val Phe Val Ala Lys Gln Val Gly Gly

115 120 125

Gly Ser Gly Val Asn Ala Met Met Asn Met Arg Gly Ser Ala Glu Asp

130 135 140

Tyr Asp Arg Trp Ala Thr Leu Phe Gly Ala Asp Ala Gln Gln Gly Thr

145 150 155 160

Ala Asp Trp Ser Trp Asp Gly Ile Leu Pro Phe Phe Lys Lys Gly Leu

165 170 175

His Phe Thr Glu Pro Pro Lys Glu Leu Thr Asp Asn Phe Pro Ser Ile

180 185 190

Lys Thr Asp Ala Ser Tyr Trp Gly Asp Ser Ser Glu Ile Tyr Ala Gly

195 200 205

Trp Pro Arg Phe Tyr Tyr Pro Gly Val Asn Pro Leu Val Glu Ala Phe

210 215 220

Lys Glu Ile Glu Gly Val Glu Phe Pro Pro Asp Ser Gly Ala Gly Gln

225 230 235 240

Pro Gly Val Phe Trp Phe Pro Thr Leu Met Asp Pro Arg Ser Thr Thr

245 250 255

Arg Ser Tyr Ala Ala Thr Gly His Tyr Leu Asn Val Asn Ala Thr Arg

260 265 270

Ser Asn Tyr His Leu Leu Val Asn Thr Leu Ala Arg Lys Leu Val Leu

275 280 285

Asn Asp Glu Leu Thr Ala Thr Gly Val Glu Phe Pro Leu Gly Asn Asn

290 295 300

Thr Leu Val Thr Val Asn Ala Lys Lys Glu Val Ile Leu Ser Ala Gly

305 310 315 320

Ala Val His Thr Pro His Leu Leu Gln Leu Ser Gly Ile Gly Pro Lys

325 330 335

Lys Leu Leu Glu Ala Gly Asn Ile Asp Val Arg Val Asp Leu Pro Gly

340 345 350

Val Gly Gln Asn Phe Gln Asp His Ser Ser Leu Ser Ala Met Asn Ile

355 360 365

Thr Leu Ser Lys Leu Ala Ser Ile His Pro Asn Pro Asn Asp Leu Val

370 375 380

Asp Gly Asn Glu Phe Lys Thr Trp Ala Glu Glu Val Trp Ala Ala Asn

385 390 395 400

Lys Thr Gly Pro Tyr Ser Leu Ala Phe Thr Asn Leu Ala Gly Trp Leu

405 410 415

Pro Phe Thr Ala Val Ser Asp Arg Ala Glu Glu Leu Ala Ala Lys Leu

420 425 430

Glu Thr Gln Asp Phe Ala Ser Leu Leu Pro Ala Gly Ala Asp Val Thr

435 440 445

Val Val Ala Gly Phe Glu Ala Gln Met Lys Ile Leu Ala Ser Gln Leu

450 455 460

Arg Ser Lys Asn Thr Ala Phe Thr Arg Leu Gln Leu Val Pro Glu His

465 470 475 480

Gly Ala Gln Gly Pro Val Ala Met Gln Ser Phe Ser Arg Gly Thr Ile

485 490 495

Asn Ile Asn Thr Thr Asp Pro Trp Asn Thr Glu Pro Val Ile Asp Tyr

500 505 510

Arg Val Leu Ser Asn Pro Val Glu Ala Asp Phe Phe Val Glu Ser Ile

515 520 525

Lys Phe Leu Arg Arg Tyr Asn Phe Glu Thr Ser Leu Ala Ala Glu Tyr

530 535 540

Asp Pro Val Glu Tyr Ala Pro Gly Ala Asp Val Thr Ser Asp Ala Asp

545 550 555 560

Leu Lys Glu Tyr Ile Ala Asn Thr Leu Ser Pro Thr Asp Tyr His Pro

565 570 575

Val Gly Thr Ala Ser Met Leu Pro Leu Glu Leu Gly Gly Val Val Asp

580 585 590

Gln Thr Leu Arg Val Tyr Gly Val Lys Asn Leu Arg Val Val Asp Ala

595 600 605

Ser Val Met Pro Met Val Pro Ser Ala Asn Thr Cys Gln Pro Thr Tyr

610 615 620

Ala Leu Ala Glu Lys Ala Ala Glu Ile Ile Lys Gln Gly Val

625 630 635

<210> 11

<211> 1923

<212> DNA

<213> fungus anthrax

<400> 11

atgatgccgt cgtttcgcca gtccaagtcc cagcctcggt ggcgagggat cgcaactgca 60

accctcctca cggcgagctg tgtcgccaac gcctacgtca tcccgcgcca gatcaagcct 120

tcgcagctcc agcaaagtta tgactatgtc attgtgggag gtggaacggc aggactgacc 180

gtggcggacc gcctgaccga ggaccccgag acgaccgtct tggtcctcga agccggtgat 240

tggggcaaca tggcagacaa tcttttggtt cgcacttcaa acagaaatgg atctttcacc 300

gacttgctgt ggcccggtct tcagtctgtg ccacagccca acctgaacgg aaggcctggc 360

aacgtcttca ttgctaagca agttggaggc ggcgcgtccg tgaacgccat gataaacatg 420

cgtggctctg cagaggacta cgaccgctgg gccaccctgt tcggatatga agctcaggag 480

ggcactgctg actggagctg ggatggcatc ttgccgttct tcaagaaggg cctccacttt 540

actgagccgc ctccggaact gaccgacaac ttcgatagcg tcaagtatga tgcctcctac 600

tggggagact cttccgacat ctatgccggt tggcctcggt tctactaccc aggcgtgagc 660

ccactcttgg aagcgttcaa ggagatcgac ggcgttgagt tcccgtccga cagtggtgct 720

ggacaggcgg gcgtttactg gttccccacg ctcatggacc ccagaactgt cacgcgctcc 780

tacgccggta ctggtcacta tctcaacgtc aacgctaccc gccccaacta ccaccttttg 840

gtcaacaccc aggctcgcag gctggtagtg gacgacgagc tctctgccac aggagtcgag 900

ttcccgttgg gaaacaacac cctgttcacc gtcaatgcaa agaaggaggt cattctctct 960

gccggtacca ttcacactcc tcacctcttg cagctgagcg gtatcggtcc caagaagatt 1020

cttgaggctg cgggcattga cgtgcgtgtc gacctacccg gtgttggcca gaacttccaa 1080

gaccattcta gtctggctga ggtgaacatc acactctcta aactcacgtc gatccacccg 1140

aacccgcggg atctggtgga tggaaacgac tttaaaaact gggccgacga ggtgtgggca 1200

gctaacaaga ccggtccata ctctatcgca ctcaccaacc tggccggctg gctccccttc 1260

accgccgtgt cggataaggc cgacgagctt gctacgaagc tggaggaaca agactttgcc 1320

agcctgctgt cggccgacgc cgatgccact gtggtcgccg gcttcgagtc gcagatgaag 1380

atcctggccg cccagatgcg ctccaagaac acggccttca cccgtttcca gctcatcgcc 1440

gaccacggcg cccagggccc cgtggcgatg caatccttca gccgcggcac catcaacatc 1500

aacacgaccg acccgctgaa cacggagccc gtgattgact accgcgccct gaccaacccc 1560

ctggaggcag acttcttcgt tgagtcgatc aagttcctcc gccgctacaa cttcgagacc 1620

tccctggcct ctgagtttgg tccggtcgag tacgtccccg gtcctgacgt cgtctcggac 1680

gaggacctca aggcctacat cgccagtgct ttgtcgccga ccgactacca tcccgtcggc 1740

acagcatcca tgctgccgct gaagttgggt ggtgtcgtcg atcaaacctt gcgtgtgtac 1800

ggagtcaaga acttgagagt agtagacgcc agtgtcatgc ccatggttcc cagtgccaac 1860

acgtgccagc ctacctacgc cctggccgag aaggctgcgg aaatcatcaa gcaaggcatc 1920

taa 1923

<210> 12

<211> 640

<212> PRT

<213> fungus anthrax

<400> 12

Met Met Pro Ser Phe Arg Gln Ser Lys Ser Gln Pro Arg Trp Arg Gly

1 5 10 15

Ile Ala Thr Ala Thr Leu Leu Thr Ala Ser Cys Val Ala Asn Ala Tyr

20 25 30

Val Ile Pro Arg Gln Ile Lys Pro Ser Gln Leu Gln Gln Ser Tyr Asp

35 40 45

Tyr Val Ile Val Gly Gly Gly Thr Ala Gly Leu Thr Val Ala Asp Arg

50 55 60

Leu Thr Glu Asp Pro Glu Thr Thr Val Leu Val Leu Glu Ala Gly Asp

65 70 75 80

Trp Gly Asn Met Ala Asp Asn Leu Leu Val Arg Thr Ser Asn Arg Asn

85 90 95

Gly Ser Phe Thr Asp Leu Leu Trp Pro Gly Leu Gln Ser Val Pro Gln

100 105 110

Pro Asn Leu Asn Gly Arg Pro Gly Asn Val Phe Ile Ala Lys Gln Val

115 120 125

Gly Gly Gly Ala Ser Val Asn Ala Met Ile Asn Met Arg Gly Ser Ala

130 135 140

Glu Asp Tyr Asp Arg Trp Ala Thr Leu Phe Gly Tyr Glu Ala Gln Glu

145 150 155 160

Gly Thr Ala Asp Trp Ser Trp Asp Gly Ile Leu Pro Phe Phe Lys Lys

165 170 175

Gly Leu His Phe Thr Glu Pro Pro Pro Glu Leu Thr Asp Asn Phe Asp

180 185 190

Ser Val Lys Tyr Asp Ala Ser Tyr Trp Gly Asp Ser Ser Asp Ile Tyr

195 200 205

Ala Gly Trp Pro Arg Phe Tyr Tyr Pro Gly Val Ser Pro Leu Leu Glu

210 215 220

Ala Phe Lys Glu Ile Asp Gly Val Glu Phe Pro Ser Asp Ser Gly Ala

225 230 235 240

Gly Gln Ala Gly Val Tyr Trp Phe Pro Thr Leu Met Asp Pro Arg Thr

245 250 255

Val Thr Arg Ser Tyr Ala Gly Thr Gly His Tyr Leu Asn Val Asn Ala

260 265 270

Thr Arg Pro Asn Tyr His Leu Leu Val Asn Thr Gln Ala Arg Arg Leu

275 280 285

Val Val Asp Asp Glu Leu Ser Ala Thr Gly Val Glu Phe Pro Leu Gly

290 295 300

Asn Asn Thr Leu Phe Thr Val Asn Ala Lys Lys Glu Val Ile Leu Ser

305 310 315 320

Ala Gly Thr Ile His Thr Pro His Leu Leu Gln Leu Ser Gly Ile Gly

325 330 335

Pro Lys Lys Ile Leu Glu Ala Ala Gly Ile Asp Val Arg Val Asp Leu

340 345 350

Pro Gly Val Gly Gln Asn Phe Gln Asp His Ser Ser Leu Ala Glu Val

355 360 365

Asn Ile Thr Leu Ser Lys Leu Thr Ser Ile His Pro Asn Pro Arg Asp

370 375 380

Leu Val Asp Gly Asn Asp Phe Lys Asn Trp Ala Asp Glu Val Trp Ala

385 390 395 400

Ala Asn Lys Thr Gly Pro Tyr Ser Ile Ala Leu Thr Asn Leu Ala Gly

405 410 415

Trp Leu Pro Phe Thr Ala Val Ser Asp Lys Ala Asp Glu Leu Ala Thr

420 425 430

Lys Leu Glu Glu Gln Asp Phe Ala Ser Leu Leu Ser Ala Asp Ala Asp

435 440 445

Ala Thr Val Val Ala Gly Phe Glu Ser Gln Met Lys Ile Leu Ala Ala

450 455 460

Gln Met Arg Ser Lys Asn Thr Ala Phe Thr Arg Phe Gln Leu Ile Ala

465 470 475 480

Asp His Gly Ala Gln Gly Pro Val Ala Met Gln Ser Phe Ser Arg Gly

485 490 495

Thr Ile Asn Ile Asn Thr Thr Asp Pro Leu Asn Thr Glu Pro Val Ile

500 505 510

Asp Tyr Arg Ala Leu Thr Asn Pro Leu Glu Ala Asp Phe Phe Val Glu

515 520 525

Ser Ile Lys Phe Leu Arg Arg Tyr Asn Phe Glu Thr Ser Leu Ala Ser

530 535 540

Glu Phe Gly Pro Val Glu Tyr Val Pro Gly Pro Asp Val Val Ser Asp

545 550 555 560

Glu Asp Leu Lys Ala Tyr Ile Ala Ser Ala Leu Ser Pro Thr Asp Tyr

565 570 575

His Pro Val Gly Thr Ala Ser Met Leu Pro Leu Lys Leu Gly Gly Val

580 585 590

Val Asp Gln Thr Leu Arg Val Tyr Gly Val Lys Asn Leu Arg Val Val

595 600 605

Asp Ala Ser Val Met Pro Met Val Pro Ser Ala Asn Thr Cys Gln Pro

610 615 620

Thr Tyr Ala Leu Ala Glu Lys Ala Ala Glu Ile Ile Lys Gln Gly Ile

625 630 635 640

<210> 13

<211> 1914

<212> DNA

<213> colletotrichum MAFF240289

<400> 13

atgccgttct ttcaacaatc caagccacag tggcttggaa tcgcatccgc ggtcctcctg 60

ggagcaagct gtgttactga cgcctacgtg attccccgcc aaatcaactc atctcagttg 120

ctgaagagct acgattatgt cattgttggc ggtggaactg caggtctgac tgtggcagac 180

cgtctgacgg aggaccccga aaccaaagtc ttggttctcg aggctgcaga ctggggcaac 240

atgactgaaa acctcaaggt ttccttcctg actcggactt ccgctttcac agacctactt 300

tggcccggtc tgcagtctgt accgcagcct ggcctgaatg gaagagtcgg caacgtcttc 360

attgccaaac aggtcggagg tggtgcgtca gtaaacgcca tgatgaacat gcgtggctcg 420

gccgaggact acgaccgttg ggctagcctc ttcggatccg aggctcagca gggaactgcc 480

gactggagct gggatggcat cttgccgttc ttcaagaagg gccttcactt cactgagcca 540

cctcctgagt tgaccgacaa ctttgacagc gtcaagtatg acgcctcttt ctggggagac 600

tcgtcagaga tctacgcggg atggccccgg ttctactacc cgggtgtcaa gccccttgtc 660

gaagccttca aggagatcga cggagtagag ttcccggccg acagcggtgc cggaaagccc 720

ggtgtcttct ggttccccac gctcatggat ccccgaaccg tcacccgctc ctatgctggc 780

accggtcact acctcaacgt taacgccact cgtccgaact atcacctttt gctcaacact 840

caggcccgca agttgatcct tgacgaccag ctctctgtca caggagttga gttcccatcg 900

ggaaacaaca gcttcgtcac tatcactgca aagaaggagg ttctccttgc tgccggtgct 960

gttcacactc cccagctctt gcagctcagc ggtatcggcc ccaagagcct tctcgaggct 1020

ggtggcattg atgtcctcgt cgacctccct ggtgtcggcc agaacttcca agaccacagc 1080

agtctctctt ccatgaacat taccctctcc aagctcactg acatccaccc caacccaacc 1140

gacctggtcg aagggaatga cttcaagact tgggccgacg aagtgtgggc agccaacaag 1200

accggcccct attctattgc attgacaaac ttggccggct ggctcccgtt caccgctgtg 1260

tctgaacggg ctgaggagct cgccaccaag ttagagcaac aggactttgc tagcctgctg 1320

ccgaccgata ccgaaaccac agttgtcgct ggtttcgaag cgcagatgaa gatcctggct 1380

gcacagatgc gttccaagga cacagcattc actcgcatgc agttgatcgc gaaccagggt 1440

tctcagggcc ccgtggcaat gcagtccttc agccgtggca ccatcaacat taatacgact 1500

gacccgtgga acaccgagcc cgttatcgac taccgctcct tgtccaaccc tcttgaggcc 1560

gatttcttcg tcgaatcaat caagttcctc cgccgttaca acttcaacac ctctttagct 1620

accgagttcg cccctgtcga gtacgctccc ggtcctgagg tcacctcgga cgaggacctc 1680

aaggcctaca ttgccggtgc catgtcgccg actgactacc accccgtggg gactgcatct 1740

atgatgcccc tgaatctggg gggtgttgtc gatcagactt tgcgggtgta cggagtgaaa 1800

aatttgagag tcgttgacgc cagtgttatg cccatggttc ctagcgccaa tacatgccag 1860

cctacctacg ctttggccga aaaggccgcc gaaatcatca agcaaggcat ctaa 1914

<210> 14

<211> 637

<212> PRT

<213> colletotrichum MAFF240289

<400> 14

Met Pro Phe Phe Gln Gln Ser Lys Pro Gln Trp Leu Gly Ile Ala Ser

1 5 10 15

Ala Val Leu Leu Gly Ala Ser Cys Val Thr Asp Ala Tyr Val Ile Pro

20 25 30

Arg Gln Ile Asn Ser Ser Gln Leu Leu Lys Ser Tyr Asp Tyr Val Ile

35 40 45

Val Gly Gly Gly Thr Ala Gly Leu Thr Val Ala Asp Arg Leu Thr Glu

50 55 60

Asp Pro Glu Thr Lys Val Leu Val Leu Glu Ala Ala Asp Trp Gly Asn

65 70 75 80

Met Thr Glu Asn Leu Lys Val Ser Phe Leu Thr Arg Thr Ser Ala Phe

85 90 95

Thr Asp Leu Leu Trp Pro Gly Leu Gln Ser Val Pro Gln Pro Gly Leu

100 105 110

Asn Gly Arg Val Gly Asn Val Phe Ile Ala Lys Gln Val Gly Gly Gly

115 120 125

Ala Ser Val Asn Ala Met Met Asn Met Arg Gly Ser Ala Glu Asp Tyr

130 135 140

Asp Arg Trp Ala Ser Leu Phe Gly Ser Glu Ala Gln Gln Gly Thr Ala

145 150 155 160

Asp Trp Ser Trp Asp Gly Ile Leu Pro Phe Phe Lys Lys Gly Leu His

165 170 175

Phe Thr Glu Pro Pro Pro Glu Leu Thr Asp Asn Phe Asp Ser Val Lys

180 185 190

Tyr Asp Ala Ser Phe Trp Gly Asp Ser Ser Glu Ile Tyr Ala Gly Trp

195 200 205

Pro Arg Phe Tyr Tyr Pro Gly Val Lys Pro Leu Val Glu Ala Phe Lys

210 215 220

Glu Ile Asp Gly Val Glu Phe Pro Ala Asp Ser Gly Ala Gly Lys Pro

225 230 235 240

Gly Val Phe Trp Phe Pro Thr Leu Met Asp Pro Arg Thr Val Thr Arg

245 250 255

Ser Tyr Ala Gly Thr Gly His Tyr Leu Asn Val Asn Ala Thr Arg Pro

260 265 270

Asn Tyr His Leu Leu Leu Asn Thr Gln Ala Arg Lys Leu Ile Leu Asp

275 280 285

Asp Gln Leu Ser Val Thr Gly Val Glu Phe Pro Ser Gly Asn Asn Ser

290 295 300

Phe Val Thr Ile Thr Ala Lys Lys Glu Val Leu Leu Ala Ala Gly Ala

305 310 315 320

Val His Thr Pro Gln Leu Leu Gln Leu Ser Gly Ile Gly Pro Lys Ser

325 330 335

Leu Leu Glu Ala Gly Gly Ile Asp Val Leu Val Asp Leu Pro Gly Val

340 345 350

Gly Gln Asn Phe Gln Asp His Ser Ser Leu Ser Ser Met Asn Ile Thr

355 360 365

Leu Ser Lys Leu Thr Asp Ile His Pro Asn Pro Thr Asp Leu Val Glu

370 375 380

Gly Asn Asp Phe Lys Thr Trp Ala Asp Glu Val Trp Ala Ala Asn Lys

385 390 395 400

Thr Gly Pro Tyr Ser Ile Ala Leu Thr Asn Leu Ala Gly Trp Leu Pro

405 410 415

Phe Thr Ala Val Ser Glu Arg Ala Glu Glu Leu Ala Thr Lys Leu Glu

420 425 430

Gln Gln Asp Phe Ala Ser Leu Leu Pro Thr Asp Thr Glu Thr Thr Val

435 440 445

Val Ala Gly Phe Glu Ala Gln Met Lys Ile Leu Ala Ala Gln Met Arg

450 455 460

Ser Lys Asp Thr Ala Phe Thr Arg Met Gln Leu Ile Ala Asn Gln Gly

465 470 475 480

Ser Gln Gly Pro Val Ala Met Gln Ser Phe Ser Arg Gly Thr Ile Asn

485 490 495

Ile Asn Thr Thr Asp Pro Trp Asn Thr Glu Pro Val Ile Asp Tyr Arg

500 505 510

Ser Leu Ser Asn Pro Leu Glu Ala Asp Phe Phe Val Glu Ser Ile Lys

515 520 525

Phe Leu Arg Arg Tyr Asn Phe Asn Thr Ser Leu Ala Thr Glu Phe Ala

530 535 540

Pro Val Glu Tyr Ala Pro Gly Pro Glu Val Thr Ser Asp Glu Asp Leu

545 550 555 560

Lys Ala Tyr Ile Ala Gly Ala Met Ser Pro Thr Asp Tyr His Pro Val

565 570 575

Gly Thr Ala Ser Met Met Pro Leu Asn Leu Gly Gly Val Val Asp Gln

580 585 590

Thr Leu Arg Val Tyr Gly Val Lys Asn Leu Arg Val Val Asp Ala Ser

595 600 605

Val Met Pro Met Val Pro Ser Ala Asn Thr Cys Gln Pro Thr Tyr Ala

610 615 620

Leu Ala Glu Lys Ala Ala Glu Ile Ile Lys Gln Gly Ile

625 630 635

<210> 15

<211> 1923

<212> DNA

<213> Pleurotus ostreatus strain RD057037

<400> 15

atgccgttcc ttcgccagtc caagtcccag cccttgtggc cccggactgc gttcacagct 60

ctccttgcag tgagctccgt cgccaacgcc ttggccgtcc ctcgccagat caagtcatct 120

cagctgctgc aaagctacga ctatgtcgtt gtcggaggtg gaacggccgg cctgaccctg 180

gcagaccgtt tgactgagga ccccgaaacg agtgtcttgg tcctcgaggc cggcaactgg 240

ggcaacatgg aggagaatct tgcggcttac ttcaccgagt cgcgatatgc caacctcatg 300

agctccctct ggcccggcgt ggagtctgtg ccgcagccca acttgaatgg aagagttggc 360

accgtctgga ttgccaagca agtcggaggc ggctcttccg tgaacgccat gatgaacatg 420

cgaggttccg ccgaggacta cgaccgctgg gcaaccctat tcggaggcga agctcagcag 480

ggagtcgctg actggagctg ggatggcatg ttgaagtcct tcaagaaggg cctccacttt 540

accgagccca ctcccgagct gaccgacaac ttcgacagct tcaaatatga tgcctcctac 600

tggggtgatt cctcagagat ctatgctggc tggcctcgct tctactaccc cggcgtgaag 660

ccgctggtgg aagcgttcaa ggagatcgac ggcgttgagt tccctcccga cagtggtgcc 720

ggacagcccg gtgtcttctg gttccccacg ctgatggacc cccggactgt cacccgctcc 780

tacgctggta ctggtcacta cctcagcgtc aacgctaccc gccccaacta ccaccttctg 840

ctcgacaccc aggctcgcaa gctggtagtt gatgacgacc tcactgccac tggcgtcgtc 900

ttcccttccg gaaacaacac cctcgtcacc gtcaacgcaa agaaggaggt cattctctct 960

gctggtaccg ttcacactcc tcacctcttg cagctgagcg gtatcggccc caagaacgtt 1020

ctcgaggctg gaggcatcga tgtccgcgtc gatcttcctg gtgttggcca gaacttccag 1080

gaccacagca gtttgtctgc tatgaacatt acgctatcca agctctcatc gattcacccg 1140

aacccgagtg acctggttga tggcaacgaa ttcaaggact gggctgaaga ggtgtgggca 1200

gccaacaaga ccggcccgta ctctctcaca ttcaccaact tggccggttg gctccccttc 1260

actgtcgtgt ctggaaaggc cgacgagctc gccaccaagt tggaacaaca agacttcgct 1320

agccttctcc ctgccgatgc cgactccacc gtcgtcgcgg gtttcgcggc gcagatgaag 1380

atcctggccg cccagatgcg ctccaagaac accgccttca cccgcatgca gctcgtccct 1440

gaccacggct cccagggccc tgtcgcgatg cagtctttca gccgcggcac catcaacatc 1500

aacacgaccg acccgtggaa caccgagccc gtgatcgact accgcgccct cagcaacccc 1560

gtcgaggccg acttcttcgt cgaggcgatc aagttcctcc gccgctacaa cttcgagacc 1620

tcgctggcca ccgagtacga gcctgtcgag tacgcccctg gcccggatgt cacttccgac 1680

gaggacctca aggcctacat cgccggtgct ctgtcgccta ccgactacca ccccgtgggc 1740

acttcgtcta tgctgccgct ggagttgggc ggtgtcgtcg accagaccct tcgcgtttac 1800

ggagtcaaga acctcagagt cgttgacgcc agtgtcatgc ccatggttcc cagtgccaac 1860

acttgccagc ccacttatgc cctggccgag aaggccgcgg aaatcattaa gcaaggtatc 1920

taa 1923

<210> 16

<211> 640

<212> PRT

<213> Pleurotus ostreatus strain RD057037

<400> 16

Met Pro Phe Leu Arg Gln Ser Lys Ser Gln Pro Leu Trp Pro Arg Thr

1 5 10 15

Ala Phe Thr Ala Leu Leu Ala Val Ser Ser Val Ala Asn Ala Leu Ala

20 25 30

Val Pro Arg Gln Ile Lys Ser Ser Gln Leu Leu Gln Ser Tyr Asp Tyr

35 40 45

Val Val Val Gly Gly Gly Thr Ala Gly Leu Thr Leu Ala Asp Arg Leu

50 55 60

Thr Glu Asp Pro Glu Thr Ser Val Leu Val Leu Glu Ala Gly Asn Trp

65 70 75 80

Gly Asn Met Glu Glu Asn Leu Ala Ala Tyr Phe Thr Glu Ser Arg Tyr

85 90 95

Ala Asn Leu Met Ser Ser Leu Trp Pro Gly Val Glu Ser Val Pro Gln

100 105 110

Pro Asn Leu Asn Gly Arg Val Gly Thr Val Trp Ile Ala Lys Gln Val

115 120 125

Gly Gly Gly Ser Ser Val Asn Ala Met Met Asn Met Arg Gly Ser Ala

130 135 140

Glu Asp Tyr Asp Arg Trp Ala Thr Leu Phe Gly Gly Glu Ala Gln Gln

145 150 155 160

Gly Val Ala Asp Trp Ser Trp Asp Gly Met Leu Lys Ser Phe Lys Lys

165 170 175

Gly Leu His Phe Thr Glu Pro Thr Pro Glu Leu Thr Asp Asn Phe Asp

180 185 190

Ser Phe Lys Tyr Asp Ala Ser Tyr Trp Gly Asp Ser Ser Glu Ile Tyr

195 200 205

Ala Gly Trp Pro Arg Phe Tyr Tyr Pro Gly Val Lys Pro Leu Val Glu

210 215 220

Ala Phe Lys Glu Ile Asp Gly Val Glu Phe Pro Pro Asp Ser Gly Ala

225 230 235 240

Gly Gln Pro Gly Val Phe Trp Phe Pro Thr Leu Met Asp Pro Arg Thr

245 250 255

Val Thr Arg Ser Tyr Ala Gly Thr Gly His Tyr Leu Ser Val Asn Ala

260 265 270

Thr Arg Pro Asn Tyr His Leu Leu Leu Asp Thr Gln Ala Arg Lys Leu

275 280 285

Val Val Asp Asp Asp Leu Thr Ala Thr Gly Val Val Phe Pro Ser Gly

290 295 300

Asn Asn Thr Leu Val Thr Val Asn Ala Lys Lys Glu Val Ile Leu Ser

305 310 315 320

Ala Gly Thr Val His Thr Pro His Leu Leu Gln Leu Ser Gly Ile Gly

325 330 335

Pro Lys Asn Val Leu Glu Ala Gly Gly Ile Asp Val Arg Val Asp Leu

340 345 350

Pro Gly Val Gly Gln Asn Phe Gln Asp His Ser Ser Leu Ser Ala Met

355 360 365

Asn Ile Thr Leu Ser Lys Leu Ser Ser Ile His Pro Asn Pro Ser Asp

370 375 380

Leu Val Asp Gly Asn Glu Phe Lys Asp Trp Ala Glu Glu Val Trp Ala

385 390 395 400

Ala Asn Lys Thr Gly Pro Tyr Ser Leu Thr Phe Thr Asn Leu Ala Gly

405 410 415

Trp Leu Pro Phe Thr Val Val Ser Gly Lys Ala Asp Glu Leu Ala Thr

420 425 430

Lys Leu Glu Gln Gln Asp Phe Ala Ser Leu Leu Pro Ala Asp Ala Asp

435 440 445

Ser Thr Val Val Ala Gly Phe Ala Ala Gln Met Lys Ile Leu Ala Ala

450 455 460

Gln Met Arg Ser Lys Asn Thr Ala Phe Thr Arg Met Gln Leu Val Pro

465 470 475 480

Asp His Gly Ser Gln Gly Pro Val Ala Met Gln Ser Phe Ser Arg Gly

485 490 495

Thr Ile Asn Ile Asn Thr Thr Asp Pro Trp Asn Thr Glu Pro Val Ile

500 505 510

Asp Tyr Arg Ala Leu Ser Asn Pro Val Glu Ala Asp Phe Phe Val Glu

515 520 525

Ala Ile Lys Phe Leu Arg Arg Tyr Asn Phe Glu Thr Ser Leu Ala Thr

530 535 540

Glu Tyr Glu Pro Val Glu Tyr Ala Pro Gly Pro Asp Val Thr Ser Asp

545 550 555 560

Glu Asp Leu Lys Ala Tyr Ile Ala Gly Ala Leu Ser Pro Thr Asp Tyr

565 570 575

His Pro Val Gly Thr Ser Ser Met Leu Pro Leu Glu Leu Gly Gly Val

580 585 590

Val Asp Gln Thr Leu Arg Val Tyr Gly Val Lys Asn Leu Arg Val Val

595 600 605

Asp Ala Ser Val Met Pro Met Val Pro Ser Ala Asn Thr Cys Gln Pro

610 615 620

Thr Tyr Ala Leu Ala Glu Lys Ala Ala Glu Ile Ile Lys Gln Gly Ile

625 630 635 640

<210> 17

<211> 1920

<212> DNA

<213> Helianthus annuus Leptodermas sp

<400> 17

atgtcgctct ttcgccaaca caagtcccag cctcgctggt caggcattac gttctctgcc 60

cttcttgccg cgagctctat cacaaatgcc ttcgttattc cgcgccatgt caactcatcc 120

cagctgctga agagttatga ttatgtcgtc gttggaggtg gtaccgcagg cctgactgtg 180

gcggaccgcc tcaccgagga ccccaatgtc aacgtcctcg tcctcgaagc cggtaacttt 240

ggtgacatgg acttcaacct agcggtcaac ttcgccacca gagttgggaa tgccacggcc 300

acatattggc ctggcctcca gtccataccg cagccaaaca tggacggcag gcctggcccg 360

gttaccattg ctagacaagt cggaggcggc tcgtctgtga acgccatgat gaacatgcgt 420

ggctctgtcg aggactacga ccgttggggc gcactgttcg gctccgaggc tcagacggga 480

gctgcagact ggagctggga tggcatcctg ccatttttca agaagggcct gcactttgcc 540

gcgccatctc cagagctcac tgacaagttc gacagtatca agtacgatgc gtcctactgg 600

ggtgactctt cggatattta cgccggctgg ccagatttct actaccctgg cgttgctccc 660

ctgctggaag cattcaagga gatcgatggc attgagttcc cagaggacag tggcgccgga 720

aaggctggcg tgttctggtt cccttcgctg atagacccca gaaccttcac tcgatcctac 780

gccgcaacag gccactatct gaacgtcaat gctacccggc caaactacca tctcttgacc 840

gacaccttgg caagcaggct cataatagac gatgacctgt gtgctaccgg tgttgagttc 900

aagtcaggca acagcactcc tgtcaccgtc aaggcagaga aggaggtcat tgtgtctgcc 960

ggtacaatcc acactcctca gcttctgcag ctgagcggta ttggtcccgc gagcctcctc 1020

gaggagggtg ggattgatgt gctcgtcaac ttgcccggtg ttggtcagaa cttccatgac 1080

cacagcaacc tggctgcgat gaacattacg ctctccaagc tcgcagacat ccaccccaac 1140

cctaacgatc tcgtcgacgg aaacgagttc aaggaatggg ccgacgagtt gtgggcagcc 1200

aacaggaccg gcccatactc catcgccttt actaacctgg ctggctggct ccccttcacc 1260

atagtcactg acaaggccga cgagatcgcc accaagctcg agtcgcagga ctacgccagc 1320

atcctgcccg ccggcgcaga ctccaccgtc atcgccggct acgaggcaca gatgaagctc 1380

ctggccgccc agatgcgctc caaggacact gccttcaccc gcctccagct gcagcccgcg 1440

cagggatccc agggcccagt cgccatgcag tccttcagcc gcggcagcat caacatcaac 1500

accaccgacc ccttcaacac ggagccaatc atcgactacc gcgccctgac gaaccccgtc 1560

gagttcgact tctttgtcga gagcatcagg tttgtccgcc gctacagctt cgagacgtcg 1620

ctcaaggaca agttcgaccc cgtcgagtac agccccggcc ccaacgtcac ctcggacgcc 1680

gacctcagga cctacatctc gaaaaacatg tcaccctccg actggcaccc cgtcggcacc 1740

tccgccatgc tgcccctgga gcttggcggt gtcgtcgacc agaccctgcg cgtgtacggc 1800

gtcaagaacc tccgtgtcgt tgacgccagc gtcatgccca tggttcccgg cgccaacaca 1860

tgccagccca cttacgctct ggctgagaag gctgcggaaa tcatcaagtc aggcatctaa 1920

<210> 18

<211> 639

<212> PRT

<213> Helianthus annuus Leptodermas sp

<400> 18

Met Ser Leu Phe Arg Gln His Lys Ser Gln Pro Arg Trp Ser Gly Ile

1 5 10 15

Thr Phe Ser Ala Leu Leu Ala Ala Ser Ser Ile Thr Asn Ala Phe Val

20 25 30

Ile Pro Arg His Val Asn Ser Ser Gln Leu Leu Lys Ser Tyr Asp Tyr

35 40 45

Val Val Val Gly Gly Gly Thr Ala Gly Leu Thr Val Ala Asp Arg Leu

50 55 60

Thr Glu Asp Pro Asn Val Asn Val Leu Val Leu Glu Ala Gly Asn Phe

65 70 75 80

Gly Asp Met Asp Phe Asn Leu Ala Val Asn Phe Ala Thr Arg Val Gly

85 90 95

Asn Ala Thr Ala Thr Tyr Trp Pro Gly Leu Gln Ser Ile Pro Gln Pro

100 105 110

Asn Met Asp Gly Arg Pro Gly Pro Val Thr Ile Ala Arg Gln Val Gly

115 120 125

Gly Gly Ser Ser Val Asn Ala Met Met Asn Met Arg Gly Ser Val Glu

130 135 140

Asp Tyr Asp Arg Trp Gly Ala Leu Phe Gly Ser Glu Ala Gln Thr Gly

145 150 155 160

Ala Ala Asp Trp Ser Trp Asp Gly Ile Leu Pro Phe Phe Lys Lys Gly

165 170 175

Leu His Phe Ala Ala Pro Ser Pro Glu Leu Thr Asp Lys Phe Asp Ser

180 185 190

Ile Lys Tyr Asp Ala Ser Tyr Trp Gly Asp Ser Ser Asp Ile Tyr Ala

195 200 205

Gly Trp Pro Asp Phe Tyr Tyr Pro Gly Val Ala Pro Leu Leu Glu Ala

210 215 220

Phe Lys Glu Ile Asp Gly Ile Glu Phe Pro Glu Asp Ser Gly Ala Gly

225 230 235 240

Lys Ala Gly Val Phe Trp Phe Pro Ser Leu Ile Asp Pro Arg Thr Phe

245 250 255

Thr Arg Ser Tyr Ala Ala Thr Gly His Tyr Leu Asn Val Asn Ala Thr

260 265 270

Arg Pro Asn Tyr His Leu Leu Thr Asp Thr Leu Ala Ser Arg Leu Ile

275 280 285

Ile Asp Asp Asp Leu Cys Ala Thr Gly Val Glu Phe Lys Ser Gly Asn

290 295 300

Ser Thr Pro Val Thr Val Lys Ala Glu Lys Glu Val Ile Val Ser Ala

305 310 315 320

Gly Thr Ile His Thr Pro Gln Leu Leu Gln Leu Ser Gly Ile Gly Pro

325 330 335

Ala Ser Leu Leu Glu Glu Gly Gly Ile Asp Val Leu Val Asn Leu Pro

340 345 350

Gly Val Gly Gln Asn Phe His Asp His Ser Asn Leu Ala Ala Met Asn

355 360 365

Ile Thr Leu Ser Lys Leu Ala Asp Ile His Pro Asn Pro Asn Asp Leu

370 375 380

Val Asp Gly Asn Glu Phe Lys Glu Trp Ala Asp Glu Leu Trp Ala Ala

385 390 395 400

Asn Arg Thr Gly Pro Tyr Ser Ile Ala Phe Thr Asn Leu Ala Gly Trp

405 410 415

Leu Pro Phe Thr Ile Val Thr Asp Lys Ala Asp Glu Ile Ala Thr Lys

420 425 430

Leu Glu Ser Gln Asp Tyr Ala Ser Ile Leu Pro Ala Gly Ala Asp Ser

435 440 445

Thr Val Ile Ala Gly Tyr Glu Ala Gln Met Lys Leu Leu Ala Ala Gln

450 455 460

Met Arg Ser Lys Asp Thr Ala Phe Thr Arg Leu Gln Leu Gln Pro Ala

465 470 475 480

Gln Gly Ser Gln Gly Pro Val Ala Met Gln Ser Phe Ser Arg Gly Ser

485 490 495

Ile Asn Ile Asn Thr Thr Asp Pro Phe Asn Thr Glu Pro Ile Ile Asp

500 505 510

Tyr Arg Ala Leu Thr Asn Pro Val Glu Phe Asp Phe Phe Val Glu Ser

515 520 525

Ile Arg Phe Val Arg Arg Tyr Ser Phe Glu Thr Ser Leu Lys Asp Lys

530 535 540

Phe Asp Pro Val Glu Tyr Ser Pro Gly Pro Asn Val Thr Ser Asp Ala

545 550 555 560

Asp Leu Arg Thr Tyr Ile Ser Lys Asn Met Ser Pro Ser Asp Trp His

565 570 575

Pro Val Gly Thr Ser Ala Met Leu Pro Leu Glu Leu Gly Gly Val Val

580 585 590

Asp Gln Thr Leu Arg Val Tyr Gly Val Lys Asn Leu Arg Val Val Asp

595 600 605

Ala Ser Val Met Pro Met Val Pro Gly Ala Asn Thr Cys Gln Pro Thr

610 615 620

Tyr Ala Leu Ala Glu Lys Ala Ala Glu Ile Ile Lys Ser Gly Ile

625 630 635

<210> 19

<211> 1884

<212> DNA

<213> Blastomyces nigrosporus

<400> 19

atggccttca gccgctccag cctcctcgtg accctcgccg ctgcctcctc cgctctcgtc 60

agcaccgctc atggcttcgc tacccctcgc tccgcctacc aggctcgcca gatctccgat 120

gcctccggcc tcctgcctgc ctacgattac gtcatcgtcg gcggtggcac cagcggcctc 180

accgtcgccg atcgcctcac cgaggatcct gataccaccg tcctcgtcct tgaggccggc 240

atcttcgccc ctaaggccga tgtcctgcct gtcgccggtg gcggcaccgg tcgccagcct 300

cgctactcct tcaagtccgc tcctcaagaa accctcggcg gcaagacctt caacgtcatg 360

ctcggcaaga tggtcggcgg ctcctccggc gtcaacgcca tgatgtccgt ccgcggctcc 420

gccgaggatt acgatcgctg gggccagctg ttcggcggtg atgcccaagg ctggtcctgg 480

gatggcctgc tgccttactt caagaagggc ctgactctca accctccagc cgccgagctg 540

gccgagcgct tcaacatctc caccgagcct tccaactggg gcaaagattc ccctatccag 600

gctagcttcc catccttcca gtatcctggc cttgagcctc tcctgtccgc cttccaagag 660

ctgcctggcg tcgagatggt cagcgattcc ggcgctggct ccgctggcgt ctactggtat 720

cctaccttca tggatcctgt caaggctgag cgctcctacg ccggcaacgc ccatctcaag 780

accgatcggc ctaactacca tgtgatcgcc gaaaccgccg tccgccgcgt cctcctcgat 840

gacaccacca ccgctaccgg cgtcgagttc tacaccggtg ctggcgtcgc caccgtcaag 900

gccgacaaag aggtcatcat ggccgctggc gccgtgcata cccctaagct cctccagctg 960

tccggcatcg gccctcgcaa ggtcctcgaa gccgctggcc tcgaaaccgt cgtcgatctc 1020

cccggcgtcg gccagaactt ccaggatcat tccaacatcg gcgctgccat cagcctcggt 1080

ggcctcgccg atatccatcc taacgccaac gatctgaccg gcgacgccgc cttcaagaaa 1140

tgggctgatg ccctctgggc cgccaaccgc accggtcctc gctctatcgc cttcggcaac 1200

ctcgccggct ggctccctct gaccgccatc tctcctgagc gcttcgccga actcgccgat 1260

gagctggaag gtcaggatca tgccgcattc ttgcctgctg atgtccatcc taccgtcgcc 1320

aagggctacg ccgctcagat gaagggcctc gctgccgcca tgcgctccga gcataccgtg 1380

ttcgcccgct tcgctgtcga tgctacccgc ggtgcctccg ctcctatcct caaccagcct 1440

ttcagccgcg gcagcgtcaa cgtcgatcct cgcgatcctt tcggcgctaa ccctgtcgtc 1500

gattaccgcg ctctcaccaa tcctgtcgaa acccgcgtcg tcgttgagat ggtgaagtgg 1560

tatcgccgct accatttcga gacttccctc aagggcttcg ccccaaccga ggccgctcct 1620

ggcgccgctg tcgtcaccga tgagcagatc gccgcttggg tccctaccgt cctcaatcct 1680

accgattacc atcctgccgg caccgccgct ctcgcccctc tcgaactcgg cggcgtggtc 1740

gatcagaccc tccgcgtcta cggcgtcaag aaactccgcg tcaccgacgc ctccatcatg 1800

ccttctctcc ctggcgccaa cacctgtcag cctacctacg ctatcgccga gaaggccgct 1860

gatatcatca agtccgaggc ctaa 1884

<210> 20

<211> 627

<212> PRT

<213> Blastomyces nigrosporus

<400> 20

Met Ala Phe Ser Arg Ser Ser Leu Leu Val Thr Leu Ala Ala Ala Ser

1 5 10 15

Ser Ala Leu Val Ser Thr Ala His Gly Phe Ala Thr Pro Arg Ser Ala

20 25 30

Tyr Gln Ala Arg Gln Ile Ser Asp Ala Ser Gly Leu Leu Pro Ala Tyr

35 40 45

Asp Tyr Val Ile Val Gly Gly Gly Thr Ser Gly Leu Thr Val Ala Asp

50 55 60

Arg Leu Thr Glu Asp Pro Asp Thr Thr Val Leu Val Leu Glu Ala Gly

65 70 75 80

Ile Phe Ala Pro Lys Ala Asp Val Leu Pro Val Ala Gly Gly Gly Thr

85 90 95

Gly Arg Gln Pro Arg Tyr Ser Phe Lys Ser Ala Pro Gln Glu Thr Leu

100 105 110

Gly Gly Lys Thr Phe Asn Val Met Leu Gly Lys Met Val Gly Gly Ser

115 120 125

Ser Gly Val Asn Ala Met Met Ser Val Arg Gly Ser Ala Glu Asp Tyr

130 135 140

Asp Arg Trp Gly Gln Leu Phe Gly Gly Asp Ala Gln Gly Trp Ser Trp

145 150 155 160

Asp Gly Leu Leu Pro Tyr Phe Lys Lys Gly Leu Thr Leu Asn Pro Pro

165 170 175

Ala Ala Glu Leu Ala Glu Arg Phe Asn Ile Ser Thr Glu Pro Ser Asn

180 185 190

Trp Gly Lys Asp Ser Pro Ile Gln Ala Ser Phe Pro Ser Phe Gln Tyr

195 200 205

Pro Gly Leu Glu Pro Leu Leu Ser Ala Phe Gln Glu Leu Pro Gly Val

210 215 220

Glu Met Val Ser Asp Ser Gly Ala Gly Ser Ala Gly Val Tyr Trp Tyr

225 230 235 240

Pro Thr Phe Met Asp Pro Val Lys Ala Glu Arg Ser Tyr Ala Gly Asn

245 250 255

Ala His Leu Lys Thr Asp Arg Pro Asn Tyr His Val Ile Ala Glu Thr

260 265 270

Ala Val Arg Arg Val Leu Leu Asp Asp Thr Thr Thr Ala Thr Gly Val

275 280 285

Glu Phe Tyr Thr Gly Ala Gly Val Ala Thr Val Lys Ala Asp Lys Glu

290 295 300

Val Ile Met Ala Ala Gly Ala Val His Thr Pro Lys Leu Leu Gln Leu

305 310 315 320

Ser Gly Ile Gly Pro Arg Lys Val Leu Glu Ala Ala Gly Leu Glu Thr

325 330 335

Val Val Asp Leu Pro Gly Val Gly Gln Asn Phe Gln Asp His Ser Asn

340 345 350

Ile Gly Ala Ala Ile Ser Leu Gly Gly Leu Ala Asp Ile His Pro Asn

355 360 365

Ala Asn Asp Leu Thr Gly Asp Ala Ala Phe Lys Lys Trp Ala Asp Ala

370 375 380

Leu Trp Ala Ala Asn Arg Thr Gly Pro Arg Ser Ile Ala Phe Gly Asn

385 390 395 400

Leu Ala Gly Trp Leu Pro Leu Thr Ala Ile Ser Pro Glu Arg Phe Ala

405 410 415

Glu Leu Ala Asp Glu Leu Glu Gly Gln Asp His Ala Ala Phe Leu Pro

420 425 430

Ala Asp Val His Pro Thr Val Ala Lys Gly Tyr Ala Ala Gln Met Lys

435 440 445

Gly Leu Ala Ala Ala Met Arg Ser Glu His Thr Val Phe Ala Arg Phe

450 455 460

Ala Val Asp Ala Thr Arg Gly Ala Ser Ala Pro Ile Leu Asn Gln Pro

465 470 475 480

Phe Ser Arg Gly Ser Val Asn Val Asp Pro Arg Asp Pro Phe Gly Ala

485 490 495

Asn Pro Val Val Asp Tyr Arg Ala Leu Thr Asn Pro Val Glu Thr Arg

500 505 510

Val Val Val Glu Met Val Lys Trp Tyr Arg Arg Tyr His Phe Glu Thr

515 520 525

Ser Leu Lys Gly Phe Ala Pro Thr Glu Ala Ala Pro Gly Ala Ala Val

530 535 540

Val Thr Asp Glu Gln Ile Ala Ala Trp Val Pro Thr Val Leu Asn Pro

545 550 555 560

Thr Asp Tyr His Pro Ala Gly Thr Ala Ala Leu Ala Pro Leu Glu Leu

565 570 575

Gly Gly Val Val Asp Gln Thr Leu Arg Val Tyr Gly Val Lys Lys Leu

580 585 590

Arg Val Thr Asp Ala Ser Ile Met Pro Ser Leu Pro Gly Ala Asn Thr

595 600 605

Cys Gln Pro Thr Tyr Ala Ile Ala Glu Lys Ala Ala Asp Ile Ile Lys

610 615 620

Ser Glu Ala

625

<210> 21

<211> 1878

<212> DNA

<213> Acremonium

<400> 21

atgcctatct cctccagcct cgtcaagacc ctcgtcgcct cctccgctct ggtcagcacc 60

gcctccgcct tcgctatccc tcgctccgtg ttccaggctc gccagatctc cgatccttcc 120

gagctgcttg aggcctacga ttacgtcatc gtcggcggtg gcaccgctgg cctcaccgtc 180

gccgatcgcc tcaccgagga tgaggatatc accgtcctcg tccttgagtc cggcagcttc 240

cctatcgccg gcaacgtcct gcctatcatc ggtggcggcg ctaagcctac catgaccttc 300

gctagcaccc ctcaagagaa cctcaacggt cgcacccaga acgtcatcct gggcaacacc 360

gtcggcggct cctccgccgt caacgccatg atcaccgctc gcggctccgc cgaggattac 420

gatcgctggg gccagctgtt cggcaagaag gatacctacg gctggaactg ggagggcctc 480

ctgccttact tcaagaaggg cctcactctc aaccctccat ccgccgagct ggccgagcgc 540

ttcaacatca cctacgatga gtcctactgg ggtgatgagg cccctatcca ggctagcttc 600

cctgcctacc agtatcctgg cctcgatcct ctcgtcagcg ccttcttcga gctgcctggc 660

gtcgagagca gccctgattc cggcgctggc ggtgccggcg tgttctggtt ccctaccttc 720

atggatccta tcaagcgcga gcgctcctac gccttcaact cccattacga gaacatcggt 780

cgcgccaact accatgtcct cgctgatacc cctggtcgcc gcgtcctcct cgataagcgc 840

cgtgccaccg gcgtcgagtt ccagaagggc aacgatacct tcaccgtcaa ggctacccaa 900

gaggtcctcc tggccgctgg cgccgtgcat acccctaagc tcctccaggt cagcggcatc 960

ggccctaaga agctcctcaa agaggccggc atcaagaccg tcgtggatct ccccggcgtc 1020

ggccagaact tccaggatca ttcctccgtc ggcgctgcca tctctctccc tggcctccgc 1080

gatatccatc ctaacgccgg cgatctcgct accgatgccg atttccgcaa ctgggcccaa 1140

gagctgtgga ccaccaaccg caccggtcct tactctatcg ccttcggcaa tatcgccggt 1200

tggctccctc tcaccgctat cacccctgat cgcttcgatg agctggctga tgagcttgag 1260

tcccagaacc atgcctccta cctgcctgct gacacccata agtccgtcgc caagggctac 1320

gccgctcaga tgcgcggtct ggtccctgcc atgcgctcca aggataccat ctggtcccgc 1380

tactacgtca acgctaccac cggcgcttct cgccctatcc tcaaccagcc tttcagccgc 1440

ggcagcgtca acatcaacac cgccgatcct ttcggcgctc gccctgtcgt cgattaccgc 1500

gctctcacca atcctgtcga aggcaaggtc ctcgtcgaga tgatcaagtg gtatcgccgc 1560

taccatttcg agacttccct gaaagagctg ggccctgtcg agtccgctcc tggcgccgat 1620

gtccagaccg atgaggaact caccgcctgg ctcgccgagg gcttctcccc ttccgattac 1680

catcctgctg gcaccgccgc catgatgcct ctcgatctcg gcggcgtcgt cgatcagacc 1740

ctccgcgtct acaaggtcaa gaacctgcgc gtcatcgatg cctccgtcat gcctgtcctg 1800

ccaggcgcca acacctgtca gcctacctac gctctcgccg agaaggccgc cgatatcatc 1860

aagtccaagg ccaagtaa 1878

<210> 22

<211> 625

<212> PRT

<213> Acremonium

<400> 22

Met Pro Ile Ser Ser Ser Leu Val Lys Thr Leu Val Ala Ser Ser Ala

1 5 10 15

Leu Val Ser Thr Ala Ser Ala Phe Ala Ile Pro Arg Ser Val Phe Gln

20 25 30

Ala Arg Gln Ile Ser Asp Pro Ser Glu Leu Leu Glu Ala Tyr Asp Tyr

35 40 45

Val Ile Val Gly Gly Gly Thr Ala Gly Leu Thr Val Ala Asp Arg Leu

50 55 60

Thr Glu Asp Glu Asp Ile Thr Val Leu Val Leu Glu Ser Gly Ser Phe

65 70 75 80

Pro Ile Ala Gly Asn Val Leu Pro Ile Ile Gly Gly Gly Ala Lys Pro

85 90 95

Thr Met Thr Phe Ala Ser Thr Pro Gln Glu Asn Leu Asn Gly Arg Thr

100 105 110

Gln Asn Val Ile Leu Gly Asn Thr Val Gly Gly Ser Ser Ala Val Asn

115 120 125

Ala Met Ile Thr Ala Arg Gly Ser Ala Glu Asp Tyr Asp Arg Trp Gly

130 135 140

Gln Leu Phe Gly Lys Lys Asp Thr Tyr Gly Trp Asn Trp Glu Gly Leu

145 150 155 160

Leu Pro Tyr Phe Lys Lys Gly Leu Thr Leu Asn Pro Pro Ser Ala Glu

165 170 175

Leu Ala Glu Arg Phe Asn Ile Thr Tyr Asp Glu Ser Tyr Trp Gly Asp

180 185 190

Glu Ala Pro Ile Gln Ala Ser Phe Pro Ala Tyr Gln Tyr Pro Gly Leu

195 200 205

Asp Pro Leu Val Ser Ala Phe Phe Glu Leu Pro Gly Val Glu Ser Ser

210 215 220

Pro Asp Ser Gly Ala Gly Gly Ala Gly Val Phe Trp Phe Pro Thr Phe

225 230 235 240

Met Asp Pro Ile Lys Arg Glu Arg Ser Tyr Ala Phe Asn Ser His Tyr

245 250 255

Glu Asn Ile Gly Arg Ala Asn Tyr His Val Leu Ala Asp Thr Pro Gly

260 265 270

Arg Arg Val Leu Leu Asp Lys Arg Arg Ala Thr Gly Val Glu Phe Gln

275 280 285

Lys Gly Asn Asp Thr Phe Thr Val Lys Ala Thr Gln Glu Val Leu Leu

290 295 300

Ala Ala Gly Ala Val His Thr Pro Lys Leu Leu Gln Val Ser Gly Ile

305 310 315 320

Gly Pro Lys Lys Leu Leu Lys Glu Ala Gly Ile Lys Thr Val Val Asp

325 330 335

Leu Pro Gly Val Gly Gln Asn Phe Gln Asp His Ser Ser Val Gly Ala

340 345 350

Ala Ile Ser Leu Pro Gly Leu Arg Asp Ile His Pro Asn Ala Gly Asp

355 360 365

Leu Ala Thr Asp Ala Asp Phe Arg Asn Trp Ala Gln Glu Leu Trp Thr

370 375 380

Thr Asn Arg Thr Gly Pro Tyr Ser Ile Ala Phe Gly Asn Ile Ala Gly

385 390 395 400

Trp Leu Pro Leu Thr Ala Ile Thr Pro Asp Arg Phe Asp Glu Leu Ala

405 410 415

Asp Glu Leu Glu Ser Gln Asn His Ala Ser Tyr Leu Pro Ala Asp Thr

420 425 430

His Lys Ser Val Ala Lys Gly Tyr Ala Ala Gln Met Arg Gly Leu Val

435 440 445

Pro Ala Met Arg Ser Lys Asp Thr Ile Trp Ser Arg Tyr Tyr Val Asn

450 455 460

Ala Thr Thr Gly Ala Ser Arg Pro Ile Leu Asn Gln Pro Phe Ser Arg

465 470 475 480

Gly Ser Val Asn Ile Asn Thr Ala Asp Pro Phe Gly Ala Arg Pro Val

485 490 495

Val Asp Tyr Arg Ala Leu Thr Asn Pro Val Glu Gly Lys Val Leu Val

500 505 510

Glu Met Ile Lys Trp Tyr Arg Arg Tyr His Phe Glu Thr Ser Leu Lys

515 520 525

Glu Leu Gly Pro Val Glu Ser Ala Pro Gly Ala Asp Val Gln Thr Asp

530 535 540

Glu Glu Leu Thr Ala Trp Leu Ala Glu Gly Phe Ser Pro Ser Asp Tyr

545 550 555 560

His Pro Ala Gly Thr Ala Ala Met Met Pro Leu Asp Leu Gly Gly Val

565 570 575

Val Asp Gln Thr Leu Arg Val Tyr Lys Val Lys Asn Leu Arg Val Ile

580 585 590

Asp Ala Ser Val Met Pro Val Leu Pro Gly Ala Asn Thr Cys Gln Pro

595 600 605

Thr Tyr Ala Leu Ala Glu Lys Ala Ala Asp Ile Ile Lys Ser Lys Ala

610 615 620

Lys

625

<210> 23

<211> 1878

<212> DNA

<213> Blastomyces nigrosporus

<400> 23

atggcccctc atgtccgcgc cagcctgctc tggaccctcg ccaccgcctc caccgctctg 60

tccttcgcta tccctcgctc cgtctaccag gctcgccagg tccagtcctc cgagctgctc 120

gatgcctacg attacgtcat cgtcggtggc ggcgctgccg gcctcaccgt cgcagatcgc 180

ctcaccgaga acctcaccac caccgtcctc gtcctcgaag ccggcgtcat gggcgatcct 240

gccgaggtcc tgcctgcctc cgccggtggc aacggctaca ccctgccagc ctggaacttc 300

cagagcgtcc ctcagcctaa cctcaacaac cgcaccagcc tcgtctggat cggcaacctc 360

gtcggcggtg gctccgccat caacgccatg atggccgctc gcggctccgc cgaggattac 420

gatcgctggg gcctgccttt cggtgatgat gatggcggct ggaactggaa gggcatcctg 480

ccttacttcc gcaagggcgt ccgtatgaac ccacctcctg ctcagctcgc caaccgcttc 540

aacatcacca tcgatgcctc caactggggc accgattctc ccgtccaggt cagcttccca 600

tccttccagt atcctggcct cgatcctctc gctaccgctt tcaccgagat gcctggcgtc 660

gagttcgtca aggattccgg cgctggcggt gccggcgtct actggttccc tactctcatg 720

tcccctgagg tcgagcgctc ctacgccggc aacgctcatt actccaacct caagcgccct 780

aactaccatc tcatggtcga aacccctgtc cgccgcgtcc tcatgaacgg caccatggcc 840

atcggcgttg agttctccac cggtgatggc gtcgccaccg tcaaggctac ccgcgaggtc 900

ctcatggccg ctggcgccat ccatactcct aaggtcctga tgctctccgg catcggccct 960

cgcaaggcca tcgagaagat cggcctcgat tccgtcgtcg atctccccgg cgtcggccag 1020

aatttccagg atcatgccaa cgtcggcgct gctatcaccc ttcctggcct gaagcagatc 1080

catcctaacg ctgatgatct cgccgaaggc ggtgatgccg atttcaagca gtggtccgat 1140

gccgtctggg ccgctaaccg caccggtcct aagtctatcg cctacggcaa cctggccggc 1200

tggctccctc tgaccgtcat ctcccctgat cgctacgatg ccctcgccag cgagcttgag 1260

tcccaggatc acgccgccta cctcggtgat gtcgatccta ccgtcgctgc cggttacgcc 1320

gtccagatga agggcctcgc cgctgccatg cgctccaagg ataccgtgtt cgcccgctac 1380

ctcctcgatg ccaccaccgg cgcctccgct cctatcctca accagccttt cagccgcggc 1440

agcgtcaccc tggatccaaa ggatcccttc ggcgccctgc ctatcgtcga ttaccgcgct 1500

ctcagcaacc ctatcgaggt cgccgtcgct gtcgagatgg tcaagtggta tcgccgcttc 1560

catttcgata ccagcctggc cgctctcaag cctaacgaga ctgcccctgg cgctggtgtc 1620

aacgccgtca ccgatgagca gatcgccgat tggctggcca ccgcttgggt ccctaccgat 1680

taccatcctg ccggaaccgc cgctctcctg cctctgaagc tcggcggtgt cgtcgatacc 1740

tctctccgcg tctacggcgt cgagaagctc cgcgtcgtgg atgccagcat catgccttct 1800

ctccctggcg gcaacacctg tcagcctacc tacgctatcg ccgagaaggc cgccgatatc 1860

atcaagtccg gcgtctaa 1878

<210> 24

<211> 625

<212> PRT

<213> Blastomyces nigrosporus

<400> 24

Met Ala Pro His Val Arg Ala Ser Leu Leu Trp Thr Leu Ala Thr Ala

1 5 10 15

Ser Thr Ala Leu Ser Phe Ala Ile Pro Arg Ser Val Tyr Gln Ala Arg

20 25 30

Gln Val Gln Ser Ser Glu Leu Leu Asp Ala Tyr Asp Tyr Val Ile Val

35 40 45

Gly Gly Gly Ala Ala Gly Leu Thr Val Ala Asp Arg Leu Thr Glu Asn

50 55 60

Leu Thr Thr Thr Val Leu Val Leu Glu Ala Gly Val Met Gly Asp Pro

65 70 75 80

Ala Glu Val Leu Pro Ala Ser Ala Gly Gly Asn Gly Tyr Thr Leu Pro

85 90 95

Ala Trp Asn Phe Gln Ser Val Pro Gln Pro Asn Leu Asn Asn Arg Thr

100 105 110

Ser Leu Val Trp Ile Gly Asn Leu Val Gly Gly Gly Ser Ala Ile Asn

115 120 125

Ala Met Met Ala Ala Arg Gly Ser Ala Glu Asp Tyr Asp Arg Trp Gly

130 135 140

Leu Pro Phe Gly Asp Asp Asp Gly Gly Trp Asn Trp Lys Gly Ile Leu

145 150 155 160

Pro Tyr Phe Arg Lys Gly Val Arg Met Asn Pro Pro Pro Ala Gln Leu

165 170 175

Ala Asn Arg Phe Asn Ile Thr Ile Asp Ala Ser Asn Trp Gly Thr Asp

180 185 190

Ser Pro Val Gln Val Ser Phe Pro Ser Phe Gln Tyr Pro Gly Leu Asp

195 200 205

Pro Leu Ala Thr Ala Phe Thr Glu Met Pro Gly Val Glu Phe Val Lys

210 215 220

Asp Ser Gly Ala Gly Gly Ala Gly Val Tyr Trp Phe Pro Thr Leu Met

225 230 235 240

Ser Pro Glu Val Glu Arg Ser Tyr Ala Gly Asn Ala His Tyr Ser Asn

245 250 255

Leu Lys Arg Pro Asn Tyr His Leu Met Val Glu Thr Pro Val Arg Arg

260 265 270

Val Leu Met Asn Gly Thr Met Ala Ile Gly Val Glu Phe Ser Thr Gly

275 280 285

Asp Gly Val Ala Thr Val Lys Ala Thr Arg Glu Val Leu Met Ala Ala

290 295 300

Gly Ala Ile His Thr Pro Lys Val Leu Met Leu Ser Gly Ile Gly Pro

305 310 315 320

Arg Lys Ala Ile Glu Lys Ile Gly Leu Asp Ser Val Val Asp Leu Pro

325 330 335

Gly Val Gly Gln Asn Phe Gln Asp His Ala Asn Val Gly Ala Ala Ile

340 345 350

Thr Leu Pro Gly Leu Lys Gln Ile His Pro Asn Ala Asp Asp Leu Ala

355 360 365

Glu Gly Gly Asp Ala Asp Phe Lys Gln Trp Ser Asp Ala Val Trp Ala

370 375 380

Ala Asn Arg Thr Gly Pro Lys Ser Ile Ala Tyr Gly Asn Leu Ala Gly

385 390 395 400

Trp Leu Pro Leu Thr Val Ile Ser Pro Asp Arg Tyr Asp Ala Leu Ala

405 410 415

Ser Glu Leu Glu Ser Gln Asp His Ala Ala Tyr Leu Gly Asp Val Asp

420 425 430

Pro Thr Val Ala Ala Gly Tyr Ala Val Gln Met Lys Gly Leu Ala Ala

435 440 445

Ala Met Arg Ser Lys Asp Thr Val Phe Ala Arg Tyr Leu Leu Asp Ala

450 455 460

Thr Thr Gly Ala Ser Ala Pro Ile Leu Asn Gln Pro Phe Ser Arg Gly

465 470 475 480

Ser Val Thr Leu Asp Pro Lys Asp Pro Phe Gly Ala Leu Pro Ile Val

485 490 495

Asp Tyr Arg Ala Leu Ser Asn Pro Ile Glu Val Ala Val Ala Val Glu

500 505 510

Met Val Lys Trp Tyr Arg Arg Phe His Phe Asp Thr Ser Leu Ala Ala

515 520 525

Leu Lys Pro Asn Glu Thr Ala Pro Gly Ala Gly Val Asn Ala Val Thr

530 535 540

Asp Glu Gln Ile Ala Asp Trp Leu Ala Thr Ala Trp Val Pro Thr Asp

545 550 555 560

Tyr His Pro Ala Gly Thr Ala Ala Leu Leu Pro Leu Lys Leu Gly Gly

565 570 575

Val Val Asp Thr Ser Leu Arg Val Tyr Gly Val Glu Lys Leu Arg Val

580 585 590

Val Asp Ala Ser Ile Met Pro Ser Leu Pro Gly Gly Asn Thr Cys Gln

595 600 605

Pro Thr Tyr Ala Ile Ala Glu Lys Ala Ala Asp Ile Ile Lys Ser Gly

610 615 620

Val

625

<210> 25

<211> 1887

<212> DNA

<213> Lasiosphaeris hirsuta

<400> 25

atgcctcgca gccatagcaa ccctatctct atgctcgcct ccgctctcct cgccttcgtc 60

gccaccagcc atgccttcag catccctcca tccacctacc gcgctcgcca gatcaccaag 120

accagccagc tgctccctgc ctacgattac gtcatcgtcg gcggtggcac ctccggcctc 180

accgtcgccg atcgcctcac cgagaatcct cataccaccg tcctcgtcct tgaggccggc 240

agcttcccta atcctgatga tgtcctgcct gtctccgatg gcggcacccg ccgtcaggat 300

aactacctct accagagcgt ccctcagact catctcaaca accagtcctt ctacgtcatc 360

ctgggcaaca tggtcggcgg ctcctccggc atcaacggca tgatgaccgc tcgcggctcc 420

gccgaggatt acgatcgctg gggcgagctg ttcggtcgcg gcagcaagca cgattggtcc 480

tggaagggcc tcctgcctta cttcaagaag tctatcagct tcttccctcc tcctgccgat 540

ctcgccaacc gcttccatat caagtacgat accagctact ggggcaagtc ctccgccgtc 600

gatgcctcct ggccttccta ccagtatcct ggcctcgatc ctctcctgtc cgccttcaaa 660

gagctgcctg atatcgagtt caccgccgat tccggcgctg gctccgctgg cgtctactgg 720

ttccctacct tcatggatcc tgtcaagcac gagcgctcct acgccaccaa cggccattac 780

tccaacgtca tccggcctaa ctaccatctc atggctgata ccccagtccg ccgcgtcctc 840

ctcaaggatg gcggcgctac cggcgtcgag cttaagaccg ataactccac cttcaccatc 900

aaggccacca aagagatcct cgtcgccgct ggcgccgtgc atacccctca gatcctccag 960

cgctccggta tcggccctca gaaggtcctc aaggccgctg gcatcaagac cctcgtcgat 1020

ctccctggcg tcggccagaa cttccaggat catcctgaga tcggcgctga gattaccctc 1080

ggcggcctcc tgggcatcca tcctaacgct catgatcttg agatcgatgc cagcttccgc 1140

aagttcgctg aggattcctg gtccgctaac cgcaccggtc cttactctat cgccttcggc 1200

aatatcgccg gctggctccc tctcacctct atcaccccta gccgctaccg cagcctctcc 1260

accgctctct ccgctcagaa ccatgccgcc tacctgctgc ctggcactca tcctaccgtc 1320

gctgccggct acagcgccca gatgcgcggc ctcgccaagg ccatgctctc ccgcgacgtc 1380

gtgttcgccc gcatcctcct gaacgccacc accggtgccg tcggtcctgt cctcaaccag 1440

cctttcagcc gcggttccat caacatcgat cccgccgatc ctttcggcgg tgcccctgtc 1500

atcgatttca acggcctcag caaccctgtc gagcgcgccg tcatggtcga gatcctcaag 1560

ttcttccgcc gctaccatta cgagacttcc ctcaagaagc tggcccctaa cgagactgcc 1620

cctggtcctg ccgtcgtcac cgatgctcag ctcgatgcct ggctgccttc cggcgtcacc 1680

ccttccgatt ggcacgctgc tggcaccgct gccatgatgc ctctcgaact cggcggcgtc 1740

gtcgatcaga ccctccgcgt ctacggcgtc aagggcctgc gcgtcatcga tggctctgtc 1800

atccctagcc tgcctggcgg caacccttgc catgccgtct acgctatcgc cgagaaggct 1860

gctgatctca tccgctccaa cgcctaa 1887

<210> 26

<211> 628

<212> PRT

<213> Lasiosphaeris hirsuta

<400> 26

Met Pro Arg Ser His Ser Asn Pro Ile Ser Met Leu Ala Ser Ala Leu

1 5 10 15

Leu Ala Phe Val Ala Thr Ser His Ala Phe Ser Ile Pro Pro Ser Thr

20 25 30

Tyr Arg Ala Arg Gln Ile Thr Lys Thr Ser Gln Leu Leu Pro Ala Tyr

35 40 45

Asp Tyr Val Ile Val Gly Gly Gly Thr Ser Gly Leu Thr Val Ala Asp

50 55 60

Arg Leu Thr Glu Asn Pro His Thr Thr Val Leu Val Leu Glu Ala Gly

65 70 75 80

Ser Phe Pro Asn Pro Asp Asp Val Leu Pro Val Ser Asp Gly Gly Thr

85 90 95

Arg Arg Gln Asp Asn Tyr Leu Tyr Gln Ser Val Pro Gln Thr His Leu

100 105 110

Asn Asn Gln Ser Phe Tyr Val Ile Leu Gly Asn Met Val Gly Gly Ser

115 120 125

Ser Gly Ile Asn Gly Met Met Thr Ala Arg Gly Ser Ala Glu Asp Tyr

130 135 140

Asp Arg Trp Gly Glu Leu Phe Gly Arg Gly Ser Lys His Asp Trp Ser

145 150 155 160

Trp Lys Gly Leu Leu Pro Tyr Phe Lys Lys Ser Ile Ser Phe Phe Pro

165 170 175

Pro Pro Ala Asp Leu Ala Asn Arg Phe His Ile Lys Tyr Asp Thr Ser

180 185 190

Tyr Trp Gly Lys Ser Ser Ala Val Asp Ala Ser Trp Pro Ser Tyr Gln

195 200 205

Tyr Pro Gly Leu Asp Pro Leu Leu Ser Ala Phe Lys Glu Leu Pro Asp

210 215 220

Ile Glu Phe Thr Ala Asp Ser Gly Ala Gly Ser Ala Gly Val Tyr Trp

225 230 235 240

Phe Pro Thr Phe Met Asp Pro Val Lys His Glu Arg Ser Tyr Ala Thr

245 250 255

Asn Gly His Tyr Ser Asn Val Ile Arg Pro Asn Tyr His Leu Met Ala

260 265 270

Asp Thr Pro Val Arg Arg Val Leu Leu Lys Asp Gly Gly Ala Thr Gly

275 280 285

Val Glu Leu Lys Thr Asp Asn Ser Thr Phe Thr Ile Lys Ala Thr Lys

290 295 300

Glu Ile Leu Val Ala Ala Gly Ala Val His Thr Pro Gln Ile Leu Gln

305 310 315 320

Arg Ser Gly Ile Gly Pro Gln Lys Val Leu Lys Ala Ala Gly Ile Lys

325 330 335

Thr Leu Val Asp Leu Pro Gly Val Gly Gln Asn Phe Gln Asp His Pro

340 345 350

Glu Ile Gly Ala Glu Ile Thr Leu Gly Gly Leu Leu Gly Ile His Pro

355 360 365

Asn Ala His Asp Leu Glu Ile Asp Ala Ser Phe Arg Lys Phe Ala Glu

370 375 380

Asp Ser Trp Ser Ala Asn Arg Thr Gly Pro Tyr Ser Ile Ala Phe Gly

385 390 395 400

Asn Ile Ala Gly Trp Leu Pro Leu Thr Ser Ile Thr Pro Ser Arg Tyr

405 410 415

Arg Ser Leu Ser Thr Ala Leu Ser Ala Gln Asn His Ala Ala Tyr Leu

420 425 430

Leu Pro Gly Thr His Pro Thr Val Ala Ala Gly Tyr Ser Ala Gln Met

435 440 445

Arg Gly Leu Ala Lys Ala Met Leu Ser Arg Asp Val Val Phe Ala Arg

450 455 460

Ile Leu Leu Asn Ala Thr Thr Gly Ala Val Gly Pro Val Leu Asn Gln

465 470 475 480

Pro Phe Ser Arg Gly Ser Ile Asn Ile Asp Pro Ala Asp Pro Phe Gly

485 490 495

Gly Ala Pro Val Ile Asp Phe Asn Gly Leu Ser Asn Pro Val Glu Arg

500 505 510

Ala Val Met Val Glu Ile Leu Lys Phe Phe Arg Arg Tyr His Tyr Glu

515 520 525

Thr Ser Leu Lys Lys Leu Ala Pro Asn Glu Thr Ala Pro Gly Pro Ala

530 535 540

Val Val Thr Asp Ala Gln Leu Asp Ala Trp Leu Pro Ser Gly Val Thr

545 550 555 560

Pro Ser Asp Trp His Ala Ala Gly Thr Ala Ala Met Met Pro Leu Glu

565 570 575

Leu Gly Gly Val Val Asp Gln Thr Leu Arg Val Tyr Gly Val Lys Gly

580 585 590

Leu Arg Val Ile Asp Gly Ser Val Ile Pro Ser Leu Pro Gly Gly Asn

595 600 605

Pro Cys His Ala Val Tyr Ala Ile Ala Glu Lys Ala Ala Asp Leu Ile

610 615 620

Arg Ser Asn Ala

625

<210> 27

<211> 1920

<212> DNA

<213> housing of a stand

<400> 27

atgtccctgt tccgccagca taagtcccag cctcgctgga ccggtatcac cttctccgct 60

ctgctcgccg cctcctccat caccaacgcc ttcgtcatcc ctcgccagat caactccgct 120

cagctcctca agtcctacga ttacgtcatc gtcggcggtg gcaccgctgg cctcaccgtc 180

gccgatcgcc tcaccgagga tcctgatgtc aacgtcctcg tccttgaggc cggcgtgttc 240

ggcgatatgg atttcaacct ccaggtcaac ttcgctaccc gcgtcggtga tgccggcgct 300

acctactggc ctggcctcca gagcgtccct cagcctggcc tgaacggcaa gcccggccag 360

gtcatcattg ccaagcaggt cggtggcggc tcctccgtca acgccatgat caacatgcgc 420

ggctccgccg aggattacga tcgctggggc gctctcttcg gctccgaggc tcaggccggc 480

accgccgatt ggtcctggga tggcatcctg ccattcttca agaagggcct ccatttcacc 540

gagcctccac ctgagctgac cgataacttc gataccgtca agtacgatcc ttcctactgg 600

ggcgattcct ccgagatcta cgccggctgg cctcgcttct actaccctag cgtcgtccct 660

ctcatggaag ccttcaaaga gctggacggc atcgagttcc ctgccgattc cggcgctggc 720

aaggccggtg tctactggtt ccctactctc caggatccac gcaccgtcac tcgctcctac 780

gccgctaccg gccattacct cgacgtcaac gctacccggc ctaactacca tctcctcacc 840

aacactctcg ccagccgcct catcgtcgat gatgatctct ccgccaccgg cgtcgagttc 900

aagtccgcca acagcaccct cgtcaccgtc aaggccgaca aagaggtcat cgtttccgcc 960

ggcaccatcc atactcctca gctgctccag ctctccggca tcggccctgc ctccgtcctc 1020

gaagccggcg gtatcgatgt cctcgtcgat ctccctggcg tcggccagaa cttccaggat 1080

cattccagcc tctccaccat gaacatcacc ctctccaaga tcaccgatat ccatcctaat 1140

ccttccgatc tcgtcgatgg caatgagttc aaggattggg ccgatgaggt ctgggccgct 1200

aaccgcaccg gtccttacac tatcgccctg tccaacctcg ccggttggct ccctttcacc 1260

gccatctccg agaaggctga tgagatcgcc accaagcttg agcagcagga tttcgccagc 1320

ctcctgcctg ccgatgctga ttccaccgtc gtcgccggtt acgaggccca gatgaagctc 1380

ctcgctgccc agatgcgctc caaggatacc gctttcaccc gcgtcatgct ccagcctgag 1440

cagggctccc agggtcctgt cgccatgcag tccttctctc gcggctccat caacatcaac 1500

accaccgatc ctttcaacac cgagccagtc atcgattacc gcagcctcac caatcctatc 1560

gaggtcgatt tcttcatcga gaacatcaag ttcgtccgcc gctacaactt cgagacttcc 1620

ctcaaggata agttcgcccc tgtcgagtac gcccctggcg ccaacgtcac ctccgatgcc 1680

gatctcaagg cctacgtcgc ctccgctctc tcccctaccg attaccatcc tgtcggcacc 1740

tccgccatga tgcctctcga actcggcggc gtcgtcgatc agaccctccg cgtctacggc 1800

gtcaagaacc tccgcgtggt cgatgccagc gtcatgccta tggtcccttc cgccaatacc 1860

tgccagccta cctacgctct cgctgagaag gccgccgaaa tcatcaagca gggcatctaa 1920

<210> 28

<211> 639

<212> PRT

<213> housing of a stand

<400> 28

Met Ser Leu Phe Arg Gln His Lys Ser Gln Pro Arg Trp Thr Gly Ile

1 5 10 15

Thr Phe Ser Ala Leu Leu Ala Ala Ser Ser Ile Thr Asn Ala Phe Val

20 25 30

Ile Pro Arg Gln Ile Asn Ser Ala Gln Leu Leu Lys Ser Tyr Asp Tyr

35 40 45

Val Ile Val Gly Gly Gly Thr Ala Gly Leu Thr Val Ala Asp Arg Leu

50 55 60

Thr Glu Asp Pro Asp Val Asn Val Leu Val Leu Glu Ala Gly Val Phe

65 70 75 80

Gly Asp Met Asp Phe Asn Leu Gln Val Asn Phe Ala Thr Arg Val Gly

85 90 95

Asp Ala Gly Ala Thr Tyr Trp Pro Gly Leu Gln Ser Val Pro Gln Pro

100 105 110

Gly Leu Asn Gly Lys Pro Gly Gln Val Ile Ile Ala Lys Gln Val Gly

115 120 125

Gly Gly Ser Ser Val Asn Ala Met Ile Asn Met Arg Gly Ser Ala Glu

130 135 140

Asp Tyr Asp Arg Trp Gly Ala Leu Phe Gly Ser Glu Ala Gln Ala Gly

145 150 155 160

Thr Ala Asp Trp Ser Trp Asp Gly Ile Leu Pro Phe Phe Lys Lys Gly

165 170 175

Leu His Phe Thr Glu Pro Pro Pro Glu Leu Thr Asp Asn Phe Asp Thr

180 185 190

Val Lys Tyr Asp Pro Ser Tyr Trp Gly Asp Ser Ser Glu Ile Tyr Ala

195 200 205

Gly Trp Pro Arg Phe Tyr Tyr Pro Ser Val Val Pro Leu Met Glu Ala

210 215 220

Phe Lys Glu Leu Asp Gly Ile Glu Phe Pro Ala Asp Ser Gly Ala Gly

225 230 235 240

Lys Ala Gly Val Tyr Trp Phe Pro Thr Leu Gln Asp Pro Arg Thr Val

245 250 255

Thr Arg Ser Tyr Ala Ala Thr Gly His Tyr Leu Asp Val Asn Ala Thr

260 265 270

Arg Pro Asn Tyr His Leu Leu Thr Asn Thr Leu Ala Ser Arg Leu Ile

275 280 285

Val Asp Asp Asp Leu Ser Ala Thr Gly Val Glu Phe Lys Ser Ala Asn

290 295 300

Ser Thr Leu Val Thr Val Lys Ala Asp Lys Glu Val Ile Val Ser Ala

305 310 315 320

Gly Thr Ile His Thr Pro Gln Leu Leu Gln Leu Ser Gly Ile Gly Pro

325 330 335

Ala Ser Val Leu Glu Ala Gly Gly Ile Asp Val Leu Val Asp Leu Pro

340 345 350

Gly Val Gly Gln Asn Phe Gln Asp His Ser Ser Leu Ser Thr Met Asn

355 360 365

Ile Thr Leu Ser Lys Ile Thr Asp Ile His Pro Asn Pro Ser Asp Leu

370 375 380

Val Asp Gly Asn Glu Phe Lys Asp Trp Ala Asp Glu Val Trp Ala Ala

385 390 395 400

Asn Arg Thr Gly Pro Tyr Thr Ile Ala Leu Ser Asn Leu Ala Gly Trp

405 410 415

Leu Pro Phe Thr Ala Ile Ser Glu Lys Ala Asp Glu Ile Ala Thr Lys

420 425 430

Leu Glu Gln Gln Asp Phe Ala Ser Leu Leu Pro Ala Asp Ala Asp Ser

435 440 445

Thr Val Val Ala Gly Tyr Glu Ala Gln Met Lys Leu Leu Ala Ala Gln

450 455 460

Met Arg Ser Lys Asp Thr Ala Phe Thr Arg Val Met Leu Gln Pro Glu

465 470 475 480

Gln Gly Ser Gln Gly Pro Val Ala Met Gln Ser Phe Ser Arg Gly Ser

485 490 495

Ile Asn Ile Asn Thr Thr Asp Pro Phe Asn Thr Glu Pro Val Ile Asp

500 505 510

Tyr Arg Ser Leu Thr Asn Pro Ile Glu Val Asp Phe Phe Ile Glu Asn

515 520 525

Ile Lys Phe Val Arg Arg Tyr Asn Phe Glu Thr Ser Leu Lys Asp Lys

530 535 540

Phe Ala Pro Val Glu Tyr Ala Pro Gly Ala Asn Val Thr Ser Asp Ala

545 550 555 560

Asp Leu Lys Ala Tyr Val Ala Ser Ala Leu Ser Pro Thr Asp Tyr His

565 570 575

Pro Val Gly Thr Ser Ala Met Met Pro Leu Glu Leu Gly Gly Val Val

580 585 590

Asp Gln Thr Leu Arg Val Tyr Gly Val Lys Asn Leu Arg Val Val Asp

595 600 605

Ala Ser Val Met Pro Met Val Pro Ser Ala Asn Thr Cys Gln Pro Thr

610 615 620

Tyr Ala Leu Ala Glu Lys Ala Ala Glu Ile Ile Lys Gln Gly Ile

625 630 635

<210> 29

<211> 1941

<212> DNA

<213> Anthrax bacteria

<400> 29

atgccattct tccgccagtc caacaagtcc cagcaggccc ctcgctggcc tggcttcgcc 60

atggccgctc tgctgaccgc ctcctccttc gccgatgcct acgtcctgcc tcgccatatc 120

aagcctagcc agctcctcaa gtcctacgat tacgtcatcg tcggcggtgg caccgctggc 180

ctcaccgtcg ccgatcgcct caccgaggat cctaagatct ccgtcctcgt cctcgaggcc 240

ggcaactggg gcaatatggc cgataacctc ctcgtctacg tcgccggacg caccggcggc 300

ttcaccgatc ctatgtggcc tggcctccag agcgtccctc agcctaacct caacggtcgc 360

cctggctccg tcctggtcgc caagcaggtc ggtggcggct cctccgtcaa cgccatgatg 420

aacatgcgcg gctccgccga ggattacgat cgctgggccg ctctcttcgg ctccgaggct 480

cgccagggca ccgccgattg gtcctgggat ggcatccttc cattcttcaa gaagggcctc 540

catttcaccg agcctccacc tgagctgacc gataacttcg attctatcaa gtacgatgcc 600

tcctactggg gcgattcctc cgatatctac gccggctggc cccgcttcta ctaccctggc 660

gtcacccctc tcgtcgaggc cttcaaagag atggaaggcg tcgagttccc tcctgattcc 720

ggtgccggcc agcctggtgt gttctggttc cctactctca tggatcctcg cagcgtcacc 780

cgctcctacg ctggcaccgg ccattacctc aacgtcaacg ctacccggcc taactaccat 840

ctcctcatcg atacccaggt ccgccgcctc ctggtcgatg atgccctctg cgccaagggt 900

gtcgaattcc ctctcggcgg caacggcaac ggtacaaccc tcgtcaccgt caaggccaag 960

aaagaggtcc tcctcgccgc tggcgccctg catacccctc atctgctcca gctctccggt 1020

atcggcccta agaacctgct cgaggctggc ggcatcgacg tccgcgtcga tctccccggc 1080

gtcggccaga acttccagga tcattccaac ctctccggcg tcaacatcac cctcaccaag 1140

ctcgcctcca tccatcctaa tcctcgcgat ctggtcgagg gctccgagtt ccaggcttgg 1200

gccgaagagg tctggcaggc caacaagacc ggtccttaca gcctcgcctt caccaacctc 1260

gctggttggc tccctttcac cgccatcacc gatcgcgccg atgagatcgc caccaagctt 1320

gagcagcagg atttcgcctc tctcctgcct gctgataccc atgccaccgt cctcgccggc 1380

ttcgaggccc agatgaagat cctggccggt cagctgcgct ccaagaacac cgctttcacc 1440

cgcttccagc tcatccctga tcatggctcc cagggtccag tcgccatgca gtccttcgct 1500

cgcggcaccg tcaatatcga taccgccgat ccttggaaca ccgagccagt catcgattac 1560

cgcgctctca ccaatcctgt cgaggccgat ttctacgtcg agtctatccg cttcctccgc 1620

cgctacaact tcgagacttc cctcgccgcc gagtacgccc ctgtcgagta cgctccaggt 1680

cctaacgtca cctccgatgc cgatctcaag gcctatatcg ccggtgctct ctcccctacc 1740

gattaccatc ctgtcggcac cgcagccatg atgcctctcg agcttggcgg cgtcgtcgat 1800

cagaccctgc gcgtctacgg cgtcaagaac ctccgcgtcg tggatgcctc cgtcatgcca 1860

atggtccctg gcgccaacac ctgtcagcct acctacgctc tcgccgagaa ggccgctgag 1920

atcatcaagc agggcatctg a 1941

<210> 30

<211> 646

<212> PRT

<213> Anthrax bacteria

<400> 30

Met Pro Phe Phe Arg Gln Ser Asn Lys Ser Gln Gln Ala Pro Arg Trp

1 5 10 15

Pro Gly Phe Ala Met Ala Ala Leu Leu Thr Ala Ser Ser Phe Ala Asp

20 25 30

Ala Tyr Val Leu Pro Arg His Ile Lys Pro Ser Gln Leu Leu Lys Ser

35 40 45

Tyr Asp Tyr Val Ile Val Gly Gly Gly Thr Ala Gly Leu Thr Val Ala

50 55 60

Asp Arg Leu Thr Glu Asp Pro Lys Ile Ser Val Leu Val Leu Glu Ala

65 70 75 80

Gly Asn Trp Gly Asn Met Ala Asp Asn Leu Leu Val Tyr Val Ala Gly

85 90 95

Arg Thr Gly Gly Phe Thr Asp Pro Met Trp Pro Gly Leu Gln Ser Val

100 105 110

Pro Gln Pro Asn Leu Asn Gly Arg Pro Gly Ser Val Leu Val Ala Lys

115 120 125

Gln Val Gly Gly Gly Ser Ser Val Asn Ala Met Met Asn Met Arg Gly

130 135 140

Ser Ala Glu Asp Tyr Asp Arg Trp Ala Ala Leu Phe Gly Ser Glu Ala

145 150 155 160

Arg Gln Gly Thr Ala Asp Trp Ser Trp Asp Gly Ile Leu Pro Phe Phe

165 170 175

Lys Lys Gly Leu His Phe Thr Glu Pro Pro Pro Glu Leu Thr Asp Asn

180 185 190

Phe Asp Ser Ile Lys Tyr Asp Ala Ser Tyr Trp Gly Asp Ser Ser Asp

195 200 205

Ile Tyr Ala Gly Trp Pro Arg Phe Tyr Tyr Pro Gly Val Thr Pro Leu

210 215 220

Val Glu Ala Phe Lys Glu Met Glu Gly Val Glu Phe Pro Pro Asp Ser

225 230 235 240

Gly Ala Gly Gln Pro Gly Val Phe Trp Phe Pro Thr Leu Met Asp Pro

245 250 255

Arg Ser Val Thr Arg Ser Tyr Ala Gly Thr Gly His Tyr Leu Asn Val

260 265 270

Asn Ala Thr Arg Pro Asn Tyr His Leu Leu Ile Asp Thr Gln Val Arg

275 280 285

Arg Leu Leu Val Asp Asp Ala Leu Cys Ala Lys Gly Val Glu Phe Pro

290 295 300

Leu Gly Gly Asn Gly Asn Gly Thr Thr Leu Val Thr Val Lys Ala Lys

305 310 315 320

Lys Glu Val Leu Leu Ala Ala Gly Ala Leu His Thr Pro His Leu Leu

325 330 335

Gln Leu Ser Gly Ile Gly Pro Lys Asn Leu Leu Glu Ala Gly Gly Ile

340 345 350

Asp Val Arg Val Asp Leu Pro Gly Val Gly Gln Asn Phe Gln Asp His

355 360 365

Ser Asn Leu Ser Gly Val Asn Ile Thr Leu Thr Lys Leu Ala Ser Ile

370 375 380

His Pro Asn Pro Arg Asp Leu Val Glu Gly Ser Glu Phe Gln Ala Trp

385 390 395 400

Ala Glu Glu Val Trp Gln Ala Asn Lys Thr Gly Pro Tyr Ser Leu Ala

405 410 415

Phe Thr Asn Leu Ala Gly Trp Leu Pro Phe Thr Ala Ile Thr Asp Arg

420 425 430

Ala Asp Glu Ile Ala Thr Lys Leu Glu Gln Gln Asp Phe Ala Ser Leu

435 440 445

Leu Pro Ala Asp Thr His Ala Thr Val Leu Ala Gly Phe Glu Ala Gln

450 455 460

Met Lys Ile Leu Ala Gly Gln Leu Arg Ser Lys Asn Thr Ala Phe Thr

465 470 475 480

Arg Phe Gln Leu Ile Pro Asp His Gly Ser Gln Gly Pro Val Ala Met

485 490 495

Gln Ser Phe Ala Arg Gly Thr Val Asn Ile Asp Thr Ala Asp Pro Trp

500 505 510

Asn Thr Glu Pro Val Ile Asp Tyr Arg Ala Leu Thr Asn Pro Val Glu

515 520 525

Ala Asp Phe Tyr Val Glu Ser Ile Arg Phe Leu Arg Arg Tyr Asn Phe

530 535 540

Glu Thr Ser Leu Ala Ala Glu Tyr Ala Pro Val Glu Tyr Ala Pro Gly

545 550 555 560

Pro Asn Val Thr Ser Asp Ala Asp Leu Lys Ala Tyr Ile Ala Gly Ala

565 570 575

Leu Ser Pro Thr Asp Tyr His Pro Val Gly Thr Ala Ala Met Met Pro

580 585 590

Leu Glu Leu Gly Gly Val Val Asp Gln Thr Leu Arg Val Tyr Gly Val

595 600 605

Lys Asn Leu Arg Val Val Asp Ala Ser Val Met Pro Met Val Pro Gly

610 615 620

Ala Asn Thr Cys Gln Pro Thr Tyr Ala Leu Ala Glu Lys Ala Ala Glu

625 630 635 640

Ile Ile Lys Gln Gly Ile

645

<210> 31

<211> 1872

<212> DNA

<213> Fusarium lambertianum

<400> 31

atgtccccta gcctcacctg ggtcctcgcc gccgtcagca ccgctctcgt cgccaccgtc 60

gatggctact ccatcagccg ctccgctcat caggcccgcc agatcaccaa gggctccgag 120

ctgctccctg cctacgatta cgtcatcgtc ggcggtggca cctccggcct caccgtcgcc 180

gatcgcctca ccgaggatgg caccaccacc gtcctcgtcc tcgaggccgg cgtgttcgcc 240

cctgatgccg atgtcctgcc tgtctggaac ggcggcaccg gtcgccagcc tcgcttcttc 300

ttcaaatccg ctcctcaaga gaacctcggc aaccagacct tcgatgtctg gctcggcaag 360

atggtcggcg gctcctccgg cgtcaacgcc atgatggcca gccgcggcag cgccctcgat 420

tacgatcgct ggggcaagct gttccctgag tccaacggct gggattggga gggcatcctg 480

ccttacttcc gcaagggcct ccatctcaac cctccagtgc ctgagctggc ctctcgcttc 540

aacatctcca cctccaccaa gtactggggc gaagattccg ccatccaggc tagcttccca 600

tcctaccagt atcctggcct cgagcatatg tcccgcgcct tctacgagct gcctggcgtc 660

gaggccgccg aggattccgg agctggcggt gccggcgtct actggttccc tactctcatg 720

gattccgtcc gctacgagcg ctcctacgcc gagaacgccc attaccgcgg cctcgctcgc 780

cctaactacc atatcgccgc cagctctcgc gtccgccgcg tcctcctgaa gaacggcgtc 840

gctaccggcg tcgagttcta cggcaaggat gatctcctga ccgtcaaggc caccaaggat 900

gtcctcatgg ccgctggcgc cgtgcatacc cctcagatcc tccagctctc cggcatcggc 960

cctaagaagc tcctccaggc cgctggtatc gaaaccctcg tcgatctccc cggcgtcggc 1020

gagaacttcc aggatcatat gtctatcgcc gctagcatca cccttgaggg cctgaagaag 1080

atccggccta atccttccga tatggtcaac ggcaccgctt tcaagaagtg ggccgacgag 1140

tcctgggccg ccaaccgcac cggtccttac agcctcgcct ggggcaacct ggccgcctgg 1200

ctgcctctct ccgccatctc tcctgatcgc tacctcgagc tggccgccga actcgagaac 1260

caggatcacg cctcctacct caagggcgac gtccatccaa ccgtcgccaa gggctacgcc 1320

gctcagatga agtacctcgc cgatgccatg cgctctaagg atgtcgtttt cgcccgctac 1380

ggtgtcgatg ccaccgctgg tgcctccgtg cctgtcctca accagcctat gtctcgcggc 1440

tccatcaccg tcgacctcaa ggatccctac aacgctaacc ctgtcgtcga tttcggcgcc 1500

ctgcgcaacc ccgtcgagcg ctctgccctg gtcgagatgg tcaagtggta tcgcaagtac 1560

aacttcgaaa ccagcctctc ctctctgtcc cctaacgaga ctgcccctgg cgtggatgtc 1620

gtgtccgatg aggatatctg ggcttggatc cctaaggctc tcaagcctac cgattaccat 1680

cctgccggct ccgccgctat gatgcctctc gagcttggcg gcgtcgtcga tcagcagctg 1740

cgcgtctacg gcgtcaagaa cctccgcgtc atcgatgcct ccatcatgcc ttctctccct 1800

tccgccaaca cctgtcagcc catgtacgcc gtcgctgaga aggctgccga tatcatcaag 1860

tccggcgtct ga 1872

<210> 32

<211> 623

<212> PRT

<213> Fusarium lambertianum

<400> 32

Met Ser Pro Ser Leu Thr Trp Val Leu Ala Ala Val Ser Thr Ala Leu

1 5 10 15

Val Ala Thr Val Asp Gly Tyr Ser Ile Ser Arg Ser Ala His Gln Ala

20 25 30

Arg Gln Ile Thr Lys Gly Ser Glu Leu Leu Pro Ala Tyr Asp Tyr Val

35 40 45

Ile Val Gly Gly Gly Thr Ser Gly Leu Thr Val Ala Asp Arg Leu Thr

50 55 60

Glu Asp Gly Thr Thr Thr Val Leu Val Leu Glu Ala Gly Val Phe Ala

65 70 75 80

Pro Asp Ala Asp Val Leu Pro Val Trp Asn Gly Gly Thr Gly Arg Gln

85 90 95

Pro Arg Phe Phe Phe Lys Ser Ala Pro Gln Glu Asn Leu Gly Asn Gln

100 105 110

Thr Phe Asp Val Trp Leu Gly Lys Met Val Gly Gly Ser Ser Gly Val

115 120 125

Asn Ala Met Met Ala Ser Arg Gly Ser Ala Leu Asp Tyr Asp Arg Trp

130 135 140

Gly Lys Leu Phe Pro Glu Ser Asn Gly Trp Asp Trp Glu Gly Ile Leu

145 150 155 160

Pro Tyr Phe Arg Lys Gly Leu His Leu Asn Pro Pro Val Pro Glu Leu

165 170 175

Ala Ser Arg Phe Asn Ile Ser Thr Ser Thr Lys Tyr Trp Gly Glu Asp

180 185 190

Ser Ala Ile Gln Ala Ser Phe Pro Ser Tyr Gln Tyr Pro Gly Leu Glu

195 200 205

His Met Ser Arg Ala Phe Tyr Glu Leu Pro Gly Val Glu Ala Ala Glu

210 215 220

Asp Ser Gly Ala Gly Gly Ala Gly Val Tyr Trp Phe Pro Thr Leu Met

225 230 235 240

Asp Ser Val Arg Tyr Glu Arg Ser Tyr Ala Glu Asn Ala His Tyr Arg

245 250 255

Gly Leu Ala Arg Pro Asn Tyr His Ile Ala Ala Ser Ser Arg Val Arg

260 265 270

Arg Val Leu Leu Lys Asn Gly Val Ala Thr Gly Val Glu Phe Tyr Gly

275 280 285

Lys Asp Asp Leu Leu Thr Val Lys Ala Thr Lys Asp Val Leu Met Ala

290 295 300

Ala Gly Ala Val His Thr Pro Gln Ile Leu Gln Leu Ser Gly Ile Gly

305 310 315 320

Pro Lys Lys Leu Leu Gln Ala Ala Gly Ile Glu Thr Leu Val Asp Leu

325 330 335

Pro Gly Val Gly Glu Asn Phe Gln Asp His Met Ser Ile Ala Ala Ser

340 345 350

Ile Thr Leu Glu Gly Leu Lys Lys Ile Arg Pro Asn Pro Ser Asp Met

355 360 365

Val Asn Gly Thr Ala Phe Lys Lys Trp Ala Asp Glu Ser Trp Ala Ala

370 375 380

Asn Arg Thr Gly Pro Tyr Ser Leu Ala Trp Gly Asn Leu Ala Ala Trp

385 390 395 400

Leu Pro Leu Ser Ala Ile Ser Pro Asp Arg Tyr Leu Glu Leu Ala Ala

405 410 415

Glu Leu Glu Asn Gln Asp His Ala Ser Tyr Leu Lys Gly Asp Val His

420 425 430

Pro Thr Val Ala Lys Gly Tyr Ala Ala Gln Met Lys Tyr Leu Ala Asp

435 440 445

Ala Met Arg Ser Lys Asp Val Val Phe Ala Arg Tyr Gly Val Asp Ala

450 455 460

Thr Ala Gly Ala Ser Val Pro Val Leu Asn Gln Pro Met Ser Arg Gly

465 470 475 480

Ser Ile Thr Val Asp Leu Lys Asp Pro Tyr Asn Ala Asn Pro Val Val

485 490 495

Asp Phe Gly Ala Leu Arg Asn Pro Val Glu Arg Ser Ala Leu Val Glu

500 505 510

Met Val Lys Trp Tyr Arg Lys Tyr Asn Phe Glu Thr Ser Leu Ser Ser

515 520 525

Leu Ser Pro Asn Glu Thr Ala Pro Gly Val Asp Val Val Ser Asp Glu

530 535 540

Asp Ile Trp Ala Trp Ile Pro Lys Ala Leu Lys Pro Thr Asp Tyr His

545 550 555 560

Pro Ala Gly Ser Ala Ala Met Met Pro Leu Glu Leu Gly Gly Val Val

565 570 575

Asp Gln Gln Leu Arg Val Tyr Gly Val Lys Asn Leu Arg Val Ile Asp

580 585 590

Ala Ser Ile Met Pro Ser Leu Pro Ser Ala Asn Thr Cys Gln Pro Met

595 600 605

Tyr Ala Val Ala Glu Lys Ala Ala Asp Ile Ile Lys Ser Gly Val

610 615 620

<210> 33

<211> 1887

<212> DNA

<213> Phialemoniopsis curvata

<400> 33

atgcctgtgc ctcgcctctt ctccctgctc accctgggcg tcgccttcgc cgccgtcggc 60

tcccatgcct tcgtcgtccc tcgctccgtc taccaggctc gccgcatcgt cgatgccagc 120

cagctcctgc ctacctacga ttacatcatc gtcggcggtg gcacctccgg cctcaccgtc 180

gccgatcgcc tcaccgagga tcctgatacc accgtcctcg tcctcgaggc cggctccatg 240

ccaatcgccg aggatgtcct gcctgtcacc ggtggcggaa cccagcgcca ggtcagctac 300

atctacgtca gcgtccctca gaagaacctc ggcggccaga tcttccccgt catcctgggc 360

aagatggtcg gcggctcctc cggcatcaac gccatgatgt ccgctcgcgg ctccgctgag 420

gattacgatc gctggggcaa cctcttcggc aagcacaaca agcacggctg gaactggcag 480

ggcctcctgc cttacttcaa gaaggctttc agcttcaacc ctcctcctgc cgatatcgtc 540

gaggaattcg atgtcaagta cgatgcctcc tactggggca ccgagtccgc tctcgatgtc 600

agcttcccat cctaccagta tcctggcctc cagcctatga tccgcgcctt ctccgagctg 660

cccggcatcg agttcacccg cgattccggc gctggcggtg ccggcgtcta ctggttccct 720

accttcatgg atcctgtcaa gcacgagcgc agctacgccg tcaacgccca ttactccaac 780

ctcggtcgcc ctaactacca tctcgccgct gataccctcg tccgccgcgt cctcctccag 840

catggctccg ccaagggcgt tgagttcact atgcagaacc agacctctcc tactcagctc 900

aaggccacca aagagatcct cgtcgccgct ggcgccgtgc atacccctaa gctgctccag 960

ctctccggta tcggccctaa gcgcgtcctc gatgctgccg gtatcgaaac cctcgtcgat 1020

ctccctggcg tcggtcagaa cttccaggat catgccaacc tgggcgctga ggtcaccctt 1080

gagggcctcc aggccatcca tcctaacggc aacgatctcg tcaccgatcc tgagttccgc 1140

aagctggccg aggatctgtg ggccaccaac cgcaccggtc cttactctat cgcctacggc 1200

aatatcgccg gctggctccc tctgaccgcc atctctcctg gccgcttctc cagcctcgcc 1260

gccgagcttg agaaccagga tcacggcgcc tacctgccac ctggcactca tcctaccgtc 1320

gtcaagggct acgccgctca gatgcgcggc ctcgcttccg ccatgcgctc caaggatacc 1380

gtgttcgccc gctacctggt caacgccacc accggtccac tcgctcctat cctcaaccag 1440

gccttcaacc gcggctccat caacgtcgat cccgccgatc ctttcgattc ccctcctctg 1500

gtcgatttca acggcctcag caaccctatc gagcgcgccg tcctggtcga gatggtcaag 1560

ttcctccgcc gctatgtctc cgagactacc ctcgccagcc tccaggtcaa cgaaacccag 1620

cctggtcctg atgtcgtgac cgatgccgag atcgatgcct ggctgccttc cgctctcacc 1680

ccttccgatt ggcacgctgc tggcaccgct gccatgatgc ctctcgagct tggcggcgtc 1740

gtcgatcaga ccctccgcgt ctacggcgtc aagaacctgc gcgtcatcga cgcctccatc 1800

atgcctagcc tgcctggcgg caacacctgt caggccgtct acgctatcgc cgagaaggcc 1860

gccgatctca tcaagtccaa cgcctga 1887

<210> 34

<211> 628

<212> PRT

<213> Phialemoniopsis curvata

<400> 34

Met Pro Val Pro Arg Leu Phe Ser Leu Leu Thr Leu Gly Val Ala Phe

1 5 10 15

Ala Ala Val Gly Ser His Ala Phe Val Val Pro Arg Ser Val Tyr Gln

20 25 30

Ala Arg Arg Ile Val Asp Ala Ser Gln Leu Leu Pro Thr Tyr Asp Tyr

35 40 45

Ile Ile Val Gly Gly Gly Thr Ser Gly Leu Thr Val Ala Asp Arg Leu

50 55 60

Thr Glu Asp Pro Asp Thr Thr Val Leu Val Leu Glu Ala Gly Ser Met

65 70 75 80

Pro Ile Ala Glu Asp Val Leu Pro Val Thr Gly Gly Gly Thr Gln Arg

85 90 95

Gln Val Ser Tyr Ile Tyr Val Ser Val Pro Gln Lys Asn Leu Gly Gly

100 105 110

Gln Ile Phe Pro Val Ile Leu Gly Lys Met Val Gly Gly Ser Ser Gly

115 120 125

Ile Asn Ala Met Met Ser Ala Arg Gly Ser Ala Glu Asp Tyr Asp Arg

130 135 140

Trp Gly Asn Leu Phe Gly Lys His Asn Lys His Gly Trp Asn Trp Gln

145 150 155 160

Gly Leu Leu Pro Tyr Phe Lys Lys Ala Phe Ser Phe Asn Pro Pro Pro

165 170 175

Ala Asp Ile Val Glu Glu Phe Asp Val Lys Tyr Asp Ala Ser Tyr Trp

180 185 190

Gly Thr Glu Ser Ala Leu Asp Val Ser Phe Pro Ser Tyr Gln Tyr Pro

195 200 205

Gly Leu Gln Pro Met Ile Arg Ala Phe Ser Glu Leu Pro Gly Ile Glu

210 215 220

Phe Thr Arg Asp Ser Gly Ala Gly Gly Ala Gly Val Tyr Trp Phe Pro

225 230 235 240

Thr Phe Met Asp Pro Val Lys His Glu Arg Ser Tyr Ala Val Asn Ala

245 250 255

His Tyr Ser Asn Leu Gly Arg Pro Asn Tyr His Leu Ala Ala Asp Thr

260 265 270

Leu Val Arg Arg Val Leu Leu Gln His Gly Ser Ala Lys Gly Val Glu

275 280 285

Phe Thr Met Gln Asn Gln Thr Ser Pro Thr Gln Leu Lys Ala Thr Lys

290 295 300

Glu Ile Leu Val Ala Ala Gly Ala Val His Thr Pro Lys Leu Leu Gln

305 310 315 320

Leu Ser Gly Ile Gly Pro Lys Arg Val Leu Asp Ala Ala Gly Ile Glu

325 330 335

Thr Leu Val Asp Leu Pro Gly Val Gly Gln Asn Phe Gln Asp His Ala

340 345 350

Asn Leu Gly Ala Glu Val Thr Leu Glu Gly Leu Gln Ala Ile His Pro

355 360 365

Asn Gly Asn Asp Leu Val Thr Asp Pro Glu Phe Arg Lys Leu Ala Glu

370 375 380

Asp Leu Trp Ala Thr Asn Arg Thr Gly Pro Tyr Ser Ile Ala Tyr Gly

385 390 395 400

Asn Ile Ala Gly Trp Leu Pro Leu Thr Ala Ile Ser Pro Gly Arg Phe

405 410 415

Ser Ser Leu Ala Ala Glu Leu Glu Asn Gln Asp His Gly Ala Tyr Leu

420 425 430

Pro Pro Gly Thr His Pro Thr Val Val Lys Gly Tyr Ala Ala Gln Met

435 440 445

Arg Gly Leu Ala Ser Ala Met Arg Ser Lys Asp Thr Val Phe Ala Arg

450 455 460

Tyr Leu Val Asn Ala Thr Thr Gly Pro Leu Ala Pro Ile Leu Asn Gln

465 470 475 480

Ala Phe Asn Arg Gly Ser Ile Asn Val Asp Pro Ala Asp Pro Phe Asp

485 490 495

Ser Pro Pro Leu Val Asp Phe Asn Gly Leu Ser Asn Pro Ile Glu Arg

500 505 510

Ala Val Leu Val Glu Met Val Lys Phe Leu Arg Arg Tyr Val Ser Glu

515 520 525

Thr Thr Leu Ala Ser Leu Gln Val Asn Glu Thr Gln Pro Gly Pro Asp

530 535 540

Val Val Thr Asp Ala Glu Ile Asp Ala Trp Leu Pro Ser Ala Leu Thr

545 550 555 560

Pro Ser Asp Trp His Ala Ala Gly Thr Ala Ala Met Met Pro Leu Glu

565 570 575

Leu Gly Gly Val Val Asp Gln Thr Leu Arg Val Tyr Gly Val Lys Asn

580 585 590

Leu Arg Val Ile Asp Ala Ser Ile Met Pro Ser Leu Pro Gly Gly Asn

595 600 605

Thr Cys Gln Ala Val Tyr Ala Ile Ala Glu Lys Ala Ala Asp Leu Ile

610 615 620

Lys Ser Asn Ala

625

<210> 35

<211> 1872

<212> DNA

<213> Fusarium lambertianum (A.o)

<400> 35

atgctcttct cactggcatt cctgagtgcc ctgtcgctgg ccacggcatc accggctgga 60

cgggcctact ccatcagccg ctccgctcat caggcccgcc agatcaccaa gggctccgag 120

ctgctccctg cctacgatta cgtcatcgtc ggcggtggca cctccggcct caccgtcgcc 180

gatcgcctca ccgaggatgg caccaccacc gtcctcgtcc tcgaggccgg cgtgttcgcc 240

cctgatgccg atgtcctgcc tgtctggaac ggcggcaccg gtcgccagcc tcgcttcttc 300

ttcaaatccg ctcctcaaga gaacctcggc aaccagacct tcgatgtctg gctcggcaag 360

atggtcggcg gctcctccgg cgtcaacgcc atgatggcca gccgcggcag cgccctcgat 420

tacgatcgct ggggcaagct gttccctgag tccaacggct gggattggga gggcatcctg 480

ccttacttcc gcaagggcct ccatctcaac cctccagtgc ctgagctggc ctctcgcttc 540

aacatctcca cctccaccaa gtactggggc gaagattccg ccatccaggc tagcttccca 600

tcctaccagt atcctggcct cgagcatatg tcccgcgcct tctacgagct gcctggcgtc 660

gaggccgccg aggattccgg agctggcggt gccggcgtct actggttccc tactctcatg 720

gattccgtcc gctacgagcg ctcctacgcc gagaacgccc attaccgcgg cctcgctcgc 780

cctaactacc atatcgccgc cagctctcgc gtccgccgcg tcctcctgaa gaacggcgtc 840

gctaccggcg tcgagttcta cggcaaggat gatctcctga ccgtcaaggc caccaaggat 900

gtcctcatgg ccgctggcgc cgtgcatacc cctcagatcc tccagctctc cggcatcggc 960

cctaagaagc tcctccaggc cgctggtatc gaaaccctcg tcgatctccc cggcgtcggc 1020

gagaacttcc aggatcatat gtctatcgcc gctagcatca cccttgaggg cctgaagaag 1080

atccggccta atccttccga tatggtcaac ggcaccgctt tcaagaagtg ggccgacgag 1140

tcctgggccg ccaaccgcac cggtccttac agcctcgcct ggggcaacct ggccgcctgg 1200

ctgcctctct ccgccatctc tcctgatcgc tacctcgagc tggccgccga actcgagaac 1260

caggatcacg cctcctacct caagggcgac gtccatccaa ccgtcgccaa gggctacgcc 1320

gctcagatga agtacctcgc cgatgccatg cgctctaagg atgtcgtttt cgcccgctac 1380

ggtgtcgatg ccaccgctgg tgcctccgtg cctgtcctca accagcctat gtctcgcggc 1440

tccatcaccg tcgacctcaa ggatccctac aacgctaacc ctgtcgtcga tttcggcgcc 1500

ctgcgcaacc ccgtcgagcg ctctgccctg gtcgagatgg tcaagtggta tcgcaagtac 1560

aacttcgaaa ccagcctctc ctctctgtcc cctaacgaga ctgcccctgg cgtggatgtc 1620

gtgtccgatg aggatatctg ggcttggatc cctaaggctc tcaagcctac cgattaccat 1680

cctgccggct ccgccgctat gatgcctctc gagcttggcg gcgtcgtcga tcagcagctg 1740

cgcgtctacg gcgtcaagaa cctccgcgtc atcgatgcct ccatcatgcc ttctctccct 1800

tccgccaaca cctgtcagcc catgtacgcc gtcgctgaga aggctgccga tatcatcaag 1860

tccggcgtct ga 1872

<210> 36

<211> 623

<212> PRT

<213> Fusarium lambertianum (A.o)

<400> 36

Met Leu Phe Ser Leu Ala Phe Leu Ser Ala Leu Ser Leu Ala Thr Ala

1 5 10 15

Ser Pro Ala Gly Arg Ala Tyr Ser Ile Ser Arg Ser Ala His Gln Ala

20 25 30

Arg Gln Ile Thr Lys Gly Ser Glu Leu Leu Pro Ala Tyr Asp Tyr Val

35 40 45

Ile Val Gly Gly Gly Thr Ser Gly Leu Thr Val Ala Asp Arg Leu Thr

50 55 60

Glu Asp Gly Thr Thr Thr Val Leu Val Leu Glu Ala Gly Val Phe Ala

65 70 75 80

Pro Asp Ala Asp Val Leu Pro Val Trp Asn Gly Gly Thr Gly Arg Gln

85 90 95

Pro Arg Phe Phe Phe Lys Ser Ala Pro Gln Glu Asn Leu Gly Asn Gln

100 105 110

Thr Phe Asp Val Trp Leu Gly Lys Met Val Gly Gly Ser Ser Gly Val

115 120 125

Asn Ala Met Met Ala Ser Arg Gly Ser Ala Leu Asp Tyr Asp Arg Trp

130 135 140

Gly Lys Leu Phe Pro Glu Ser Asn Gly Trp Asp Trp Glu Gly Ile Leu

145 150 155 160

Pro Tyr Phe Arg Lys Gly Leu His Leu Asn Pro Pro Val Pro Glu Leu

165 170 175

Ala Ser Arg Phe Asn Ile Ser Thr Ser Thr Lys Tyr Trp Gly Glu Asp

180 185 190

Ser Ala Ile Gln Ala Ser Phe Pro Ser Tyr Gln Tyr Pro Gly Leu Glu

195 200 205

His Met Ser Arg Ala Phe Tyr Glu Leu Pro Gly Val Glu Ala Ala Glu

210 215 220

Asp Ser Gly Ala Gly Gly Ala Gly Val Tyr Trp Phe Pro Thr Leu Met

225 230 235 240

Asp Ser Val Arg Tyr Glu Arg Ser Tyr Ala Glu Asn Ala His Tyr Arg

245 250 255

Gly Leu Ala Arg Pro Asn Tyr His Ile Ala Ala Ser Ser Arg Val Arg

260 265 270

Arg Val Leu Leu Lys Asn Gly Val Ala Thr Gly Val Glu Phe Tyr Gly

275 280 285

Lys Asp Asp Leu Leu Thr Val Lys Ala Thr Lys Asp Val Leu Met Ala

290 295 300

Ala Gly Ala Val His Thr Pro Gln Ile Leu Gln Leu Ser Gly Ile Gly

305 310 315 320

Pro Lys Lys Leu Leu Gln Ala Ala Gly Ile Glu Thr Leu Val Asp Leu

325 330 335

Pro Gly Val Gly Glu Asn Phe Gln Asp His Met Ser Ile Ala Ala Ser

340 345 350

Ile Thr Leu Glu Gly Leu Lys Lys Ile Arg Pro Asn Pro Ser Asp Met

355 360 365

Val Asn Gly Thr Ala Phe Lys Lys Trp Ala Asp Glu Ser Trp Ala Ala

370 375 380

Asn Arg Thr Gly Pro Tyr Ser Leu Ala Trp Gly Asn Leu Ala Ala Trp

385 390 395 400

Leu Pro Leu Ser Ala Ile Ser Pro Asp Arg Tyr Leu Glu Leu Ala Ala

405 410 415

Glu Leu Glu Asn Gln Asp His Ala Ser Tyr Leu Lys Gly Asp Val His

420 425 430

Pro Thr Val Ala Lys Gly Tyr Ala Ala Gln Met Lys Tyr Leu Ala Asp

435 440 445

Ala Met Arg Ser Lys Asp Val Val Phe Ala Arg Tyr Gly Val Asp Ala

450 455 460

Thr Ala Gly Ala Ser Val Pro Val Leu Asn Gln Pro Met Ser Arg Gly

465 470 475 480

Ser Ile Thr Val Asp Leu Lys Asp Pro Tyr Asn Ala Asn Pro Val Val

485 490 495

Asp Phe Gly Ala Leu Arg Asn Pro Val Glu Arg Ser Ala Leu Val Glu

500 505 510

Met Val Lys Trp Tyr Arg Lys Tyr Asn Phe Glu Thr Ser Leu Ser Ser

515 520 525

Leu Ser Pro Asn Glu Thr Ala Pro Gly Val Asp Val Val Ser Asp Glu

530 535 540

Asp Ile Trp Ala Trp Ile Pro Lys Ala Leu Lys Pro Thr Asp Tyr His

545 550 555 560

Pro Ala Gly Ser Ala Ala Met Met Pro Leu Glu Leu Gly Gly Val Val

565 570 575

Asp Gln Gln Leu Arg Val Tyr Gly Val Lys Asn Leu Arg Val Ile Asp

580 585 590

Ala Ser Ile Met Pro Ser Leu Pro Ser Ala Asn Thr Cys Gln Pro Met

595 600 605

Tyr Ala Val Ala Glu Lys Ala Ala Asp Ile Ile Lys Ser Gly Val

610 615 620

<210> 37

<211> 1881

<212> DNA

<213> Phialemoniopsis curvata(A.o)

<400> 37

atgctcttct cactggcatt cctgagtgcc ctgtcgctgg ccacggcatc accggctgga 60

cgggccgtcg tccctcgctc cgtctaccag gctcgccgca tcgtcgatgc cagccagctc 120

ctgcctacct acgattacat catcgtcggc ggtggcacct ccggcctcac cgtcgccgat 180

cgcctcaccg aggatcctga taccaccgtc ctcgtcctcg aggccggctc catgccaatc 240

gccgaggatg tcctgcctgt caccggtggc ggaacccagc gccaggtcag ctacatctac 300

gtcagcgtcc ctcagaagaa cctcggcggc cagatcttcc ccgtcatcct gggcaagatg 360

gtcggcggct cctccggcat caacgccatg atgtccgctc gcggctccgc tgaggattac 420

gatcgctggg gcaacctctt cggcaagcac aacaagcacg gctggaactg gcagggcctc 480

ctgccttact tcaagaaggc tttcagcttc aaccctcctc ctgccgatat cgtcgaggaa 540

ttcgatgtca agtacgatgc ctcctactgg ggcaccgagt ccgctctcga tgtcagcttc 600

ccatcctacc agtatcctgg cctccagcct atgatccgcg ccttctccga gctgcccggc 660

atcgagttca cccgcgattc cggcgctggc ggtgccggcg tctactggtt ccctaccttc 720

atggatcctg tcaagcacga gcgcagctac gccgtcaacg cccattactc caacctcggt 780

cgccctaact accatctcgc cgctgatacc ctcgtccgcc gcgtcctcct ccagcatggc 840

tccgccaagg gcgttgagtt cactatgcag aaccagacct ctcctactca gctcaaggcc 900

accaaagaga tcctcgtcgc cgctggcgcc gtgcataccc ctaagctgct ccagctctcc 960

ggtatcggcc ctaagcgcgt cctcgatgct gccggtatcg aaaccctcgt cgatctccct 1020

ggcgtcggtc agaacttcca ggatcatgcc aacctgggcg ctgaggtcac ccttgagggc 1080

ctccaggcca tccatcctaa cggcaacgat ctcgtcaccg atcctgagtt ccgcaagctg 1140

gccgaggatc tgtgggccac caaccgcacc ggtccttact ctatcgccta cggcaatatc 1200

gccggctggc tccctctgac cgccatctct cctggccgct tctccagcct cgccgccgag 1260

cttgagaacc aggatcacgg cgcctacctg ccacctggca ctcatcctac cgtcgtcaag 1320

ggctacgccg ctcagatgcg cggcctcgct tccgccatgc gctccaagga taccgtgttc 1380

gcccgctacc tggtcaacgc caccaccggt ccactcgctc ctatcctcaa ccaggccttc 1440

aaccgcggct ccatcaacgt cgatcccgcc gatcctttcg attcccctcc tctggtcgat 1500

ttcaacggcc tcagcaaccc tatcgagcgc gccgtcctgg tcgagatggt caagttcctc 1560

cgccgctatg tctccgagac taccctcgcc agcctccagg tcaacgaaac ccagcctggt 1620

cctgatgtcg tgaccgatgc cgagatcgat gcctggctgc cttccgctct caccccttcc 1680

gattggcacg ctgctggcac cgctgccatg atgcctctcg agcttggcgg cgtcgtcgat 1740

cagaccctcc gcgtctacgg cgtcaagaac ctgcgcgtca tcgacgcctc catcatgcct 1800

agcctgcctg gcggcaacac ctgtcaggcc gtctacgcta tcgccgagaa ggccgccgat 1860

ctcatcaagt ccaacgcctg a 1881

<210> 38

<211> 626

<212> PRT

<213> Phialemoniopsis curvata(A.o)

<400> 38

Met Leu Phe Ser Leu Ala Phe Leu Ser Ala Leu Ser Leu Ala Thr Ala

1 5 10 15

Ser Pro Ala Gly Arg Ala Val Val Pro Arg Ser Val Tyr Gln Ala Arg

20 25 30

Arg Ile Val Asp Ala Ser Gln Leu Leu Pro Thr Tyr Asp Tyr Ile Ile

35 40 45

Val Gly Gly Gly Thr Ser Gly Leu Thr Val Ala Asp Arg Leu Thr Glu

50 55 60

Asp Pro Asp Thr Thr Val Leu Val Leu Glu Ala Gly Ser Met Pro Ile

65 70 75 80

Ala Glu Asp Val Leu Pro Val Thr Gly Gly Gly Thr Gln Arg Gln Val

85 90 95

Ser Tyr Ile Tyr Val Ser Val Pro Gln Lys Asn Leu Gly Gly Gln Ile

100 105 110

Phe Pro Val Ile Leu Gly Lys Met Val Gly Gly Ser Ser Gly Ile Asn

115 120 125

Ala Met Met Ser Ala Arg Gly Ser Ala Glu Asp Tyr Asp Arg Trp Gly

130 135 140

Asn Leu Phe Gly Lys His Asn Lys His Gly Trp Asn Trp Gln Gly Leu

145 150 155 160

Leu Pro Tyr Phe Lys Lys Ala Phe Ser Phe Asn Pro Pro Pro Ala Asp

165 170 175

Ile Val Glu Glu Phe Asp Val Lys Tyr Asp Ala Ser Tyr Trp Gly Thr

180 185 190

Glu Ser Ala Leu Asp Val Ser Phe Pro Ser Tyr Gln Tyr Pro Gly Leu

195 200 205

Gln Pro Met Ile Arg Ala Phe Ser Glu Leu Pro Gly Ile Glu Phe Thr

210 215 220

Arg Asp Ser Gly Ala Gly Gly Ala Gly Val Tyr Trp Phe Pro Thr Phe

225 230 235 240

Met Asp Pro Val Lys His Glu Arg Ser Tyr Ala Val Asn Ala His Tyr

245 250 255

Ser Asn Leu Gly Arg Pro Asn Tyr His Leu Ala Ala Asp Thr Leu Val

260 265 270

Arg Arg Val Leu Leu Gln His Gly Ser Ala Lys Gly Val Glu Phe Thr

275 280 285

Met Gln Asn Gln Thr Ser Pro Thr Gln Leu Lys Ala Thr Lys Glu Ile

290 295 300

Leu Val Ala Ala Gly Ala Val His Thr Pro Lys Leu Leu Gln Leu Ser

305 310 315 320

Gly Ile Gly Pro Lys Arg Val Leu Asp Ala Ala Gly Ile Glu Thr Leu

325 330 335

Val Asp Leu Pro Gly Val Gly Gln Asn Phe Gln Asp His Ala Asn Leu

340 345 350

Gly Ala Glu Val Thr Leu Glu Gly Leu Gln Ala Ile His Pro Asn Gly

355 360 365

Asn Asp Leu Val Thr Asp Pro Glu Phe Arg Lys Leu Ala Glu Asp Leu

370 375 380

Trp Ala Thr Asn Arg Thr Gly Pro Tyr Ser Ile Ala Tyr Gly Asn Ile

385 390 395 400

Ala Gly Trp Leu Pro Leu Thr Ala Ile Ser Pro Gly Arg Phe Ser Ser

405 410 415

Leu Ala Ala Glu Leu Glu Asn Gln Asp His Gly Ala Tyr Leu Pro Pro

420 425 430

Gly Thr His Pro Thr Val Val Lys Gly Tyr Ala Ala Gln Met Arg Gly

435 440 445

Leu Ala Ser Ala Met Arg Ser Lys Asp Thr Val Phe Ala Arg Tyr Leu

450 455 460

Val Asn Ala Thr Thr Gly Pro Leu Ala Pro Ile Leu Asn Gln Ala Phe

465 470 475 480

Asn Arg Gly Ser Ile Asn Val Asp Pro Ala Asp Pro Phe Asp Ser Pro

485 490 495

Pro Leu Val Asp Phe Asn Gly Leu Ser Asn Pro Ile Glu Arg Ala Val

500 505 510

Leu Val Glu Met Val Lys Phe Leu Arg Arg Tyr Val Ser Glu Thr Thr

515 520 525

Leu Ala Ser Leu Gln Val Asn Glu Thr Gln Pro Gly Pro Asp Val Val

530 535 540

Thr Asp Ala Glu Ile Asp Ala Trp Leu Pro Ser Ala Leu Thr Pro Ser

545 550 555 560

Asp Trp His Ala Ala Gly Thr Ala Ala Met Met Pro Leu Glu Leu Gly

565 570 575

Gly Val Val Asp Gln Thr Leu Arg Val Tyr Gly Val Lys Asn Leu Arg

580 585 590

Val Ile Asp Ala Ser Ile Met Pro Ser Leu Pro Gly Gly Asn Thr Cys

595 600 605

Gln Ala Val Tyr Ala Ile Ala Glu Lys Ala Ala Asp Leu Ile Lys Ser

610 615 620

Asn Ala

625

Claims

1. A method for producing glucuronic acid, comprising the steps of: a flavin-bound glucose dehydrogenase having glucose-6-dehydrogenase activity is allowed to act on glucose in the presence of a mediator to produce glucuronic acid.

2. A method for producing a glucuronic acid derivative, comprising the steps of: a glucuronic acid derivative is produced by allowing a flavin-bound glucose dehydrogenase having glucose-6-dehydrogenase activity to act on a glucose derivative in the presence of a mediator.

3. The method for producing a glucuronic acid derivative according to claim 2, wherein the glucose derivative is an amino sugar or an N-acetyl compound thereof, a glucoside, or a glucose analog.

4. The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of claims 1 to 3, wherein the flavin-bound glucose dehydrogenase is any one of the following proteins (i) to (iii):

(i) a protein having an amino acid sequence represented by SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38,

(ii) a protein having glucose-6-dehydrogenase activity, which has an amino acid sequence in which 1 to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38,

5. The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of claims 1 to 4, wherein the flavin-bound glucose dehydrogenase has the following properties (1) to (8):

(1) the function is as follows: takes flavin as coenzyme to catalyze the dehydrogenation (oxidation) reaction of hydroxymethyl at 6-position of glucose,

(2) solubility: the water-soluble polymer is water-soluble,

(3) pH stability: is stable at a pH of at least 5.5 to 8.7,

(4) thermal stability: is stable at a temperature of at least 35 ℃,

(5) substrate specificity: the action on glucose is set to be 100%, the action on maltose, xylose and galactose is less than 2.0%,

(6) km value with respect to glucose: the concentration of the active carbon is more than 30mM,

(7) molecular weight: 64 to 66kDa, calculated from the amino acid sequence after removal of the signal,

(8) glucose oxidase activity: and cannot be detected.

6. The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of claims 1 to 5, wherein the flavin-bound glucose dehydrogenase is derived from a microorganism belonging to the genus anthrax, the genus Microchaeta, the genus Ascophyllum, the genus Husky, the genus Acremonium, the genus Lasiosphaeris, the genus Fusarium, or the genus Phelomitopsis.

7. The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of claims 1 to 6, wherein a recombinant microorganism into which a gene encoding a flavin-bound glucose dehydrogenase is introduced is used as the flavin-bound glucose dehydrogenase.

8. The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to claim 7, wherein the gene encoding the flavin-bound glucose dehydrogenase is a gene composed of any one of the following DNAs (a) to (e):

(a) a DNA having the base sequence represented by SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37,

(b) a DNA encoding a protein having glucose-6-dehydrogenase activity which has a base sequence in which 1 to several bases are deleted, substituted or added from the base sequence represented by SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37,

(c) a DNA encoding a protein having glucose-6-dehydrogenase activity and having a nucleotide sequence having 80% or more sequence homology to the nucleotide sequence represented by SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37,

(d) a DNA which hybridizes under stringent conditions to a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 and which encodes a protein having glucose-6-dehydrogenase activity,

(e) (iv) a DNA encoding the protein of the following (i), (ii) or (iii),

(ii) a protein having glucose-6-dehydrogenase activity which has an amino acid sequence comprising 1 to several amino acid residues deleted, substituted or inserted from the amino acid sequence represented by SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38,

9. The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of claims 1 to 8, wherein an oxidase is further caused to act.

10. A flavin-bound glucose dehydrogenase having glucose-6-dehydrogenase activity, which is any one of the following proteins (i) to (iii):

11. A catalyst for producing glucuronic acid or a glucuronic acid derivative, which comprises a flavin-bound glucose dehydrogenase protein having glucose-6-dehydrogenase activity.