Glycosyltransferase and its application in the preparation of rebaudioside E

Abstract

The present invention discloses a glycosyltransferase and its application in the preparation of rebaudioside E. The amino acid sequence of the glycosyltransferase of the present invention contains at least amino acid residue differences selected from the following groups compared with SEQ ID NO: 4: (1) the amino acid residues at positions 429, 433, 435, 446, 448, and 449; (2) deletion of the amino acid residues at positions 1-8; the 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 present invention has screened out a glycosyltransferase with better catalytic effect and better stability, optimized the process conditions for the enzymatic synthesis of Reb E, which is beneficial to the industrial production of Reb E; solved the problem of the high price of the glycosyl donor UDPG / ADPG, and reduced the production cost.

Description

Technical Field

[0001] The present invention belongs to the field of biosynthesis. Specifically, the present invention relates to a glycosyltransferase and its application in the preparation of rebaudioside E. Background Art

[0002] Steviol glycosides (also known as steviol glucosides) are natural sweeteners extracted from the leaves of the herbaceous plant Stevia rebaudiana Bertoni of the Compositae family, accounting for 10%-20% of the dry weight of the leaves and being a mixture of various glycosides. Steviol glycosides have the advantages of being natural (from the natural plant Stevia rebaudiana Bertoni), having high sweetness, low calories, being economical to use, having good stability, and high safety, but they have the drawback of a bitter aftertaste, which limits their application in the fields of food, beverages, etc. The essential reason for the bitter aftertaste of steviol glycosides is caused by their internal molecular structure. The more sugar groups are attached to the R1 and R2 groups in steviol glycosides, the better the taste.

[0003] The structural formula of steviol glycosides (steviol glycoside compounds) is as follows:

[0004]

[0005] The corresponding compounds of its substituents are shown in Table 1.

[0006] Table 1 Steviol glycoside compounds isolated from Stevia rebaudiana Bertoni

[0007]

[0008]

[0009] The above-mentioned steviol glycoside compounds have a common aglycone: steviol, and the difference lies in the number and type of sugar groups attached to the C-13 and C-19 positions, mainly including stevioside, rebaudioside A (Rebaudioside A, Reb A, RA), rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E (Rebaudioside E, Reb E, RE), dulcoside, steviolbioside, etc.

[0010] Among them, rebaudioside E has no bitter aftertaste and its sweetness is similar to that of sucrose. Structurally, it has one more glucose group on the C19-side chain of the skeleton compared to stevioside, but its content in the dry leaves of Stevia rebaudiana Bertoni is very low (far less than 1%). Directly separating rebaudioside E from steviol glycosides by conventional physical means is difficult and the yield is extremely low. In addition, the process for enriching rebaudioside E is cumbersome. After extraction, it requires multiple column chromatography, desalting, decolorization, and recrystallization, and a large amount of wastewater is generated during the production process, and its production cost is relatively high, which is not suitable for large-scale industrial production.

[0011] The biotransformation method mainly uses glycosyltransferases to catalyze the transfer of glycosyl groups from activated donor molecules to receptor molecules, generating a rich variety of glycoside compounds. Common glycosyl donors include monosaccharides, disaccharides, polysaccharides, phosphosugars, uridine diphosphate-glucose, etc. The glycosyl donor for the preparation of Rebaudioside E by the biotransformation method is generally uridine diphosphate-glucose (UDP-glucose) or adenosine diphosphate-glucose (ADP-glucose), but their prices are expensive, and the catalytic activity of glycosyltransferases (such as β-1,2-glycosyltransferase) is relatively low, resulting in problems such as high production costs and low production efficiency. Summary of the Invention

[0012] The technical problem to be solved by the present invention is to overcome the defect of low enzyme activity when the existing glucosyltransferase is applied to the biocatalytic preparation of Reb E, and to provide a glycosyltransferase and its application in the preparation of Rebaudioside E. The glycosyltransferase of the present invention has good catalytic effect and good stability, and jointly catalyzes the synthesis of Reb E with sucrose synthase (SUS) to achieve a cascade reaction; at the same time, UDPG / ADPG regeneration is achieved through sucrose and UDP / ADP, solving the problem of the high price of the glycosyl donor UDPG / ADPG; and further optimizing the process conditions for the enzyme-catalyzed synthesis of Reb E, providing more choices for optimizing the process conditions for large-scale industrial production, and being conducive to realizing industrial production.

[0013] The present invention solves the above technical problems through the following technical solutions:

[0014] The first aspect of the present invention provides a glycosyltransferase, the amino acid sequence of which contains at least amino acid residue differences selected from the following groups compared with SEQ ID NO:4:

[0015] (1) having one or more of the following amino acid residue differences:

[0016] The amino acid residue at position 429 is D;

[0017] The amino acid residue at position 433 is D;

[0018] The amino acid residue at position 435 is V;

[0019] The amino acid residue at position 446 is S;

[0020] The amino acid residue at position 448 is K; and

[0021] The amino acid residue at position 449 is S;

[0022] (2) having one or more of the following amino acid residue differences:

[0023] The amino acid residues at positions 1-8 are deleted;

[0024] The amino acid residue at position 9 is M;

[0025] The amino acid residue at position 10 is A;

[0026] The amino acid residue at position 11 is T;

[0027] The amino acid residue at position 12 is N;

[0028] The amino acid residue at position 16 is L;

[0029] The amino acid residue at position 22 is A;

[0030] The amino acid residue at position 23 is Y;

[0031] The amino acid residue at position 26 is I;

[0032] The amino acid residue at position 27 is S;

[0033] The amino acid residue at position 31 is N;

[0034] The amino acid residue at position 42 is L;

[0035] The amino acid residue at position 46 is C;

[0036] The amino acid residue at position 49 is R;

[0037] The amino acid residue at position 54 is S;

[0038] The amino acid residue at position 56 is I;

[0039] The amino acid residue at position 58 is K;

[0040] The amino acid residue at position 64 is A;

[0041] The amino acid residue at position 65 is D;

[0042] The amino acid residue at position 70 is I;

[0043] The amino acid residue at position 73 is Q;

[0044] The amino acid residue at position 96 is P; and

[0045] The amino acid residue at position 106 is K.

[0046] In some embodiments of the present invention, in (1), the amino acid sequence of the glycosyltransferase further comprises one or more amino acid residue differences selected from the following compared to SEQ ID NO:4:

[0047] The amino acid residue at position 399 is E;

[0048] The amino acid residue at position 400 is A;

[0049] The amino acid residue at position 403 is S;

[0050] The amino acid residue at position 405 is V;

[0051] The amino acid residue at position 406 is T;

[0052] The amino acid residue at position 408 is E;

[0053] The amino acid residue at position 419 is E;

[0054] The amino acid residue at position 422 is K;

[0055] The amino acid residue at position 423 is N;

[0056] The amino acid residue at position 425 is K;

[0057] The amino acid residue at position 426 is S; and

[0058] The amino acid residue at position 427 is I.

[0059] In some embodiments of the present invention, the amino acid sequence of the glycosyltransferase further comprises one or more amino acid residue differences selected from the following compared to SEQ ID NO:4:

[0060] The amino acid residue at position 373 is K;

[0061] The amino acid residue at position 375 is M;

[0062] The amino acid residue at position 385 is V;

[0063] The amino acid residue at position 388 is D;

[0064] The amino acid residue at position 391 is K;

[0065] The amino acid residue at position 392 is I; and

[0066] The amino acid residue at position 395 is G.

[0067] In some embodiments of the present invention, the amino acid sequence of the glycosyltransferase further comprises one or more amino acid residue differences at one or more residue positions selected from the following compared to SEQ ID NO:4:

[0068] The amino acid residue at position 309 is E;

[0069] The amino acid residue at position 315 is I;

[0070] The amino acid residue at position 317 is E;

[0071] The amino acid residue at position 324 is K;

[0072] The amino acid residue at position 325 is F;

[0073] The amino acid residue at position 326 is A;

[0074] The amino acid residue at position 329 is P;

[0075] The amino acid residue at position 330 is R;

[0076] The amino acid residue at position 364 is I;

[0077] The amino acid residue at position 365 is H; and

[0078] The amino acid residue at position 366 is N.

[0079] In some embodiments of the present invention, the glycosyltransferase does not contain one or more amino acid differences at positions 210-257 compared to SEQ ID NO:4.

[0080] In some specific embodiments of the present invention, the glycosyltransferase does not contain one or more amino acid residue differences at positions 259-306 compared to SEQ ID NO:4.

[0081] In some specific embodiments of the present invention, the glycosyltransferase does not contain one or more amino acid residue differences at positions 111-202 compared to SEQ ID NO:4.

[0082] In some specific embodiments of the present invention, the glycosyltransferase does not contain one or more amino acid residue differences at positions 259-306 and 111-202 compared to SEQ ID NO:4.

[0083] In some specific embodiments of the present invention, the amino acid sequence of the glycosyltransferase is as shown in SEQ ID NO:32.

[0084] In some specific embodiments of the present invention, the amino acid sequence of the glycosyltransferase is as shown in SEQ ID NO:38.

[0085] In some specific embodiments of the present invention, the amino acid sequence of the glycosyltransferase is as shown in SEQ ID NO:30.

[0086] In some specific embodiments of the present invention, the amino acid sequence of the glycosyltransferase is as shown in SEQ ID NO:28.

[0087] In some specific embodiments of the present invention, the amino acid sequence of the glycosyltransferase is as shown in SEQ ID NO: 36.

[0088] The second aspect of the present invention provides an isolated nucleic acid encoding the glycosyltransferase as described in the first aspect.

[0089] The third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the second aspect.

[0090] The fourth aspect of the present invention provides a transformant comprising the nucleic acid as described in the second aspect or the recombinant expression vector as described in the third aspect.

[0091] The fifth aspect of the present invention provides a method for preparing the glycosyltransferase as described in the first aspect, the method comprising culturing the transformant as described in the fourth aspect under conditions suitable for expressing the glycosyltransferase.

[0092] The sixth aspect of the present invention provides a method for preparing rebaudioside E, the method comprising: transferring a glycosyl group from an activated glycosyl donor to a glycosyl acceptor by a glycosyltransferase;

[0093] 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.

[0094] In some embodiments of the present invention, the uridine diphosphate glucose and / or adenosine diphosphate glucose is generated by the decomposition synthesis of sucrose.

[0095] In the present invention, the decomposition synthesis of sucrose means that in the presence of uridine diphosphate and / or adenosine diphosphate, one molecule of sucrose is decomposed by sucrose synthase to generate one molecule of fructose and one molecule of uridine diphosphate glucose and / or adenosine diphosphate glucose.

[0096] In some specific embodiments of the present 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.

[0097] In some embodiments of the present invention, the forms of use of the glycosyltransferase and the sucrose synthase are crude enzyme solution, pure enzyme, immobilized enzyme or cells expressing the glycosyltransferase and the sucrose synthase.

[0098] In the present invention, the host cell of the cells expressing the glycosyltransferase and the sucrose synthase can be conventional in the art, such as E. coli; those skilled in the art can culture the cells by conventional technical means and obtain the glycosyltransferase and sucrose synthase.

[0099] In some embodiments of the present invention, the mass ratio of the cells expressing the glycosyltransferase to stevioside is 3:(9 - 30), preferably 3:20.

[0100] In some embodiments of the present invention, the mass ratio of the cells expressing the sucrose synthase to sucrose is 3:(150 - 300), preferably 3:200.

[0101] In some embodiments of the present invention, the mass ratio of the sucrose to stevioside is (0.5 - 3):1, preferably 2:1.

[0102] In some embodiments of the present invention, the mass ratio of the sucrose:uridine diphosphate glucose or adenosine diphosphate glucose is (500 - 3000):1, preferably 2000:1.

[0103] In some embodiments of the present invention, the concentration of stevioside 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 °C.

[0104] In some specific embodiments of the present invention, every 10 mL of the reaction system contains 1.5 mL of glycosyltransferase, 0.3 mL of sucrose synthase, 2 g of sucrose, 1 g of stevioside, 1 mg of uridine diphosphate or adenosine diphosphate, the pH is 5.5, and the reaction temperature is 60 °C.

[0105] The seventh aspect of the present invention provides an enzyme combination, which includes the glycosyltransferase as described in the first aspect and the sucrose synthase with the amino acid sequence as shown in SEQ ID NO:24.

[0106] In some embodiments of the present 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.

[0107] In some specific embodiments of the present invention, the nucleotide sequence encoding the sucrose synthase is as shown in SEQ ID NO:23.

[0108] The eighth aspect of the present invention provides an application of the glycosyltransferase as described in the first aspect or the enzyme combination as described in the seventh aspect in the preparation of rebaudioside D or rebaudioside E.

[0109] In some embodiments of the present invention, the rebaudioside D is prepared from rebaudioside A.

[0110] In some embodiments of the present invention, the rebaudioside E is prepared from stevioside.

[0111] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0112] The reagents and raw materials used in the present invention are all commercially available.

[0113] The positive and progressive effects of the present invention are as follows:

[0114] The present invention has screened out glycosyltransferases with better catalytic effects and better stability, optimized the technological conditions for the enzymatic synthesis of Reb E, which is conducive to the industrial production of Reb E; solved the problem of the high price of glycosyl donors UDPG / ADPG, and reduced the production cost. Description of the Drawings

[0115] Figure 1 It shows the synthetic route for preparing Rebaudioside E from Stevioside.

[0116] Figure 2 It shows a screenshot of the chromatogram of the results using the HPLC detection method of the present invention. The retention time of the Stevioside reference substance is 12.761 min.

[0117] Figure 3 It shows a screenshot of the chromatogram of the results using the HPLC detection method of the present invention. The retention time of the Rebaudioside E reference substance is 11.757 min.

[0118] Figure 4 It is a screenshot of the HPLC chromatogram of the experimental results of the catalytic synthesis of Reb E by Enz.5 in Example 5. Detailed Embodiments

[0119] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0120] The experimental methods in the present invention are all conventional methods unless otherwise specified. The gene cloning operation can specifically refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0121] The amino acid abbreviation symbols in the present invention are all conventional in the art unless otherwise specified. The specific amino acids corresponding to the abbreviation symbols are shown in Table 2.

[0122] Table 2 Amino Acid Alphabet

[0123]

[0124] The codons corresponding to the said amino acids are also conventional in the art. The specific correspondence between amino acids and codons is shown in Table 3.

[0125] Table 3 Amino Acid Codon Table

[0126]

[0127]

[0128] The schematic diagram of the route of the present invention is as Figure 1 shown.

[0129] The KOD Mix enzyme was purchased from TOYOBO CO., LTD. The DpnI enzyme was purchased from Invitrogen (Shanghai) Trading Co., Ltd.; The E. coli Trans10 and E. coli BL21(DE3) competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. The reaction substrate stevioside was purchased from Bidepharm (purity 95%). Sucrose was purchased from Sangon Biotech (Shanghai) Co., Ltd. The Reb E reference substance was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0130] HPLC detection method for conversion rate: Chromatographic column: ZORBAX Eclipse plus C18 (4.6 mm * 150 mm, 3.5 μm). Mobile phase: 0.1% TFA aqueous solution was used as mobile phase A, and 0.1% TFA acetonitrile solution was used as mobile phase B, and gradient elution was performed according to Table 4 below. Detection wavelength: 210 nm; Flow rate: 1 mL / min; Injection volume: 20 μL; Column temperature: 35 °C. As Figure 2 shown, the peak time of stevioside: 12.761 min; As Figure 3 shown, the peak time of Reb E: 11.757 min.

[0131] Table 4 Gradient elution

[0132] 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

[0133] Example 1 Construction of β-1,2-glycosyltransferase mutant library

[0134] Totally synthesize the β-1,2-glycosyltransferase (β-1,2-GT enzyme) gene numbered Enz.1 as shown in SEQ ID NO:1, and ligate it onto the pET28a plasmid vector to obtain the recombinant plasmid pET28a-Enz.1. The gene synthesis company is Sangon Biotech (Shanghai) Co., Ltd. (No. 698, Xiangmin Road, Songjiang District, Shanghai). The amino acid sequence of Enz.1 is as shown in SEQ ID NO:2.

[0135] Fully synthesize the β-1,2-glycosyltransferase (β-1,2-GT enzyme) gene numbered Enz.2 as shown in SEQ ID NO:3, and ligate it to the pET28a plasmid vector to obtain the recombinant plasmid pET28a-Enz.2. The gene synthesis company is Sangon Biotech (Shanghai) Co., Ltd. (No. 698, Xiangmin Road, Songjiang District, Shanghai). The amino acid sequence of Enz.2 is as shown in SEQ ID NO:4.

[0136] Using the pET28a-Enz.1 plasmid as a template, perform PCR amplification to obtain Fragment 1 with the primer sequences Enz.X-F (X = 3 - 10) and Km-R in Table 5; using the pET28a-Enz.2 plasmid as a template, perform PCR amplification to obtain Fragment 2 with the primer sequences Enz.X-R (X = 3 - 10) and Km-F in Table 5. Use Novozymes homologous recombination enzyme (Exnase II, 5X CEII) to recombine Fragment 1 and Fragment 2 and ligate them to the pET28a plasmid vector. After ligation, transform it into E. coli Trans10 competent cells, coat it on LB medium containing 50 μg / mL kanamycin, and culture it overnight at 37°C; pick a single colony into an LB test tube (Km resistant), culture it for 8 - 10 h, extract the plasmid for sequencing transformation and perform sequencing verification to obtain each mutant recombinant plasmid pET28a-Enz.3 - pET28a-Enz.10.

[0137] Table 5 Primer sequence table

[0138]

[0139]

[0140] In the table: F is the forward primer and R is the reverse primer.

[0141] The PCR amplification reaction system is:

[0142] KOD Mix: 25 μL

[0143] ddH 2 O: 20 μL

[0144] Primer: 2 μL * 2

[0145] Template: 1 μL

[0146] The amplification program is as follows:

[0147] (1) 98°C for 3 min

[0148] (2) 98°C for 10 s

[0149] (3) 55°C for 5 s

[0150] (4) 68 °C for 5 s / kbp

[0151] (5) 68 °C for 5 min

[0152] (6) Incubate at 12 °C

[0153] (2) - (4) Cycle 34 times.

[0154] Example 2 Preparation of β-1,2-glycosyltransferase

[0155] 1. Protein expression:

[0156] The correctly sequenced recombinant plasmids (pET28a-Enz.1 - pET28a-Enz.10) described in Example 1 were separately transformed into the host E. coli BL21(DE3) competent cells to obtain genetically engineered strains containing the recombinant plasmids. Single colonies were separately picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37 °C for 4 h. Transferred at an inoculation amount of 2% (v / v) to 50 mL of fresh TB liquid medium containing 50 μg / mL kanamycin as well, and cultured with shaking at 37 °C until the OD 600 reached about 0.8, then IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.1 mM, and induced to culture at 25 °C for 20 h. After the culture was completed, the culture solution was centrifuged at 4000 rpm for 20 min, the supernatant was discarded, and the cells were collected. Stored at -20 °C for later use.

[0157] 2. Obtaining crude enzyme solution:

[0158] Prepare a 50 mM phosphate buffer (PBS) at pH 5.5, and suspend the above-obtained cells according to 1:10 (M / V, g / mL) respectively, and then homogenize using a high-pressure homogenizer (homogenize at 550 Mbar for 1.5 min); the homogenized enzyme solutions were centrifuged at 12000 rpm for 2 min respectively to obtain crude enzyme solutions of each β-1,2-glycosyltransferase.

[0159] Example 3 Preparation of sucrose synthase SUS

[0160] Totally synthesize the sucrose synthase (SUS) gene as shown in SEQ ID NO:23, and ligate it onto the pET28a plasmid vector to obtain the recombinant plasmid pET28a-SUS. The gene synthesis company is Sangon Biotech (Shanghai) Co., Ltd. (No. 698, Xiangmin Road, Songjiang District, Shanghai).

[0161] The plasmid pET28a-SUS was transformed into the host E. coli BL21(DE3) competent cells to obtain an engineered strain containing the sucrose synthase gene. A single colony was picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37 °C for 4 h. It was transferred to 50 mL of fresh TB liquid medium containing 50 μg / mL kanamycin at an inoculation amount of 2% (v / v), and cultured with shaking at 37 °C until the OD 600 reached about 0.8. IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 25 °C for 20 h. After the culture was completed, the culture solution was centrifuged at 4000 rpm for 20 min, the supernatant was discarded, and the cells were collected. They were stored at -20 °C for later use.

[0162] A 50 mM phosphate buffer solution with pH 5.5 was prepared. The cells obtained above were suspended at a ratio of 1:10 (M / V, g / mL), and subjected to high-pressure homogenization (550 Mbar, 1.5 min). Then, it was centrifuged at 12000 rpm for 2 min to obtain a crude sucrose synthase enzyme solution.

[0163] Example 4 Screening of β-1,2-glycosyltransferase mutants

[0164] In a 1 mL reaction system, 150 μL of the crude β-1,2-glycosyltransferase enzyme solution prepared in Example 2, a final concentration of 100 g / L of stevioside (STV), a final concentration of 0.1 g / L of UDP / ADP, a final concentration of 200 g / L of sucrose, and 30 μL of the crude sucrose synthase enzyme solution were added respectively. Finally, 50 mM PBS with pH 6.0 was added to a final volume of 1 mL. The prepared reaction system was placed in a metal bath and reacted at 60 °C and 600 rpm for 30 min. 10 μL of the reaction solution was added to 990 μL of hydrochloric acid with pH 2-3, vortexed, centrifuged at 13000 rpm for 10 min, and the supernatant was analyzed by HPLC for the concentration of Reb E. The experimental results obtained using the HPLC detection method of the present invention are shown in Table 6.

[0165] Table 6 Screening of β-1,2-glycosyltransferase mutants

[0166] Enzyme number 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

[0167] As can be seen from the preliminary screening results in Table 6: when ADP is used as the catalytic substrate for sucrose synthase, the activity of Enz.5 is the highest, followed by Enz.6; when UDP is used as the catalytic substrate for sucrose synthase, the activity of Enz.8 is the best, followed by Enz.9. The activities of Enz.5 and Enz.8, Enz.9 are close, and the catalytic effect of Enz.4 is also better than that of the enzyme in the control group. From the experimental data, it can be seen that the activities of Enz.5 catalyzing ADP or UDP as substrates are relatively high. When ADP is used as the catalytic substrate for sucrose synthase, the yield of RebE catalyzed by Enz.5 is much higher than that of Reb E when UDP is used as the substrate. Therefore, subsequently, ADP is used as the catalytic substrate for sucrose synthase, and Enz.5 catalyzes the glycosyl transfer reaction to prepare rebaudioside E.

[0168] Example 5 Enzyme Enz.5 Catalyzed Synthesis of Reb E

[0169] In a 10 mL reaction system, 1.5 mL of the crude enzyme solution of β-1,2-glycosyltransferase Enz.5 prepared by the method described in Example 2, 0.3 mL of the crude enzyme solution of sucrose synthase prepared by the method described in Example 3, the final concentration of stevioside was 100 g / L, the final concentration of sucrose was 200 g / L, and the final concentration of ADP was 0.1 g / L. Finally, 50 mM PBS at pH 5.5 was added to a final volume of 10 mL. The prepared reaction system was placed in a metal bath and reacted at 60 °C and 600 rpm for 7 h. 10 μL of the reaction solution was added to 990 μL of hydrochloric acid with a pH of 2-3, vortexed, and centrifuged at 13,000 rpm for 10 min. The supernatant was analyzed by HPLC for the concentration of Reb E. The results are as Figure 4 shown, and the concentration of RebE was 68.62%. SEQUENCE LISTING <110> Yikeleai Biotechnology (Shanghai) Co., Ltd. <120> Glycosyltransferase and Its Application in the Preparation of 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 Methionine, Alanine, Threonine, Asparagine, Leucine, Arginine, Valine, Leucine, Methionine, Phenylalanine, Proline, Tryptophan, Leucine, Alanine, Tyrosine, Glycine 1 5 10 15 Histidine, Isoleucine, Serine, Proline, Phenylalanine, Leucine, Asparagine, Isoleucine, Alanine, Lysine, Glutamine, Leucine, Alanine, Aspartic acid, Arginine, Glycine 20 25 30 Phenylalanine, Leucine, Isoleucine, Tyrosine, Leucine, Cysteine, Serine, Threonine, Arginine, Isoleucine, Asparagine, Leucine, Glutamic acid, Serine, Isoleucine, Isoleucine 35 40 45 Lysine, Lysine, Isoleucine, Proline, Glutamic acid, Lysine, Tyrosine, Alanine, Aspartic acid, Serine, Isoleucine, Histidine, Leucine, Isoleucine, Glutamic acid, Leucine 50 55 60 Glutamine, Leucine, Proline, Glutamic acid, Leucine, Proline, Glutamic acid, Leucine, Proline, Proline, Histidine, Tyrosine, Histidine, Threonine, Threonine, Asparagine 65 70 75 80 Glycine, Leucine, Proline, Proline, Histidine, Leucine, Asparagine, Proline, Threonine, Leucine, Histidine, Lysine, Alanine, Leucine, Lysine, Methionine 85 90 95 Serine, Lysine, Proline, Asparagine, Phenylalanine, Serine, Arginine, Isoleucine, Leucine, Glutamine, Asparagine, Leucine, Lysine, Proline, Aspartic acid, Leucine 100 105 110 Leucine, Isoleucine, Tyrosine, Aspartic acid, Valine, Leucine, Glutamine, Proline, Tryptophan, Alanine, Glutamic acid, Histidine, Valine, Alanine, Asparagine, Glutamic acid 115 120 125 Glutamine, Glycine, Isoleucine, Proline, Alanine, Glycine, Lysine, Leucine, Leucine, Valine, Serine, Cysteine, Alanine, Alanine, Valine, Phenylalanine 130 135 140 Serine, Tyrosine, Phenylalanine, Phenylalanine, Serine, Phenylalanine, Arginine, Lysine, Asparagine, Proline, Glycine, Valine, Glutamic acid, Phenylalanine, Proline, Phenylalanine 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 atgcaccatc atcatgaagg cgtgagcgac cagaccctga gagtaacgat gtttccgtgg 60 cttgggctgg gtcatgttaa cccgtttttg cgtatcgcta aacaactggc cgatcgtggt 120 cttgggctgg gtcatgttaa cccgtttttg cgtatcgcta aacaactggc cgatcgtggt 120 ttcgttatct atttagttag taccgctatt aacctcgaaa tgatcaaaaa gagaatcccg 180 ttcgttatct atttagttag taccgctatt aacctcgaaa tgatcaaaaa gagaatcccg 180 gagaaataca gtaatagcat ccatctggtt gagctgcgcc tgccagaatt accggaactg 240 gagaaataca gtaatagcat ccatctggtt gagctgcgcc tgccagaatt accggaactg 240 ccaccacatt accatactac caacggttta ccaccgcatc tgaacaaaac cctgcacaag 300 ccaccacatt accatactac caacggttta ccaccgcatc tgaacaaaac cctgcacaag 300 gcactgaaga tgagcgctcc caactttagc aagatccttc aaaatattaa gccggacctg 360 gcactgaaga tgagcgctcc caactttagc aagatccttc aaaatattaa gccggacctg 360 gtcctttacg attttctggt tccgtgggca gaaaaagtcg cgcttgaaca gggcatcccg 420 gtcctttacg attttctggt tccgtgggca gaaaaagtcg cgcttgaaca gggcatcccg 420 gctgttccat tgctaaccag tggtgcggca ctgttcagct actttttcaa cttcctgaag 480 gctgttccat tgctaaccag tggtgcggca ctgttcagct actttttcaa cttcctgaag 480 cgaccgggtg aagagtttcc gtttgaggca atccgcctgt cgaagcgaga acaggataag 540 cgaccgggtg aagagtttcc gtttgaggca atccgcctgt cgaagcgaga acaggataag 540 atgcgcgaga tgtttggaac agagccgcct gaagaagatt ttttagcgcc ggcccaggcc 600 atgcgcgaga tgtttggaac agagccgcct gaagaagatt ttttagcgcc ggcccaggcc 600 ggtatcatgc tgatgtgcac gagccgcgta attgaggcta agtacctgga ctattgtacc 660 ggtatcatgc tgatgtgcac gagccgcgta attgaggcta agtacctgga ctattgtacc 660 gaactgacca atgtaaaagt tgttccggtt ggtccgccgt ttcaggatcc gctgaccgaa 720 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 ctggatagcc cgcgttcatg gcgtgcattc ctgcgtcatt tagtcgcatc tcagcgtgac 120 tcatggctac ataccgattt acagcacgcg tgcaagacgt ttcgtgaaca gcctccggaa 180 tcatggctac ataccgattt acagcacgcg tgcaagacgt ttcgtgaaca gcctccggaa 180 ggctatcctg aagatattgg ttggctggca gattttattg cgcattgcca ggaagcgatc 240 ggctatcctg aagatattgg ttggctggca gattttattg cgcattgcca ggaagcgatc 240 ttccgggatc cgtggatggt ttttgcgtgg cgtctacgtc caggtgtttg ggagtatgtg 300 ttccgggatc cgtggatggt ttttgcgtgg cgtctacgtc caggtgtttg ggagtatgtg 300 cgcatacatg tagaacagct ggcggtggag gagctgagca ctgatgaata tctgcaagcc 360 cgcatacatg tagaacagct ggcggtggag gagctgagca ctgatgaata tctgcaagcc 360 aaagaacaac ttgttggctt aggtgcagaa ggtgaagctg ttctgacggt ggatttcgaa 420 aaagaacaac ttgttggctt aggtgcagaa ggtgaagctg ttctgacggt ggatttcgaa 420 gattttcgtc cggtgagcca gcgtttaaaa gacgagagca ccattggtga tggtcttacc 480 gattttcgtc cggtgagcca gcgtttaaaa gacgagagca ccattggtga tggtcttacc 480 catctgaatc gtcatttagc aggtcgcatc tggactgatt tagcagcagg tcgtagtgct 540 catctgaatc gtcatttagc aggtcgcatc tggactgatt tagcagcagg tcgtagtgct 540 attctggaat ttctgggcct gcatcgtctg gataaccaga atctgatgct gagcaacggc 600 attctggaat ttctgggcct gcatcgtctg gataaccaga atctgatgct gagcaacggc 600 aataccgatt ttgactcttt acgtcaaacc gtacaatatc tgggcacctt accaagagaa 660 aataccgatt ttgactcttt acgtcaaacc gtacaatatc tgggcacctt accaagagaa 660 actccgtggg cagagtttcg tgaagacatg cgtcgtcgtg gttttgaacc cggttggggc 720 actccgtggg cagagtttcg tgaagacatg cgtcgtcgtg gttttgaacc cggttggggc 720 aacaccgcgg gccgtgttcg cgaaaccatg cgtctgctga tggatctgct tgactctccg 780 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 ccgcctgaag aagatttttt agcgccggcc caggccggta tcatgctgat gtgcacgagc 600 cgcgtaattg aggctaagta cctggactat tgtaccgaac tgaccaatgt aaaagttgtt 660 cgcgtaattg aggctaagta cctggactat tgtaccgaac tgaccaatgt aaaagttgtt 660 ccggttggtc cgccgtttca ggatccgctg accgaagata ttgacgaccc cgaactgatg 720 ccggttggtc cgccgtttca ggatccgctg accgaagata ttgacgaccc cgaactgatg 720 gattggttag ataccaaacc cgaacatagt gttgtctatg tgtcgtttgg cagcgaagcg 780 gattggttag ataccaaacc cgaacatagt gttgtctatg tgtcgtttgg cagcgaagcg 780 ttcctgagcc gtgaagatat ggaagaagtc gcgttcggcc tggagctgag cggcgtgaac 840 ttcctgagcc gtgaagatat ggaagaagtc gcgttcggcc tggagctgag cggcgtgaac 840 tttatctggg ttgcacgctt tccgaaaggc gaagaacagc gtctggaaga cgttctgcca 900 tttatctggg ttgcacgctt tccgaaaggc gaagaacagc gtctggaaga cgttctgcca 900 aaaggcttcc tggaacgcgt tggtgatcgt ggtcgcgttc tggaccatct ggtgccgcag 960 aaaggcttcc tggaacgcgt tggtgatcgt ggtcgcgttc tggaccatct ggtgccgcag 960 gcccatattc tgaaccatcc gagcacgggt ggcttcatct ctcattgcgg ttggaacagc 1020 gcccatattc tgaaccatcc gagcacgggt ggcttcatct ctcattgcgg ttggaacagc 1020 gtcatggaaa gcattgattt cggcgttccg atcattgcga tgccgatgca gtgggatcag 1080 gtcatggaaa gcattgattt cggcgttccg atcattgcga tgccgatgca gtgggatcag 1080 ccgattaacg cgagactgct tgtggaatta ggcgtggcag tggagatccc gcgtgatgaa 1140 ccgattaacg cgagactgct tgtggaatta ggcgtggcag tggagatccc gcgtgatgaa 1140 gatggccggg tccaccgcgc cgaaattgcc cgtgtcctga aagatgtgat ttcgggcccg 1200 gatggccggg tccaccgcgc cgaaattgcc cgtgtcctga aagatgtgat ttcgggcccg 1200 actggtgaga tactgcgcgc gaaagtacgc gacattagcg cacgcctgag agcgagacgc 1260 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

1. A glycosyltransferase, characterized in that, the amino acid sequence of the glycosyltransferase is as shown in 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.

2. An isolated nucleic acid, characterized in that, the nucleic acid encodes the glycosyltransferase according to claim 1.

3. A recombinant expression vector comprising the nucleic acid according to claim 2.

4. A transformant comprising the nucleic acid according to claim 2 or the recombinant expression vector according to claim 3.

5. A method for preparing the glycosyltransferase according to claim 1, characterized in that, the method comprises culturing the transformant according to claim 4 under conditions suitable for expressing the glycosyltransferase.

6. A method for preparing rebaudioside E, characterized in that, the method comprises: the glycosyltransferase transferring a glycosyl group from an activated glycosyl donor to a glycosyl acceptor; wherein, the glycosyltransferase is as described in claim 1; the glycosyl acceptor is stevioside; the glycosyl donor is uridine diphosphate glucose and / or adenosine diphosphate glucose.

7. The method according to claim 6, characterized in that, the uridine diphosphate glucose and / or adenosine diphosphate glucose is generated by catalyzing the decomposition and synthesis of sucrose by sucrose synthase; the amino acid sequence of the sucrose synthase is as shown in SEQ ID NO:

24.

8. The method according to claim 7, characterized in that, the nucleotide sequence encoding the sucrose synthase is as shown in SEQ IDNO:

23.

9. The method according to claim 7, characterized in that, the forms of use of the glycosyltransferase and the sucrose synthase are crude enzyme solution, pure enzyme, immobilized enzyme or cells expressing the glycosyltransferase and the sucrose synthase.

10. The method according to claim 9, characterized in that, the mass ratio of the cells expressing the glycosyltransferase to stevioside is 3:(9 - 30); the mass ratio of the cells expressing the sucrose synthase to sucrose is 3:(150 - 300); the mass ratio of the sucrose to stevioside is (0.5 - 3):1; the mass ratio of the sucrose:uridine diphosphate glucose or adenosine diphosphate glucose is (500 - 3000):

1.

11. The method according to claim 10, characterized in that, the mass ratio of the cells expressing the glycosyltransferase to stevioside is 3:20; the mass ratio of the cells expressing the sucrose synthase to sucrose is 3:200; the mass ratio of the sucrose to stevioside is 2:1; the mass ratio of the sucrose:uridine diphosphate glucose or adenosine diphosphate glucose is 2000:

1.

12. The method according to any one of claims 6 - 11, characterized in that, the concentration of stevioside in the reaction system used in the method is 50 - 250 g / L, the pH is 5 - 8, and the reaction temperature is 20 - 60°C.

13. The method according to claim 9, characterized in that, The reaction system used in the method contains per 10 mL: 1.5 mL of glycosyltransferase, 0.3 mL of sucrose synthase, 2 g of sucrose, 1 g of stevioside, 1 mg of uridine diphosphate or adenosine diphosphate, with a pH of 5.5 and a reaction temperature of 60 °C.

14. An enzyme composition, characterized in that the enzyme composition comprises the glycosyltransferase as described in claim 1 and a sucrose synthase with an amino acid sequence as shown in SEQ ID NO:

24.

15. The enzyme composition according to claim 14, characterized in that the nucleotide sequence of the sucrose synthase is as shown in SEQ ID NO: 23; and / or, the mass ratio of the sucrose synthase to the glycosyltransferase is 1:(3 - 10).

16. The enzyme composition according to claim 15, characterized in that the mass ratio of the sucrose synthase to the glycosyltransferase is 1:

5.

17. Use of the glycosyltransferase as described in claim 1 or the enzyme composition according to any one of claims 14 - 16 in the preparation of rebaudioside D or rebaudioside E.

18. The use according to claim 17, characterized in that the rebaudioside D is prepared from rebaudioside A; the rebaudioside E is prepared from stevioside.

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