CN115725528A - Glycosyl transferase and application thereof - Google Patents

Glycosyl transferase and application thereof Download PDF

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CN115725528A
CN115725528A CN202111006803.4A CN202111006803A CN115725528A CN 115725528 A CN115725528 A CN 115725528A CN 202111006803 A CN202111006803 A CN 202111006803A CN 115725528 A CN115725528 A CN 115725528A
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glycosyltransferase
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吴燕
田振华
郑孝富
王舒
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Ecolab Biotechnology Shanghai Co ltd
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Abstract

The invention provides a glycosyl transferase and application thereof. The glycosyltransferase comprises an amino acid residue difference at a residue position selected from one or more of: the 14 th amino acid is I; l represents amino acid 189; amino acid 257 is A, C, L, M, S or V; the 265 th amino acid is E or A; the amino acid at position 273 is G; amino acid 302 is G; amino acid 324 is G; amino acid 347 is G; amino acid E at position 451; amino acid 455 is D or C; and has a glycosyltransferase activity not lower than that shown by the amino acid sequence of SEQ ID NO. 2. The glycosyltransferase has high enzyme activity and good stability; compared with a glycosyltransferase parent, the catalytic activity of the complex is obviously improved and the conversion rate is obviously improved when the complex is used for preparing stevioside. The invention solves the problem of high price of glycosyl donors UDPG and ADPG, and also provides a plurality of selection possibilities of substrates.

Description

Glycosyl transferase and application thereof
Technical Field
The invention relates to glycosyltransferase and application thereof in glycosylation reaction of stevioside.
Background
Steviol glycosides (also called Steviol glycosides) are natural sweeteners extracted from stevia rebaudiana leaves of herbal plants of the family asteraceae, are mixtures of a plurality of glycosides, and different Steviol glycosides have great difference in taste quality. The stevioside has the advantages of pure nature (from pure natural plant stevia rebaudiana), high sweetness (250-450 times of cane sugar), low calorie (only 1/300 of white sugar), economical use (the cost is only one third of cane sugar), good stability (heat resistance, acid resistance, alkali resistance, difficult decomposition phenomenon), high safety (no toxic or side effect) and the like, and has potential curative effects of resisting hyperglycemia, hypertension, inflammation, tumor, diarrhea and the like.
The structural formula of stevioside (stevioside compound) is as follows:
Figure BDA0003237510550000011
serial number Compound (I) R 1 R 2
1 Steviol H H
2 Steviol monoglycoside H β-Glc
3 Steviol bisglycosides H β-Glc-β-Glc(2-1)
4 Rubusoside 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 Dukoside A β-Glc β-Glc-α-Rha(2-1)
The stevioside compounds have the following common aglycone: steviol (Steviol), which is distinguished by the number and type of glycosyl groups attached at the C-13 and C-19 positions, mainly comprises eight glycosides such as Stevioside (Stevioside), rebaudioside A (Rebaudianide A, reb A), rebaudioside B, rebaudioside C, rebaudioside D (Rebaudianide D, reb D), rebaudioside E, dulcoside, steviolbioside, and the like. Stevia leaves can accumulate as much as 10-20% (dry weight basis) steviol glycosides. The major 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 and F, steviolbioside and rubusoside, are found at much lower levels (approximately 0-0.2%).
Although stevioside is a high-intensity sweetener, the stevioside has the defect of post-bitterness, and the application of the stevioside in the fields of foods, beverages and the like with high requirements on taste is severely limited. The essential reason for causing the bitter taste after the stevioside is caused by the intrinsic molecular structure, R in the stevioside 1 And R 2 The more the number of glycosyl groups attached to the group, the better the mouthfeel. Typically, stevioside is found to be 110-270 times sweeter than sucrose, with rebaudioside a ranging from 150 to 320 times, however, even in a highly purified state, stevioside still has undesirable taste attributes such as bitter taste, sweet aftertaste, licorice taste, and the like.
Rebaudioside D is stevioside with the most application potential, has high sweetness which is about 300-350 times of that of cane sugar compared with other stevioside, has pure sweetness, is closer to the cane sugar in mouthfeel, has no bitter taste and licorice peculiar smell, has good stability, and is an ideal natural high-power sweetener product. The rebaudioside D content in stevia leaves is very low (less than 5%), a large amount of stevia raw materials are needed for producing rebaudioside D by adopting an extraction method, the process for enriching rebaudioside D is complicated, column passing, desalting, decoloring and recrystallizing are needed for many times after extraction, a large amount of waste water is generated in the production process, the production cost is high, and the method is not suitable for industrial large-scale production.
The prior method for synthesizing rebaudioside D by a biological enzyme method needs to add expensive UDP-glucose as one of substrates, and generates rebaudioside D by catalysis under the action of UDP-glucosyltransferase (UGT for short) and taking stevioside or rebaudioside A as the substrate. But due to the extremely high selling price of UDP-glucose, the feasibility of industrially preparing the rebaudioside D is almost completely limited, the economy is poor, and the market competitiveness is lacked.
Rebaudioside M (RebM) has better taste properties, but its content of dry weight of the leaves is less than 0.1%, resulting in high separation cost and high price. Biocatalytic approaches to high rebaudioside M concentrations have attracted attention from researchers. It is reported that recombinant enzymes derived from stevia rebaudiana can catalyze rebaudioside D to produce rebaudioside M, but the yield is low. Rebaudioside D is used as a substrate, and rebaudioside M can be obtained through a microbial enzyme production catalysis method. However, the following problems mainly exist in the bio-enzyme catalysis method at present: (1) The cost of producing rebaudioside M by bio-enzyme catalysis of rebaudioside D is high, and the enzyme-catalyzed yield needs to be further optimized; (2) The glycosyl transferase used for catalysis is not easy to separate from the product and recycle, and is volatile; (3) The natural plants have high rebaudioside A content and very low rebaudioside D content, and the direct conversion of rebaudioside A into rebaudioside D at low cost is also an urgent problem to be solved.
Glucosyltransferases are enzymes that transfer only glucose groups in an enzymatic reaction by catalyzing the transfer of the glucose residue of a glycosyl donor to a glycosyl acceptor molecule, thereby modulating the activity of the acceptor molecule. UDP-glucosyltransferase is one of glucosyltransferases, and UDP-glucose is used as a glycosyl donor and is present in almost all organisms.
UDP-glucose, also referred to simply as UDP-glucose or UDPG, is a short name for uridine diphosphate glucose (uridine diphosphate glucose), a vitamin consisting of uridine diphosphate and glucose, which can be regarded as "active glucose", widely distributed in cells of plants, animals and microorganisms, and as a donor of glucose groups in the synthesis of sucrose, starch, glycogen and other oligosaccharides and polysaccharides, which is the most common one of the glycosyl donors.
Nowadays, with the wide application of the natural sweetener stevioside and the increasing development of the biological catalysis technology, glucosyltransferases are increasingly applied to the field of biological catalysis preparation of stevioside. Enzymes used in the field of biological enzyme method preparation of stevioside at present often have the defects of low enzyme activity, poor stability and the like, so that the cost for preparing the stevioside by applying the enzymes to industrial mass production is higher. Therefore, there is a need to modify glucosyltransferase to obtain modified enzyme with higher enzyme activity and better stability, so as to better serve for industrial mass production.
Disclosure of Invention
The invention aims to solve the technical problem that the prior glucosyltransferase has low enzyme activity and poor stability when being applied to the biological catalytic preparation of stevioside, so that the glucosyltransferase has the defects of low conversion rate and the like when being applied to the catalysis of the stevioside, and therefore the glucosyltransferase and the application thereof in the preparation of the stevioside are provided. The Glycosyltransferase (GT) of the invention has high enzyme activity and good stability; compared with a glycosyltransferase parent, the method has the advantages that when the method is used for preparing stevioside (such as rebaudioside A, rebaudioside D or rebaudioside M), the catalytic activity is obviously improved, and the conversion rate is obviously improved, so that the reaction cost is reduced, and the method is favorable for industrial production.
To solve the above technical problem, the first aspect of the present invention provides a glycosyltransferase comprising an amino acid residue difference at a residue position selected from one or more of:
the 14 th amino acid is I;
l at amino acid position 189;
amino acid 257 is A, C, L, M, S or V;
the 265 th amino acid is E or A;
the amino acid at position 273 is G;
amino acid 302 is G;
amino acid 324 is G;
amino acid 347 is G;
amino acid E at position 451;
amino acid 455 is D or C;
and has glycosyltransferase activity not lower than that shown by the amino acid sequence of SEQ ID NO. 2.
In the present invention, the difference may be obtained by mutation on the amino acid sequence shown in SEQ ID NO. 2, or may be obtained by mutation on the basis of other amino acid sequences, and the final mutation result is within the scope of the present invention as long as the difference is obtained by comparing the amino acid sequence shown in SEQ ID NO. 2.
Preferably, the glycosyltransferase differs in amino acid residue from SEQ ID NO 2 by an amount selected from the group consisting of:
(1) The 265 th amino acid is E; or the like, or, alternatively,
amino acid 257 is A, and amino acid 451 is E; or the like, or, alternatively,
the 265 th amino acid is E, and the 451 th amino acid is E;
(2) Amino acid 14 is I, amino acid 257 is A and amino acid 451 is E; or
Amino acid 257 is A, amino acid 451 is E, and amino acid 189 is L; or the like, or, alternatively,
amino acid 257 is A, amino acid 451 is E, and amino acid 273 is G; or the like, or a combination thereof,
amino acid 257 is A, amino acid 451 is E, and amino acid 302 is G; or
C at amino acid 257 and E at amino acid 451; or
L at amino acid position 257 and E at amino acid position 451; or
M at amino acid 257 and E at amino acid 451; or
Amino acid 257 is S, and amino acid 451 is E; or
V at amino acid 257 and E at amino acid 451; or
Amino acid 257 is A, amino acid 451 is E, and amino acid 265 is A;
(3) Amino acid 257 is A, amino acid 451 is E, amino acid 189 is L, and amino acid 14 is I; or the like, or a combination thereof,
an amino acid A at position 257, an amino acid E at position 451, an amino acid L at position 189 and an amino acid G at position 273; or the like, or a combination thereof,
amino acid 257 is A, amino acid 451 is E, amino acid 189 is L, and amino acid 324 is G; or the like, or, alternatively,
a 257 th amino acid is a, a 451 th amino acid is E, a 189 th amino acid is L, and a 347 th amino acid is G; or the like, or a combination thereof,
(3) A at amino acid 257, E at amino acid 451, L at amino acid 189, and D or C at amino acid 455.
In order to solve the above technical problem, the second aspect of the present invention provides an isolated nucleic acid encoding the glycosyltransferase according to the first aspect of the invention.
In order to solve the above technical problems, the third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid according to the second aspect of the present invention.
In order to solve the above technical problems, a fourth aspect of the present invention provides a transformant which is a host cell comprising the nucleic acid according to the second aspect of the present invention or the recombinant expression vector according to the third aspect of the present invention.
The host cell may be conventional in the art, preferably Escherichia coli (Escherichia coli) such as E.coli BL21 (DE 3).
In order to solve the above-mentioned technical problems, a fifth aspect of the present invention provides a method for producing the glycosyltransferase according to the first aspect of the present invention, the method comprising culturing the transformant according to the fourth aspect of the present invention under conditions suitable for expression of the glycosyltransferase.
After the glycosyltransferase is expressed on the transformant, the glycosyltransferase can be extracted by means of the conventional technical means in the field, for example, a crude enzyme solution can be prepared, the crude enzyme solution can be subjected to conventional concentration and replacement after preparation, and the crude enzyme solution can be further subjected to one or more purification steps such as ion exchange chromatography, affinity chromatography, hydrophobic chromatography, molecular sieve chromatography and the like to purify the glycosyltransferase. In a preferred embodiment, the following steps are used: (1) Inoculating the transformant containing the glycosyltransferase into a culture medium containing antibiotics, such as an LB culture medium, and performing shake culture to obtain a seed solution; (2) Transferring the seed liquid in the step (1) to a culture medium containing antibiotics, such as TB culture medium, and carrying out shaking culture; (3) Adding IPTG into the culture medium in the step (2) for overnight induction, and collecting thalli after centrifugation; (4) And (4) washing and re-suspending the thalli collected in the step (3), crushing and centrifuging to obtain a crude enzyme solution containing the glycosyltransferase.
In order to solve the above technical problems, a sixth aspect of the present invention provides a composition comprising the glycosyltransferase according to the first aspect of the invention.
In order to solve the above technical problem, a seventh aspect of the present invention provides a method for the glycosylation of a substrate, the method comprising providing at least one substrate, a glycosyltransferase according to the first aspect of the present invention, and contacting the substrate with the glycosyltransferase under conditions such that the substrate is glycosylated to produce at least one glycosylation product.
In order to solve the above technical problems, an eighth aspect of the present invention provides a method for preparing rebaudioside a, comprising the steps of: reacting stevioside with a glycosyl donor in the presence of a glycosyltransferase according to the first aspect of the invention to obtain rebaudioside A.
In a preferred embodiment, the glycosyltransferase is present as glycosyltransferase bacteria, crude enzyme solution, pure enzyme solution, or immobilized enzyme.
In a preferred embodiment, the concentration of stevioside is 1-150g/L, preferably 100g/L.
In a preferred embodiment, the mass ratio of the glycosyltransferase thalli to stevioside is 1 (3-10), preferably 3.
In a preferred embodiment, the glycosyl donor is UDP-glucose and/or ADP-glucose.
Preferably, it is 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.
The concentration of said UDP or said ADP is preferably 0.05-0.2g/L, such as 0.1g/L.
In a preferred embodiment, the reaction solvent for the reaction has a pH of 5 to 8, preferably 6.
In a preferred embodiment, the pH is controlled by a buffer solution, preferably a phosphate buffer solution.
In a preferred embodiment, the speed of rotation of the reaction is between 500 and 1000rpm, preferably 600rpm.
In a preferred embodiment, the temperature of the reaction is 20 to 90 ℃, preferably 60 ℃.
In order to solve the above technical problems, the ninth aspect of the present invention provides a method for preparing rebaudioside D, which comprises the step of preparing rebaudioside a according to the preparation method described in the eighth aspect of the present invention.
In preparing rebaudioside D, a β -1, 2-glycosyltransferase is used in addition to the glycosyltransferase according to the first aspect of the invention.
In order to solve the above technical problems, a tenth aspect of the present invention provides a method for preparing rebaudioside M, comprising the step of preparing rebaudioside a according to the preparation method of the eighth aspect of the present invention.
In a preferred embodiment, the method comprises providing a stevioside substrate, a glycosyl donor, and a glycosyltransferase as previously described, and reacting the stevioside substrate, glycosyl donor, and glycosyltransferase as previously described under conditions such that rebaudioside D or rebaudioside M is produced.
In order to solve the above technical problems, an eleventh aspect of the present invention provides a glycosyltransferase according to the first aspect of the present invention for use in the preparation of a steviol glycoside.
The steviol glycoside is preferably rebaudioside a, rebaudioside D or rebaudioside M.
"glycosyltransferases" in the context of the present invention include NDP-glycosyltransferases, including, but not limited to, UDP-glucose-dependent glycosyltransferases (UDP-glycosyltransferase; UGT), ADP-glucose-dependent glycosyltransferases (ADP-glycosyltransferase; AGT), CDP-glucose-dependent glycosyltransferases (CDP-glycosyltransferase; CGT), GDP-glucose-dependent glycosyltransferases (GDP-glycosyltransferase; GGT), TDP-glucose-dependent glycosyltransferases (TDP-glycosyltransferase; TGT), and IDP-glucose-dependent glycosyltransferases (IDP-glycosyltransferase; IGT).
The sucrose synthase of the present invention is referred to as sucrose synthase (EC 2.4.1.1.13, SUS) or simply SuSy/SS, etc.
The Glycosyltransferase (GT) of the invention has high enzyme activity and good stability; compared with a glycosyltransferase parent, the method has the advantages that when the method is used for preparing stevioside (such as rebaudioside A, rebaudioside D or rebaudioside M), the catalytic activity is obviously improved, and the conversion rate is obviously improved, so that the reaction cost is reduced, and the method is favorable for industrial production. According to the invention, the RA, RD and RM are synthesized by jointly using Glycosyltransferase (GT) and sucrose synthase for catalysis, so that a cascade reaction is realized, not only can the UDPG regeneration be realized by using sucrose and UDP, but also the ADPG regeneration can be realized by using sucrose and ADPG, the problem that glycosyl donors UDPG and ADPG are expensive is solved, various selection possibilities of substrates are provided, more process condition optimization choices are provided for further realizing large-scale industrial production, and the realization of large-scale industrialization is facilitated.
Drawings
FIG. 1 shows a schematic route for preparing rebaudioside A, rebaudioside D and rebaudioside M from stevioside in an embodiment of the invention.
FIG. 2 shows the retention times of stevioside, rebaudioside A controls using HPLC detection method 1; the retention time of stevioside is 12.761min, and the retention time of rebaudioside A is 12.377min.
FIG. 3 is a graph of rebaudioside A control using HPLC detection method 2 with a retention time of 14.186min.
FIG. 4 is a graph of rebaudioside D control using HPLC detection method 2 with a retention time of 11.821min.
FIG. 5 is a graph of rebaudioside M control using HPLC detection method 2 with a retention time of 12.316min.
FIG. 6 is a graph showing the activity of Enz.7 in Table 6 for catalytically synthesizing RA.
FIG. 7 is a graph showing the activity of Enz.10 for catalytic synthesis of RA in Table 8.
FIG. 8 is a graph showing the activity of Enz.45 in the catalytic synthesis of RA under the condition where ADP is nucleoside diphosphate in Table 10.
FIG. 9 is a graph showing the activity of Enz.45 for catalytically synthesizing RM under the condition that UDP is nucleoside diphosphate in Table 11.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the invention thereto. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.
The experimental methods in the invention are conventional methods unless otherwise specified, and the gene cloning operation can be specifically referred to the molecular cloning experimental guidance compiled by J. Sambruka et al.
The abbreviations of the amino acids in the present invention are those conventional in the art unless otherwise specified, and the amino acids corresponding to the specific abbreviations are shown in Table 1.
TABLE 1
Figure BDA0003237510550000081
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
Figure BDA0003237510550000082
The route of the invention is schematically shown in fig. 1.
KOD Mix enzyme was purchased from TOYOBO co., ltd., and DpnI enzyme was purchased from yiwei fundi (shanghai) trade ltd; coli Trans10 competent cells were purchased from the beijing dingguoshang biotechnology ltd, and e coli BL21 (DE 3) competent cells were purchased from the beijing dingguoshang biotechnology ltd. Sucrose was purchased from a biological organism. Stevioside, a reaction substrate for the first, second and third rounds of screening, was obtained from Biyu medicine (purity 95%), RA60, a reaction substrate for RM synthesis, was obtained from Chenopodium (RA content 60%, stevioside content about 30%, product specification TSG90/RA 60). Sucrose was purchased from Biotechnology engineering (Shanghai) GmbH. Reb a control was purchased from mcelin. Reb D and Reb M controls were purchased from national stevia rebaudiana trade ltd, sinensland.
HPLC detection method 1: and (3) chromatographic column: agilent 5TC-C18 (2) (250X 4.6 mm). Mobile phase: 0.1% aqueous TFA as mobile phase A,0.1% acetonitrile TFA as mobile phase B, gradient elution was performed as in Table 3 below. Detection wavelength: 210nm; flow rate: 1ml/min; sample injection volume: 20 mu l of the mixture; column temperature: at 40 ℃. As shown in FIG. 2, stevioside retention time was 12.76min and Reb A retention time was 12.38min.
TABLE 3
Time (minutes) Mobile phase A% Mobile phase B%
0.00 70 30
15.00 60 40
20.00 30 70
25.00 30 70
25.10 70 30
32.00 70 30
HPLC detection method 2: and (3) chromatographic column: ZORBAXeclipse plus C18 (4.6 mm 150mm,3.5 um). Mobile phase: 0.1% aqueous TFA as mobile phase A,0.1% acetonitrile TFA as mobile phase B, gradient elution was performed as follows in Table 4. Detection wavelength: 210nm; flow rate: 1ml/min; sample injection volume: 20 mu l of the mixture; column temperature: 35 ℃ is carried out. As shown in fig. 3, reb a peak time: 14.186min; as shown in fig. 4, reb D peak time: 11.821min; as shown in fig. 5, reb M peak time: 12.316min.
TABLE 4
Figure BDA0003237510550000091
Figure BDA0003237510550000101
EXAMPLE 1 first round construction of a library of beta-1, 3-glycosyltransferase mutants
The total synthesis of beta-1, 3-glycosyltransferase (beta-1, 3-GT enzyme) gene with number Enz.1 as shown in SEQ ID NO:1, which has been linked to pET28a plasmid vector to obtain recombinant plasmid pET28a-Enz.1, the GenBank of China, min-Lu 698, shanghai, was the company of Industrial bioengineering (Shanghai). The amino acid sequence of Enz.1 is shown in SEQ ID NO. 2.
pET28a-Enz.1 plasmid is used as a template, a primer sequence shown in a table 5 is adopted, KOD enzyme is adopted for PCR amplification, and gene fragments and vector fragments of target mutants Enz.2-Enz.8 are obtained.
TABLE 5
Figure BDA0003237510550000102
The PCR amplification reaction system is as follows:
KOD Mix：25μL
ddH 2 O：20μL
primer: 2 μ L2
Template: 1 μ L
The amplification procedure was as follows:
(1)98℃3min
(2)98℃10s
(3)55℃5s
(4)68℃5s/kbp
(5)68℃5min
(6) Heat preservation at 12 DEG C
(2) The cycle of (4) to (4) is 34 times.
And (3) carrying out DpnI digestion on the PCR product, and carrying out gel running and gel recovery to obtain a target DNA fragment. Two fragments of homologous recombinase (Exnase II,5 XCE II) of Novoxan are connected to the pET28a plasmid vector to obtain recombinant plasmids pET28 a-Enz.2-pET 28a-Enz.8 of each mutant. After ligation, E.coli Trans10 competent cells were transformed, plated on LB medium containing 50. Mu.g/mL kanamycin, and cultured overnight at 37 ℃; picking single colony to LB test tube (Km resistance), culturing for 8-10h, extracting plasmid and sequencing.
EXAMPLE 2 preparation of beta-1, 3-glycosyltransferase mutants
1. Protein expression of the mutated vector was performed:
transforming the recombinant plasmid with correct sequencing into host E.coli BL21 (DE 3) competent cells to obtain the genetic engineering strain containing point mutation. A single colony was inoculated into 5ml of LB liquid medium containing 50. Mu.g/ml kanamycin and shake-cultured at 37 ℃ for 4 hours. Inoculating the mixture to 50ml of fresh TB liquid medium containing 50. Mu.g/ml kanamycin at an inoculation amount of 2% (v/v), and performing shake culture at 37 ℃ until OD is reached 600 When the concentration reached about 0.8, IPTG (Isopropyl-. Beta. -D-thiogalactoside) was added to a final concentration of 0.1mM, and induction culture was carried out at 25 ℃ for 20 hours. After the culture was completed, the culture solution was centrifuged at 4000rpm for 20min, and the supernatant was discarded to collect the cells. Storing at-20 deg.C for use.
2. Obtaining a reaction enzyme solution:
50mM Phosphate Buffer Solution (PBS) at pH6.0 was prepared, and the obtained cells were suspended at 1; and (3) treating the homogenized beta-1, 3-GT enzyme liquid at 80 ℃ for 15min, and centrifuging at 12000rpm for 2min to obtain the reaction enzyme liquid.
Example 3 preparation of sucrose synthase SUS
The sucrose synthase (SUS) gene of SEQ ID NO:49, numbered Enz.47, was synthesized in its entirety and ligated to pET28a plasmid vector to obtain recombinant plasmid pET28a-SUS. The gene synthesis company is the company of Industrial bioengineering (Shanghai) GmbH (Songjiang Kong Min-Lu 698, shanghai).
The plasmid pET28a-SUS was transformed into E.coli BL21 (DE 3) -competent cells as a host, to obtain an engineered strain containing the Enz.47 gene. A single colony was selected and inoculated into 5ml of LB liquid medium containing 50. Mu.g/ml kanamycin and shake-cultured at 37 ℃ for 4 hours. Transferred to 50ml of fresh TB liquid medium containing 50. Mu.g/ml kanamycin at an inoculum size of 2v/v%, and shake-cultured to OD at 37 ℃ with shaking 600 When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.1mM, and induction culture was carried out at 25 ℃ for 20 hours. After the culture was completed, the culture was centrifuged at 10000rpm for 10min, and the supernatant was discarded to collect the cells. Storing at-20 deg.C for use.
A50mM Phosphate Buffer Solution (PBS) with pH6.0 was prepared, the Enz.47 cell obtained above was suspended in (M/V) 1.
EXAMPLE 4 first round screening of beta-1, 3-glycosyltransferase mutants
In 1mL reaction system, 150. Mu.L of reaction enzyme liquid of beta-1, 3-glycosyltransferase is added, the final concentration of stevioside (stevioside content 95%, obtained medicine) is 100g/L, the final concentration of UDP is 0.1g/L, the final concentration of sucrose is 200g/L, the reaction enzyme liquid of sucrose synthase is 30. Mu.L, and finally 50mM phosphate buffer solution with pH6.0 is added to the final volume of 1mL. The prepared reaction system is placed in a metal bath, the reaction is carried out for 60min at 60 ℃ and 600rpm, the reaction solution is diluted by 100 times, 10 mu L of the reaction solution is added into 990 mu L of hydrochloric acid with pH value of 2-3, the mixture is vortexed and centrifuged at 13000rpm for 10min, and the supernatant is subjected to HPLC analysis to analyze the concentration of Reb A (see the Reb A percent in the table 6 for details, which represents the percent of Reb A in the reaction solution). The experimental results obtained using HPLC detection method 1 are shown in table 6.
TABLE 6
Enzyme numbering Mutation point Codons RA％
Enz.1 / / 47.826
Enz.2 V14I ATC 39.214
Enz.3 E99L CTA 41.663
Enz.4 L257A GCG 43.981
Enz.5 Q451E GAG 39.01
Enz.6 Q265E GAA 50.444
Enz.7 L257A-Q451E GCG-GAG 52.81
Enz.8 Q265E-Q451E GAA-GAG 52.16
From the preliminary screening results in table 6, it can be seen that: the activity of Enz.7 is improved to the maximum extent, compared with the Enz.1 of the starting sequence, the activity of Enz.6 and Enz.8 is improved by 10 percent, and the improvement extent is between 5 and 10 percent. A second round of mutations was subsequently performed based on enz.7. FIG. 6 is an HPLC plot of the activity of Enz.7 in Table 6 for the catalytic synthesis of RA.
EXAMPLE 5 construction of a second round of a library of beta-1, 3-glycosyltransferase mutants
And connecting the gene coding Enz.7 obtained in the first round with a vector pET28a to obtain a pET28a-Enz.7 recombinant plasmid, and performing PCR amplification by using KOD enzyme by using the pET28a-Enz.7 as a template and a primer sequence shown in table 7 to obtain gene fragments and vector fragments of target mutants Enz.9-16 and Enz.18-Enz.35.
TABLE 7
Figure BDA0003237510550000131
Figure BDA0003237510550000141
Wherein NNK is conventional in the art, i.e., N represents A, T, G or C; k represents G or T.
The PCR amplification reaction system is as follows:
KOD Mix：25μL；
ddH 2 O：20μL；
primer: 2 μ L x 2;
template: 1 μ L.
The PCR amplification procedure was as follows:
(1)98℃3min
(2)98℃10s
(3)55℃5s
(4)68℃5s/kbp
(5)68℃5min
(6) Heat preservation at 12 DEG C
(2) The (4) to (4) cycles 34 times.
DpnI digestion of the PCR product and gel running and gel recovery. Ligation was performed using two fragment homologous recombinases of Novowed (Exnase II,5 XCE II). Coli Trans10 competent cells, plated in LB medium containing 50 μ g/mL kanamycin, and cultured overnight at 37 ℃; selecting a single colony to an LB test tube (Km resistance), culturing for 8-10h, and extracting a plasmid for sequencing identification.
EXAMPLE 6 preparation of second round beta-1, 3-glycosyltransferase mutants
1. Protein expression of the mutational vector was performed:
the recombinant plasmid with correct sequencing in example 5 was transformed into competent cells of the host e.coli BL21 (DE 3), to obtain a genetically engineered strain containing a point mutation. A single colony was inoculated into 5ml of LB liquid medium containing 50. Mu.g/ml kanamycin and shake-cultured at 37 ℃ for 4 hours. Transferred to 50ml of fresh TB liquid medium containing 50. Mu.g/ml kanamycin at an inoculum size of 2% (v/v), and shake-cultured at 37 ℃Nourished to OD 600 When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.1mM, and induction culture was carried out at 25 ℃ for 20 hours. After the culture was completed, the culture solution was centrifuged at 4000rpm for 20min, and the supernatant was discarded to collect the cells. Storing at-20 deg.C for use.
2. Obtaining a reaction enzyme solution:
the collected cells were suspended in PBS (50 mM, pH 6.0) at a concentration of 1 (M/V, g/mL) and then homogenized using a high-pressure homogenizer (550 Mbar homogenization for 1.5 min); and (3) treating the homogenized beta-1, 3-glycosyltransferase enzyme solution at 80 ℃ for 15min, and centrifuging at 12000rpm for 2min to obtain the reaction enzyme solution. Storing at-4 deg.C for use.
Example 7 second round screening of mutants
Taking stevioside (with stevioside content of 95 percent, a finished medicine) as a substrate, adding 150 mu L of reaction enzyme liquid of a beta-1, 3-glycosyltransferase mutant into a 1mL reaction system, wherein the final concentration of the stevioside is 100g/L, the final concentration of UDP is 0.1g/L, the final concentration of sucrose is 200g/L, sucrose synthase is 30 mu L, and finally adding 50mM phosphate buffer with pH value of 6.0 to the final volume of 1mL. The prepared reaction system is placed in a metal bath, reacts for 60min at 60 ℃ and 600rpm, is diluted by 100 times, and is subjected to HPLC analysis to analyze the concentration of Reb A. The experimental results obtained using HPLC detection method 1 are shown in table 8.
TABLE 8
Figure BDA0003237510550000151
Figure BDA0003237510550000161
Note: enz.18-Enz.29 were obtained by NNK from GT001-257-F/R, and Enz.30-Enz.35 were obtained by NNK from GT 001-265-F/R.
From the results in table 8, it can be seen that: the enzyme activities of Enz.9, enz.10, enz.12, enz.13, enz.18, enz.21, enz.22, enz.25, enz.27 and Enz.30 are higher than that of Enz.1, wherein the activity of Enz.10 is the highest and exceeds Enz.1 percent or so. FIG. 7 is an HPLC plot of Enz.10 catalytic synthesis of RA activity in Table 8. A new round of mutation was subsequently performed on the basis of Enz.10 and screened.
EXAMPLE 8 third round of construction of a library of beta-1, 3-glycosyltransferase mutants
And connecting the gene coding Enz.10 obtained in the second round with a vector pET28a to obtain a pET28a-Enz.10 recombinant plasmid, and performing PCR amplification on a target DNA fragment and a vector fragment by using KOD enzyme by using the pET28a-Enz.10 as a template and the primer sequences shown in Table 9.
TABLE 9
Figure BDA0003237510550000162
Figure BDA0003237510550000171
The PCR amplification reaction system is as follows:
KOD Mix：25μL；
ddH 2 O：20μL；
primer: 2 μ L x 2;
template: 1 μ L.
The PCR amplification procedure was as follows:
(1)98℃3min
(2)98℃10s
(3)55℃5s
(4)68℃5s/kbp
(5)68℃5min
(6) Heat preservation at 12 DEG C
(2) The cycle of (4) to (4) is 34 times.
The PCR product was subjected to DpnI digestion and run and gel recovery. Two piece homologous recombination enzymes of Novozan (Exnase II,5X CE II) are connected to the pET28a plasmid vector to obtain recombinant plasmids pET28 a-Enz.36-pET 28a-Enz.45. Coli Trans10 competent cells, plated in LB medium containing 50 μ g/mL kanamycin, and cultured overnight at 37 ℃; picking single colony to LB test tube (Km resistance), culturing for 8-10h, extracting plasmid and sequencing.
EXAMPLE 9 preparation of the third round of beta-1, 3-glycosyltransferase mutants
1. Protein expression of the mutational vector was performed:
the recombinant plasmid with correct sequencing in example 8 was transformed into host e.coli BL21 (DE 3) competent cells to obtain a genetically engineered strain containing a point mutation. A single colony was inoculated into 5ml of LB liquid medium containing 50. Mu.g/ml kanamycin and shake-cultured at 37 ℃ for 4 hours. Was inoculated at 2% (v/v) into 50ml of fresh TB broth containing 50. Mu.g/ml kanamycin, and shake-cultured at 37 ℃ to OD 600 When the concentration reached about 0.8, IPTG was added to a final concentration of 0.1mM, and induction culture was carried out at 25 ℃ for 20 hours. After the culture was completed, the culture solution was centrifuged at 4000rpm for 20min, and the supernatant was discarded to collect the cells. Storing at-20 deg.C for use.
2. Obtaining a reaction enzyme solution:
the collected cells were suspended in PBS (50 mM, pH 6.0) at a concentration of 1 (M/V, g/mL) and then homogenized by a high-pressure homogenizer (550 Mbar homogenization for 1.5 min); and (3) treating the homogenized beta-1, 3-GT enzyme solution at 80 ℃ for 15min, and centrifuging at 12000rpm for 2min to obtain a reaction enzyme solution. Storing at-4 deg.C for use.
EXAMPLE 10 third round of mutant screening
Taking stevioside (with stevioside content of 95 percent, obtained medicine) as a substrate, adding 150 mu L of reaction enzyme liquid of the beta-1, 3-glycosyltransferase mutant into 1mL of a reaction system, wherein the final concentration of the stevioside is 100g/L, the final concentration of UDP or ADP is 0.1g/L, the final concentration of sucrose is 200g/L, the final concentration of sucrose synthase is 30 mu L, and finally adding 50mM phosphate buffer solution with pH6.0 to the final volume of 1mL. Placing the prepared reaction system in a metal bath, reacting the UDP group for 60min at 60 ℃ and 600rpm, adding 10 mu L of reaction liquid into 990 mu L of hydrochloric acid with pH of 2-3, vortexing, centrifuging at 13000rpm for 10min, and carrying out HPLC analysis on the supernatant for the concentration of Reb A; the ADP group was reacted for 20min, 10. Mu.L of the reaction mixture was added to 990. Mu.L of hydrochloric acid of pH2 to 3, vortexed, centrifuged at 13000rpm for 10min, and the supernatant was subjected to HPLC analysis for the concentration of Reb A (see Reb A% in Table 10). The experimental results obtained using HPLC detection method 1 are shown in table 10.
TABLE 10
Figure BDA0003237510550000181
From the preliminary screening results in table 10, it can be seen that: (1) UDP group: the enzyme activities of the mutants Enz.36, enz.37, enz.41, enz.43, enz.44 and Enz.45 are higher than that of Enz.1, wherein the activity of Enz.45 is the highest and exceeds the enzyme activity of Enz.1 by about 12%. (2) ADP group: the enzyme activities of the mutants Enz.36, enz.37, enz.41, enz.43, enz.44 and Enz.45 are higher than that of Enz.1, wherein the activity of Enz.45 is the highest and exceeds the enzyme activity of Enz.1 by about 8.8%. And (3) the ADP group has higher activity than the UDP group. FIG. 8 is an HPLC chart showing the activity of Enz.45 in the catalytic synthesis of RA with ADP as nucleoside diphosphate in Table 10.
EXAMPLE 11 preparation of beta-1, 2-glycosyltransferase
A set of beta-1, 2-glycosyltransferase genes is synthesized according to the gene of beta-1, 2-glycosyltransferase (enzyme number Enz.17) shown as a nucleotide sequence SEQ ID NO:51, and the genes are connected on a pET28a plasmid vector to obtain a recombinant plasmid pET28a-Enz.17. Gene Synthesis Co: biometrics (Shanghai) Ltd.
The plasmid pET28a-Enz.17 is transformed into a host E.coli BL21 (DE 3) competent cell to obtain an engineering strain containing the beta-1, 2-glycosyltransferase gene.
After the engineering bacteria containing beta-1, 2-glycosyltransferase gene is activated by plating and streaking, a single colony is selected and inoculated into 5ml LB liquid culture medium containing 50 mug/ml kanamycin, and shake culture is carried out for 12h at 37 ℃. Was inoculated into 50ml of fresh LB liquid medium containing 50. Mu.g/ml of kanamycin at an inoculum size of 2v/v%, shake-cultured at 37 ℃ until OD600 reached 0.6 to 0.8, IPTG was added to a final concentration of 0.1mM, and induction-cultured at 24 ℃ for 22 hours. And after the culture is finished, centrifuging the culture solution at 10000rpm for 10min, removing the supernatant, collecting the thalli, and storing the thalli in an ultralow temperature refrigerator at the temperature of 20 ℃ below zero for later use.
A50mM Phosphate Buffer Solution (PBS) with pH6.0 was prepared, collected cells were suspended in (M/V) 1.
The amino acid sequence of the beta-1, 2-glycosyltransferase prepared in this example is shown in SEQ ID NO 52.
EXAMPLE 12 RM Synthesis reaction
Reb A60 (the content of Reb A is 60%) is used as a substrate, 150 mu L of reaction enzyme liquid of a beta-1, 3-glycosyltransferase mutant, 120 mu L of reaction enzyme liquid of beta-1, 2-glycosyltransferase, 100g/L of RA60 final concentration, 0.1g/L of UDP or ADP final concentration, 200g/L of sucrose final concentration, 30 mu L of sucrose synthase reaction enzyme liquid are added into a 1mL reaction system, and finally 50mM phosphate buffer solution with pH6.0 is added until the volume reaches 1mL. The prepared reaction system is placed in a metal bath and reacts for 3.5h at 60 ℃ and 600rpm, 10 mu L of reaction liquid is taken, 990 mu L of hydrochloric acid with pH2-3 is added to the reaction liquid for vortex, 13000rpm is centrifuged for 10min, the concentration of Reb A, an intermediate product Reb D and a product Reb M is analyzed by HPLC, and the experimental results obtained by using an HPLC detection method 2 are shown in Table 11 (using UDP) and Table 12 (using ADP).
TABLE 11
Enzyme RA％ RD％ RM％
Enz.36 5.56 10.23 84.21
Enz.41 7.49 10.37 82.15
Enz.44 8.48 11.56 79.96
Enz.45 7.47 9.85 82.67
TABLE 12
Enzymes RA％ RD％ RM％
Enz.36 2.21 3.55 94.24
Enz.41 1.17 4.06 94.77
Enz.44 1.56 3.97 94.48
Enz.45 1.74 4.25 94.01
The results in tables 11 and 12 show that the four mutants of Enz.36, enz.41, enz.44 and Enz.45 are all suitable for synthesis of RM by using RA60 as a raw material, and the conversion rate can reach more than 80% after reaction for 3.5h under UDP condition; the reaction is carried out for 3.5h under the ADP condition, and the RM conversion rate can reach more than 94 percent. FIG. 9 is an HPLC plot of the activity of Enz.45 in catalyzing the synthesis of RM under the conditions of nucleoside diphosphate UDP in Table 11.
SEQUENCE LISTING
<110> cheque Korea of Korea Biotechnology (Shanghai)
<120> glycosyltransferase and application thereof
<130> P21015971C
<160> 52
<170> PatentIn version 3.5
<210> 1
<211> 1374
<212> DNA
<213> Artificial Sequence
<220>
<223> nucleotide sequence of beta-1, 3-glycosyltransferase
<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 ggtggttcgt 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 gtaa 1374
<210> 2
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> amino acid sequence of beta-1, 3-glycosyltransferase
<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 Val 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> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-V14I-F
<400> 3
cgtcgtatca ttatgtttcc ggttccgttc 30
<210> 4
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-V14I-R
<400> 4
gaaacataat gatacgacga cgacgcacgg tag 33
<210> 5
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-E99L-F
<400> 5
cgcggatcta ttccgtaagg agctggaaat c 31
<210> 6
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-E99L-R
<400> 6
ccttacggaa tagatccgcg ccgtgtttgt ta 32
<210> 7
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-L257A-F
<400> 7
gcactagcct ggcggatact gatccgagca ccg 33
<210> 8
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-L257A-R
<400> 8
cggatcagta tccgccaggc tagtgctcga agcc 34
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-Q451E-F
<400> 9
gaacgcctgg agagctatat tagcagcctg 30
<210> 10
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-Q451E-R
<400> 10
ctaatatagc tctccaggcg ttccaggctc tcg 33
<210> 11
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-Q265E-F
<400> 11
ccgccgaatg gctggaccag cagccgcc 28
<210> 12
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-Q265E-R
<400> 12
gtccagccat tcggcggtgc tcggatcagt atc 33
<210> 13
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Km-F
<400> 13
gcccgacatt atcgcgagc 19
<210> 14
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Km-R
<400> 14
gggtataaat gggctcgcg 19
<210> 15
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-I189L-F
<400> 15
gttaaagatc tccgtcgcgc gtattcgca 29
<210> 16
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-I189L-R
<400> 16
cgcgacggag atctttaacc ttcagcatc 29
<210> 17
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-T254G-F
<400> 17
cttcgagcgg tagcctgctg gatactgatc c 31
<210> 18
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-T254G-R
<400> 18
cagcaggcta ccgctcgaag ccctataatg c 31
<210> 19
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-S273G-F
<400> 19
gcagccgccg ggcagcgtgc tgtacgttgg 30
<210> 20
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-S273G-R
<400> 20
cacgctgccc ggcggctgct ggtccagc 28
<210> 21
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-K302G-F
<400> 21
gttgcgagcg gacaaagctt tctgtggaac g 31
<210> 22
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-K302G-R
<400> 22
gaaagctttg tccgctcgca accagaccac gc 32
<210> 23
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-K392I-F
<400> 23
gaggttctgc gtgtgggcgt ttatctggag 30
<210> 24
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-K392I-R
<400> 24
cgcccacacg cagaacctcg ctcatgta 28
<210> 25
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-N408R-F
<400> 25
gatcattagg gcggttaggc gtgtgatgg 29
<210> 26
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-N408R-R
<400> 26
ctaaccgccc taatgatctc accacggat 29
<210> 27
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-S455L-F
<400> 27
ctatattctc agcctgtaag cttgcggc 28
<210> 28
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-S455L-R
<400> 28
cttacaggct gagaatatag ctctgcaggc gt 32
<210> 29
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-257-F
<220>
<221> misc_feature
<222> (8)..(9)
<223> n is a, c, g, or t
<400> 29
tagcctgnnk gatactgatc cgagcaccg 29
<210> 30
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-257-R
<220>
<221> misc_feature
<222> (11)..(12)
<223> n is a, c, g, or t
<400> 30
atcagtatcm nncaggctag tgctcgaagc c 31
<210> 31
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-265-F
<220>
<221> misc_feature
<222> (8)..(9)
<223> n is a, c, g, or t
<400> 31
caccgccnnk tggctggacc agcagccgc 29
<210> 32
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-265-R
<220>
<221> misc_feature
<222> (10)..(11)
<223> n is a, c, g, or t
<400> 32
tccagccamn nggcggtgct cggatcagta 30
<210> 33
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-S455D-F
<400> 33
acgcctgcag agctatattg acagcctgta agcttgcgg 39
<210> 34
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-S455D-R
<400> 34
cgcaagctta caggctgtca atatagctct gcaggcgt 38
<210> 35
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-S455C-F
<400> 35
acgcctgcag agctatattt gcagcctgta agcttgcgg 39
<210> 36
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-S455C-R
<400> 36
cgcaagctta caggctgcaa atatagctct gcaggcgt 38
<210> 37
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-G316S-F
<400> 37
cttcgtgaag agttatgagt ggattgagct 30
<210> 38
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-G316S-R
<400> 38
taactcttca cgaagcccgg acgaacc 27
<210> 39
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-L378W-F
<400> 39
tctttagcga tttcggttgg gatcagccgc tgaacgcg 38
<210> 40
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-L378W-R
<400> 40
cgcgttcagc ggctgatccc aaccgaaatc gctaaagat 39
<210> 41
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-L378R-F
<400> 41
gcgatttcgg tcgggatcag ccgctgaacg c 31
<210> 42
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-L378R-R
<400> 42
gcgttcagcg gctgatcccg accgaaatcg ctaaagat 38
<210> 43
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-K347G-F
<400> 43
aagaagtgct ggcgcacggg gcgattggtg cgttctgg 38
<210> 44
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-K347G-R
<400> 44
gaacgcacca atcgccccgt gcgccagcac ttcttgt 37
<210> 45
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-V309N-F
<400> 45
tgtgggtgaa tcgtccgggc ttcgtgaag 29
<210> 46
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-V309N-R
<400> 46
gcccggacga ttcacccaca gaaagctttg t 31
<210> 47
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-P324G-F
<400> 47
agtggattga gctgctgggg gatggttttc tgggtgaa 38
<210> 48
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> GT001-P324G-R
<400> 48
aaaaccatcc cccagcagct caatccactc ataactcttc acga 44
<210> 49
<211> 2412
<212> DNA
<213> Artificial Sequence
<220>
<223> nucleotide sequence of sucrose synthase
<400> 49
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 ag 2412
<210> 50
<211> 804
<212> PRT
<213> Artificial Sequence
<220>
<223> amino acid sequence of sucrose synthase
<400> 50
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> 51
<211> 1353
<212> DNA
<213> Artificial Sequence
<220>
<223> nucleotide sequence of beta-1, 2-glycosyltransferase
<400> 51
atgcaccatc atcatgaagg cgtgagcgac cagaccctga gagtaacgat gtttccgtgg 60
cttgggctgg gtcatgttaa cccgtttttg cgtatcgcta aacaactggc cgatcgtggt 120
ttcgttatct atttagttag taccgctatt aacctcgaaa tgatcaaaaa gagaatcccg 180
gagaaataca gtaatagcat ccatctggtt gagctgcgcc tgccagaatt accggaactg 240
ccaccacatt accatactac caacggttta ccaccgcatc tgaacaaaac cctgcacaag 300
gcactgaaga tgagcgctcc caactttagc aagatccttc aaaatattaa gccggacctg 360
gtcctttacg attttctggt tccgtgggca gaaaaagtcg cgcttgaaca gggcatcccg 420
gctgttccat tgctaaccag tggtgcggca ctgttcagct actttttcaa cttcctgaag 480
cgaccgggtg aagagtttcc gtttgaggca atccgcctgt cgaagcgaga acaggataag 540
atgcgcgaga tgtttggaac agagccgcct gaagaagatt ttttagcgcc ggcccaggcc 600
ggtatcatgc tgatgtgcac gagccgcgta attgaggcta agtacctgga ctattgtacc 660
gaactgacca atgtaaaagt tgttccggtt ggtccgccgt ttcaggatcc gctgaccgaa 720
gatattgacg accccgaact gatggattgg ttagatacca aacccgaaca tagtgttgtc 780
tatgtgtcgt ttggcagcga agcgttcctg agccgtgaag atatggaaga agtcgcgttc 840
ggcctggagc tgagcggcgt gaactttatc tgggttgcac gctttccgaa aggcgaagaa 900
cagcgtctgg aagacgttct gccaaaaggc ttcctggaac gcgttggtga tcgtggtcgc 960
gttctggacc atctggtgcc gcaggcccat attctgaacc atccgagcac gggtggcttc 1020
atctctcatt gcggttggaa cagcgtcatg gaaagcattg atttcggcgt tccgatcatt 1080
gcgatgccga tgcagtggga tcagccgatt aacgcgagac tgcttgtgga attaggcgtg 1140
gcagtggaga tcccgcgtga tgaagatggc cgggtccacc gcgccgaaat tgcccgtgtc 1200
ctgaaagatg tgatttcggg cccgactggt gagatactgc gcgcgaaagt acgcgacatt 1260
agcgcacgcc tgagagcgag acgcgaggag gaaatgaacg cagcggcgga agaactgata 1320
cagctgtgtc gcaaccgcaa cgcctacaag taa 1353
<210> 52
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> amino acid sequence of beta-1, 2-glycosyltransferase
<400> 52
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

Claims (11)

1. A glycosyltransferase comprising an amino acid residue difference at a residue position selected from one or more of:
the 14 th amino acid is I;
l represents amino acid 189;
amino acid 257 is A, C, L, M, S or V;
the 265 th amino acid is E or A;
the amino acid at position 273 is G;
amino acid 302 is G;
amino acid 324 is G;
amino acid 347 is G;
amino acid E at position 451;
amino acid 455 is D or C;
and has a glycosyltransferase activity not lower than that shown by the amino acid sequence of SEQ ID NO. 2.
2. The glycosyltransferase of claim 1, wherein the glycosyltransferase differs in amino acid residue from SEQ ID NO:2 selected from the group consisting of:
(1) The 265 th amino acid is E; or the like, or, alternatively,
amino acid 257 is A, and amino acid 451 is E; or the like, or, alternatively,
the 265 th amino acid is E, and the 451 th amino acid is E;
(2) Amino acid 14 is I, amino acid 257 is A and amino acid 451 is E; or
Amino acid 257 is A, amino acid 451 is E, and amino acid 189 is L; or the like, or, alternatively,
amino acid 257 is A, amino acid 451 is E, and amino acid 273 is G; or the like, or a combination thereof,
an amino acid A at position 257, an amino acid E at position 451, and an amino acid G at position 302; or
Amino acid 257 is C, and amino acid 451 is E; or
L at amino acid 257 and E at amino acid 451; or
M at amino acid 257 and E at amino acid 451; or
Amino acid 257 is S, amino acid 451 is E; or
Amino acid 257 is V, amino acid 451 is E; or
Amino acid 257 is A, amino acid 451 is E, and amino acid 265 is A;
(3) Amino acid 257 is A, amino acid 451 is E, amino acid 189 is L, and amino acid 14 is I; or the like, or a combination thereof,
amino acid 257 is A, amino acid 451 is E, amino acid 189 is L, and amino acid 273 is G; or the like, or, alternatively,
amino acid 257 is A, amino acid 451 is E, amino acid 189 is L, and amino acid 324 is G; or the like, or a combination thereof,
a 257 th amino acid is a, a 451 th amino acid is E, a 189 th amino acid is L, and a 347 th amino acid is G; or the like, or, alternatively,
a at amino acid 257, E at amino acid 451, L at amino acid 189, and D or C at amino acid 455.
3. An isolated nucleic acid encoding the glycosyltransferase of claim 1 or 2.
4. A recombinant expression vector comprising the nucleic acid of claim 3.
5. A transformant which is a host cell comprising the nucleic acid of claim 3 or the recombinant expression vector of claim 4; preferably, the host cell is Escherichia coli (Escherichia coli) such as e.coli BL21 (DE 3).
6. A method of producing the glycosyltransferase of claim 1 or 2, comprising culturing the transformant of claim 5 under conditions suitable for expression of the glycosyltransferase.
7. A composition comprising the glycosyltransferase of claim 1 or 2.
8. A method for the glycosylation of a substrate, said method comprising providing at least one substrate, a glycosyltransferase of claim 1 or 2, and contacting said substrate with said glycosyltransferase under conditions such that said substrate is glycosylated to produce at least one glycosylation product.
9. The preparation method of rebaudioside A is characterized by comprising the following steps: reacting stevioside with a glycosyl donor in the presence of the glycosyltransferase of claim 1 or 2 to obtain rebaudioside a;
preferably, 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 concentration of stevioside is 1-150g/L, preferably 100g/L;
the mass ratio of the glycosyltransferase thalli to stevioside is 1 (3-10), preferably 3;
the glycosyl donor is UDP-glucose and/or ADP-glucose; preferably via UDP or ADP in the presence of sucrose and sucrose synthase, the concentration of sucrose is preferably 100-300g/L such as 200g/L, the concentration of UDP or ADP is preferably 0.05-0.2g/L such as 0.1g/L;
the reaction solvent for the reaction has a pH of 5 to 8, preferably 6;
the pH is controlled by a buffer solution, preferably a phosphoric acid buffer solution;
the rotating speed of the reaction is 500-1000rpm, preferably 600rpm;
the temperature of the reaction is 20 to 90 ℃, preferably 60 ℃.
10. A method for preparing rebaudioside D or rebaudioside M, comprising the step of preparing rebaudioside a according to the preparation method of claim 9.
11. Use of a glycosyltransferase of claim 1 or 2 in the preparation of a steviol glycoside; the steviol glycoside is preferably rebaudioside a, rebaudioside D or rebaudioside M.
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