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CN116555210A - Glycosyltransferases and their use in the preparation of rebaudioside E - Google Patents

Glycosyltransferases and their use in the preparation of rebaudioside E Download PDF

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CN116555210A
CN116555210A CN202210114711.6A CN202210114711A CN116555210A CN 116555210 A CN116555210 A CN 116555210A CN 202210114711 A CN202210114711 A CN 202210114711A CN 116555210 A CN116555210 A CN 116555210A
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amino acid
leu
acid residue
glu
val
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吴燕
郑孝富
田振华
王舒
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Ecolab Biotechnology Shanghai Co ltd
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Ecolab Biotechnology Shanghai Co ltd
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Abstract

The invention discloses glycosyltransferases and their use in the preparation of rebaudioside E. The amino acid sequence of the glycosyltransferase of the invention comprises at least the amino acid residue differences compared to SEQ ID NO. 4 selected from the group consisting of: (1) Amino acid residues 429, 433, 435, 446, 448 and 449; (2) deletion of amino acid residues 1 to 8; amino acid residues at positions 9, 10, 11, 12, 16, 22, 23, 26, 27, 31, 42, 46, 49, 54, 56, 58, 64, 65, 70, 73, 96 and 106. The glycosyltransferase with good catalytic effect and good stability is screened out, the process condition for synthesizing the Reb E by an enzyme method is optimized, and the industrial production of the Reb E is facilitated; solves the problem of high price of glycosyl donor UDPG/ADPG, and reduces the production cost.

Description

Glycosyltransferases and their use in the preparation of rebaudioside E
Technical Field
The invention belongs to the field of biosynthesis, and particularly relates to glycosyltransferase and application thereof in preparing rebaudioside E.
Background
Stevioside (Steviol glycosides, also known as steviol glycoside) is a natural sweetener extracted from leaves of stevia rebaudiana Bertoni of Compositae, and is 10% -20% of dry weight of leaves, and is a mixture of various glycosides. Stevioside has the advantages of pure nature (from pure natural plant stevia), high sweetness, low calorie, economy in use, good stability, high safety and the like, but has the defect of bitter taste after the use, and limits the application of stevioside in the fields of food, beverage and the like. The intrinsic reason for the bitter taste after steviol glycoside is that the intrinsic molecular structure of steviol glycoside causes that the more the number of the sugar groups connected on R1 and R2 groups in steviol glycoside, the better the taste.
Steviol glycosides (steviol glycoside compounds) have the following structural formula:
the compounds corresponding to the substituents are shown in Table 1.
TABLE 1 steviol glycosides isolated from stevia rebaudiana
The steviol glycoside compounds have a common aglycone: steviol (Steviol), differing in the number and type of glycosyl groups attached at the C-13 and C-19 positions, mainly includes Stevioside (Stevioside), rebaudioside a (rebaudiosid a, reba a, RA), rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E (rebaudiosid E, RE), dulcoside, steviolbioside, etc.
The taste of the rebaudioside E is free from bitter taste, the sweetness is similar to that of sucrose, the structure of the rebaudioside E is more than that of stevioside on a C19 side chain on a framework, but the content of the rebaudioside E in dry leaves of the stevia is very small (much less than 1 percent), and the rebaudioside E is directly separated from the stevioside by a conventional physical means, so that the difficulty is high and the yield is very low. In addition, the process for enriching the rebaudioside E is complicated, multiple column passes, desalination, decoloration and recrystallization are needed after extraction, and a large amount of wastewater is generated in the production process, so that the method has high production cost and is not suitable for industrial mass production.
The bioconversion method mainly uses glycosyltransferase to catalyze glycosyl to transfer from an activated donor molecule to an acceptor molecule, so as to generate various glycoside compounds. Common glycosyl donors include monosaccharides, disaccharides, polysaccharides, phosphate sugars, uridine diphosphate-glucose, and the like. The glycosyl donor for preparing rebaudioside E by the bioconversion method is generally uridine diphosphate-glucose (UDP-glucose) or adenosine diphosphate-glucose (ADP-glucose), but the glycosyl donor is expensive, and the catalytic activity of glycosyltransferase (such as beta-1, 2-glycosyltransferase) is relatively low, so that the production cost is high, and the production efficiency is low.
Disclosure of Invention
The invention aims to overcome the defect of low enzyme activity when the existing glycosyltransferase is applied to biocatalysis preparation of Reb E, and provides the glycosyltransferase and the application thereof in preparation of rebaudioside E. The glycosyltransferase has good catalytic effect and stability, and can be used for synthesizing Reb E by combining with sucrose synthase (SUS) to realize cascade reaction; meanwhile, the regeneration of UDPG/ADPG is realized through sucrose and UDP/ADP, so that the problem of high price of glycosyl donor UDPG/ADPG is solved; and further optimizes the process conditions for synthesizing the Reb E by enzyme catalysis, provides more choices for optimizing the process conditions for realizing large-scale industrial production, and is favorable for realizing industrial production.
The invention solves the technical problems by the following technical proposal:
in a first aspect the present invention provides a glycosyltransferase having an amino acid sequence which comprises at least the amino acid residue differences compared to SEQ ID NO. 4 selected from the group consisting of:
(1) Having one or more of the following amino acid residue differences:
the amino acid residue at position 429 is D;
the amino acid residue at position 433 is D;
the amino acid residue at position 435 is V;
the amino acid residue at position 446 is S;
the amino acid residue at position 448 is K; and
the 449 amino acid residue is S;
(2) Having one or more of the following amino acid residue differences:
deletion of amino acid residues at positions 1 to 8;
the amino acid residue at position 9 is M;
the amino acid residue at position 10 is A;
the amino acid residue at position 11 is T;
the amino acid residue at position 12 is N;
the amino acid residue at position 16 is L;
the amino acid residue at position 22 is A;
the amino acid residue at position 23 is Y;
the amino acid residue at position 26 is I;
the amino acid residue at position 27 is S;
the amino acid residue at position 31 is N;
the amino acid residue at position 42 is L;
the amino acid residue at position 46 is C;
the amino acid residue at position 49 is R;
the amino acid residue at position 54 is S;
amino acid residue at position 56 is I;
the amino acid residue at position 58 is K;
the amino acid residue at position 64 is A;
the amino acid residue at position 65 is D;
the amino acid residue at position 70 is I;
the amino acid residue at position 73 is Q;
the amino acid residue at position 96 is P; and
the amino acid residue at position 106 is K.
In some embodiments of the invention, in (1), the amino acid sequence of the glycosyltransferase further comprises one or more amino acid residue differences compared to SEQ ID NO. 4 selected from the group consisting of:
the amino acid residue at position 399 is E;
the amino acid residue at position 400 is A;
the amino acid residue at position 403 is S;
the amino acid residue at position 405 is V;
the amino acid residue at position 406 is T;
the amino acid residue at position 408 is E;
the amino acid residue at position 419 is E;
the 422 th amino acid residue is K;
the amino acid residue at position 423 is N;
the amino acid residue at position 425 is K;
the amino acid residue at position 426 is S; and
the amino acid residue at position 427 is I.
In some embodiments of the invention, the amino acid sequence of the glycosyltransferase further comprises one or more amino acid residue differences compared to SEQ ID NO. 4 selected from the group consisting of:
the amino acid residue at position 373 is K;
amino acid residue at position 375 is M;
the amino acid residue at position 385 is V;
the amino acid residue at position 388 is D;
the amino acid residue at position 391 is K;
the amino acid residue at position 392 is I; and
the amino acid residue at position 395 is G.
In some embodiments of the invention, the amino acid sequence of the glycosyltransferase further comprises amino acid residue differences at one or more residue positions selected from the group consisting of:
amino acid residue 309 is E;
amino acid residue at position 315 is I;
the amino acid residue at position 317 is E;
the amino acid residue at position 324 is K;
the amino acid residue at position 325 is F;
the amino acid residue at position 326 is A;
the amino acid residue at position 329 is P;
amino acid residue at position 330 is R;
amino acid residue at position 364 is I;
the amino acid residue at position 365 is H; and
the amino acid residue at position 366 is N.
In some embodiments of the invention, the glycosyltransferase does not comprise one or more amino acid differences from positions 210 to 257 as compared to SEQ ID NO. 4.
In some embodiments of the invention, the glycosyltransferase does not comprise one or more amino acid residue differences from position 259 to position 306 as compared to SEQ ID NO. 4.
In some embodiments of the invention, the glycosyltransferase does not comprise one or more amino acid residue differences from positions 111 to 202 as compared to SEQ ID NO. 4.
In some embodiments of the invention, the glycosyltransferase does not comprise one or more amino acid residue differences from position 259 to position 306 and from position 111 to position 202 as compared to SEQ ID NO. 4.
In some embodiments of the invention, the amino acid sequence of the glycosyltransferase is shown as SEQ ID NO. 32.
In some embodiments of the invention, the amino acid sequence of the glycosyltransferase is shown in SEQ ID NO. 38.
In some embodiments of the invention, the amino acid sequence of the glycosyltransferase is shown as SEQ ID NO. 30.
In some embodiments of the invention, the amino acid sequence of the glycosyltransferase is shown as SEQ ID NO. 28.
In some embodiments of the invention, the amino acid sequence of the glycosyltransferase is shown as SEQ ID NO. 36.
In a second aspect the invention provides an isolated nucleic acid encoding a glycosyltransferase according to the first aspect.
In a third aspect the present invention provides a recombinant expression vector comprising a nucleic acid as described in the second aspect.
In a fourth aspect the present invention provides a transformant comprising a nucleic acid as described in the second aspect or a recombinant expression vector as described in the third aspect.
In a fifth aspect the present invention provides a method of preparing a glycosyltransferase according to the first aspect, the method comprising culturing a transformant according to the fourth aspect under conditions suitable for expression of the glycosyltransferase.
A sixth aspect of the invention provides a method of preparing rebaudioside E, the method comprising: glycosyltransferases transfer a glycosyl group on an activated glycosyl donor to a glycosyl acceptor;
wherein the glycosyltransferase is as described in the first aspect; the glycosyl acceptor is stevioside; the glycosyl donor is uridine diphosphate glucose and/or adenosine diphosphate glucose.
In some embodiments of the invention, the uridine diphosphate glucose and/or adenosine diphosphate glucose are produced by the destructive synthesis of sucrose.
In the invention, the decomposition and synthesis of sucrose means: in the presence of uridine diphosphate and/or adenosine diphosphate, a molecule of sucrose is decomposed by sucrose synthase to produce a molecule of fructose and a molecule of uridine diphosphate glucose and/or adenosine diphosphate glucose.
In some embodiments of the invention, the amino acid sequence of the sucrose synthase is as shown in SEQ ID NO. 24; the nucleotide sequence encoding the sucrose synthase is preferably as shown in SEQ ID NO. 23.
In some embodiments of the invention, the glycosyltransferase and the sucrose synthase are used in the form of a crude enzyme solution, a pure enzyme, an immobilized enzyme, or a cell expressing the glycosyltransferase and the sucrose synthase.
In the present invention, the host cell of the cell expressing the glycosyltransferase and the sucrose synthase may be conventional in the art, e.g., e.coli; the person skilled in the art can culture the cells and obtain glycosyltransferases and sucrose synthases by conventional means.
In some embodiments of the invention, the mass ratio of cells expressing the glycosyltransferase to stevioside is 3 (9-30), preferably 3:20.
In some embodiments of the invention, the mass ratio of the cell expressing the sucrose synthase to sucrose is 3 (150-300), preferably 3:200.
In some embodiments of the invention, the mass ratio of sucrose to stevioside is (0.5-3): 1, preferably 2:1.
In some embodiments of the invention, the sucrose to glucose uridine diphosphate or glucose adenosine diphosphate mass ratio is (500-3000): 1, preferably 2000:1.
In some embodiments of the invention, the method uses a reaction system with stevioside concentration of 50-250 g/L, pH of 5-8 and reaction temperature of 20-90 ℃.
In some embodiments of the invention, the reaction system comprises 1.5mL of glycosyltransferase, 0.3mL of sucrose synthase, 2g of sucrose, 1g of stevioside, 1mg of uridine diphosphate or adenosine diphosphate, pH 5.5, and reaction temperature of 60℃per 10mL of reaction system.
In a seventh aspect the present invention provides an enzyme combination comprising a glycosyltransferase according to the first aspect and a sucrose synthase having an amino acid sequence as shown in SEQ ID NO. 24.
In some embodiments of the invention, the sucrose synthase and the glycosyltransferase are used in a mass ratio of 1 (3-10), preferably in a mass ratio of 1:5.
In some embodiments of the invention, the nucleotide sequence encoding the sucrose synthase is shown in SEQ ID NO. 23.
An eighth aspect of the invention provides the use of a glycosyltransferase as described in the first aspect or an enzyme combination as described in the seventh aspect for the preparation of rebaudioside D or rebaudioside E.
In some embodiments of the invention, the rebaudioside D is produced by rebaudioside a.
In some embodiments of the invention, the rebaudioside E is produced by stevioside.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that:
the glycosyltransferase with good catalytic effect and good stability is screened out, the process condition for synthesizing the Reb E by an enzyme method is optimized, and the industrial production of the Reb E is facilitated; solves the problem of high price of glycosyl donor UDPG/ADPG, and reduces the production cost.
Drawings
FIG. 1 shows a synthetic route for preparing rebaudioside E from stevioside.
FIG. 2 shows a graph cut of the results of the HPLC detection method of the present invention, with a retention time of 12.761min for stevioside control.
FIG. 3 shows a profile screenshot of the results using the HPLC detection method of the present invention with a rebaudioside E control retention time of 11.757min.
FIG. 4 is a plot of the HPLC plot of the experimental results of the Enz.5 catalytic synthesis of Reb E in example 5.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
The experimental methods in the invention are all conventional methods unless otherwise specified, and specific reference is made to the "molecular cloning Experimental guidelines" by J.Sam Broker et al for gene cloning operations.
Amino acid shorthand symbols in the invention are conventional in the art unless otherwise specified, and amino acids corresponding to specific shorthand symbols are shown in table 2.
TABLE 2 amino acid alphabet
The codons corresponding to the amino acids are also conventional in the art, and the correspondence of specific amino acids to codons is shown in table 3.
TABLE 3 amino acid codon table
The route of the invention is schematically shown in figure 1.
KOD Mix enzyme was purchased from TOYOBO CO. LTD.DpnI enzyme was purchased from Yingwei Jiegui (Shanghai) trade Co., ltd; competent cells of E.coli Trans10 and E.coli BL21 (DE 3) were purchased from Beijing Ding Guo Changchun Biotechnology Limited. The reaction substrate stevioside was purchased from pichia pastoris (purity 95%). Sucrose was purchased from biological engineering (Shanghai) Inc. Reb E controls were purchased from shanghai source leaf biotechnology limited.
Conversion HPLC detection method: chromatographic column: ZORBAXEclipse plus C18 (4.6 mm. Times.150 mm,3.5 μm). Mobile phase: the aqueous 0.1% TFA solution was mobile phase A and the acetonitrile 0.1% TFA solution was mobile phase B, and the gradient elution was performed as shown in Table 4 below. Detection wavelength: 210nm; flow rate: 1mL/min; sample injection volume: 20. Mu.L; column temperature: 35 ℃. As shown in fig. 2, stevioside peak time: 12.761min; as shown in fig. 3, reb E peak time: 11.757min.
TABLE 4 gradient elution
Time(min) A％ B％
0.00 90 10
15.00 60 40
20.00 0 100
24.00 0 100
24.10 90 10
32.00 90 10
EXAMPLE 1 construction of library of beta-1, 2-glycosyltransferase mutants
The beta-1, 2-glycosyltransferase (beta-1, 2-GT enzyme) gene with the number of Enz.1 shown in SEQ ID NO. 1 is totally synthesized and is connected to a pET28a plasmid vector to obtain a recombinant plasmid pET28a-Enz.1, and a gene synthesis company is a biological engineering (Shanghai) stock company (Shanghai Songjiang region Min road 698). The amino acid sequence of Enz.1 is shown as SEQ ID NO. 2.
The beta-1, 2-glycosyltransferase (beta-1, 2-GT enzyme) enzyme gene with the number of Enz.2 shown in SEQ ID NO. 3 is totally synthesized and is connected to a pET28a plasmid vector to obtain a recombinant plasmid pET28a-Enz.2, and a gene synthesis company is a biological engineering (Shanghai) stock company (Shanghai Songjiang region Min road 698). The amino acid sequence of Enz.2 is shown in SEQ ID NO. 4.
PCR amplification was performed using pET28a-Enz.1 plasmid as a template, and the primer sequences Enz.X-F (X=3 to 10) and Km-R in Table 5, respectively, to obtain fragment one; PCR was performed using pET28a-Enz.2 plasmid as a template, and the primer sequences Enz.X-R (X=3 to 10) and Km-F in Table 5 were used to obtain fragment two. Fragments one and two were recombined using a norvirally homologous recombinase (Exnase II,5 xce II) and ligated into pET28a plasmid vector. After ligation, transformed into E.coli Trans10 competent cells, plated on LB medium containing 50. Mu.g/mL of kananamycin, and cultured overnight at 37 ℃; and (3) picking single colonies to an LB test tube (Km resistance), culturing for 8-10 hours, extracting plasmids, carrying out sequencing conversion and sequencing verification, and obtaining recombinant plasmids pET28 a-Enz.3-pET 28a-Enz.10 of each mutant.
TABLE 5 primer sequence listing
In the table: f is a forward primer, and R is a reverse primer.
The PCR amplification reaction system is as follows:
KOD Mix：25μL
ddH 2 O：20μL
primer: 2 mu L2
And (3) a template: 1 mu L
The amplification procedure was as follows:
(1)98℃3min
(2)98℃10s
(3)55℃5s
(4)68℃5s/kbp
(5)68℃5min
(6) Heat preservation at 12 DEG C
(2) And (4) circulating 34 times.
Example 2 preparation of beta-1, 2-glycosyltransferase
1. Protein expression:
the recombinant plasmids (pET 28 a-Enz.1-pET 28 a-Enz.10) with correct sequencing described in example 1 were transformed into competent cells of the host E.coli BL21 (DE 3) respectively, and genetically engineered strains containing the recombinant plasmids were obtained. Single colonies were individually picked and inoculated into 5mL LB liquid medium containing 50. Mu.g/mL kanamycin, and shake-cultured at 37℃for 4 hours. Transfer to 50mL fresh TB liquid medium also containing 50. Mu.g/mL kanamycin at 2% (v/v) inoculum size, shake culture to OD at 37 ℃ 600 When the concentration reaches about 0.8, IPTG (isopropyl-. Beta. -D-thiogalactoside) is added to the final concentration of 0.1mM, and the culture is induced at 25℃for 20 hours. After the completion of the culture, the culture broth was centrifuged at 4000rpm for 20 minutes, and the supernatant was discarded to collect the cells. Preserving at-20 ℃ for standby.
2. Obtaining crude enzyme liquid:
50mM Phosphate Buffer (PBS) having pH of 5.5 was prepared, and the cells obtained above were suspended at a ratio of 1:10 (M/V, g/mL), and homogenized by a high-pressure homogenizer (550 Mbar homogenization for 1.5 min); and (3) respectively centrifuging the homogenized enzyme solutions at 12000rpm for 2min to obtain crude enzyme solutions of the beta-1, 2-glycosyltransferase.
EXAMPLE 3 preparation of sucrose synthase SUS
The sucrose synthase (SUS) gene shown in SEQ ID NO. 23 was synthesized and ligated to the pET28a plasmid vector to obtain recombinant plasmid pET28a-SUS. The gene synthesis company is biological engineering (Shanghai) stock limited company (Shanghai city, songjiang region Min Ji Lu 698).
Plasmid pET28a-SUS is transformed into host E.coli BL21 (DE 3) competent cells to obtain the engineering strain containing sucrose synthase gene. Single colonies were picked and inoculated into 5mL LB liquid medium containing 50. Mu.g/mL kanamycinShake culturing for 4 hr at 37deg.C. Transfer to 50mL fresh TB liquid medium also containing 50. Mu.g/mL kanamycin at 2% (v/v) inoculum size, shake culture to OD at 37 ℃ 600 When about 0.8 was reached, IPTG was added to a final concentration of 0.1mM and the culture was induced at 25℃for 20 hours. After the completion of the culture, the culture broth was centrifuged at 4000rpm for 20 minutes, and the supernatant was discarded to collect the cells. Preserving at-20 ℃ for standby.
50mM phosphate buffer solution with pH of 5.5 is prepared, the bacterial cells obtained above are suspended according to the ratio of 1:10 (M/V, g/mL), high-pressure homogenization is carried out (550 Mbar, 1.5 min), and then the sucrose synthase crude enzyme solution is obtained after centrifugation at 12000rpm for 2 min.
EXAMPLE 4 screening of beta-1, 2-glycosyltransferase mutants
In a 1mL reaction system, 150. Mu.L of the crude enzyme solution of the beta-1, 2-glycosyltransferase prepared in example 2, 100g/L of Stevioside (STV) and 0.1g/L of UDP/ADP are added, 200g/L of sucrose and 30. Mu.L of the crude enzyme solution of the sucrose synthase are added, and finally 50mM of PBS with pH of 6.0 is added to a final volume of 1mL. The prepared reaction system was placed in a metal bath, reacted at 60℃and 600rpm for 30 minutes, 10. Mu.L of the reaction solution was added to 990. Mu.L of hydrochloric acid having pH2-3, vortexed, centrifuged at 13000rpm for 10 minutes, and the supernatant was analyzed for the concentration of Reb E by HPLC. The experimental results obtained using the HPLC detection method of the present invention are shown in Table 6.
TABLE 6 screening of beta-1, 2-glycosyltransferase mutants
Enzyme numbering Nucleotide sequence Amino acid sequence Reb E％(ADP) Reb E％(UDP)
Enz.1 (control) 1 2 2.327 5.501
Enz.2 (control) 3 4 70.84 33.209
Enz.3 25 26 45.26 33.232
Enz.4 27 28 66.242 35.340
Enz.5 29 30 71.663 42.059
Enz.6 31 32 71.465 35.349
Enz.7 33 34 4.032 33.098
Enz.8 35 36 9.232 44.386
Enz.9 37 38 16.527 44.190
Enz.10 39 40 0.848 2.217
From the preliminary screening results in table 6, it can be seen that: when ADP is used as a sucrose synthase catalytic substrate, the activity of Enz.5 is the highest, and Enz.6 times; when UDP is used as a substrate for catalyzing sucrose synthase, the activity of Enz.8 is best, enz.9 times, the activity of Enz.5 is close to that of Enz.8 and Enz.9, and the catalysis effect of Enz.4 is better than that of the enzyme of a control group. From experimental data, the activity of Enz.5 for catalyzing ADP as a substrate or UDP as a substrate is relatively high, and when ADP is used as a sucrose synthase for catalyzing the substrate, the yield of the Enz.5 for catalyzing and generating the Reb E is far higher than that when UDP is used as the substrate. Thus, ADP is used as a catalytic substrate of sucrose synthase, enz.5 catalyzes glycosyl transfer reaction to prepare rebaudioside E.
EXAMPLE 5 enzyme Enz.5 catalytic Synthesis of Reb E
In a 10mL reaction system, 1.5mL of the crude enzyme solution of beta-1, 2-glycosyltransferase Enz.5 prepared by the method described in example 2, 0.3mL of the crude enzyme solution of sucrose synthase prepared by the method described in example 3, 100g/L stevioside, 200g/L sucrose and 0.1g/L ADP were added, and finally 50mM PBS with pH 5.5 was added to a final volume of 10mL. The prepared reaction system was placed in a metal bath, reacted at 60℃and 600rpm for 7 hours, 10. Mu.L of the reaction solution was added to 990. Mu.L of hydrochloric acid having pH2-3, vortexed, centrifuged at 13000rpm for 10 minutes, and the supernatant was analyzed for the concentration of Reb E by HPLC, as shown in FIG. 4, and the concentration of Reb E was 68.62%.
SEQUENCE LISTING
<110> chess Ke Lai Biotechnology (Shanghai) stock Co., ltd
<120> glycosyltransferase and its use in preparing rebaudioside E
<130> P210110234C
<160> 40
<170> PatentIn version 3.5
<210> 1
<211> 1329
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.1
<400> 1
atggcgacca acctgcgtgt tctgatgttc ccgtggctgg cgtacggcca catcagcccg 60
ttcctgaaca tcgcgaaaca gctggcggat cgtggtttcc tgatctatct gtgctccacc 120
cgcatcaacc tggaatctat catcaagaaa atcccggaaa aatacgcgga ttctatccat 180
ctgatcgaac ttcagctgcc ggagctgccg gaactgccgc cgcactatca caccactaac 240
ggtctgccgc cgcatctgaa cccgaccctg cacaaagcgc tgaaaatgtc taaaccgaac 300
ttcagccgca tcttgcagaa cctgaaaccg gacctgctga tctacgatgt gctccagccg 360
tgggcggaac acgtggcgaa cgaacagggc atcccggctg gcaaactgct ggtttcttgc 420
gcggcggttt tctcctactt tttctctttc cgtaaaaatc cgggcgttga atttccgttc 480
ccggcgatcc acctgccgga agtggaaaaa gttaaaatcc gtgaaatcct ggctaaagaa 540
ccggaagaag gcggccgtct ggacgaaggc aacaaacaga tgatgctgat gtgcacttct 600
cgtaccattg aagctaaata cattgattac tgcaccgaac tgtgcaactg gaaagttgtt 660
ccggttggtc cgccgttcca ggatctgatc actaacgatg cggataacaa agaactgatc 720
gattggctgg gcaccaaacc ggaaaactcc accgtgttcg ttagcttcgg ctccgaatac 780
ttcctgagca aagaagatat ggaagaaatt gctttcgctc tggaagcatc taacgttaac 840
ttcatctggg ttgtgcgttt cccgaaaggc gaagaacgta acctggaaga tgcactgccg 900
gaaggcttcc tggaacgtat tggtgaacgt ggtcgcgttc tggacaaatt cgcgccgcag 960
ccgcgcatcc tgaaccaccc gagcaccggc ggtttcatct ctcactgcgg ttggaacagc 1020
gttatggaaa gcatcgactt cggtgtgccg atcatcgcga tgccgatcca caacgatcag 1080
ccgatcaacg ctaaactgat ggttgaactg ggcgttgcgg ttgaaatcgt tcgtgatgat 1140
gatggtaaaa tccaccgcgg cgaaatcgcg gaagcactga aaagcgttgt gaccggtgaa 1200
accggcgaaa tcctgcgtgc gaaagttcgt gaaatcagca aaaacctgaa atccatccgt 1260
gacgaagaaa tggacgcggt tgctgaagaa ctgatccagc tgtgccgtaa ctctaacaaa 1320
agcaaataa 1329
<210> 2
<211> 442
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.1
<400> 2
Met Ala Thr Asn Leu Arg Val Leu Met Phe Pro Trp Leu Ala Tyr Gly
1 5 10 15
His Ile Ser Pro Phe Leu Asn Ile Ala Lys Gln Leu Ala Asp Arg Gly
20 25 30
Phe Leu Ile Tyr Leu Cys Ser Thr Arg Ile Asn Leu Glu Ser Ile Ile
35 40 45
Lys Lys Ile Pro Glu Lys Tyr Ala Asp Ser Ile His Leu Ile Glu Leu
50 55 60
Gln Leu Pro Glu Leu Pro Glu Leu Pro Pro His Tyr His Thr Thr Asn
65 70 75 80
Gly Leu Pro Pro His Leu Asn Pro Thr Leu His Lys Ala Leu Lys Met
85 90 95
Ser Lys Pro Asn Phe Ser Arg Ile Leu Gln Asn Leu Lys Pro Asp Leu
100 105 110
Leu Ile Tyr Asp Val Leu Gln Pro Trp Ala Glu His Val Ala Asn Glu
115 120 125
Gln Gly Ile Pro Ala Gly Lys Leu Leu Val Ser Cys Ala Ala Val Phe
130 135 140
Ser Tyr Phe Phe Ser Phe Arg Lys Asn Pro Gly Val Glu Phe Pro Phe
145 150 155 160
Pro Ala Ile His Leu Pro Glu Val Glu Lys Val Lys Ile Arg Glu Ile
165 170 175
Leu Ala Lys Glu Pro Glu Glu Gly Gly Arg Leu Asp Glu Gly Asn Lys
180 185 190
Gln Met Met Leu Met Cys Thr Ser Arg Thr Ile Glu Ala Lys Tyr Ile
195 200 205
Asp Tyr Cys Thr Glu Leu Cys Asn Trp Lys Val Val Pro Val Gly Pro
210 215 220
Pro Phe Gln Asp Leu Ile Thr Asn Asp Ala Asp Asn Lys Glu Leu Ile
225 230 235 240
Asp Trp Leu Gly Thr Lys Pro Glu Asn Ser Thr Val Phe Val Ser Phe
245 250 255
Gly Ser Glu Tyr Phe Leu Ser Lys Glu Asp Met Glu Glu Ile Ala Phe
260 265 270
Ala Leu Glu Ala Ser Asn Val Asn Phe Ile Trp Val Val Arg Phe Pro
275 280 285
Lys Gly Glu Glu Arg Asn Leu Glu Asp Ala Leu Pro Glu Gly Phe Leu
290 295 300
Glu Arg Ile Gly Glu Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln
305 310 315 320
Pro Arg Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys
325 330 335
Gly Trp Asn Ser Val Met Glu Ser Ile Asp Phe Gly Val Pro Ile Ile
340 345 350
Ala Met Pro Ile His Asn Asp Gln Pro Ile Asn Ala Lys Leu Met Val
355 360 365
Glu Leu Gly Val Ala Val Glu Ile Val Arg Asp Asp Asp Gly Lys Ile
370 375 380
His Arg Gly Glu Ile Ala Glu Ala Leu Lys Ser Val Val Thr Gly Glu
385 390 395 400
Thr Gly Glu Ile Leu Arg Ala Lys Val Arg Glu Ile Ser Lys Asn Leu
405 410 415
Lys Ser Ile Arg Asp Glu Glu Met Asp Ala Val Ala Glu Glu Leu Ile
420 425 430
Gln Leu Cys Arg Asn Ser Asn Lys Ser Lys
435 440
<210> 3
<211> 1350
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.2
<400> 3
atgcaccatc atcatgaagg cgtgagcgac cagaccctga gagtaacgat gtttccgtgg 60
cttgggctgg gtcatgttaa cccgtttttg cgtatcgcta aacaactggc cgatcgtggt 120
ttcgttatct atttagttag taccgctatt aacctcgaaa tgatcaaaaa gagaatcccg 180
gagaaataca gtaatagcat ccatctggtt gagctgcgcc tgccagaatt accggaactg 240
ccaccacatt accatactac caacggttta ccaccgcatc tgaacaaaac cctgcacaag 300
gcactgaaga tgagcgctcc caactttagc aagatccttc aaaatattaa gccggacctg 360
gtcctttacg attttctggt tccgtgggca gaaaaagtcg cgcttgaaca gggcatcccg 420
gctgttccat tgctaaccag tggtgcggca ctgttcagct actttttcaa cttcctgaag 480
cgaccgggtg aagagtttcc gtttgaggca atccgcctgt cgaagcgaga acaggataag 540
atgcgcgaga tgtttggaac agagccgcct gaagaagatt ttttagcgcc ggcccaggcc 600
ggtatcatgc tgatgtgcac gagccgcgta attgaggcta agtacctgga ctattgtacc 660
gaactgacca atgtaaaagt tgttccggtt ggtccgccgt ttcaggatcc gctgaccgaa 720
gatattgacg accccgaact gatggattgg ttagatacca aacccgaaca tagtgttgtc 780
tatgtgtcgt ttggcagcga agcgttcctg agccgtgaag atatggaaga agtcgcgttc 840
ggcctggagc tgagcggcgt gaactttatc tgggttgcac gctttccgaa aggcgaagaa 900
cagcgtctgg aagacgttct gccaaaaggc ttcctggaac gcgttggtga tcgtggtcgc 960
gttctggacc atctggtgcc gcaggcccat attctgaacc atccgagcac gggtggcttc 1020
atctctcatt gcggttggaa cagcgtcatg gaaagcattg atttcggcgt tccgatcatt 1080
gcgatgccga tgcagtggga tcagccgatt aacgcgagac tgcttgtgga attaggcgtg 1140
gcagtggaga tcccgcgtga tgaagatggc cgggtccacc gcgccgaaat tgcccgtgtc 1200
ctgaaagatg tgatttcggg cccgactggt gagatactgc gcgcgaaagt acgcgacatt 1260
agcgcacgcc tgagagcgag acgcgaggag gaaatgaacg cagcggcgga agaactgata 1320
cagctgtgtc gcaaccgcaa cgcctacaag 1350
<210> 4
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.2
<400> 4
Met His His His His Glu Gly Val Ser Asp Gln Thr Leu Arg Val Thr
1 5 10 15
Met Phe Pro Trp Leu Gly Leu Gly His Val Asn Pro Phe Leu Arg Ile
20 25 30
Ala Lys Gln Leu Ala Asp Arg Gly Phe Val Ile Tyr Leu Val Ser Thr
35 40 45
Ala Ile Asn Leu Glu Met Ile Lys Lys Arg Ile Pro Glu Lys Tyr Ser
50 55 60
Asn Ser Ile His Leu Val Glu Leu Arg Leu Pro Glu Leu Pro Glu Leu
65 70 75 80
Pro Pro His Tyr His Thr Thr Asn Gly Leu Pro Pro His Leu Asn Lys
85 90 95
Thr Leu His Lys Ala Leu Lys Met Ser Ala Pro Asn Phe Ser Lys Ile
100 105 110
Leu Gln Asn Ile Lys Pro Asp Leu Val Leu Tyr Asp Phe Leu Val Pro
115 120 125
Trp Ala Glu Lys Val Ala Leu Glu Gln Gly Ile Pro Ala Val Pro Leu
130 135 140
Leu Thr Ser Gly Ala Ala Leu Phe Ser Tyr Phe Phe Asn Phe Leu Lys
145 150 155 160
Arg Pro Gly Glu Glu Phe Pro Phe Glu Ala Ile Arg Leu Ser Lys Arg
165 170 175
Glu Gln Asp Lys Met Arg Glu Met Phe Gly Thr Glu Pro Pro Glu Glu
180 185 190
Asp Phe Leu Ala Pro Ala Gln Ala Gly Ile Met Leu Met Cys Thr Ser
195 200 205
Arg Val Ile Glu Ala Lys Tyr Leu Asp Tyr Cys Thr Glu Leu Thr Asn
210 215 220
Val Lys Val Val Pro Val Gly Pro Pro Phe Gln Asp Pro Leu Thr Glu
225 230 235 240
Asp Ile Asp Asp Pro Glu Leu Met Asp Trp Leu Asp Thr Lys Pro Glu
245 250 255
His Ser Val Val Tyr Val Ser Phe Gly Ser Glu Ala Phe Leu Ser Arg
260 265 270
Glu Asp Met Glu Glu Val Ala Phe Gly Leu Glu Leu Ser Gly Val Asn
275 280 285
Phe Ile Trp Val Ala Arg Phe Pro Lys Gly Glu Glu Gln Arg Leu Glu
290 295 300
Asp Val Leu Pro Lys Gly Phe Leu Glu Arg Val Gly Asp Arg Gly Arg
305 310 315 320
Val Leu Asp His Leu Val Pro Gln Ala His Ile Leu Asn His Pro Ser
325 330 335
Thr Gly Gly Phe Ile Ser His Cys Gly Trp Asn Ser Val Met Glu Ser
340 345 350
Ile Asp Phe Gly Val Pro Ile Ile Ala Met Pro Met Gln Trp Asp Gln
355 360 365
Pro Ile Asn Ala Arg Leu Leu Val Glu Leu Gly Val Ala Val Glu Ile
370 375 380
Pro Arg Asp Glu Asp Gly Arg Val His Arg Ala Glu Ile Ala Arg Val
385 390 395 400
Leu Lys Asp Val Ile Ser Gly Pro Thr Gly Glu Ile Leu Arg Ala Lys
405 410 415
Val Arg Asp Ile Ser Ala Arg Leu Arg Ala Arg Arg Glu Glu Glu Met
420 425 430
Asn Ala Ala Ala Glu Glu Leu Ile Gln Leu Cys Arg Asn Arg Asn Ala
435 440 445
Tyr Lys
450
<210> 5
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.3-F
<400> 5
gagcacgggt ggtttcatct ctcactgcg 29
<210> 6
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.3-R
<400> 6
agatgaaacc acccgtgctc ggatggttc 29
<210> 7
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.4-F
<400> 7
gtgggatcag ccgatcaacg cta 23
<210> 8
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.4-R
<400> 8
gttgatcggc tgatcccact gca 23
<210> 9
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.5-F
<400> 9
gaaattgccg aagcactgaa aagcgttg 28
<210> 10
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.5-R
<400> 10
ttcagtgctt cggcaatttc ggcgcggtgg 30
<210> 11
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.6-F
<400> 11
gagacgcgac gaagaaatgg acgcgg 26
<210> 12
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.6-R
<400> 12
ccatttcttc gtcgcgtctc gctctcaggc 30
<210> 13
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.7-F
<400> 13
ctcccaactt cagccgcatc ttgc 24
<210> 14
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.7-R
<400> 14
gatgcggctg aagttgggag cgctcatct 29
<210> 15
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.8-F
<400> 15
gttctgccag aaggcttcct ggaacgta 28
<210> 16
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.8-R
<400> 16
ggaagccttc tggcagaacg tcttccaga 29
<210> 17
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.9-F
<400> 17
ctaaaccgaa ctttagcaag atccttcaaa 30
<210> 18
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.9-R
<400> 18
cttgctaaag ttcggtttag acattt 26
<210> 19
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.10-F
<400> 19
gaaaactccg ttgtctatgt gtcgtttg 28
<210> 20
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.10-R
<400> 20
catagacaac ggagttttcc ggtttggtg 29
<210> 21
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Km-F
<400> 21
gcccgacatt atcgcgagc 19
<210> 22
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Km-R
<400> 22
gggtataaat gggctcgcg 19
<210> 23
<211> 2415
<212> DNA
<213> Artificial Sequence
<220>
<223> SUS
<400> 23
atgcaccatc atcatcatca tggcggtagc ggcatgattg aagtactgcg ccaacagctg 60
ctggatagcc cgcgttcatg gcgtgcattc ctgcgtcatt tagtcgcatc tcagcgtgac 120
tcatggctac ataccgattt acagcacgcg tgcaagacgt ttcgtgaaca gcctccggaa 180
ggctatcctg aagatattgg ttggctggca gattttattg cgcattgcca ggaagcgatc 240
ttccgggatc cgtggatggt ttttgcgtgg cgtctacgtc caggtgtttg ggagtatgtg 300
cgcatacatg tagaacagct ggcggtggag gagctgagca ctgatgaata tctgcaagcc 360
aaagaacaac ttgttggctt aggtgcagaa ggtgaagctg ttctgacggt ggatttcgaa 420
gattttcgtc cggtgagcca gcgtttaaaa gacgagagca ccattggtga tggtcttacc 480
catctgaatc gtcatttagc aggtcgcatc tggactgatt tagcagcagg tcgtagtgct 540
attctggaat ttctgggcct gcatcgtctg gataaccaga atctgatgct gagcaacggc 600
aataccgatt ttgactcttt acgtcaaacc gtacaatatc tgggcacctt accaagagaa 660
actccgtggg cagagtttcg tgaagacatg cgtcgtcgtg gttttgaacc cggttggggc 720
aacaccgcgg gccgtgttcg cgaaaccatg cgtctgctga tggatctgct tgactctccg 780
agcccagctg ccctggagag cttcctggat cgcatcccga tgattagcaa cgttctgatc 840
gtgagcattc acggatggtt tgcgcaggac aaggttctgg gtcgtccgga cactggtggt 900
caggtcgtgt atattctgga tcaggcccgt gcactggaac gcgaaatgcg taaccgcctg 960
cgccaacagg gtgttgatgt ggagccgcgc attttgattg cgacccgttt aatcccggaa 1020
agtgatggca cgacttgtga ccagcgtctg gagcctgtcc atggtgccga gaatgtgcag 1080
attctgcgcg ttccgtttcg ctatgaggat ggtcgtattc acccgcattg gatctcacgc 1140
ttcaaggttt ggccgtatct tgaacgctat gcaagggatc tggaacgcga agttaaggcc 1200
gaattaggta gtcgtccaga tctgatcatc ggcaactata gcgacggtgg gctggttgca 1260
accatcctgt cagaaaaatt aggtgttacg cagtgcaaca ttgcacatgc cctggagaaa 1320
agcaagtacc cggggtccga tctgcattgg ccgctgtatg aacaggacca tcactttgcg 1380
tgtcagttta ccgcggatct gatcgcgatg aatgcagcag acatcatcgt gacgagcaca 1440
taccaggaaa ttgcaggtaa tgaccgcgag gttggtcaat atgaatctca ccaggactat 1500
actttaccgg gcttgtatcg tgtcgagaat ggtattgacg tgttcgatag caagtttaac 1560
attgtgagtc cgggcgcaga tccgagtacg tattttagct atgcccgtca tgaagaacgc 1620
ttctcgtcgc tgtggccaga aatcgaaagt ctgctgtttg gccgcgaacc aggtccggat 1680
attcgtggtg ttctcgaaga tcctcagaaa ccgattattc tgtcggtggc ccgtatggat 1740
cgcatcaaga acctgagcgg tctggccgaa ctgtatggtc ggagtgcgcg cttacgtagc 1800
ctggccaatt tggtgatcat cggtggtcat gttgatgtac aggccagtat ggatgcagaa 1860
gaacgcgaag aaatccgtcg tatgcacgag atcatggacc gctaccagct ggatggtcag 1920
atgcgttggg tgggatcgca tctggataaa cgcgtcgtgg gcgaattgta tcgtgtagtg 1980
gcggatggac gtggcgtttt tgtgcaacca gccctgtttg aggcgttcgg cctgaccgtg 2040
attgaggcaa tgagcagtgg cctgccagtg tttgcgaccc gccacggtgg tccgctggaa 2100
atcatcgaag acggcgttag cggcttccat attgatccca acgaccctga agcggtagca 2160
gaaaaactgg ccgacttcct ggaagcagcg cgtgaacgtc cgaagtattg ggaggaaatt 2220
agccaggcgg ctcttgcgcg cgtcagcgaa cgttacacgt gggagcgcta tgcggaacgc 2280
ttgatgacca tcgcgcgttg cttcggcttt tggcgcttcg ttctgtcacg cgaatcacag 2340
gtcatggaac gctatctgca aatgttccgc cacctgcaat ggcgcccgct ggctcatgcc 2400
gtaccgatgg agtaa 2415
<210> 24
<211> 804
<212> PRT
<213> Artificial Sequence
<220>
<223> SUS
<400> 24
Met His His His His His His Gly Gly Ser Gly Met Ile Glu Val Leu
1 5 10 15
Arg Gln Gln Leu Leu Asp Ser Pro Arg Ser Trp Arg Ala Phe Leu Arg
20 25 30
His Leu Val Ala Ser Gln Arg Asp Ser Trp Leu His Thr Asp Leu Gln
35 40 45
His Ala Cys Lys Thr Phe Arg Glu Gln Pro Pro Glu Gly Tyr Pro Glu
50 55 60
Asp Ile Gly Trp Leu Ala Asp Phe Ile Ala His Cys Gln Glu Ala Ile
65 70 75 80
Phe Arg Asp Pro Trp Met Val Phe Ala Trp Arg Leu Arg Pro Gly Val
85 90 95
Trp Glu Tyr Val Arg Ile His Val Glu Gln Leu Ala Val Glu Glu Leu
100 105 110
Ser Thr Asp Glu Tyr Leu Gln Ala Lys Glu Gln Leu Val Gly Leu Gly
115 120 125
Ala Glu Gly Glu Ala Val Leu Thr Val Asp Phe Glu Asp Phe Arg Pro
130 135 140
Val Ser Gln Arg Leu Lys Asp Glu Ser Thr Ile Gly Asp Gly Leu Thr
145 150 155 160
His Leu Asn Arg His Leu Ala Gly Arg Ile Trp Thr Asp Leu Ala Ala
165 170 175
Gly Arg Ser Ala Ile Leu Glu Phe Leu Gly Leu His Arg Leu Asp Asn
180 185 190
Gln Asn Leu Met Leu Ser Asn Gly Asn Thr Asp Phe Asp Ser Leu Arg
195 200 205
Gln Thr Val Gln Tyr Leu Gly Thr Leu Pro Arg Glu Thr Pro Trp Ala
210 215 220
Glu Phe Arg Glu Asp Met Arg Arg Arg Gly Phe Glu Pro Gly Trp Gly
225 230 235 240
Asn Thr Ala Gly Arg Val Arg Glu Thr Met Arg Leu Leu Met Asp Leu
245 250 255
Leu Asp Ser Pro Ser Pro Ala Ala Leu Glu Ser Phe Leu Asp Arg Ile
260 265 270
Pro Met Ile Ser Asn Val Leu Ile Val Ser Ile His Gly Trp Phe Ala
275 280 285
Gln Asp Lys Val Leu Gly Arg Pro Asp Thr Gly Gly Gln Val Val Tyr
290 295 300
Ile Leu Asp Gln Ala Arg Ala Leu Glu Arg Glu Met Arg Asn Arg Leu
305 310 315 320
Arg Gln Gln Gly Val Asp Val Glu Pro Arg Ile Leu Ile Ala Thr Arg
325 330 335
Leu Ile Pro Glu Ser Asp Gly Thr Thr Cys Asp Gln Arg Leu Glu Pro
340 345 350
Val His Gly Ala Glu Asn Val Gln Ile Leu Arg Val Pro Phe Arg Tyr
355 360 365
Glu Asp Gly Arg Ile His Pro His Trp Ile Ser Arg Phe Lys Val Trp
370 375 380
Pro Tyr Leu Glu Arg Tyr Ala Arg Asp Leu Glu Arg Glu Val Lys Ala
385 390 395 400
Glu Leu Gly Ser Arg Pro Asp Leu Ile Ile Gly Asn Tyr Ser Asp Gly
405 410 415
Gly Leu Val Ala Thr Ile Leu Ser Glu Lys Leu Gly Val Thr Gln Cys
420 425 430
Asn Ile Ala His Ala Leu Glu Lys Ser Lys Tyr Pro Gly Ser Asp Leu
435 440 445
His Trp Pro Leu Tyr Glu Gln Asp His His Phe Ala Cys Gln Phe Thr
450 455 460
Ala Asp Leu Ile Ala Met Asn Ala Ala Asp Ile Ile Val Thr Ser Thr
465 470 475 480
Tyr Gln Glu Ile Ala Gly Asn Asp Arg Glu Val Gly Gln Tyr Glu Ser
485 490 495
His Gln Asp Tyr Thr Leu Pro Gly Leu Tyr Arg Val Glu Asn Gly Ile
500 505 510
Asp Val Phe Asp Ser Lys Phe Asn Ile Val Ser Pro Gly Ala Asp Pro
515 520 525
Ser Thr Tyr Phe Ser Tyr Ala Arg His Glu Glu Arg Phe Ser Ser Leu
530 535 540
Trp Pro Glu Ile Glu Ser Leu Leu Phe Gly Arg Glu Pro Gly Pro Asp
545 550 555 560
Ile Arg Gly Val Leu Glu Asp Pro Gln Lys Pro Ile Ile Leu Ser Val
565 570 575
Ala Arg Met Asp Arg Ile Lys Asn Leu Ser Gly Leu Ala Glu Leu Tyr
580 585 590
Gly Arg Ser Ala Arg Leu Arg Ser Leu Ala Asn Leu Val Ile Ile Gly
595 600 605
Gly His Val Asp Val Gln Ala Ser Met Asp Ala Glu Glu Arg Glu Glu
610 615 620
Ile Arg Arg Met His Glu Ile Met Asp Arg Tyr Gln Leu Asp Gly Gln
625 630 635 640
Met Arg Trp Val Gly Ser His Leu Asp Lys Arg Val Val Gly Glu Leu
645 650 655
Tyr Arg Val Val Ala Asp Gly Arg Gly Val Phe Val Gln Pro Ala Leu
660 665 670
Phe Glu Ala Phe Gly Leu Thr Val Ile Glu Ala Met Ser Ser Gly Leu
675 680 685
Pro Val Phe Ala Thr Arg His Gly Gly Pro Leu Glu Ile Ile Glu Asp
690 695 700
Gly Val Ser Gly Phe His Ile Asp Pro Asn Asp Pro Glu Ala Val Ala
705 710 715 720
Glu Lys Leu Ala Asp Phe Leu Glu Ala Ala Arg Glu Arg Pro Lys Tyr
725 730 735
Trp Glu Glu Ile Ser Gln Ala Ala Leu Ala Arg Val Ser Glu Arg Tyr
740 745 750
Thr Trp Glu Arg Tyr Ala Glu Arg Leu Met Thr Ile Ala Arg Cys Phe
755 760 765
Gly Phe Trp Arg Phe Val Leu Ser Arg Glu Ser Gln Val Met Glu Arg
770 775 780
Tyr Leu Gln Met Phe Arg His Leu Gln Trp Arg Pro Leu Ala His Ala
785 790 795 800
Val Pro Met Glu
<210> 25
<211> 1329
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.3
<400> 25
atggcgacca acctgcgtgt tctgatgttc ccgtggctgg cgtacggcca catcagcccg 60
ttcctgaaca tcgcgaaaca gctggcggat cgtggtttcc tgatctatct gtgctccacc 120
cgcatcaacc tggaatctat catcaagaaa atcccggaaa aatacgcgga ttctatccat 180
ctgatcgaac ttcagctgcc ggagctgccg gaactgccgc cgcactatca caccactaac 240
ggtctgccgc cgcatctgaa cccgaccctg cacaaagcgc tgaaaatgtc taaaccgaac 300
ttcagccgca tcttgcagaa cctgaaaccg gacctgctga tctacgatgt gctccagccg 360
tgggcggaac acgtggcgaa cgaacagggc atcccggctg gcaaactgct ggtttcttgc 420
gcggcggttt tctcctactt tttctctttc cgtaaaaatc cgggcgttga atttccgttc 480
ccggcgatcc acctgccgga agtggaaaaa gttaaaatcc gtgaaatcct ggctaaagaa 540
ccggaagaag gcggccgtct ggacgaaggc aacaaacaga tgatgctgat gtgcacttct 600
cgtaccattg aagctaaata cattgattac tgcaccgaac tgtgcaactg gaaagttgtt 660
ccggttggtc cgccgttcca ggatctgatc actaacgatg cggataacaa agaactgatc 720
gattggctgg gcaccaaacc ggaaaactcc accgtgttcg ttagcttcgg ctccgaatac 780
ttcctgagca aagaagatat ggaagaaatt gctttcgctc tggaagcatc taacgttaac 840
ttcatctggg ttgtgcgttt cccgaaaggc gaagaacgta acctggaaga tgcactgccg 900
aaaggcttcc tggaacgcgt tggtgatcgt ggtcgcgttc tggaccatct ggtgccgcag 960
gcccatattc tgaaccatcc gagcacgggt ggtttcatct ctcactgcgg ttggaacagc 1020
gttatggaaa gcatcgactt cggtgtgccg atcatcgcga tgccgatcca caacgatcag 1080
ccgatcaacg ctaaactgat ggttgaactg ggcgttgcgg ttgaaatcgt tcgtgatgat 1140
gatggtaaaa tccaccgcgg cgaaatcgcg gaagcactga aaagcgttgt gaccggtgaa 1200
accggcgaaa tcctgcgtgc gaaagttcgt gaaatcagca aaaacctgaa atccatccgt 1260
gacgaagaaa tggacgcggt tgctgaagaa ctgatccagc tgtgccgtaa ctctaacaaa 1320
agcaaataa 1329
<210> 26
<211> 442
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.3
<400> 26
Met Ala Thr Asn Leu Arg Val Leu Met Phe Pro Trp Leu Ala Tyr Gly
1 5 10 15
His Ile Ser Pro Phe Leu Asn Ile Ala Lys Gln Leu Ala Asp Arg Gly
20 25 30
Phe Leu Ile Tyr Leu Cys Ser Thr Arg Ile Asn Leu Glu Ser Ile Ile
35 40 45
Lys Lys Ile Pro Glu Lys Tyr Ala Asp Ser Ile His Leu Ile Glu Leu
50 55 60
Gln Leu Pro Glu Leu Pro Glu Leu Pro Pro His Tyr His Thr Thr Asn
65 70 75 80
Gly Leu Pro Pro His Leu Asn Pro Thr Leu His Lys Ala Leu Lys Met
85 90 95
Ser Lys Pro Asn Phe Ser Arg Ile Leu Gln Asn Leu Lys Pro Asp Leu
100 105 110
Leu Ile Tyr Asp Val Leu Gln Pro Trp Ala Glu His Val Ala Asn Glu
115 120 125
Gln Gly Ile Pro Ala Gly Lys Leu Leu Val Ser Cys Ala Ala Val Phe
130 135 140
Ser Tyr Phe Phe Ser Phe Arg Lys Asn Pro Gly Val Glu Phe Pro Phe
145 150 155 160
Pro Ala Ile His Leu Pro Glu Val Glu Lys Val Lys Ile Arg Glu Ile
165 170 175
Leu Ala Lys Glu Pro Glu Glu Gly Gly Arg Leu Asp Glu Gly Asn Lys
180 185 190
Gln Met Met Leu Met Cys Thr Ser Arg Thr Ile Glu Ala Lys Tyr Ile
195 200 205
Asp Tyr Cys Thr Glu Leu Cys Asn Trp Lys Val Val Pro Val Gly Pro
210 215 220
Pro Phe Gln Asp Leu Ile Thr Asn Asp Ala Asp Asn Lys Glu Leu Ile
225 230 235 240
Asp Trp Leu Gly Thr Lys Pro Glu Asn Ser Thr Val Phe Val Ser Phe
245 250 255
Gly Ser Glu Tyr Phe Leu Ser Lys Glu Asp Met Glu Glu Ile Ala Phe
260 265 270
Ala Leu Glu Ala Ser Asn Val Asn Phe Ile Trp Val Val Arg Phe Pro
275 280 285
Lys Gly Glu Glu Arg Asn Leu Glu Asp Ala Leu Pro Lys Gly Phe Leu
290 295 300
Glu Arg Val Gly Asp Arg Gly Arg Val Leu Asp His Leu Val Pro Gln
305 310 315 320
Ala His Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys
325 330 335
Gly Trp Asn Ser Val Met Glu Ser Ile Asp Phe Gly Val Pro Ile Ile
340 345 350
Ala Met Pro Ile His Asn Asp Gln Pro Ile Asn Ala Lys Leu Met Val
355 360 365
Glu Leu Gly Val Ala Val Glu Ile Val Arg Asp Asp Asp Gly Lys Ile
370 375 380
His Arg Gly Glu Ile Ala Glu Ala Leu Lys Ser Val Val Thr Gly Glu
385 390 395 400
Thr Gly Glu Ile Leu Arg Ala Lys Val Arg Glu Ile Ser Lys Asn Leu
405 410 415
Lys Ser Ile Arg Asp Glu Glu Met Asp Ala Val Ala Glu Glu Leu Ile
420 425 430
Gln Leu Cys Arg Asn Ser Asn Lys Ser Lys
435 440
<210> 27
<211> 1353
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.4
<400> 27
atgcaccatc atcatgaagg cgtgagcgac cagaccctga gagtaacgat gtttccgtgg 60
cttgggctgg gtcatgttaa cccgtttttg cgtatcgcta aacaactggc cgatcgtggt 120
ttcgttatct atttagttag taccgctatt aacctcgaaa tgatcaaaaa gagaatcccg 180
gagaaataca gtaatagcat ccatctggtt gagctgcgcc tgccagaatt accggaactg 240
ccaccacatt accatactac caacggttta ccaccgcatc tgaacaaaac cctgcacaag 300
gcactgaaga tgagcgctcc caactttagc aagatccttc aaaatattaa gccggacctg 360
gtcctttacg attttctggt tccgtgggca gaaaaagtcg cgcttgaaca gggcatcccg 420
gctgttccat tgctaaccag tggtgcggca ctgttcagct actttttcaa cttcctgaag 480
cgaccgggtg aagagtttcc gtttgaggca atccgcctgt cgaagcgaga acaggataag 540
atgcgcgaga tgtttggaac agagccgcct gaagaagatt ttttagcgcc ggcccaggcc 600
ggtatcatgc tgatgtgcac gagccgcgta attgaggcta agtacctgga ctattgtacc 660
gaactgacca atgtaaaagt tgttccggtt ggtccgccgt ttcaggatcc gctgaccgaa 720
gatattgacg accccgaact gatggattgg ttagatacca aacccgaaca tagtgttgtc 780
tatgtgtcgt ttggcagcga agcgttcctg agccgtgaag atatggaaga agtcgcgttc 840
ggcctggagc tgagcggcgt gaactttatc tgggttgcac gctttccgaa aggcgaagaa 900
cagcgtctgg aagacgttct gccaaaaggc ttcctggaac gcgttggtga tcgtggtcgc 960
gttctggacc atctggtgcc gcaggcccat attctgaacc atccgagcac gggtggcttc 1020
atctctcatt gcggttggaa cagcgtcatg gaaagcattg atttcggcgt tccgatcatt 1080
gcgatgccga tgcagtggga tcagccgatc aacgctaaac tgatggttga actgggcgtt 1140
gcggttgaaa tcgttcgtga tgatgatggt aaaatccacc gcggcgaaat cgcggaagca 1200
ctgaaaagcg ttgtgaccgg tgaaaccggc gaaatcctgc gtgcgaaagt tcgtgaaatc 1260
agcaaaaacc tgaaatccat ccgtgacgaa gaaatggacg cggttgctga agaactgatc 1320
cagctgtgcc gtaactctaa caaaagcaaa taa 1353
<210> 28
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.4
<400> 28
Met His His His His Glu Gly Val Ser Asp Gln Thr Leu Arg Val Thr
1 5 10 15
Met Phe Pro Trp Leu Gly Leu Gly His Val Asn Pro Phe Leu Arg Ile
20 25 30
Ala Lys Gln Leu Ala Asp Arg Gly Phe Val Ile Tyr Leu Val Ser Thr
35 40 45
Ala Ile Asn Leu Glu Met Ile Lys Lys Arg Ile Pro Glu Lys Tyr Ser
50 55 60
Asn Ser Ile His Leu Val Glu Leu Arg Leu Pro Glu Leu Pro Glu Leu
65 70 75 80
Pro Pro His Tyr His Thr Thr Asn Gly Leu Pro Pro His Leu Asn Lys
85 90 95
Thr Leu His Lys Ala Leu Lys Met Ser Ala Pro Asn Phe Ser Lys Ile
100 105 110
Leu Gln Asn Ile Lys Pro Asp Leu Val Leu Tyr Asp Phe Leu Val Pro
115 120 125
Trp Ala Glu Lys Val Ala Leu Glu Gln Gly Ile Pro Ala Val Pro Leu
130 135 140
Leu Thr Ser Gly Ala Ala Leu Phe Ser Tyr Phe Phe Asn Phe Leu Lys
145 150 155 160
Arg Pro Gly Glu Glu Phe Pro Phe Glu Ala Ile Arg Leu Ser Lys Arg
165 170 175
Glu Gln Asp Lys Met Arg Glu Met Phe Gly Thr Glu Pro Pro Glu Glu
180 185 190
Asp Phe Leu Ala Pro Ala Gln Ala Gly Ile Met Leu Met Cys Thr Ser
195 200 205
Arg Val Ile Glu Ala Lys Tyr Leu Asp Tyr Cys Thr Glu Leu Thr Asn
210 215 220
Val Lys Val Val Pro Val Gly Pro Pro Phe Gln Asp Pro Leu Thr Glu
225 230 235 240
Asp Ile Asp Asp Pro Glu Leu Met Asp Trp Leu Asp Thr Lys Pro Glu
245 250 255
His Ser Val Val Tyr Val Ser Phe Gly Ser Glu Ala Phe Leu Ser Arg
260 265 270
Glu Asp Met Glu Glu Val Ala Phe Gly Leu Glu Leu Ser Gly Val Asn
275 280 285
Phe Ile Trp Val Ala Arg Phe Pro Lys Gly Glu Glu Gln Arg Leu Glu
290 295 300
Asp Val Leu Pro Lys Gly Phe Leu Glu Arg Val Gly Asp Arg Gly Arg
305 310 315 320
Val Leu Asp His Leu Val Pro Gln Ala His Ile Leu Asn His Pro Ser
325 330 335
Thr Gly Gly Phe Ile Ser His Cys Gly Trp Asn Ser Val Met Glu Ser
340 345 350
Ile Asp Phe Gly Val Pro Ile Ile Ala Met Pro Met Gln Trp Asp Gln
355 360 365
Pro Ile Asn Ala Lys Leu Met Val Glu Leu Gly Val Ala Val Glu Ile
370 375 380
Val Arg Asp Asp Asp Gly Lys Ile His Arg Gly Glu Ile Ala Glu Ala
385 390 395 400
Leu Lys Ser Val Val Thr Gly Glu Thr Gly Glu Ile Leu Arg Ala Lys
405 410 415
Val Arg Glu Ile Ser Lys Asn Leu Lys Ser Ile Arg Asp Glu Glu Met
420 425 430
Asp Ala Val Ala Glu Glu Leu Ile Gln Leu Cys Arg Asn Ser Asn Lys
435 440 445
Ser Lys
450
<210> 29
<211> 1353
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.5
<400> 29
atgcaccatc atcatgaagg cgtgagcgac cagaccctga gagtaacgat gtttccgtgg 60
cttgggctgg gtcatgttaa cccgtttttg cgtatcgcta aacaactggc cgatcgtggt 120
ttcgttatct atttagttag taccgctatt aacctcgaaa tgatcaaaaa gagaatcccg 180
gagaaataca gtaatagcat ccatctggtt gagctgcgcc tgccagaatt accggaactg 240
ccaccacatt accatactac caacggttta ccaccgcatc tgaacaaaac cctgcacaag 300
gcactgaaga tgagcgctcc caactttagc aagatccttc aaaatattaa gccggacctg 360
gtcctttacg attttctggt tccgtgggca gaaaaagtcg cgcttgaaca gggcatcccg 420
gctgttccat tgctaaccag tggtgcggca ctgttcagct actttttcaa cttcctgaag 480
cgaccgggtg aagagtttcc gtttgaggca atccgcctgt cgaagcgaga acaggataag 540
atgcgcgaga tgtttggaac agagccgcct gaagaagatt ttttagcgcc ggcccaggcc 600
ggtatcatgc tgatgtgcac gagccgcgta attgaggcta agtacctgga ctattgtacc 660
gaactgacca atgtaaaagt tgttccggtt ggtccgccgt ttcaggatcc gctgaccgaa 720
gatattgacg accccgaact gatggattgg ttagatacca aacccgaaca tagtgttgtc 780
tatgtgtcgt ttggcagcga agcgttcctg agccgtgaag atatggaaga agtcgcgttc 840
ggcctggagc tgagcggcgt gaactttatc tgggttgcac gctttccgaa aggcgaagaa 900
cagcgtctgg aagacgttct gccaaaaggc ttcctggaac gcgttggtga tcgtggtcgc 960
gttctggacc atctggtgcc gcaggcccat attctgaacc atccgagcac gggtggcttc 1020
atctctcatt gcggttggaa cagcgtcatg gaaagcattg atttcggcgt tccgatcatt 1080
gcgatgccga tgcagtggga tcagccgatt aacgcgagac tgcttgtgga attaggcgtg 1140
gcagtggaga tcccgcgtga tgaagatggc cgggtccacc gcgccgaaat tgccgaagca 1200
ctgaaaagcg ttgtgaccgg tgaaaccggc gaaatcctgc gtgcgaaagt tcgtgaaatc 1260
agcaaaaacc tgaaatccat ccgtgacgaa gaaatggacg cggttgctga agaactgatc 1320
cagctgtgcc gtaactctaa caaaagcaaa taa 1353
<210> 30
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.5
<400> 30
Met His His His His Glu Gly Val Ser Asp Gln Thr Leu Arg Val Thr
1 5 10 15
Met Phe Pro Trp Leu Gly Leu Gly His Val Asn Pro Phe Leu Arg Ile
20 25 30
Ala Lys Gln Leu Ala Asp Arg Gly Phe Val Ile Tyr Leu Val Ser Thr
35 40 45
Ala Ile Asn Leu Glu Met Ile Lys Lys Arg Ile Pro Glu Lys Tyr Ser
50 55 60
Asn Ser Ile His Leu Val Glu Leu Arg Leu Pro Glu Leu Pro Glu Leu
65 70 75 80
Pro Pro His Tyr His Thr Thr Asn Gly Leu Pro Pro His Leu Asn Lys
85 90 95
Thr Leu His Lys Ala Leu Lys Met Ser Ala Pro Asn Phe Ser Lys Ile
100 105 110
Leu Gln Asn Ile Lys Pro Asp Leu Val Leu Tyr Asp Phe Leu Val Pro
115 120 125
Trp Ala Glu Lys Val Ala Leu Glu Gln Gly Ile Pro Ala Val Pro Leu
130 135 140
Leu Thr Ser Gly Ala Ala Leu Phe Ser Tyr Phe Phe Asn Phe Leu Lys
145 150 155 160
Arg Pro Gly Glu Glu Phe Pro Phe Glu Ala Ile Arg Leu Ser Lys Arg
165 170 175
Glu Gln Asp Lys Met Arg Glu Met Phe Gly Thr Glu Pro Pro Glu Glu
180 185 190
Asp Phe Leu Ala Pro Ala Gln Ala Gly Ile Met Leu Met Cys Thr Ser
195 200 205
Arg Val Ile Glu Ala Lys Tyr Leu Asp Tyr Cys Thr Glu Leu Thr Asn
210 215 220
Val Lys Val Val Pro Val Gly Pro Pro Phe Gln Asp Pro Leu Thr Glu
225 230 235 240
Asp Ile Asp Asp Pro Glu Leu Met Asp Trp Leu Asp Thr Lys Pro Glu
245 250 255
His Ser Val Val Tyr Val Ser Phe Gly Ser Glu Ala Phe Leu Ser Arg
260 265 270
Glu Asp Met Glu Glu Val Ala Phe Gly Leu Glu Leu Ser Gly Val Asn
275 280 285
Phe Ile Trp Val Ala Arg Phe Pro Lys Gly Glu Glu Gln Arg Leu Glu
290 295 300
Asp Val Leu Pro Lys Gly Phe Leu Glu Arg Val Gly Asp Arg Gly Arg
305 310 315 320
Val Leu Asp His Leu Val Pro Gln Ala His Ile Leu Asn His Pro Ser
325 330 335
Thr Gly Gly Phe Ile Ser His Cys Gly Trp Asn Ser Val Met Glu Ser
340 345 350
Ile Asp Phe Gly Val Pro Ile Ile Ala Met Pro Met Gln Trp Asp Gln
355 360 365
Pro Ile Asn Ala Arg Leu Leu Val Glu Leu Gly Val Ala Val Glu Ile
370 375 380
Pro Arg Asp Glu Asp Gly Arg Val His Arg Ala Glu Ile Ala Glu Ala
385 390 395 400
Leu Lys Ser Val Val Thr Gly Glu Thr Gly Glu Ile Leu Arg Ala Lys
405 410 415
Val Arg Glu Ile Ser Lys Asn Leu Lys Ser Ile Arg Asp Glu Glu Met
420 425 430
Asp Ala Val Ala Glu Glu Leu Ile Gln Leu Cys Arg Asn Ser Asn Lys
435 440 445
Ser Lys
450
<210> 31
<211> 1353
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.6
<400> 31
atgcaccatc atcatgaagg cgtgagcgac cagaccctga gagtaacgat gtttccgtgg 60
cttgggctgg gtcatgttaa cccgtttttg cgtatcgcta aacaactggc cgatcgtggt 120
ttcgttatct atttagttag taccgctatt aacctcgaaa tgatcaaaaa gagaatcccg 180
gagaaataca gtaatagcat ccatctggtt gagctgcgcc tgccagaatt accggaactg 240
ccaccacatt accatactac caacggttta ccaccgcatc tgaacaaaac cctgcacaag 300
gcactgaaga tgagcgctcc caactttagc aagatccttc aaaatattaa gccggacctg 360
gtcctttacg attttctggt tccgtgggca gaaaaagtcg cgcttgaaca gggcatcccg 420
gctgttccat tgctaaccag tggtgcggca ctgttcagct actttttcaa cttcctgaag 480
cgaccgggtg aagagtttcc gtttgaggca atccgcctgt cgaagcgaga acaggataag 540
atgcgcgaga tgtttggaac agagccgcct gaagaagatt ttttagcgcc ggcccaggcc 600
ggtatcatgc tgatgtgcac gagccgcgta attgaggcta agtacctgga ctattgtacc 660
gaactgacca atgtaaaagt tgttccggtt ggtccgccgt ttcaggatcc gctgaccgaa 720
gatattgacg accccgaact gatggattgg ttagatacca aacccgaaca tagtgttgtc 780
tatgtgtcgt ttggcagcga agcgttcctg agccgtgaag atatggaaga agtcgcgttc 840
ggcctggagc tgagcggcgt gaactttatc tgggttgcac gctttccgaa aggcgaagaa 900
cagcgtctgg aagacgttct gccaaaaggc ttcctggaac gcgttggtga tcgtggtcgc 960
gttctggacc atctggtgcc gcaggcccat attctgaacc atccgagcac gggtggcttc 1020
atctctcatt gcggttggaa cagcgtcatg gaaagcattg atttcggcgt tccgatcatt 1080
gcgatgccga tgcagtggga tcagccgatt aacgcgagac tgcttgtgga attaggcgtg 1140
gcagtggaga tcccgcgtga tgaagatggc cgggtccacc gcgccgaaat tgcccgtgtc 1200
ctgaaagatg tgatttcggg cccgactggt gagatactgc gcgcgaaagt acgcgacatt 1260
agcgcacgcc tgagagcgag acgcgacgaa gaaatggacg cggttgctga agaactgatc 1320
cagctgtgcc gtaactctaa caaaagcaaa taa 1353
<210> 32
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.6
<400> 32
Met His His His His Glu Gly Val Ser Asp Gln Thr Leu Arg Val Thr
1 5 10 15
Met Phe Pro Trp Leu Gly Leu Gly His Val Asn Pro Phe Leu Arg Ile
20 25 30
Ala Lys Gln Leu Ala Asp Arg Gly Phe Val Ile Tyr Leu Val Ser Thr
35 40 45
Ala Ile Asn Leu Glu Met Ile Lys Lys Arg Ile Pro Glu Lys Tyr Ser
50 55 60
Asn Ser Ile His Leu Val Glu Leu Arg Leu Pro Glu Leu Pro Glu Leu
65 70 75 80
Pro Pro His Tyr His Thr Thr Asn Gly Leu Pro Pro His Leu Asn Lys
85 90 95
Thr Leu His Lys Ala Leu Lys Met Ser Ala Pro Asn Phe Ser Lys Ile
100 105 110
Leu Gln Asn Ile Lys Pro Asp Leu Val Leu Tyr Asp Phe Leu Val Pro
115 120 125
Trp Ala Glu Lys Val Ala Leu Glu Gln Gly Ile Pro Ala Val Pro Leu
130 135 140
Leu Thr Ser Gly Ala Ala Leu Phe Ser Tyr Phe Phe Asn Phe Leu Lys
145 150 155 160
Arg Pro Gly Glu Glu Phe Pro Phe Glu Ala Ile Arg Leu Ser Lys Arg
165 170 175
Glu Gln Asp Lys Met Arg Glu Met Phe Gly Thr Glu Pro Pro Glu Glu
180 185 190
Asp Phe Leu Ala Pro Ala Gln Ala Gly Ile Met Leu Met Cys Thr Ser
195 200 205
Arg Val Ile Glu Ala Lys Tyr Leu Asp Tyr Cys Thr Glu Leu Thr Asn
210 215 220
Val Lys Val Val Pro Val Gly Pro Pro Phe Gln Asp Pro Leu Thr Glu
225 230 235 240
Asp Ile Asp Asp Pro Glu Leu Met Asp Trp Leu Asp Thr Lys Pro Glu
245 250 255
His Ser Val Val Tyr Val Ser Phe Gly Ser Glu Ala Phe Leu Ser Arg
260 265 270
Glu Asp Met Glu Glu Val Ala Phe Gly Leu Glu Leu Ser Gly Val Asn
275 280 285
Phe Ile Trp Val Ala Arg Phe Pro Lys Gly Glu Glu Gln Arg Leu Glu
290 295 300
Asp Val Leu Pro Lys Gly Phe Leu Glu Arg Val Gly Asp Arg Gly Arg
305 310 315 320
Val Leu Asp His Leu Val Pro Gln Ala His Ile Leu Asn His Pro Ser
325 330 335
Thr Gly Gly Phe Ile Ser His Cys Gly Trp Asn Ser Val Met Glu Ser
340 345 350
Ile Asp Phe Gly Val Pro Ile Ile Ala Met Pro Met Gln Trp Asp Gln
355 360 365
Pro Ile Asn Ala Arg Leu Leu Val Glu Leu Gly Val Ala Val Glu Ile
370 375 380
Pro Arg Asp Glu Asp Gly Arg Val His Arg Ala Glu Ile Ala Arg Val
385 390 395 400
Leu Lys Asp Val Ile Ser Gly Pro Thr Gly Glu Ile Leu Arg Ala Lys
405 410 415
Val Arg Asp Ile Ser Ala Arg Leu Arg Ala Arg Arg Asp Glu Glu Met
420 425 430
Asp Ala Val Ala Glu Glu Leu Ile Gln Leu Cys Arg Asn Ser Asn Lys
435 440 445
Ser Lys
450
<210> 33
<211> 1353
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.7
<400> 33
atgcaccatc atcatgaagg cgtgagcgac cagaccctga gagtaacgat gtttccgtgg 60
cttgggctgg gtcatgttaa cccgtttttg cgtatcgcta aacaactggc cgatcgtggt 120
ttcgttatct atttagttag taccgctatt aacctcgaaa tgatcaaaaa gagaatcccg 180
gagaaataca gtaatagcat ccatctggtt gagctgcgcc tgccagaatt accggaactg 240
ccaccacatt accatactac caacggttta ccaccgcatc tgaacaaaac cctgcacaag 300
gcactgaaga tgagcgctcc caactttagc aagatccttc aaaatattaa gccggacctg 360
gtcctttacg attttctggt tccgtgggca gaaaaagtcg cgcttgaaca gggcatcccg 420
gctgttccat tgctaaccag tggtgcggca ctgttcagct actttttcaa cttcctgaag 480
cgaccgggtg aagagtttcc gtttgaggca atccgcctgt cgaagcgaga acaggataag 540
atgcgcgaga tgtttggaac agagccgcct gaagaagatt ttttagcgcc ggcccaggcc 600
ggtatcatgc tgatgtgcac gagccgtacc attgaagcta aatacattga ttactgcacc 660
gaactgtgca actggaaagt tgttccggtt ggtccgccgt tccaggatct gatcactaac 720
gatgcggata acaaagaact gatcgattgg ctgggcacca aaccggaaaa ctccaccgtg 780
ttcgttagct tcggctccga atacttcctg agcaaagaag atatggaaga aattgctttc 840
gctctggaag catctaacgt taacttcatc tgggttgtgc gtttcccgaa aggcgaagaa 900
cgtaacctgg aagatgcact gccggaaggc ttcctggaac gtattggtga acgtggtcgc 960
gttctggaca aattcgcgcc gcagccgcgc atcctgaacc acccgagcac cggcggtttc 1020
atctctcact gcggttggaa cagcgttatg gaaagcatcg acttcggtgt gccgatcatc 1080
gcgatgccga tccacaacga tcagccgatc aacgctaaac tgatggttga actgggcgtt 1140
gcggttgaaa tcgttcgtga tgatgatggt aaaatccacc gcggcgaaat cgcggaagca 1200
ctgaaaagcg ttgtgaccgg tgaaaccggc gaaatcctgc gtgcgaaagt tcgtgaaatc 1260
agcaaaaacc tgaaatccat ccgtgacgaa gaaatggacg cggttgctga agaactgatc 1320
cagctgtgcc gtaactctaa caaaagcaaa taa 1353
<210> 34
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.7
<400> 34
Met His His His His Glu Gly Val Ser Asp Gln Thr Leu Arg Val Thr
1 5 10 15
Met Phe Pro Trp Leu Gly Leu Gly His Val Asn Pro Phe Leu Arg Ile
20 25 30
Ala Lys Gln Leu Ala Asp Arg Gly Phe Val Ile Tyr Leu Val Ser Thr
35 40 45
Ala Ile Asn Leu Glu Met Ile Lys Lys Arg Ile Pro Glu Lys Tyr Ser
50 55 60
Asn Ser Ile His Leu Val Glu Leu Arg Leu Pro Glu Leu Pro Glu Leu
65 70 75 80
Pro Pro His Tyr His Thr Thr Asn Gly Leu Pro Pro His Leu Asn Lys
85 90 95
Thr Leu His Lys Ala Leu Lys Met Ser Ala Pro Asn Phe Ser Lys Ile
100 105 110
Leu Gln Asn Ile Lys Pro Asp Leu Val Leu Tyr Asp Phe Leu Val Pro
115 120 125
Trp Ala Glu Lys Val Ala Leu Glu Gln Gly Ile Pro Ala Val Pro Leu
130 135 140
Leu Thr Ser Gly Ala Ala Leu Phe Ser Tyr Phe Phe Asn Phe Leu Lys
145 150 155 160
Arg Pro Gly Glu Glu Phe Pro Phe Glu Ala Ile Arg Leu Ser Lys Arg
165 170 175
Glu Gln Asp Lys Met Arg Glu Met Phe Gly Thr Glu Pro Pro Glu Glu
180 185 190
Asp Phe Leu Ala Pro Ala Gln Ala Gly Ile Met Leu Met Cys Thr Ser
195 200 205
Arg Thr Ile Glu Ala Lys Tyr Ile Asp Tyr Cys Thr Glu Leu Cys Asn
210 215 220
Trp Lys Val Val Pro Val Gly Pro Pro Phe Gln Asp Leu Ile Thr Asn
225 230 235 240
Asp Ala Asp Asn Lys Glu Leu Ile Asp Trp Leu Gly Thr Lys Pro Glu
245 250 255
Asn Ser Thr Val Phe Val Ser Phe Gly Ser Glu Tyr Phe Leu Ser Lys
260 265 270
Glu Asp Met Glu Glu Ile Ala Phe Ala Leu Glu Ala Ser Asn Val Asn
275 280 285
Phe Ile Trp Val Val Arg Phe Pro Lys Gly Glu Glu Arg Asn Leu Glu
290 295 300
Asp Ala Leu Pro Glu Gly Phe Leu Glu Arg Ile Gly Glu Arg Gly Arg
305 310 315 320
Val Leu Asp Lys Phe Ala Pro Gln Pro Arg Ile Leu Asn His Pro Ser
325 330 335
Thr Gly Gly Phe Ile Ser His Cys Gly Trp Asn Ser Val Met Glu Ser
340 345 350
Ile Asp Phe Gly Val Pro Ile Ile Ala Met Pro Ile His Asn Asp Gln
355 360 365
Pro Ile Asn Ala Lys Leu Met Val Glu Leu Gly Val Ala Val Glu Ile
370 375 380
Val Arg Asp Asp Asp Gly Lys Ile His Arg Gly Glu Ile Ala Glu Ala
385 390 395 400
Leu Lys Ser Val Val Thr Gly Glu Thr Gly Glu Ile Leu Arg Ala Lys
405 410 415
Val Arg Glu Ile Ser Lys Asn Leu Lys Ser Ile Arg Asp Glu Glu Met
420 425 430
Asp Ala Val Ala Glu Glu Leu Ile Gln Leu Cys Arg Asn Ser Asn Lys
435 440 445
Ser Lys
450
<210> 35
<211> 1353
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.8
<400> 35
atgcaccatc atcatgaagg cgtgagcgac cagaccctga gagtaacgat gtttccgtgg 60
cttgggctgg gtcatgttaa cccgtttttg cgtatcgcta aacaactggc cgatcgtggt 120
ttcgttatct atttagttag taccgctatt aacctcgaaa tgatcaaaaa gagaatcccg 180
gagaaataca gtaatagcat ccatctggtt gagctgcgcc tgccagaatt accggaactg 240
ccaccacatt accatactac caacggttta ccaccgcatc tgaacaaaac cctgcacaag 300
gcactgaaga tgagcgctcc caactttagc aagatccttc aaaatattaa gccggacctg 360
gtcctttacg attttctggt tccgtgggca gaaaaagtcg cgcttgaaca gggcatcccg 420
gctgttccat tgctaaccag tggtgcggca ctgttcagct actttttcaa cttcctgaag 480
cgaccgggtg aagagtttcc gtttgaggca atccgcctgt cgaagcgaga acaggataag 540
atgcgcgaga tgtttggaac agagccgcct gaagaagatt ttttagcgcc ggcccaggcc 600
ggtatcatgc tgatgtgcac gagccgcgta attgaggcta agtacctgga ctattgtacc 660
gaactgacca atgtaaaagt tgttccggtt ggtccgccgt ttcaggatcc gctgaccgaa 720
gatattgacg accccgaact gatggattgg ttagatacca aacccgaaca tagtgttgtc 780
tatgtgtcgt ttggcagcga agcgttcctg agccgtgaag atatggaaga agtcgcgttc 840
ggcctggagc tgagcggcgt gaactttatc tgggttgcac gctttccgaa aggcgaagaa 900
cagcgtctgg aagacgttct gccagaaggc ttcctggaac gtattggtga acgtggtcgc 960
gttctggaca aattcgcgcc gcagccgcgc atcctgaacc acccgagcac cggcggtttc 1020
atctctcact gcggttggaa cagcgttatg gaaagcatcg acttcggtgt gccgatcatc 1080
gcgatgccga tccacaacga tcagccgatc aacgctaaac tgatggttga actgggcgtt 1140
gcggttgaaa tcgttcgtga tgatgatggt aaaatccacc gcggcgaaat cgcggaagca 1200
ctgaaaagcg ttgtgaccgg tgaaaccggc gaaatcctgc gtgcgaaagt tcgtgaaatc 1260
agcaaaaacc tgaaatccat ccgtgacgaa gaaatggacg cggttgctga agaactgatc 1320
cagctgtgcc gtaactctaa caaaagcaaa taa 1353
<210> 36
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.8
<400> 36
Met His His His His Glu Gly Val Ser Asp Gln Thr Leu Arg Val Thr
1 5 10 15
Met Phe Pro Trp Leu Gly Leu Gly His Val Asn Pro Phe Leu Arg Ile
20 25 30
Ala Lys Gln Leu Ala Asp Arg Gly Phe Val Ile Tyr Leu Val Ser Thr
35 40 45
Ala Ile Asn Leu Glu Met Ile Lys Lys Arg Ile Pro Glu Lys Tyr Ser
50 55 60
Asn Ser Ile His Leu Val Glu Leu Arg Leu Pro Glu Leu Pro Glu Leu
65 70 75 80
Pro Pro His Tyr His Thr Thr Asn Gly Leu Pro Pro His Leu Asn Lys
85 90 95
Thr Leu His Lys Ala Leu Lys Met Ser Ala Pro Asn Phe Ser Lys Ile
100 105 110
Leu Gln Asn Ile Lys Pro Asp Leu Val Leu Tyr Asp Phe Leu Val Pro
115 120 125
Trp Ala Glu Lys Val Ala Leu Glu Gln Gly Ile Pro Ala Val Pro Leu
130 135 140
Leu Thr Ser Gly Ala Ala Leu Phe Ser Tyr Phe Phe Asn Phe Leu Lys
145 150 155 160
Arg Pro Gly Glu Glu Phe Pro Phe Glu Ala Ile Arg Leu Ser Lys Arg
165 170 175
Glu Gln Asp Lys Met Arg Glu Met Phe Gly Thr Glu Pro Pro Glu Glu
180 185 190
Asp Phe Leu Ala Pro Ala Gln Ala Gly Ile Met Leu Met Cys Thr Ser
195 200 205
Arg Val Ile Glu Ala Lys Tyr Leu Asp Tyr Cys Thr Glu Leu Thr Asn
210 215 220
Val Lys Val Val Pro Val Gly Pro Pro Phe Gln Asp Pro Leu Thr Glu
225 230 235 240
Asp Ile Asp Asp Pro Glu Leu Met Asp Trp Leu Asp Thr Lys Pro Glu
245 250 255
His Ser Val Val Tyr Val Ser Phe Gly Ser Glu Ala Phe Leu Ser Arg
260 265 270
Glu Asp Met Glu Glu Val Ala Phe Gly Leu Glu Leu Ser Gly Val Asn
275 280 285
Phe Ile Trp Val Ala Arg Phe Pro Lys Gly Glu Glu Gln Arg Leu Glu
290 295 300
Asp Val Leu Pro Glu Gly Phe Leu Glu Arg Ile Gly Glu Arg Gly Arg
305 310 315 320
Val Leu Asp Lys Phe Ala Pro Gln Pro Arg Ile Leu Asn His Pro Ser
325 330 335
Thr Gly Gly Phe Ile Ser His Cys Gly Trp Asn Ser Val Met Glu Ser
340 345 350
Ile Asp Phe Gly Val Pro Ile Ile Ala Met Pro Ile His Asn Asp Gln
355 360 365
Pro Ile Asn Ala Lys Leu Met Val Glu Leu Gly Val Ala Val Glu Ile
370 375 380
Val Arg Asp Asp Asp Gly Lys Ile His Arg Gly Glu Ile Ala Glu Ala
385 390 395 400
Leu Lys Ser Val Val Thr Gly Glu Thr Gly Glu Ile Leu Arg Ala Lys
405 410 415
Val Arg Glu Ile Ser Lys Asn Leu Lys Ser Ile Arg Asp Glu Glu Met
420 425 430
Asp Ala Val Ala Glu Glu Leu Ile Gln Leu Cys Arg Asn Ser Asn Lys
435 440 445
Ser Lys
450
<210> 37
<211> 1329
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.9
<400> 37
atggcgacca acctgcgtgt tctgatgttc ccgtggctgg cgtacggcca catcagcccg 60
ttcctgaaca tcgcgaaaca gctggcggat cgtggtttcc tgatctatct gtgctccacc 120
cgcatcaacc tggaatctat catcaagaaa atcccggaaa aatacgcgga ttctatccat 180
ctgatcgaac ttcagctgcc ggagctgccg gaactgccgc cgcactatca caccactaac 240
ggtctgccgc cgcatctgaa cccgaccctg cacaaagcgc tgaaaatgtc taaaccgaac 300
tttagcaaga tccttcaaaa tattaagccg gacctggtcc tttacgattt tctggttccg 360
tgggcagaaa aagtcgcgct tgaacagggc atcccggctg ttccattgct aaccagtggt 420
gcggcactgt tcagctactt tttcaacttc ctgaagcgac cgggtgaaga gtttccgttt 480
gaggcaatcc gcctgtcgaa gcgagaacag gataagatgc gcgagatgtt tggaacagag 540
ccgcctgaag aagatttttt agcgccggcc caggccggta tcatgctgat gtgcacgagc 600
cgcgtaattg aggctaagta cctggactat tgtaccgaac tgaccaatgt aaaagttgtt 660
ccggttggtc cgccgtttca ggatccgctg accgaagata ttgacgaccc cgaactgatg 720
gattggttag ataccaaacc cgaacatagt gttgtctatg tgtcgtttgg cagcgaagcg 780
ttcctgagcc gtgaagatat ggaagaagtc gcgttcggcc tggagctgag cggcgtgaac 840
tttatctggg ttgcacgctt tccgaaaggc gaagaacagc gtctggaaga cgttctgcca 900
aaaggcttcc tggaacgcgt tggtgatcgt ggtcgcgttc tggaccatct ggtgccgcag 960
gcccatattc tgaaccatcc gagcacgggt ggcttcatct ctcattgcgg ttggaacagc 1020
gtcatggaaa gcattgattt cggcgttccg atcattgcga tgccgatgca gtgggatcag 1080
ccgattaacg cgagactgct tgtggaatta ggcgtggcag tggagatccc gcgtgatgaa 1140
gatggccggg tccaccgcgc cgaaattgcc cgtgtcctga aagatgtgat ttcgggcccg 1200
actggtgaga tactgcgcgc gaaagtacgc gacattagcg cacgcctgag agcgagacgc 1260
gaggaggaaa tgaacgcagc ggcggaagaa ctgatacagc tgtgtcgcaa ccgcaacgcc 1320
tacaagtaa 1329
<210> 38
<211> 442
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.9
<400> 38
Met Ala Thr Asn Leu Arg Val Leu Met Phe Pro Trp Leu Ala Tyr Gly
1 5 10 15
His Ile Ser Pro Phe Leu Asn Ile Ala Lys Gln Leu Ala Asp Arg Gly
20 25 30
Phe Leu Ile Tyr Leu Cys Ser Thr Arg Ile Asn Leu Glu Ser Ile Ile
35 40 45
Lys Lys Ile Pro Glu Lys Tyr Ala Asp Ser Ile His Leu Ile Glu Leu
50 55 60
Gln Leu Pro Glu Leu Pro Glu Leu Pro Pro His Tyr His Thr Thr Asn
65 70 75 80
Gly Leu Pro Pro His Leu Asn Pro Thr Leu His Lys Ala Leu Lys Met
85 90 95
Ser Lys Pro Asn Phe Ser Lys Ile Leu Gln Asn Ile Lys Pro Asp Leu
100 105 110
Val Leu Tyr Asp Phe Leu Val Pro Trp Ala Glu Lys Val Ala Leu Glu
115 120 125
Gln Gly Ile Pro Ala Val Pro Leu Leu Thr Ser Gly Ala Ala Leu Phe
130 135 140
Ser Tyr Phe Phe Asn Phe Leu Lys Arg Pro Gly Glu Glu Phe Pro Phe
145 150 155 160
Glu Ala Ile Arg Leu Ser Lys Arg Glu Gln Asp Lys Met Arg Glu Met
165 170 175
Phe Gly Thr Glu Pro Pro Glu Glu Asp Phe Leu Ala Pro Ala Gln Ala
180 185 190
Gly Ile Met Leu Met Cys Thr Ser Arg Val Ile Glu Ala Lys Tyr Leu
195 200 205
Asp Tyr Cys Thr Glu Leu Thr Asn Val Lys Val Val Pro Val Gly Pro
210 215 220
Pro Phe Gln Asp Pro Leu Thr Glu Asp Ile Asp Asp Pro Glu Leu Met
225 230 235 240
Asp Trp Leu Asp Thr Lys Pro Glu His Ser Val Val Tyr Val Ser Phe
245 250 255
Gly Ser Glu Ala Phe Leu Ser Arg Glu Asp Met Glu Glu Val Ala Phe
260 265 270
Gly Leu Glu Leu Ser Gly Val Asn Phe Ile Trp Val Ala Arg Phe Pro
275 280 285
Lys Gly Glu Glu Gln Arg Leu Glu Asp Val Leu Pro Lys Gly Phe Leu
290 295 300
Glu Arg Val Gly Asp Arg Gly Arg Val Leu Asp His Leu Val Pro Gln
305 310 315 320
Ala His Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys
325 330 335
Gly Trp Asn Ser Val Met Glu Ser Ile Asp Phe Gly Val Pro Ile Ile
340 345 350
Ala Met Pro Met Gln Trp Asp Gln Pro Ile Asn Ala Arg Leu Leu Val
355 360 365
Glu Leu Gly Val Ala Val Glu Ile Pro Arg Asp Glu Asp Gly Arg Val
370 375 380
His Arg Ala Glu Ile Ala Arg Val Leu Lys Asp Val Ile Ser Gly Pro
385 390 395 400
Thr Gly Glu Ile Leu Arg Ala Lys Val Arg Asp Ile Ser Ala Arg Leu
405 410 415
Arg Ala Arg Arg Glu Glu Glu Met Asn Ala Ala Ala Glu Glu Leu Ile
420 425 430
Gln Leu Cys Arg Asn Arg Asn Ala Tyr Lys
435 440
<210> 39
<211> 1329
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.10
<400> 39
atggcgacca acctgcgtgt tctgatgttc ccgtggctgg cgtacggcca catcagcccg 60
ttcctgaaca tcgcgaaaca gctggcggat cgtggtttcc tgatctatct gtgctccacc 120
cgcatcaacc tggaatctat catcaagaaa atcccggaaa aatacgcgga ttctatccat 180
ctgatcgaac ttcagctgcc ggagctgccg gaactgccgc cgcactatca caccactaac 240
ggtctgccgc cgcatctgaa cccgaccctg cacaaagcgc tgaaaatgtc taaaccgaac 300
ttcagccgca tcttgcagaa cctgaaaccg gacctgctga tctacgatgt gctccagccg 360
tgggcggaac acgtggcgaa cgaacagggc atcccggctg gcaaactgct ggtttcttgc 420
gcggcggttt tctcctactt tttctctttc cgtaaaaatc cgggcgttga atttccgttc 480
ccggcgatcc acctgccgga agtggaaaaa gttaaaatcc gtgaaatcct ggctaaagaa 540
ccggaagaag gcggccgtct ggacgaaggc aacaaacaga tgatgctgat gtgcacttct 600
cgtaccattg aagctaaata cattgattac tgcaccgaac tgtgcaactg gaaagttgtt 660
ccggttggtc cgccgttcca ggatctgatc actaacgatg cggataacaa agaactgatc 720
gattggctgg gcaccaaacc ggaaaactcc gttgtctatg tgtcgtttgg cagcgaagcg 780
ttcctgagcc gtgaagatat ggaagaagtc gcgttcggcc tggagctgag cggcgtgaac 840
tttatctggg ttgcacgctt tccgaaaggc gaagaacagc gtctggaaga cgttctgcca 900
aaaggcttcc tggaacgcgt tggtgatcgt ggtcgcgttc tggaccatct ggtgccgcag 960
gcccatattc tgaaccatcc gagcacgggt ggcttcatct ctcattgcgg ttggaacagc 1020
gtcatggaaa gcattgattt cggcgttccg atcattgcga tgccgatgca gtgggatcag 1080
ccgattaacg cgagactgct tgtggaatta ggcgtggcag tggagatccc gcgtgatgaa 1140
gatggccggg tccaccgcgc cgaaattgcc cgtgtcctga aagatgtgat ttcgggcccg 1200
actggtgaga tactgcgcgc gaaagtacgc gacattagcg cacgcctgag agcgagacgc 1260
gaggaggaaa tgaacgcagc ggcggaagaa ctgatacagc tgtgtcgcaa ccgcaacgcc 1320
tacaagtaa 1329
<210> 40
<211> 442
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.10
<400> 40
Met Ala Thr Asn Leu Arg Val Leu Met Phe Pro Trp Leu Ala Tyr Gly
1 5 10 15
His Ile Ser Pro Phe Leu Asn Ile Ala Lys Gln Leu Ala Asp Arg Gly
20 25 30
Phe Leu Ile Tyr Leu Cys Ser Thr Arg Ile Asn Leu Glu Ser Ile Ile
35 40 45
Lys Lys Ile Pro Glu Lys Tyr Ala Asp Ser Ile His Leu Ile Glu Leu
50 55 60
Gln Leu Pro Glu Leu Pro Glu Leu Pro Pro His Tyr His Thr Thr Asn
65 70 75 80
Gly Leu Pro Pro His Leu Asn Pro Thr Leu His Lys Ala Leu Lys Met
85 90 95
Ser Lys Pro Asn Phe Ser Arg Ile Leu Gln Asn Leu Lys Pro Asp Leu
100 105 110
Leu Ile Tyr Asp Val Leu Gln Pro Trp Ala Glu His Val Ala Asn Glu
115 120 125
Gln Gly Ile Pro Ala Gly Lys Leu Leu Val Ser Cys Ala Ala Val Phe
130 135 140
Ser Tyr Phe Phe Ser Phe Arg Lys Asn Pro Gly Val Glu Phe Pro Phe
145 150 155 160
Pro Ala Ile His Leu Pro Glu Val Glu Lys Val Lys Ile Arg Glu Ile
165 170 175
Leu Ala Lys Glu Pro Glu Glu Gly Gly Arg Leu Asp Glu Gly Asn Lys
180 185 190
Gln Met Met Leu Met Cys Thr Ser Arg Thr Ile Glu Ala Lys Tyr Ile
195 200 205
Asp Tyr Cys Thr Glu Leu Cys Asn Trp Lys Val Val Pro Val Gly Pro
210 215 220
Pro Phe Gln Asp Leu Ile Thr Asn Asp Ala Asp Asn Lys Glu Leu Ile
225 230 235 240
Asp Trp Leu Gly Thr Lys Pro Glu Asn Ser Val Val Tyr Val Ser Phe
245 250 255
Gly Ser Glu Ala Phe Leu Ser Arg Glu Asp Met Glu Glu Val Ala Phe
260 265 270
Gly Leu Glu Leu Ser Gly Val Asn Phe Ile Trp Val Ala Arg Phe Pro
275 280 285
Lys Gly Glu Glu Gln Arg Leu Glu Asp Val Leu Pro Lys Gly Phe Leu
290 295 300
Glu Arg Val Gly Asp Arg Gly Arg Val Leu Asp His Leu Val Pro Gln
305 310 315 320
Ala His Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys
325 330 335
Gly Trp Asn Ser Val Met Glu Ser Ile Asp Phe Gly Val Pro Ile Ile
340 345 350
Ala Met Pro Met Gln Trp Asp Gln Pro Ile Asn Ala Arg Leu Leu Val
355 360 365
Glu Leu Gly Val Ala Val Glu Ile Pro Arg Asp Glu Asp Gly Arg Val
370 375 380
His Arg Ala Glu Ile Ala Arg Val Leu Lys Asp Val Ile Ser Gly Pro
385 390 395 400
Thr Gly Glu Ile Leu Arg Ala Lys Val Arg Asp Ile Ser Ala Arg Leu
405 410 415
Arg Ala Arg Arg Glu Glu Glu Met Asn Ala Ala Ala Glu Glu Leu Ile
420 425 430
Gln Leu Cys Arg Asn Arg Asn Ala Tyr Lys
435 440

Claims (11)

1. Glycosyltransferase, characterized in that its amino acid sequence comprises at least the amino acid residue differences compared to SEQ ID No. 4 selected from the group consisting of:
(1) Having one or more of the following amino acid residue differences:
the amino acid residue at position 429 is D;
the amino acid residue at position 433 is D;
the amino acid residue at position 435 is V;
the amino acid residue at position 446 is S;
the amino acid residue at position 448 is K; and
the 449 amino acid residue is S;
(2) Having one or more of the following amino acid residue differences:
deletion of amino acid residues at positions 1 to 8;
the amino acid residue at position 9 is M;
the amino acid residue at position 10 is A;
the amino acid residue at position 11 is T;
the amino acid residue at position 12 is N;
the amino acid residue at position 16 is L;
the amino acid residue at position 22 is A;
the amino acid residue at position 23 is Y;
the amino acid residue at position 26 is I;
the amino acid residue at position 27 is S;
the amino acid residue at position 31 is N;
the amino acid residue at position 42 is L;
the amino acid residue at position 46 is C;
the amino acid residue at position 49 is R;
the amino acid residue at position 54 is S;
amino acid residue at position 56 is I;
the amino acid residue at position 58 is K;
the amino acid residue at position 64 is A;
the amino acid residue at position 65 is D;
the amino acid residue at position 70 is I;
the amino acid residue at position 73 is Q;
the amino acid residue at position 96 is P; and
the amino acid residue at position 106 is K.
2. The glycosyltransferase of claim 1, wherein the glycosyltransferase comprises,
(1) The amino acid sequence of the glycosyltransferase further comprises one or more amino acid residue differences compared to SEQ ID No. 4 selected from the group consisting of:
the amino acid residue at position 399 is E;
the amino acid residue at position 400 is A;
the amino acid residue at position 403 is S;
the amino acid residue at position 405 is V;
the amino acid residue at position 406 is T;
the amino acid residue at position 408 is E;
the amino acid residue at position 419 is E;
the 422 th amino acid residue is K;
the amino acid residue at position 423 is N;
the amino acid residue at position 425 is K;
the amino acid residue at position 426 is S; and
the amino acid residue at position 427 is I;
preferably, the amino acid sequence of the glycosyltransferase further comprises one or more amino acid residue differences compared to SEQ ID NO. 4 selected from the group consisting of:
the amino acid residue at position 373 is K;
amino acid residue at position 375 is M;
the amino acid residue at position 385 is V;
the amino acid residue at position 388 is D;
the amino acid residue at position 391 is K;
the amino acid residue at position 392 is I; and
the amino acid residue at position 395 is G;
more preferably, the amino acid sequence of the glycosyltransferase further comprises amino acid residue differences at one or more residue positions selected from the following positions compared to SEQ ID NO: 4:
amino acid residue 309 is E;
amino acid residue at position 315 is I;
the amino acid residue at position 317 is E;
the amino acid residue at position 324 is K;
the amino acid residue at position 325 is F;
the amino acid residue at position 326 is A;
the amino acid residue at position 329 is P;
amino acid residue at position 330 is R;
amino acid residue at position 364 is I;
the amino acid residue at position 365 is H; and
the amino acid residue at position 366 is N.
3. The glycosyltransferase of claim 1 or 2, wherein the glycosyltransferase does not comprise one or more amino acid differences from positions 210 to 257 as compared to SEQ ID No. 4;
preferably, the amino acid sequence of the glycosyltransferase is shown as SEQ ID NO. 32; or, as shown in SEQ ID NO. 38; or as shown in SEQ ID NO. 30; or, as shown in SEQ ID NO. 28; or, as shown in SEQ ID NO. 36.
4. An isolated nucleic acid encoding the glycosyltransferase of any one of claims 1-3.
5. A recombinant expression vector comprising the nucleic acid of claim 4.
6. A transformant comprising the nucleic acid of claim 4 or the recombinant expression vector of claim 5.
7. A method of preparing the glycosyltransferase of any one of claims 1-3, comprising culturing the transformant of claim 6 under conditions suitable for expression of the glycosyltransferase.
8. A method of preparing rebaudioside E, the method comprising: glycosyltransferases transfer a glycosyl group on an activated glycosyl donor to a glycosyl acceptor;
wherein the glycosyltransferase is as claimed in any one of claims 1 to 3; the glycosyl acceptor is stevioside; the glycosyl donor is uridine diphosphate glucose and/or adenosine diphosphate glucose;
preferably, the uridine diphosphate glucose and/or adenosine diphosphate glucose are produced by the decomposition synthesis of sucrose; the amino acid sequence of the sucrose synthase is shown as SEQ ID NO. 24, and the nucleotide sequence for encoding the sucrose synthase is preferably shown as SEQ ID NO. 23;
more preferably, the glycosyltransferase and the sucrose synthase are used in the form of a crude enzyme solution, a pure enzyme, an immobilized enzyme or a cell expressing the glycosyltransferase and the sucrose synthase;
even more preferably, the mass ratio of cells expressing the glycosyltransferase to stevioside is 3 (9-30), preferably 3:20; the mass ratio of the cell expressing the sucrose synthase to sucrose is 3 (150-300), preferably 3:200; the mass ratio of the sucrose to the stevioside is (0.5-3) 1, preferably 2:1; the mass ratio of the sucrose to the uridine diphosphate glucose or the adenosine diphosphate glucose is (500-3000) 1, preferably 2000:1.
9. The method according to claim 8, wherein the stevioside concentration in the reaction system used in the method is 50-250 g/L, the pH is 5-8, and the reaction temperature is 20-90 ℃;
preferably, each 10mL of the reaction system comprises: 1.5mL of glycosyltransferase, 0.3mL of sucrose synthase, 2g of sucrose, 1g of stevioside, 1mg of uridine diphosphate or adenosine diphosphate, pH 5.5, and reaction temperature of 60 ℃.
10. An enzyme combination comprising a glycosyltransferase according to any one of claims 1 to 3 and a sucrose synthase having an amino acid sequence as shown in SEQ ID No. 24;
preferably, the nucleotide sequence of the sucrose synthase is shown as SEQ ID NO. 23; and/or the sucrose synthase and the glycosyltransferase are used in a mass ratio of 1 (3-10), preferably in a mass ratio of 1:5.
11. Use of the glycosyltransferase of any one of claims 1-3 or the enzyme combination of claim 10 in the preparation of rebaudioside D or rebaudioside E;
preferably, the rebaudioside D is prepared by rebaudioside a; the rebaudioside E is prepared from stevioside.
CN202210114711.6A 2022-01-30 2022-01-30 Glycosyltransferases and their use in the preparation of rebaudioside E Pending CN116555210A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109750072A (en) * 2019-01-31 2019-05-14 南京工业大学 Method for preparing rebaudioside E by enzyme method
CN112375750A (en) * 2020-12-02 2021-02-19 南京工业大学 Glycosyltransferase mutant and method for catalytically synthesizing rebaudioside A by using same
CN112805295A (en) * 2018-07-30 2021-05-14 科德克希思公司 Engineering glycosyltransferases and methods of glycosylation of steviol glycosides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112805295A (en) * 2018-07-30 2021-05-14 科德克希思公司 Engineering glycosyltransferases and methods of glycosylation of steviol glycosides
CN109750072A (en) * 2019-01-31 2019-05-14 南京工业大学 Method for preparing rebaudioside E by enzyme method
CN112375750A (en) * 2020-12-02 2021-02-19 南京工业大学 Glycosyltransferase mutant and method for catalytically synthesizing rebaudioside A by using same

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