CN116334162A - Preparation method and application of rebaudioside I - Google Patents

Abstract

The invention discloses a preparation method and application of rebaudioside I. A method of preparing rebaudioside I by reacting rebaudioside a with a glycosyl donor in the presence of a glycosyltransferase comprising an amino acid residue difference at a residue position selected from one or more of the following compared to SEQ ID No. 2: amino acid at position 12 is Q; amino acid at position 118 is S; amino acid 265 is E; amino acid 271 is A; amino acid 308 is N; amino acid 333 is K; amino acid 347 is P; amino acid at position 418 is D; amino acid 455 is R; and has a glycosyltransferase activity not lower than that shown by the amino acid sequence of SEQ ID NO. 2. The preparation method provided by the invention has better catalytic effect and higher stability, optimizes the process conditions for synthesizing the rebaudioside I by an enzyme method, and solves the problem of high price of glycosyl donor UDPG (and/or ADPG).

Description

Preparation method and application of rebaudioside I

Technical Field

The invention belongs to the field of enzyme catalysis, and particularly relates to a preparation method of rebaudioside I, application of glycosyltransferase in preparing rebaudioside I, a catalytic enzyme composition and application of the catalytic enzyme composition in preparing rebaudioside I.

Background

Steviol glycoside (Steviol glycosides, also known as steviol glycoside) is a natural sweetener extracted from stevia rebaudiana leaves of the family Compositae, and is a mixture of various glycosides, and different steviol glycosides have great differences in taste quality. Stevioside has the advantages of pure nature (from pure natural plant stevia rebaudiana Bertoni), high sweetness (250-450 times of sucrose), low calorie (only 1/300 of that of white sugar), economical use (only one third of that of sucrose), good stability (heat resistance, acid resistance, alkali resistance, difficult decomposition phenomenon), high safety (no toxic or side effect), and the like, and has the potential curative effects of hyperglycemia resistance, hypertension resistance, inflammation resistance, tumor resistance, diarrhea resistance and the like.

Steviol glycosides (steviol glycoside compounds) have the following structural formula:

sequence number	Compounds of formula (I)	R 1	R 2
				1	Steviol (stevia rebaudiana)	H	H
2	Steviol monosaccharides	H	β-Glc
				3	Steviol disaccharide glycoside	H	β-Glc-β-Glc(2-1)
4	Sweet tea glycoside	β-Glc	β-Glc
				5	Stevioside (STV)	β-Glc	β-Glc-β-Glc(2-1)
6	Rebaudioside A (RA)	β-Glc	β-Glc-β-Glc(2-1)-β-Glc(3-1)
				7	Rebaudioside B (RB)	H	β-Glc-β-Glc(2-1)-β-Glc(3-1)
8	Rebaudioside C (RC)	β-Glc	β-Glc-α-Rha(2-1)-β-Glc(3-1)
				9	Rebaudioside D (RD)	β-Glc-β-Glc(2-1)	β-Glc-β-Glc(2-1)-β-Glc(3-1)
10	Rebaudioside E (RE)	β-Glc-β-Glc(2-1)	β-Glc-β-Glc(2-1)
				11	Rebaudioside F (RF)	β-Glc	β-Glc-α-Xly(2-1)-β-Glc(3-1)
12	Rebaudioside M (RM)	β-Glc-β-Glc(2-1)-β-Glc(3-1)	β-Glc-β-Glc(2-1)-β-Glc(3-1)
				13	Du Keer glycoside A	β-Glc	β-Glc-α-Rha(2-1)
14	Rebaudioside I (RI)	β-Glc-β-Glc(3-1)	β-Glc-β-Glc(2-1)-β-Glc(3-1)

The steviol glycoside compounds have a common aglycone: steviol (Steviol), which differs 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, rebaudioside I, dulcoside, steviolbioside, and the like. Stevia leaves are capable of accumulating up to 10-20% (dry weight basis) steviol glycosides. The main glycosides found in stevia leaves are rebaudioside a (2-10%), stevioside (2-10%) and rebaudioside C (1-2%), other glycosides such as rebaudiosides B, D, E, F and I, steviolbioside and rubusoside are found at much lower levels (about 0-0.2%). The rebaudioside A has the highest sweetness, and the sweetness is similar to that of sucrose, so that the rebaudioside A is a novel natural sweetener with high sweetness, low calorie, easy dissolution, heat resistance and stability, and the content or purity of the natural sweetener is also a main index for measuring the quality of stevioside. In addition, rebaudioside a is also an important intermediate for synthesizing stevioside compounds such as Rebaudioside D (rebaudiosides D, rebad, RD), rebaudioside E (rebaudiosides E, RE), rebaudioside M (rebaudiosides M, rebam, RM), rebaudioside I (rebaudiosides I, rebai, RI), and the like.

Glucosyltransferases are enzymes that transfer only glucosyl groups in an enzymatic reaction, the mechanism of action of which is to catalyze the transfer of the glucose residues of a glycosyl donor to a glycosyl acceptor molecule, thereby modulating the activity of the acceptor molecule.

"nucleoside" refers to glycosylamines (glycosamines) comprising a nucleobase (i.e., a nitrogenous base) and a 5-carbon sugar (e.g., ribose or deoxyribose). Non-limiting examples of nucleosides include cytidine (cytidine), uridine (uridine), adenosine (adenosine), guanosine (guanosine), thymidine (thytidine), and inosine (inosine).

"nucleoside diphosphate (nucleoside diphosphate)" refers to a glycosylamine comprising nucleobases (i.e., nitrogenous bases), 5-carbon sugars (e.g., ribose or deoxyribose), and diphosphate (i.e., pyrophosphate) moieties. "nucleoside diphosphate" may be abbreviated "NDP". Non-limiting examples of nucleoside diphosphates include Cytidine Diphosphate (CDP), uridine Diphosphate (UDP), adenosine Diphosphate (ADP), guanosine Diphosphate (GDP), thymidine Diphosphate (TDP), and Inosine Diphosphate (IDP).

UDP-glucose is an abbreviation of uridine diphosphate glucose (uridine diphosphate glucose), also referred to as UDP-glucose or UDPG for short, is a vitamin composed of uridine diphosphate and glucose, can be regarded as "active glucose", is widely distributed in cells of plants, animals and microorganisms, and is a most common glycosyl donor in the synthesis of sucrose, starch, glycogen and other oligosaccharides and polysaccharides. ADP-glucose, TDP-glucose, dTDP-glucose (deoxythymidine diphosphate glucose), CDP-glucose, IDP-glucose, GDP-glucose or the like are also commonly used as sugar donor compounds in addition to UDP-glucose. These glycosyl donors are expensive and are not suitable for industrial production.

Sucrose synthase (SUS, also abbreviated as SuSy/SS et al, EC2.4.1.13), which reversibly catalyzes the chemical reaction NDP-glucose+D-fructose to NDP and sucrose, was first identified in wheat (Triticum aestivum) germ as belonging to the glycosyltransferase-4 subfamily. Each protein exists in the form of a tetramer. Each subunit has a molecular weight of around 90 kDa. The gene size is generally 5.9kb, and the cDNA size is about 2.7 kb. The full length of the encoded amino acid sequence is about 800 amino acid residues (Ross and Davies 1992). Sucrose synthetases have different affinities for NDP, which in turn are UDP > ADP > dTDP > CDP > GDP.

Ohta et al (J.appl. Glycosyc.57, 199-209 (2010)) reported the first isolation of Rebaudioside I (Rebaudioside I, reb I, RI) from stevia rebaudiana. InfraPrakash et al (molecular 2014,19,17345-17355) reported for the first time a biosynthetic method for preparing RI using RA as the starting material and a mutant UGT76G1-R11-F12 of the glycosyltransferase UGT76G1, which method uses UDP-glucose as the glycosyl donor at pH7.5, mgCl ₂ Under the condition of 30 ℃ shaking flask reaction, the RI yield is 22.5%. Infra Prakash is published in patent application CN106795523A by the inventor under GenBank accession number AAR06912.1 of the glucosyltransferase UGT76G1, and discloses DNA sequences. CN110914445A selects mutant of glucosyltransferase UGT76G1 for catalyzing preparation of RI from RA under pH7.0 and MgCl condition ₂ 10.3mM, temperature 35 ℃. CN106795523a reports sweetness data and content of RICompositions of RI.

Only glucosyltransferases or mutants thereof having RI-catalyzing activity for RA synthesis are disclosed in the above prior art, but these enzymes have low activity and are not suitable for industrial production.

Disclosure of Invention

The invention aims to solve the technical problem that the existing glucosyltransferase has low enzyme activity when being applied to the biocatalytic preparation of RI, and provides a preparation method and application of rebaudioside I. The glycosyltransferase and sucrose synthase (SUS) are combined to catalyze and synthesize RI, so that cascade reaction is realized; wherein, sucrose and UDP (and/or ADP) generate UDPG (and/or ADPG) under SUS catalysis, thereby realizing regeneration of UDPG (and/or ADPG) and solving the problem of high price of glycosyl donor UDPG (and/or ADPG); the invention further optimizes the process conditions for synthesizing RI by enzyme catalysis, provides more choices for optimizing the process conditions for realizing large-scale industrial production, and is favorable for realizing industrial production.

In a first aspect, the present invention provides a method for preparing rebaudioside I by reacting rebaudioside A with a glycosyl donor in the presence of a glycosyltransferase comprising an amino acid residue difference at a residue position selected from one or more of the following compared to SEQ ID NO: 2:

amino acid at position 12 is Q;

amino acid at position 118 is S;

amino acid 265 is E;

amino acid 271 is A;

amino acid 308 is N;

amino acid 333 is K;

amino acid 347 is P;

amino acid at position 418 is D;

amino acid 455 is R;

and has a glycosyltransferase activity not lower than that shown by the amino acid sequence of SEQ ID NO. 2.

Preferably, the glycosyltransferase differs from SEQ ID NO. 2 by at least the amino acid residue at position 308 by N; more preferably, amino acid 271 is A.

In a preferred embodiment, the glycosyltransferase differs from SEQ ID NO. 2 by the amino acid residue: amino acid 308 is N and amino acid 12 is Q.

In a preferred embodiment, the glycosyltransferase differs from SEQ ID NO. 2 by the amino acid residue: amino acid 308 is N and amino acid 118 is S.

In a preferred embodiment, the glycosyltransferase differs from SEQ ID NO. 2 by the amino acid residue: amino acid 308 is N and amino acid 418 is D.

In a preferred embodiment, the glycosyltransferase differs from SEQ ID NO. 2 by the amino acid residue: amino acid 308 is N and amino acid 455 is R.

In a preferred embodiment, the glycosyltransferase differs from SEQ ID NO. 2 by the amino acid residue: amino acid 308 is N and amino acid 271 is A.

In a preferred embodiment, the glycosyltransferase differs from SEQ ID NO. 2 by the amino acid residue: amino acid 308 is N, amino acid 271 is A and amino acid 265 is E.

In a preferred embodiment, the glycosyltransferase differs from SEQ ID NO. 2 by the amino acid residue: amino acid 308 is N, amino acid 333 is K, and amino acid 347 is P.

In the preparation method, the glycosyltransferase is preferably in the form of glycosyltransferase thallus, crude enzyme liquid, pure enzyme liquid or immobilized enzyme;

in the preparation method, the glycosyl donor is preferably UDP-glucose and/or ADP-glucose.

In the preparation method, the concentration of the rebaudioside A is preferably 1-150g/L, such as 100g/L, 50g/L, 80g/L, 120g/L.

In the preparation method, the mass ratio of the glycosyltransferase thallus to the rebaudioside A is preferably 1 (3-10), such as 3:20, 1:5 and 1:8.

In the production method, the pH of the reaction solvent of the reaction is preferably 5 to 8, more preferably 5.5 to 6.

In the production method, the temperature of the reaction system of the reaction is preferably 20 to 90 ℃, for example 60 ℃, 30 ℃, 50 ℃, 70 ℃.

In the preparation method, the reaction time of the reaction is preferably 5 to 30 hours, for example 24 hours, 10 hours, 15 hours, 20 hours, 28 hours.

In the preparation method, the glycosyl donor is preferably prepared by UDP and/or ADP in the presence of sucrose and sucrose synthase.

The concentration of sucrose is preferably 100-300g/L, for example 200g/L, 150g/L, 250g/L.

The concentration of UDP or ADP is preferably 0.05-0.2g/L, for example 0.1g/L.

In the preparation method, the pH is preferably controlled by a buffer solution, for example, a phosphate buffer solution.

In the preparation method, the rotational speed of the reaction is preferably 500 to 1000rpm, for example 600rpm, 800rpm.

A second aspect of the present invention provides the use of a glycosyltransferase as defined in the first aspect of the present invention for the preparation of rebaudioside I.

In a third aspect the present invention provides a catalytic enzyme composition comprising a glycosyltransferase as defined in the first aspect of the invention and a sucrose synthase having a sequence as shown in SEQ ID NO. 4.

The mass ratio of the glycosyltransferase to sucrose synthase in the catalytic enzyme composition is preferably (2-10): 1, e.g. 5:1, 3:1, 8:1.

In a fourth aspect, the present invention provides the use of a catalytic enzyme composition according to the third aspect of the invention for the preparation of rebaudioside I.

The final concentration as used herein refers to the final concentration of the substance in the initial reaction system unless otherwise specified.

The invention has the beneficial effects that: the preparation method of the rebaudioside I has good catalytic effect and good stability, optimizes the process condition of synthesizing the rebaudioside I by an enzyme method, solves the problem of high price of glycosyl donor UDPG (and/or ADPG), provides more process condition optimization choices for realizing large-scale industrial production, and is beneficial to realizing industrial production.

Drawings

FIG. 1 is a synthetic route for preparing rebaudioside A and rebaudioside I from stevioside.

FIG. 2 is a graph of rebaudioside A controls using the HPLC detection method of the present application.

FIG. 3 is a graph of a rebaudioside I control using the HPLC detection method of the present application.

FIG. 4 is an HPLC chart of example 5 for 8 hours of reaction.

FIG. 5 shows an HPLC chromatogram of the reaction for 24 hours in example 5.

Detailed Description

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 1.

TABLE 1

The codons corresponding to the amino acids are also conventional in the art, and the correspondence between specific amino acids and codons is shown in table 2.

TABLE 2

The route of the present invention is schematically shown in FIG. 1.

KOD Mix enzyme was purchased from TOYOBO CO., LTD., dpnI enzyme from Injersey (Shanghai) trade Co., ltd; e.coli Trans10 competent cells were purchased from Beijing Ding state, biotechnology Limited, and E.coli BL21 (DE 3) competent cells were purchased from Beijing Ding state, biotechnology limited. RA60 (where RA content was 60% and stevioside content was about 30%) was purchased from morning light, product specification TSG90/RA60, sucrose was purchased from Biotechnology (Shanghai) Co., ltd. Reb a and Reb I controls were purchased from microphone.

Conversion HPLC detection method: chromatographic column: ZORBAXEclipse plus C18 (4.6 mm. Times.150 mm,3.5 um). 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 3 below. Detection wavelength: 210nm; flow rate: 1ml/min; sample injection volume: 20. Mu.L; column temperature: 35 ℃. As shown in fig. 2, reb a peak time: 14.504min; as shown in fig. 3, reb I off-peak time: 14.216min.

TABLE 3 Table 3

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 first round construction of library of beta-1, 3-glycosyltransferase mutants

The beta-1, 3-glycosyltransferase (beta-1, 3-GT enzyme) enzyme gene with the number of Enz.1 shown in SEQ ID NO. 1 is totally synthesized, and the gene is connected on 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 city Pingjiang region Xiang Min way 698). The amino acid sequence of Enz.1 is shown as SEQ ID NO. 2. The recombinant plasmid is transformed into competent cells of host escherichia coli BL21 (DE 3) to obtain an engineering strain containing the aminotransferase Enz.1 gene.

Similarly, the genes of aminotransferases Enz.2 to Enz.14 obtained by site-directed mutagenesis of the Enz.1 gene in Table 4 were ligated to the vector pET28a at the cleavage sites NdeI and HindIII, to obtain recombinant plasmids containing the genes of aminotransferases Enz.2 to Enz.14, respectively. Each recombinant plasmid was transformed into competent cells of E.coli BL21 (DE 3) as a host to obtain engineering strains Enz.2 to Enz.14 containing the transaminase gene shown in Table 4.

TABLE 4 Table 4

EXAMPLE 2 preparation of beta-1, 3-glycosyltransferase mutants

1. Protein expression of the mutation vector was performed:

single colonies of the strain of example 1 were inoculated into 5mL LB liquid medium containing 50. Mu.g/mL kanamycin, respectively, 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 a reaction enzyme solution:

50mM Phosphate Buffer (PBS) having pH of 6.0 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 the enzyme solutions of the beta-1, 3-glycosyltransferase reactions.

EXAMPLE 3 preparation of sucrose synthase SUS

A sucrose synthase (SUS) gene shown in SEQ ID NO. 3 and numbered Enz.15 was synthesized, and the gene was ligated to a pET28a plasmid vector to obtain a recombinant plasmid pET28a-SUS. The gene synthesis company is biological engineering (Shanghai) stock limited company (Shanghai city, songjiang region Min Ji Lu 698). The amino acid sequence of sucrose synthase is shown as SEQ ID NO. 4.

Plasmid pET28a-SUS was transformed into host E.coli BL21 (DE 3) competent cells to obtain an engineering strain containing the Enz.15 gene. Single colonies were 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 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 (PBS) with pH of 6.0 is prepared, the bacterial cells obtained above are suspended according to a ratio of 1:10 (M/V, g/mL), and then high-pressure homogenization (550 Mbar, 1.5 min) is carried out, and centrifugation is carried out for 2min at 12000rpm, thus obtaining the sucrose synthase reaction enzyme solution.

EXAMPLE 4 screening of beta-1, 3-glycosyltransferase mutants

To 1mL of the reaction system, 150. Mu.L of the beta. -1, 3-glycosyltransferase prepared in example 2, 50g/L of RA60, 0.1g/L of ADP, 200g/L of sucrose and 30. Mu.L of the sucrose synthase enzyme prepared in example 3 were added, and finally 50mM of pH6.0 phosphate buffer was 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 a pH of 2 to 3, vortexed, centrifuged at 13000rpm for 10 minutes, and the supernatant was analyzed for the concentration of Reb I by HPLC. The experimental results obtained using the HPLC detection method are shown in table 5.

TABLE 5

Enzyme numbering	RI％(RA→RI)
		Enz.1	2.886
Enz.2	0.555
		Enz.3	1.705
Enz.4	1.324
		Enz.5	0.886
Enz.6	1.427
		Enz.7	4.139
Enz.8	4.014
		Enz.9	3.628
Enz.10	3.910
		Enz.11	4.235
Enz.12	4.646
		Enz.13	3.546
Enz.14	1.939

From the preliminary screening results in table 5, it can be seen that: enz.12 was the highest in enzyme activity, so Enz.12 was subsequently selected for the experiment.

EXAMPLE 5 enzymatic Enz.12 catalytic Synthesis of RI

In 1mL reaction system, 150. Mu.L of Enz.12 reaction enzyme solution, 30. Mu.L of sucrose synthase reaction enzyme solution, 100g/L of RA60 final concentration, 200g/L of sucrose final concentration and 0.1g/L of ADP final concentration were added, and finally PBS (50 mM, pH 5.5) was added to a final volume of 1mL. The prepared reaction system was placed in a metal bath, reacted at 60℃and 600rpm, 10. Mu.L of the reaction solution was taken at 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h and 24h, respectively, and added to 990. Mu.L of hydrochloric acid having a pH of 2 to 3, vortexed, centrifuged at 13000rpm for 10min, and the supernatant was subjected to HPLC analysis. The experimental results obtained using the HPLC detection method are shown in table 6.

TABLE 6

Time/h	1	2	3	4	5	6	7	8	24
										RI％	17.62	26.18	39.47	46.92	55.86	61.70	67.71	71.36	97.81

The reaction results in table 6 show: after 8h of reaction, the peak area ratio of RI reaches 71%, after 24h of reaction, the peak area ratio of RI reaches 97%, and the reaction is basically complete.

FIG. 4 is an HPLC chart of the results of the 8-hour reaction in Table 6. FIG. 5 is an HPLC chart showing the results of 24-hour reaction in Table 6.

SEQUENCE LISTING

<110> chess Ke Lai Biotechnology (Shanghai) stock Co., ltd

<120> preparation method and application of rebaudioside I

<130> P21017497C

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1371

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.1

<400> 1

atgccgaaca ctaacccaac taccgtgcgt cgtcgtcgtg ttattatgtt tccggttccg 60

ttcccgggcc acttaaaccc gatgctgcaa ctggcgaacg tgctgtaccg tagaggtttt 120

gaaatcacca ttctgcacac caacttcaac gccccgaaaa ccagccttta tccgcacttc 180

cagtttcgtt ttatcttgga caacgatccg caaccggagt ggttacgcaa cctgccgacg 240

actggtccgg gcgtgggtgc aagaatcccg gtaattaaca aacacggcgc ggatgaattc 300

cgtaaggagc tggaaatctg catgcgggat actccgagtg acgaggaagt tgcttgcgtg 360

attaccgatg cgctgtggta cttcgcgcaa ccggtggcgg acagcctgaa tctgaaacgt 420

ctggttctgc agaccgggag cctgtttaac ttccactgcc tggtgtgtct gccgaaattt 480

ctggagttgg gctacctgga tccggaaact aaacatcgtc cggatgaacc ggtggtaggt 540

ttcccgatgc tgaaggttaa agatatccgt cgcgcgtatt cgcacattca agaatcgaaa 600

ccaattctga tgaagatggt tgaagaaacc cgtgccagca gcggtgtgat ttggaacagc 660

gctaaagagc tggaggaaag cgagctggaa accattcagc gtgaaattcc ggcgccgagc 720

ttcctgcttc cgctgccgaa gcattatagg gcttcgagca ctagcctgct ggatactgat 780

ccgagcaccg cccaatggct ggaccagcag ccgccgagca gcgtgctgta cgttggcttt 840

ggcagccaga gctcgctgga ccccgcagat ttcctggaga ttgcgcgtgg tctggttgcg 900

agcaaacaaa gctttctgtg gctggttcgt ccgggcttcg tgaagggtta tgagtggatt 960

gagctgctgc cggatggttt tctgggtgaa aaaggtcgta tcgtgaagtc tgctccgcaa 1020

caagaagtgc tggcgcacaa ggcgattggt gcgttctgga cccacggcgg ttggaacggc 1080

accatggagg ccgtgtgcga aggcgtgccg atgatcttta gcgatttcgg tctggatcag 1140

ccgctgaacg cgcgttacat gagcgaggtt ctgcatgtgg gcgtttatct ggagaacggc 1200

ttcatccgtg gtgagatcat taatgcggtt aggcgtgtga tggttgaccc tgagggtgag 1260

gttatgcgcc aaaacgcgcg taaattgaag gataagttgg atcgaagcat tgctcccggt 1320

ggcagcagct acgagagcct ggaacgcctg cagagctata ttagcagcct g 1371

<210> 2

<211> 457

<212> PRT

<213> Artificial Sequence

<220>

<223> Enz.1

<400> 2

Met Pro Asn Thr Asn Pro Thr Thr Val Arg Arg Arg Arg Val Ile Met

1 5 10 15

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

20 25 30

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

35 40 45

Phe Asn Ala Pro Lys Thr Ser Leu Tyr Pro His Phe Gln Phe Arg Phe

50 55 60

Ile Leu Asp Asn Asp Pro Gln Pro Glu Trp Leu Arg Asn Leu Pro Thr

65 70 75 80

Thr Gly Pro Gly Val Gly Ala Arg Ile Pro Val Ile Asn Lys His Gly

85 90 95

Ala Asp Glu Phe Arg Lys Glu Leu Glu Ile Cys Met Arg Asp Thr Pro

100 105 110

Ser Asp Glu Glu Val Ala Cys Val Ile Thr Asp Ala Leu Trp Tyr Phe

115 120 125

Ala Gln Pro Val Ala Asp Ser Leu Asn Leu Lys Arg Leu Val Leu Gln

130 135 140

Thr Gly Ser Leu Phe Asn Phe His Cys Leu Val Cys Leu Pro Lys Phe

145 150 155 160

Leu Glu Leu Gly Tyr Leu Asp Pro Glu Thr Lys His Arg Pro Asp Glu

165 170 175

Pro Val Val Gly Phe Pro Met Leu Lys Val Lys Asp Ile Arg Arg Ala

180 185 190

Tyr Ser His Ile Gln Glu Ser Lys Pro Ile Leu Met Lys Met Val Glu

195 200 205

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

210 215 220

Glu Glu Ser Glu Leu Glu Thr Ile Gln Arg Glu Ile Pro Ala Pro Ser

225 230 235 240

Phe Leu Leu Pro Leu Pro Lys His Tyr Arg Ala Ser Ser Thr Ser Leu

245 250 255

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

260 265 270

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

275 280 285

Ala Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Ala Ser Lys Gln Ser

290 295 300

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

305 310 315 320

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

325 330 335

Ser Ala Pro Gln Gln Glu Val Leu Ala His Lys Ala Ile Gly Ala Phe

340 345 350

Trp Thr His Gly Gly Trp Asn Gly Thr Met Glu Ala Val Cys Glu Gly

355 360 365

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

370 375 380

Arg Tyr Met Ser Glu Val Leu His Val Gly Val Tyr Leu Glu Asn Gly

385 390 395 400

Phe Ile Arg Gly Glu Ile Ile Asn Ala Val Arg Arg Val Met Val Asp

405 410 415

Pro Glu Gly Glu Val Met Arg Gln Asn Ala Arg Lys Leu Lys Asp Lys

420 425 430

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

435 440 445

Arg Leu Gln Ser Tyr Ile Ser Ser Leu

450 455

<210> 3

<211> 2415

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.15

<400> 3

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> 4

<211> 804

<212> PRT

<213> Artificial Sequence

<220>

<223> Enz.15

<400> 4

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

Claims (10)

1. A method of preparing rebaudioside I by reacting rebaudioside a with a glycosyl donor in the presence of a glycosyltransferase, wherein the glycosyltransferase comprises an amino acid residue difference at a residue position selected from one or more of the following compared to SEQ ID No. 2:

amino acid at position 12 is Q;

amino acid at position 118 is S;

amino acid 265 is E;

amino acid 271 is A;

amino acid 308 is N;

amino acid 333 is K;

amino acid 347 is P;

amino acid at position 418 is D; a kind of electronic device with high-pressure air-conditioning system

Amino acid 455 is R;

and has a glycosyltransferase activity not lower than that shown by the amino acid sequence of SEQ ID NO. 2.

2. The method of claim 1, wherein the glycosyltransferase differs from SEQ ID No. 2 by at least the amino acid residue at position 308 by N; preferably selected from any one of the following groups:

amino acid 308 is N and amino acid 12 is Q;

amino acid 308 is N and amino acid 118 is S;

amino acid 308 is N and amino acid 418 is D;

amino acid 308 is N and amino acid 455 is R;

amino acid 308 is N and amino acid 271 is A;

amino acid 308 is N, amino acid 271 is A and amino acid 265 is E; a kind of electronic device with high-pressure air-conditioning system

Amino acid 308 is N, amino acid 333 is K, and amino acid 347 is P.

3. The preparation method according to claim 1 or 2, wherein the preparation method satisfies one or more of the following conditions:

the glycosyltransferase exists in the form of glycosyltransferase thalli, crude enzyme liquid, pure enzyme liquid or immobilized enzyme;

the glycosyl donor is UDP-glucose and/or ADP-glucose;

the concentration of the rebaudioside A is 1-150g/L, preferably 100g/L;

the mass ratio of the glycosyltransferase thallus to the rebaudioside A is 1 (3-10), preferably 3:20;

the pH of the reaction solvent of the reaction is 5 to 8, preferably 5.5 to 6;

the temperature of the reaction system of the reaction is 20-90 ℃, preferably 60 ℃;

the reaction time of the reaction is 5 to 30 hours, preferably 24 hours.

4. A method of preparation according to claim 3, wherein the glycosyl donor is prepared by UDP and/or ADP in the presence of sucrose and sucrose synthase.

5. The process according to claim 4, wherein the concentration of sucrose is 100-300g/L, preferably 200g/L, and the concentration of UDP or ADP is 0.05-0.2g/L, preferably 0.1g/L.

6. A method of preparation according to claim 3, wherein the pH is controlled by a buffer solution, preferably a phosphate buffer solution;

and/or the rotational speed of the reaction is 500-1000rpm, preferably 600rpm.

7. Use of a glycosyltransferase as defined in claim 1 or 2 for the preparation of rebaudioside I.

8. A catalytic enzyme composition comprising a glycosyltransferase as defined in claim 1 or 2 and a sucrose synthase having a sequence as set forth in SEQ ID No. 4.

9. Catalytic enzyme composition according to claim 8, wherein the mass ratio of glycosyltransferase to sucrose synthase is (2-10): 1, preferably 5:1.

10. The use of the catalytic enzyme composition of claim 8 or 9 in the preparation of rebaudioside I.

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