CN115725528B - Glycosyltransferase and application thereof - Google Patents

Glycosyltransferase and application thereof Download PDF

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CN115725528B
CN115725528B CN202111006803.4A CN202111006803A CN115725528B CN 115725528 B CN115725528 B CN 115725528B CN 202111006803 A CN202111006803 A CN 202111006803A CN 115725528 B CN115725528 B CN 115725528B
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吴燕
田振华
郑孝富
王舒
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Yikelai Biotechnology Group Co ltd
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Abstract

The invention provides glycosyltransferase and application thereof. Glycosyltransferases comprise amino acid residue differences at residue positions selected from one or more of the following compared to SEQ ID No. 2: amino acid at position 14 is I; amino acid 189 is L; amino acid 257 is A, C, L, M, S or V; amino acid 265 is E or A; amino acid 273 is G; amino acid at position 302 is G; amino acid at position 324 is G; amino acid 347 is G; amino acid 451 is E; amino acid 455 is D or C; and has a glycosyltransferase enzyme activity not lower than that shown by the amino acid sequence of SEQ ID NO. 2. The glycosyltransferase of the invention has high enzyme activity and good stability; compared with glycosyltransferase parent, the stevioside has obviously improved catalytic activity and conversion rate. 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

Glycosyltransferase and application thereof
Technical Field
The invention relates to glycosyltransferase and application thereof in a stevioside glycosylation reaction.
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)
The steviol glycoside compounds have a common aglycone: steviol (Steviol), differing in the number and type of glycosyl groups attached at the C-13 and C-19 positions, mainly includes eight glycosides of Stevioside (Stevioside), rebaudioside a (rebaudiosid a, reba a), rebaudioside B, rebaudioside C, rebaudioside D (rebaudiosid D), rebaudioside E, dulcoside, steviolbioside, etc. 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 rebaudioside B, D, E and F, steviolbioside and rubusoside, were found at much lower levels (about 0-0.2%).
Although steviol glycosides areHigh-intensity sweetener has the defect of bitter taste after the taste, and severely limits the application of the high-intensity sweetener in the fields of foods, beverages and the like with high requirements on taste. The intrinsic cause of the bitter taste after steviol glycoside is that the R in steviol glycoside is caused by the intrinsic molecular structure 1 And R is 2 The more the number of linked glycosyl groups on the group, the better the taste. Typically, stevioside is found to be 110-270 times sweeter than sucrose, with rebaudioside a 150-320 times, however, even in a highly purified state, steviol glycosides still have undesirable taste attributes such as bitter taste, sweet aftertaste, licorice taste, etc.
The rebaudioside D is the stevioside with the most application potential, has high sweetness which is 300-350 times that of the sucrose compared with other steviosides, has pure sweetness, has the taste similar to the sucrose, has no bitter taste and liquorice peculiar smell, has good stability and is an ideal natural high-power sweetener product. The content of the rebaudioside D in the stevia rebaudiana leaves is extremely small (less than 5%), a large amount of stevia rebaudiana raw materials are required for producing the rebaudioside D by adopting an extraction method, the process for enriching the rebaudioside D is complex, multiple column passes, desalination, decoloration and recrystallization are required after extraction, a large amount of wastewater is generated in the production process, and the production cost is high, so that the method is not suitable for industrial mass production.
The current method for synthesizing the rebaudioside D by the biological enzyme method needs to add expensive UDP-glucose as one of substrates, and uses stevioside or rebaudioside A as the substrate to catalyze and generate the rebaudioside D under the action of UDP-glucosyltransferase (UGT for short). However, due to the extremely high selling price of UDP-glucose, the feasibility of industrially preparing the rebaudioside D is almost completely limited, the economical efficiency is poor, and the market competitiveness is lacking.
Rebaudioside M (Rebaudioside M) has better mouthfeel characteristics, but its content is less than 0.1% of the dry weight of the leaves, resulting in high separation costs and high prices. Biocatalytic methods have attracted attention from the scholars to obtain high concentrations of rebaudioside M. It is reported that a stevia-derived recombinase can catalyze rebaudioside D to produce rebaudioside M, but in lower yields. The method has the advantages that the rebaudioside D is used as a substrate, and the rebaudioside M can be obtained through a microbial enzyme-producing catalysis method, so that compared with a traditional extraction method, the method not only improves the production flow, but also reduces the pollution to the environment, and improves the yield of the target product rebaudioside M. However, the following problems mainly exist in the bio-enzyme catalysis method at present: (1) The cost of producing rebaudioside M with bioenzyme catalyzed rebaudioside D is high and the enzyme catalyzed yield is to be further optimized; (2) The glycosyltransferase used for catalysis is not easy to separate from the product and recycle, and is easy to inactivate; (3) The direct conversion of rebaudioside a to rebaudioside D at low cost is also a challenge to be addressed by the very high rebaudioside a content and very low rebaudioside D content of natural plants.
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. UDP-glucosyltransferase is one of the glucosyltransferases, and UDP-glucose is used as a glycosyl donor, and is present in almost all organisms.
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.
Today, with the widespread use of steviol, a natural sweetener, and the increasing development of biocatalytic technology, glucosyltransferases are increasingly being used in the field of biocatalytic preparation of steviol glycosides. The existing enzyme used in the field of the biological enzyme preparation method of stevioside often has the defects of low enzyme activity, poor stability and the like, so that the cost for preparing stevioside by industrial mass production is high. Therefore, it is necessary to modify the glucosyltransferase to obtain a modified enzyme with higher enzyme activity and better stability so as to better serve industrial mass production.
Disclosure of Invention
The invention aims to solve the technical problems that the existing glycosyltransferase has low enzyme activity and poor stability when being applied to the biocatalysis preparation of stevioside, so that the conversion rate is not high when being used for catalyzing stevioside, and the like, so the invention provides the glycosyltransferase and the application thereof in the preparation of stevioside. The Glycosyltransferase (GT) has high enzyme activity and good stability; when the stevioside is used for preparing stevioside (such as rebaudioside A, rebaudioside D or rebaudioside M), compared with a glycosyltransferase parent, the catalytic activity is obviously improved, and the conversion rate is obviously improved, so that the reaction cost is reduced, and the industrial production is facilitated.
In order 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 following compared to SEQ ID No. 2:
amino acid at position 14 is I;
amino acid 189 is L;
amino acid 257 is A, C, L, M, S or V;
amino acid 265 is E or A;
amino acid 273 is G;
amino acid at position 302 is G;
amino acid at position 324 is G;
amino acid 347 is G;
amino acid 451 is E;
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.
In the present invention, the difference may be obtained by mutation of the amino acid sequence shown in SEQ ID NO. 2, or may be obtained by mutation based on other amino acid sequences, and the final mutation result falls within the scope of the present invention as long as the final mutation result has the above-mentioned difference compared with the amino acid sequence shown in SEQ ID NO. 2.
Preferably, the glycosyltransferase differs from SEQ ID NO. 2 by an amino acid residue selected from the group consisting of:
(1) Amino acid 265 is E; or alternatively, the first and second heat exchangers may be,
257 th amino acid is A, 451 th amino acid is E; or alternatively, the first and second heat exchangers may be,
amino acid 265 is E, amino acid 451 is E;
(2) Amino acid 14 is I, amino acid 257 is A and amino acid 451 is E; or (b)
257 amino acids A, 451 amino acid E and 189 amino acids L; or alternatively, the first and second heat exchangers may be,
257 th amino acid is A, 451 th amino acid is E and 273 th amino acid is G; or alternatively, the first and second heat exchangers may be,
257 amino acids A, 451 amino acid E and 302 amino acid G; or (b)
257 th amino acid is C and 451 rd amino acid is E; or (b)
257 th amino acid is L and 451 rd amino acid is E; or (b)
257 th amino acid is M and 451 th amino acid is E; or (b)
The 257 th amino acid is S and the 451 rd amino acid is E; or (b)
257 th amino acid is V and 451 th amino acid is E; or (b)
257 th amino acid is A, 451 th amino acid is E and 265 th amino acid is A;
(3) 257 amino acids A, 451 amino acid E, 189 amino acids L and 14 amino acids I; or alternatively, the first and second heat exchangers may be,
257 th amino acid is A, 451 th amino acid is E, 189 th amino acid is L and 273 th amino acid is G; or alternatively, the first and second heat exchangers may be,
257 amino acids A, 451 amino acid E, 189 amino acids L and 324 amino acids G; or alternatively, the first and second heat exchangers may be,
257 amino acids A, 451 amino acid E, 189 amino acids L and 347 amino acids G; or alternatively, the first and second heat exchangers may be,
(3) 257 amino acids A, 451 amino acid E, 189 amino acid L and 455 amino acid D or C.
In order to solve the above technical problem, the second aspect of the present invention provides an isolated nucleic acid encoding a glycosyltransferase according to the first aspect of the present invention.
In order to solve the above technical problem, 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 problem, the 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 E.coli (Escherichia coli) such as E.coli BL21 (DE 3).
In order to solve the above technical problem, a fifth aspect of the present invention provides a method for producing the glycosyltransferase according to the first aspect of the present invention, which comprises culturing the transformant according to the fourth aspect of the present invention under conditions suitable for expression of the glycosyltransferase.
After the transformant expresses the glycosyltransferase, the glycosyltransferase can be extracted by adopting 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 displacement after being prepared, and the crude enzyme solution can be further subjected to one or more of 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 may be employed: (1) Inoculating the transformant containing the glycosyltransferase into a culture medium containing antibiotics such as LB (LB) culture medium for shaking culture to obtain seed solution; (2) Transferring the seed solution in (1) to a medium containing antibiotics such as TB medium for shaking culture; (3) Adding IPTG into the culture medium in the step (2) to induce overnight, centrifuging and collecting thalli; (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 problem, the sixth aspect of the present invention provides a composition comprising the glycosyltransferase according to the first aspect of the present invention.
In order to solve the above technical problem, a seventh aspect of the present invention provides a method for 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 glycosylated product.
In order to solve the above technical problems, an eighth aspect of the present invention provides a method for preparing rebaudioside a, the method comprising the steps of: reacting stevioside with a glycosyl donor in the presence of a glycosyl transferase according to the first aspect of the invention to obtain rebaudioside A.
In a preferred embodiment, the glycosyltransferase is in the form of glycosyltransferase bacteria, crude enzyme solution, pure enzyme solution, or immobilized enzyme.
In a preferred embodiment, the stevioside is present in a concentration of 1 to 150g/L, preferably 100g/L.
In a preferred embodiment, the mass ratio of glycosyltransferase to stevioside is 1 (3-10), preferably 3:20.
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 UDP or ADP is preferably 0.05-0.2g/L, e.g. 0.1g/L.
In a preferred embodiment, the reaction solvent of 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 rotational speed of the reaction is 500-1000rpm, preferably 600rpm.
In a preferred embodiment, the temperature of the reaction is 20-90 ℃, preferably 60 ℃.
In order to solve the technical problem, a 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 according to the eighth aspect of the present invention.
In preparing rebaudioside D, a beta-1, 2-glycosyltransferase is used in addition to the glycosyltransferase of the first aspect of the invention.
In order to solve the technical problem, a tenth aspect of the present invention provides a method for preparing rebaudioside M, which comprises the step of preparing rebaudioside a according to the preparation method according to 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 described above, and reacting the stevioside substrate, glycosyl donor, and glycosyltransferase as described above under conditions such that rebaudioside D or rebaudioside M is produced.
In order to solve the technical problem, an eleventh aspect of the present invention provides a use of the glycosyltransferase according to the first aspect of the present invention in preparing steviol glycoside.
The steviol glycoside is preferably rebaudioside a, rebaudioside D or rebaudioside M.
"glycosyltransferase" in the context of the present invention includes NDP-glycosyltransferases, including but not limited to UDP-glucose dependent glycosyltransferases (UDP-glycosyltransferases; UGT), ADP-glucose dependent glycosyltransferases (ADP-glycosyltransferases; AGT), CDP-glucose dependent glycosyltransferases (CDP-glycosyltransferases; CGT), GDP-glucose dependent glycosyltransferases (GDP-glycosyltransferases; GGT), TDP-glucose dependent glycosyltransferases (TDP-glycosyltransferases; TGT) and IDP-glucose dependent glycosyltransferases (IDP-glycosyltransferases; IGT).
The sucrose synthase of the present invention is referred to as sucrose synthase (EC 2.4.1.1.13, SUS) also abbreviated as SuSy/SS, etc.
The Glycosyltransferase (GT) has high enzyme activity and good stability; when the stevioside is used for preparing stevioside (such as rebaudioside A, rebaudioside D or rebaudioside M), compared with a glycosyltransferase parent, the catalytic activity is obviously improved, and the conversion rate is obviously improved, so that the reaction cost is reduced, and the industrial production is facilitated. The invention combines Glycosyltransferase (GT) and sucrose synthase to catalyze and synthesize RA, RD and RM, realizes cascade reaction, can realize UDPG regeneration by using sucrose and UDP, can realize ADPG regeneration by using sucrose and ADP, solves the problem of high price of glycosyl donors UDPG and ADPG, provides a plurality of options of substrates, provides more options for optimizing process conditions for further realizing large-scale industrial production, and is more beneficial to realizing large-scale industrialization.
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 retention times for stevioside, rebaudioside A controls using HPLC detection method 1; the retention time of stevioside was 12.761min and the retention time of rebaudioside a was 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 of the activity of Enz.7 in Table 6 in the catalytic synthesis of RA.
FIG. 7 is a graph of the activity of Enz.10 in Table 8 in the catalytic synthesis of RA.
FIG. 8 is a graph showing the activity of Enz.45 in the catalytic synthesis of RA under the condition that ADP is nucleoside diphosphate in Table 10.
FIG. 9 is a graph showing the activity of Enz.45 in the catalytic synthesis of RM under the condition of nucleoside diphosphate UDP in Table 11.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
The experimental methods in the invention are all conventional methods unless otherwise specified, and specific reference is made to the "molecular cloning Experimental guidelines" by J.Sam Broker et al for gene cloning operations.
Amino acid shorthand symbols in the invention are conventional in the art unless otherwise specified, and amino acids corresponding to specific shorthand symbols are shown in table 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 invention is schematically shown in figure 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. Sucrose was purchased from an organism. The substrate stevioside used in the first, second and third rounds of screening was purchased from Pichia pastoris (purity 95%), and the substrate RA60 used in the synthesis of RM was purchased from Chenopodiaceae (where RA content was 60%, stevioside content was about 30%, product specification TSG90/RA 60). Sucrose was purchased from biological engineering (Shanghai) Inc. Reb a control was purchased from microphone. Reb D and Reb M controls were purchased from Qingdao Sida stevia International trade company.
HPLC detection method 1: chromatographic column: agilent 5TC-C18 (2) (250X 4.6 mm). 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 μl; column temperature: 40 ℃. As shown in FIG. 2, stevioside retention time was 12.76min and Reb A retention time was 12.38min.
TABLE 3 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: 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 4 below. Detection wavelength: 210nm; flow rate: 1ml/min; sample injection volume: 20 μl; column temperature: 35 ℃. As shown in fig. 3, reb a peak time: 14.186min; as shown in fig. 4, reb D off-peak time: 11.821min; as shown in fig. 5, reb M off-peak time: 12.316min.
TABLE 4 Table 4
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.
PCR amplification was performed using the pET28a-Enz.1 plasmid as a template and the primer sequences shown in Table 5 and KOD enzymes to obtain the gene fragments and vector fragments of the target mutants Enz.2 to Enz.8.
TABLE 5
The PCR amplification reaction system is as follows:
KOD Mix：25μL
ddH 2 O：20μL
primer: 2 mu L2
And (3) a template: 1 mu L
The amplification procedure was as follows:
(1)98℃3min
(2)98℃10s
(3)55℃5s
(4)68℃5s/kbp
(5)68℃5min
(6) Heat preservation at 12 DEG C
(2) And (4) circulating 34 times.
And (3) performing DpnI digestion on the PCR product, and performing gel running and gel recovery to obtain the target DNA fragment. Each mutant recombinant plasmid pET28 a-Enz.2-pET 28a-Enz.8 was obtained by ligating two fragments of homologous recombinase (Exnase II,5 XCE II) to the pET28a plasmid vector. After ligation, transformed into E.coli Trans10 competent cells, plated on LB medium containing 50. Mu.g/mL of kananamycin, and cultured overnight at 37 ℃; single colonies were picked up to LB tubes (Km resistance), cultured for 8-10h, and plasmids were extracted for sequencing.
EXAMPLE 2 preparation of beta-1, 3-glycosyltransferase mutants
1. Protein expression of the mutation vector was performed:
and transforming the recombinant plasmid with correct sequence into a host E.coli BL21 (DE 3) competent cell to obtain the genetically engineered strain containing the point mutation. 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 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) at pH6.0 was prepared, and the cells obtained above were suspended at a ratio of 1:10 (M/V, g/mL), and homogenized using a high-pressure homogenizer (550 Mbar homogenization for 1.5 min); and (3) carrying out treatment at 80 ℃ for 15min and centrifugation at 12000rpm for 2min on the homogenized beta-1, 3-GT enzyme solution to obtain a reaction enzyme solution.
EXAMPLE 3 preparation of sucrose synthase SUS
A sucrose synthase (SUS) gene shown in SEQ ID NO. 49 and numbered Enz.47 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).
Plasmid pET28a-SUS was transformed into host E.coli BL21 (DE 3) competent cells to obtain an engineering strain containing the Enz.47 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 2v/v% inoculum size, shake culture to OD at 37 ℃ 600 When reaching 0.6-0.8, 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 10000rpm for 10min, and the supernatant was discarded to collect the cells. Preserving at-20 ℃ for standby.
50mM Phosphate Buffer (PBS) at pH6.0 was prepared, the Enz.47 cells obtained above were suspended at a ratio of (M/V) 1:5, and after high-pressure homogenization (550-600 Mbar, 1 min), the suspension was centrifuged at 12000rpm for 2min to obtain a crude enzyme solution, and the crude enzyme solution was centrifuged to obtain a supernatant as a reactive enzyme solution of sucrose synthase SUS (enzyme number Enz.47, amino acid sequence shown in SEQ ID NO: 50).
EXAMPLE 4 first round screening of beta-1, 3-glycosyltransferase mutants
In a 1mL reaction system, 150 mu L of beta-1, 3-glycosyltransferase reaction enzyme solution, 100g/L of stevioside (stevioside content is 95%, and medicine is obtained), 0.1g/L of UDP (user datagram protocol) and 200g/L of sucrose final concentration, 30 mu L of sucrose synthase reaction enzyme solution and finally 50mM of pH6.0 phosphate buffer solution are added to a final volume of 1mL. The prepared reaction system was placed in a metal bath, reacted at 60℃and 600rpm for 60 minutes, the reaction solution was diluted 100 times, 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 subjected to HPLC analysis for the concentration of Reb A (see Table 6 for details, reb A%, which represents the percentage 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 maximally, compared with the starting sequence Enz.1, the activity of Enz.6 and Enz.8 is improved by 10 percent, and the improvement range is between 5 and 10 percent. A second round of mutation was subsequently performed based on Enz.7. FIG. 6 is an HPLC plot of Enz.7 catalytic synthesis RA activity in Table 6.
EXAMPLE 5 construction of second round library of beta-1, 3-glycosyltransferase mutants
And (3) connecting the gene encoding Enz.7 obtained in the first round with a vector pET28a to obtain a pET28a-Enz.7 recombinant plasmid, and carrying out PCR amplification by using KOD enzyme by using pET28a-Enz.7 as a template and using primer sequences shown in Table 7 to obtain gene fragments and vector fragments of target mutants Enz.9-16 and Enz.18-Enz.35.
TABLE 7
/>
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 μl×2;
and (3) a template: 1 mul.
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) And (4) circulating 34 times.
The PCR product was subjected to DpnI digestion and run-out and gel recovery. Ligation was performed using a Norpraise two-fragment homologous recombinase (Exnase II,5 XCE II). The ligation was transformed into E.coli Trans10 competent cells, plated on LB medium containing 50. Mu.g/mL of calicheamicin, and incubated overnight at 37 ℃; single colonies were picked up to LB tubes (Km resistance), cultured for 8-10h, and plasmids were extracted for sequencing identification.
EXAMPLE 6 preparation of second round beta-1, 3-glycosyltransferase mutants
1. Protein expression of the mutation vector was performed:
the recombinant plasmid with correct sequence in example 5 was transformed into host E.coli BL21 (DE 3) competent cells to obtain genetically engineered strains containing point mutations. 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 reaching 0.6-0.8, 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.
2. Obtaining a reaction enzyme solution:
the collected cells were suspended at 1:10 (M/V, g/mL) using PBS (50 mM, pH 6.0), and 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 a reaction enzyme solution. Preserving at-4 ℃ for standby.
EXAMPLE 7 second round screening of mutants
Stevioside (stevioside content is 95%, medicine is obtained) is taken as a substrate, 150 mu L of beta-1, 3-glycosyltransferase mutant reaction enzyme solution is added into a 1mL reaction system, the final concentration of stevioside is 100g/L, the final concentration of UDP is 0.1g/L, the final concentration of sucrose is 200g/L, the sucrose synthase is 30 mu L, and finally 50mM phosphate buffer solution with pH of 6.0 is added into the reaction system to a final volume of 1mL. The prepared reaction system was placed in a metal bath, reacted at 600rpm for 60 minutes at 60℃and diluted 100 times, and the concentration of Reb A was analyzed by HPLC. The experimental results obtained using HPLC detection method 1 are shown in table 8.
TABLE 8
Note that: enz.18 to Enz.29 are obtained by NNK with GT001-257-F/R, and Enz.30 to Enz.35 are obtained by NNK with 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 that of Enz.1 by about 8 percent. FIG. 7 is an HPLC plot of Enz.10 catalytic synthesis RA activity in Table 8. A new round of mutation and screening was subsequently performed on the basis of Enz.10.
EXAMPLE 8 construction of third round of beta-1, 3-glycosyltransferase mutant library
The gene encoding Enz.10 obtained in the second round was ligated to vector pET28a to obtain pET28a-Enz.10 recombinant plasmid, and the target DNA fragment and the vector fragment were amplified by PCR using KOD enzyme using pET28a-Enz.10 as a template and the primer sequences shown in Table 9.
TABLE 9
The PCR amplification reaction system is as follows:
KOD Mix：25μL；
ddH 2 O：20μL；
primer: 2 μl×2;
and (3) a template: 1 mul.
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) And (4) circulating 34 times.
The PCR product was subjected to DpnI digestion and run-out and gel recovery. The two fragments of homologous recombinases of Norflua (Exnase II,5 XCE II) are adopted to be connected to a pET28a plasmid vector, so that recombinant plasmids pET28 a-Enz.36-pET 28a-Enz.45 are obtained. The ligation was transformed into E.coli Trans10 competent cells, plated on LB medium containing 50. Mu.g/mL of calicheamicin, and incubated overnight at 37 ℃; single colonies are picked up to LB test tubes (Km resistance), cultured for 8-10 hours, and plasmids are extracted for sequencing.
EXAMPLE 9 preparation of third round of beta-1, 3-glycosyltransferase mutant
1. Protein expression of the mutation vector was performed:
the recombinant plasmid with correct sequence in example 8 was transformed into host E.coli BL21 (DE 3) competent cells to obtain genetically engineered strains containing point mutations. 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 sizeShake culturing at 37deg.C to OD 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.
2. Obtaining a reaction enzyme solution:
the collected cells were suspended at 1:10 (M/V, g/mL) using PBS (50 mM, pH 6.0), and homogenized using a high-pressure homogenizer (550 Mbar homogenization for 1.5 min); and (3) carrying out treatment at 80 ℃ for 15min and centrifugation at 12000rpm for 2min on the homogenized beta-1, 3-GT enzyme solution to obtain a reaction enzyme solution. Preserving at-4 ℃ for standby.
EXAMPLE 10 third round of mutant screening
Stevioside (stevioside content is 95%, medicine is obtained) is taken as a substrate, 150 mu L of beta-1, 3-glycosyltransferase mutant reaction enzyme solution is added into a 1mL reaction system, the final concentration of stevioside is 100g/L, the final concentration of UDP or ADP is 0.1g/L, the final concentration of sucrose is 200g/L, 30 mu L of sucrose synthase is added, and finally 50mM phosphate buffer solution with pH of 6.0 is added to a final volume of 1mL. Placing the prepared reaction system in a metal bath, reacting UDP groups at 60 ℃ and 600rpm for 60min, adding 10 mu L of reaction liquid into 990 mu L of hydrochloric acid with pH of 2-3, swirling, centrifuging at 13000rpm for 10min, and carrying out HPLC analysis on the concentration of Reb A on the supernatant; ADP group reaction was performed for 20min, 10. Mu.L of the reaction mixture was added to 990. Mu.L of hydrochloric acid having pH2-3, vortexed, centrifuged at 13000rpm for 10min, and the supernatant was subjected to HPLC analysis for the concentration of Reb A (see Table 10 for details). The experimental results obtained using HPLC detection method 1 are shown in table 10.
Table 10
From the preliminary screening results in Table 10, it can be seen that: (1) UDP group: the enzyme activities of mutants Enz.36, enz.37, enz.41, enz.43, enz.44 and Enz.45 were higher than Enz.1, with Enz.45 having the highest activity, about 12% higher than Enz.1. (2) ADP group: the enzyme activities of mutants Enz.36, enz.37, enz.41, enz.43, enz.44 and Enz.45 were higher than Enz.1, with Enz.45 having the highest activity, about 8.8% higher than Enz.1. (3) ADP group activity was higher than UDP group. FIG. 8 is an HPLC plot of Enz.45 catalytic synthesis of RA activity with ADP as nucleoside diphosphate in Table 10.
EXAMPLE 11 preparation of beta-1, 2-glycosyltransferase
According to the gene of beta-1, 2-glycosyltransferase (enzyme number Enz.17) shown in nucleotide sequence SEQ ID NO. 51, a set of beta-1, 2-glycosyltransferase genes are synthesized through total genes, and the genes are connected on a pET28a plasmid vector to obtain a recombinant plasmid pET28a-Enz.17. Gene synthesis Co: bioengineering (Shanghai) Co., ltd.
Plasmid pET28a-Enz.17 is transformed into host E.coli BL21 (DE 3) competent cells to obtain engineering strains containing beta-1, 2-glycosyltransferase genes.
After streaking and activating engineering bacteria containing beta-1, 2-glycosyltransferase genes by a plate, single colonies are selected and inoculated into 5ml LB liquid medium containing 50 mug/ml kanamycin, and shake culture is carried out for 12 hours at 37 ℃. Transfer to 50ml fresh LB liquid medium containing 50. Mu.g/ml kanamycin at 2v/v%, shake culture at 37℃until OD600 reaches 0.6-0.8, adding IPTG to its final concentration of 0.1mM, and induction culture at 24℃for 22h. After the culture is finished, the culture solution is centrifuged at 10000rpm for 10min, the supernatant is discarded, and the thalli are collected and stored in an ultralow temperature refrigerator at-20 ℃ for standby.
50mM Phosphate Buffer (PBS) at pH6.0 was prepared, the collected cells were suspended at a ratio of (M/V) of 1:10, and then homogenized at high pressure (550 to 600Mbar, 1 min), centrifuged at 12000rpm for 2min, and the supernatant was collected to obtain a crude enzyme solution of beta-1, 2-glycosyltransferase.
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 taken as a substrate, 150 mu L of beta-1, 3-glycosyltransferase mutant reaction enzyme solution, 120 mu L of beta-1, 2-glycosyltransferase reaction enzyme solution, 100g/L of RA60 final concentration, 0.1g/L of UDP or ADP final concentration, 200g/L of sucrose final concentration and 30 mu L of sucrose synthase reaction enzyme solution are added into a 1mL reaction system, and finally 50mM phosphate buffer solution with pH of 6.0 is added into the reaction system to a final volume of 1mL. The prepared reaction system was placed in a metal bath and reacted at 60℃and 600rpm for 3.5 hours, 10. Mu.L of the reaction solution was added to 990. Mu.L of hydrochloric acid at pH2-3 to conduct vortex swirling, centrifugation at 13000rpm for 10 minutes, and the supernatant was subjected to HPLC analysis of the concentrations of Reb A, intermediate Reb D and product Reb M, and the experimental results obtained using HPLC detection method 2 are shown in Table 11 (using UDP) and Table 12 (using ADP).
TABLE 11
Enzymes 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
As shown in the results of Table 11 and Table 12, the four mutants Enz.36, enz.41, enz.44 and Enz.45 are all suitable for being applied to the synthesis of RM by taking RA60 as a raw material, and the conversion rate of reaction for 3.5 hours under UDP condition can reach more than 80 percent; the reaction is carried out for 3.5 hours under the ADP condition, and the RM conversion rate can reach more than 94 percent. FIG. 9 is an HPLC plot of Enz.45 catalytic synthesis RM activity under the condition of UDP nucleoside diphosphate in Table 11.
SEQUENCE LISTING
<110> chess Ke Lai Biotechnology (Shanghai) stock Co., ltd
<120> a glycosyltransferase and its use
<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 (17)

1. A glycosyltransferase, characterized in that the glycosyltransferase differs from SEQ ID No. 2 by an amino acid residue selected from the group consisting of:
257 amino acids A, 451 amino acid E and 189 amino acids L; or alternatively, the first and second heat exchangers may be,
257 amino acids A, 451 amino acid E, 189 amino acids L and 14 amino acids I; or alternatively, the first and second heat exchangers may be,
257 th amino acid is A, 451 th amino acid is E, 189 th amino acid is L and 273 th amino acid is G; or alternatively, the first and second heat exchangers may be,
257 amino acids A, 451 amino acid E, 189 amino acids L and 324 amino acids G; or alternatively, the first and second heat exchangers may be,
257 amino acids A, 451 amino acid E, 189 amino acids L and 347 amino acids G; or alternatively, the first and second heat exchangers may be,
257 amino acids A, 451 amino acid E, 189 amino acid L and 455 amino acid D or C.
2. An isolated nucleic acid encoding the glycosyltransferase of claim 1.
3. A recombinant expression vector comprising the nucleic acid of claim 2.
4. A transformant which is a host cell comprising the nucleic acid of claim 2 or the recombinant expression vector of claim 3.
5. The transformant according to claim 4, wherein the host cell is Escherichia coliEscherichia coli）。
6. The transformant according to claim 5, wherein the Escherichia coli isE.coli BL21（DE3）。
7. A method of making the glycosyltransferase of claim 1, comprising culturing the transformant of any one of claims 4-6 under conditions suitable for expression of the glycosyltransferase.
8. A composition comprising the glycosyltransferase of claim 1.
9. A method for glycosylation of a substrate, the method comprising providing at least one substrate, the glycosyltransferase of claim 1, and contacting the substrate with the glycosyltransferase under conditions such that the substrate is glycosylated to produce at least one glycosylation product.
10. A method for preparing rebaudioside a, comprising the steps of: the rebaudioside a is obtained by reacting stevioside with a glycosyl donor in the presence of the glycosyl transferase of claim 1.
11. The method of preparation of claim 10, wherein the method of preparation meets one or more of the following conditions:
the glycosyltransferase exists in the form of glycosyltransferase thalli, crude enzyme liquid, pure enzyme or immobilized enzyme;
the concentration of stevioside is 1-150 g/L;
the mass ratio of the glycosyltransferase thalli to stevioside is 1 (3-10);
the glycosyl donor is UDP-glucose and/or ADP-glucose;
the pH of the reaction solvent of the reaction is 5-8;
the pH is controlled by a buffer solution;
the rotational speed of the reaction is 500-1000 rpm;
the temperature of the reaction system of the reaction is 20-90 ℃.
12. The method of claim 11, wherein the purified enzyme is present as a purified enzyme solution.
13. The method of preparation of claim 11, wherein the method of preparation meets one or more of the following conditions:
the concentration of stevioside is 100 g/L;
the mass ratio of the glycosyltransferase thalli to stevioside is 3:20;
the UDP-glucose and/or ADP-glucose is prepared by UDP and/or ADP in the presence of sucrose and sucrose synthase, wherein the concentration of sucrose is 100-300g/L, and the concentration of UDP or ADP is 0.05-0.2 g/L;
the pH of the reaction solvent of the reaction is 6;
the buffer solution is phosphoric acid buffer solution;
the rotational speed of the reaction was 600 rpm;
the temperature of the reaction system of the reaction was 60 ℃.
14. The method according to claim 13, wherein the sucrose concentration in the method is 200. 200g/L and the UDP or ADP concentration is 0.1. 0.1g/L.
15. A process for the preparation of rebaudioside D or rebaudioside M comprising the step of preparing rebaudioside a according to the process of any one of claims 10-14.
16. Use of the glycosyltransferase of claim 1 for the preparation of steviol glycosides.
17. The use of claim 16, wherein the steviol glycoside is rebaudioside a, rebaudioside D, or rebaudioside M.
CN202111006803.4A 2021-08-30 2021-08-30 Glycosyltransferase and application thereof Active CN115725528B (en)

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