CN114875054B - Method for preparing glycosylated stevioside compound by enzymatic method and derivative thereof - Google Patents

Method for preparing glycosylated stevioside compound by enzymatic method and derivative thereof Download PDF

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CN114875054B
CN114875054B CN202210732311.1A CN202210732311A CN114875054B CN 114875054 B CN114875054 B CN 114875054B CN 202210732311 A CN202210732311 A CN 202210732311A CN 114875054 B CN114875054 B CN 114875054B
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rebaudioside
glycosyltransferase
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stevioside
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贾红华
李艳
潘华祎
肖亮
林磊
韦萍
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Nanjing Tech University
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Abstract

The invention discloses a method for enzymatically glycosylating stevioside compounds and derivatives thereof, which comprises the steps of transferring glycosyl of a glycosyl donor to C-6 'of a first glycosyl connected with C19 of the stevioside compounds and/or transferring glycosyl of the glycosyl donor to C-2' of the first glycosyl connected with C19 of the stevioside compounds in the presence of glycosyl transferase. The glycosylated derivative has increased sweet taste and reduced bitter taste compared with corresponding substrate, and improves taste quality, and is a potential high-quality sweetener, and can be used as natural sweetener, dietary supplement and pharmaceutical ingredient in food. The method is high in yield, good in effect, simple in operation, few in product byproducts, green, environment-friendly and pollution-free, low in production cost and suitable for industrial production in the sweetener quality experiment of the stevioside compound optimized by the enzyme method at present.

Description

Method for preparing glycosylated stevioside compound by enzymatic method and derivative thereof
Technical Field
The invention belongs to the technical field of biocatalytic conversion, and relates to a method for glycosylating stevioside compounds by glycosyltransferase, derivatives thereof and application thereof.
Background
Stevioside is a glycoside extracted from the leaves of stevia rebaudiana Bertoni of Compositae, has the characteristics of high sweetness and low heat energy, and is a natural sweetener. The chemical structure of the diterpenoid compound is diterpenoid steviol which is taken as a basic framework [1] . Steviol glycosides are mainly found in stevia rebaudiana leaves, which contain rubusoside (Rub), stevioside (St), rebaudioside A (RA), rebaudioside B (RB), rebaudioside D (RD), rebaudioside E (RE), rebaudioside M (RM), etc. In addition to the advantages of high sweetness and low calorie, stevioside is considered to have important pharmacological activities such as antioxidant, antibacterial, antifungal, antiviral, antitumor, diuretic, stomach protecting (antidiarrheal), analgesic, immunoregulatory, blood pressure and blood sugar lowering, tumor growth inhibiting, cell regeneration promoting, and vascular strengthening [2] . Stevioside as sweetener for adjuvant treatment of obesity, diabetes (type II), hypertension, cardiovascular and cerebrovascular diseases, hypoglycemia, and dental caries [2-3] . Therefore, steviol glycoside compounds are used as not only sweeteners in the food industry but also as adjuvant therapeutic agents in health care drugs [6] . While the steviol glycoside industry is in vigorous development, the bitter aftertaste in mouthfeel is impeding its commercial use [4] 。
Numerous documents report that increasing the amount of beta-glucose carried on aglycones can enhance the sweetness of steviol glycosides and improve the mouthfeel to reduce bitter taste [5] . Or extending the beta-glucose on C19 (R1) can also reduce the bitter taste. The enzyme modification commonly used at present is glycosylation of stevioside compounds, and cyclodextrin glycosyltransferase and UDP-glycosyltransferase are generally adopted. The former has low regioselectivity to cause confusion of products, and a specific product can be obtained by partial glycoside hydrolysis with specific glycosidase, so that the operation difficulty is increased, and the production cost is higher; and the latter has high regioselectivity, high purity of the obtained product, reduced operation flow of byproducts and reduced production cost.
UDP-glycosyltransferases typically immobilise on glycosylation to catalyze some type of glycosidic linkage, such as UGT76G1 catalyzing a 1, 3-beta-D-glycidic glycosidic linkage, and UGTSL2 catalyzing a 1, 2-beta-D-glycidic glycosidic linkage. However, both catalyze not only the C19 glycosylation but also the C13 glycosylation, reducing the efficiency of extending the glycoside chain at the C19 position. In addition, UGTSL2 catalyzes 1, 2-beta-D-glycidic glycosidic bond of stevioside compound and also accompanies by-product of 1, 6-beta-D-glycidic glycosidic bond, which results in reduced product purity, increased difficulty in extracting product and complicated process.
The glycosyl donor is indispensable in the glycosylation reaction process of the UDP-glycosyltransferase, and the main glycosyl donor comprises UDPG, UDP-galactose, UDP-glucuronic acid and the like, wherein the UDPG has the most extensive application, but the UDPG has high price and is not suitable for large-scale application in industrial production. Coupling reaction of sucrose synthase with glycosyltransferase enables in situ regeneration cycle of UDPG by adding sucrose during glycosylation reaction. A double enzyme coupling system is established by utilizing sucrose synthase and glycosyltransferase, and when double enzymes catalyze, sucrose is catalyzed by the sucrose synthase to crack to generate UDPG and fructose [7] . The glycosylation reaction is then catalyzed by glycosyltransferases. The UDP generated in the glycosylation process can be utilized by sucrose synthase and converted into UDPG again, so that cyclic utilization is realized, and meanwhile, the inhibition of accumulation of UDP on enzyme activity is avoided. Adopts a double enzyme coupling system, and only needs to be addedThe inexpensive sucrose is added as a substrate without expensive UDPG, so that the aim of controlling the production cost can be achieved.
1、Putnik,P.,Bezuk,I.,Barba,F.J.,Lorenzo,J.M.,I.,&D.K.(2020).Sugar reduction:Stevia rebaudiana Bertoni as a natural sweetener.In Francisco J.B.,Predrag,P.,&Danijela,B.K.(Eds.),Agri-food Industry Strategies for Healthy Diets and Sustainability,(pp.123-152).Academic Press.
2、Lemus-Mondaca,R.,Vega-Galvez,A.,Rojas,P.,Stucken,K.,Delporte,C.,Valenzuela-Barra,G.,…Pasten,A.(2018).Antioxidant,antimicrobial and anti-inflammatory potential of Stevia rebaudiana leaves:effect of different drying methods.Journal of Applied Research on Medicinal and Aromatic Plants,11,37-46.
3、Ahmad,J.,Khan,I.,Blundell,R.,Azzopardi,J.,&Mahomoodally,M.F.(2020).Stevia rebaudiana Bertoni.:an updated review of its health benefits,industrial applications and safety.Trends in Food Science and Technology,100,177-189.
4、Yildiz,M.,&Karhan,M.(2021).Characteristics of some beverages adjusted with stevia extract,and persistence of steviol glycosides in the mouth after consumption.International Journal of Gastronomy and Food Science,24,100326.
5、Gerwig,G.J.,te Poele,E.M.,Dijkhuizen,L.,&Kamerling,J.P.(2016).Stevia glycosides:chemical and enzymatic modifications of their carbohydrate moieties to improve the sweet-tasting quality.In Baker,D.C.(Ed.),Advances in Carbohydrate Chemistry and Biochemistry,73,(pp.1-72).Academic Press.
6、Wang,J.,Zhao,H.,Wang,Y.,Lau,H.,Zhou,W.,Chen,C.,&Tan,S.(2020).Areview of stevia as a potential healthcare product:up-to-date functional characteristics,administrative standards and engineering techniques.Trends in Food Science and Technology,103,264-281.
7、Chen,L.,Sun,P.,Zhou,F.,Li,Y.,Chen,K.,Jia,H.,…Ouyang,P.(2018).Synthesis of rebaudioside D,using glycosyltransferase UGTSL2 and in situ UDP-glucose regeneration.Food Chemistry,259,286-291.
Disclosure of Invention
The invention aims to solve the problem of providing a method for enzymatic glycosylation of stevioside compounds, derivatives and application thereof, wherein the method utilizes glycosyltransferase (VvUGT, lbUGT, nsUGT, ntUGT) from blue fruit trees, medlar, grapes or tobacco to couple with sucrose synthase to realize enzymatic efficient glycosylation of stevioside compounds. The application method improves the taste of the stevioside compound, improves the sweetness of the stevioside compound, is suitable for various stevioside compounds, has the advantages of high reaction conversion rate, high yield, strong specificity, few by-products, simple operation flow, environment friendliness, no pollution and low production cost, and is suitable for industrial production. The glycosylated products of Rub1G, rub1G, st1G, st G, RA1G, RM2 and RM1G are added with sweet taste and reduced bitter taste compared with the corresponding substrates, and are potential high-quality sweeteners.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for enzymatically glycosylating a steviol glycoside compound, in the presence of a glycosyltransferase, transferring the glycosyl of a glycosyl donor to the C-6 'of the C19 linked first glycosyl of the steviol glycoside compound and/or transferring the glycosyl of a glycosyl donor to the C2' of the C19 linked first glycosyl of the steviol glycoside compound.
The method for enzymatically glycosylating stevioside compounds comprises the following steps:
1) Constructing engineering bacteria: cloning glycosyltransferase genes and sucrose synthase genes, constructing vectors for co-expression or separate expression of double enzymes, and transferring the vectors into host bacteria to obtain engineering bacteria of UDP-glycosyltransferase and/or sucrose synthase;
2) Preparation of the glycosylation product: fermenting the constructed engineering bacteria to produce enzyme, and performing ultrasonic crushing or high-pressure crushing on the thallus obtained by fermentation to obtain crude enzyme liquid of glycosyltransferase and sucrose synthase;
3) Carrying out glycosylation reaction on the crude enzyme solution, a glycosylation substrate and sucrose in a conversion system; the glycosyltransferase catalyzes a substrate to carry out a conversion reaction to prepare a glycosylation product; sucrose synthase catalyzes the production of UDP-glucose as a glycosyl donor from UDP produced by this process.
The glycosyltransferase is VvUGT derived from grape (Vitis vinifera), lbUGT derived from Lycium barbarum (Lycium barbarum), nsUGT derived from Lanternaria (Nyssa sinensis), or NtUGT derived from tobacco (Nicotiana tomentosiformis).
Carrying out glycosylation reaction on the glycosyltransferase crude enzyme liquid, stevioside compounds and glycosyl donors in a conversion system, wherein the adding amount of the glycosyltransferase is 0.1-5 g/L, the adding amount of the stevioside compounds is 0.1-1000 g/L, the adding amount of the glycosyl donors is 0.1-3000 g/L, the reaction temperature is 20-80 ℃, the reaction time is 2-96 h, and the reaction pH is 5-10.
The steviol glycoside compounds include, but are not limited to, rubusoside, stevioside, rebaudioside A, rebaudioside B, rebaudioside D, rebaudioside E, and rebaudioside M.
Transferring the glycosyl of the glycosyl donor to the C-6' of the first glycosyl linked to C19 of the steviol glycoside in the presence of glycosyl transferase VvUGT or LbUGT or NsUGT or NtUGT, thereby forming a first glycosylation product;
in the presence of glycosyltransferase VvUGT or LbUGT or NsUGT or NtUGT, and with a beta-glucoside or first glycosylation product attached to the C-6 'of the C19 linked first glucosyl group of the steviol glycoside compound, transferring the glycosyl group of the glycosyl donor to the C-2' of the C19 linked first glucosyl group of the steviol glycoside compound, thereby forming a second glycosylation product.
The amino acid sequence of the VvUGT is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 2; the LbUGT has an amino acid sequence shown as SEQ ID NO. 3, and a nucleotide sequence shown as SEQ ID NO. 4; the amino acid sequence of the NsUGT is shown as SEQ ID NO. 5, and the nucleotide sequence is shown as SEQ ID NO. 6; the amino acid sequence of the NtUGT is shown as SEQ ID NO. 7, and the nucleotide sequence is shown as SEQ ID NO. 8.
The sucrose synthase includes, but is not limited to, sucrose synthase StSUS, and the nucleotide sequence of the sucrose synthase StSUS is shown in SEQ ID NO. 9.
A glycosyltransferase VvUGT derived from grape (Vitis vinifera) has an amino acid sequence shown in SEQ ID NO. 1; the nucleotide sequence of the code VvUGT enzyme is shown as SEQ ID NO. 2.
The glycosyltransferase VvUGT has the following enzymatic properties:
(1) Maximum enzyme activity was reached at 45℃and pH 8;
(2) Under the condition of the existence of a substrate, the enzyme activity below 35 ℃ can retain more than 95 percent of the enzyme activity in 2 hours;
(3) In the presence of a substrate, the enzyme activity of pH 7-9 can be maintained for more than 75% in 2 hours;
(4) Metal ions (EDTA, zn) are additionally added 2+ ,Mg 2+ ,Ca 2+ ,Ni 2+ ,Mn 2+ ,Cu 2+ And Ba 2+ ) The enzyme activity all shows a decreasing trend, zn 2+ ,Ni 2+ And Cu 2+ Directly inactivating the enzyme;
the kinetic parameters Km of glycosyltransferase VvUGT for the substrates rubusoside, stevioside, rebaudioside A, E were 1.0mM, 2.4mM, 7.3mM and 25mM, respectively.
In the glycosylation reaction, the crude enzyme solution is subjected to bioconversion in a buffer solution with pH of 6-8, the reaction temperature is 20-50 ℃, the reaction time is 1-36h, the initial reaction concentration of a substrate (rubusoside, stevioside and rebaudioside A, E, D, M) is 1-50g/L, the mass ratio of sucrose to rubusoside is 1-15, the total protein concentration of the crude enzyme solution is 2-10mg/mL, and the crude enzyme activity ratio of sucrose synthase to glycosyltransferase is 1-20. Rubusoside is used as a substrate, the VvUGT is used for catalyzing for 36 hours, the rubusoside conversion rate is 100%, the rubus 1G yield is 45%, and the rubus 2G yield is 40%; stevioside is used as a substrate, the VvUGT is used for catalyzing for 36 hours, the stevioside rate is 80%, the St1G yield is 35%, and the St2G yield is 27%; rebaudioside a is a substrate VvUGT for 36h, rebaudioside a conversion is 60%, RA1G yield is 30%, RM2 yield is 18%; rebaudioside E is a substrate, catalyzed for 36h when the glycosyltransferase is VvUGT, rebaudioside E conversion is 20%, st2G yield is 13%; rebaudioside D, M is free of neogenesis products. Glycosyltransferase VvUGT preferentially catalyzes rubusoside and produces Rub1G and Rub2G.
A glycosyltransferase LbUGT derived from Lycium barbarum (Lycium barbarum) has an amino acid sequence shown in SEQ ID NO. 3 and a nucleotide sequence shown in SEQ ID NO. 4;
The glycosyltransferase LbUGT has the following enzymatic properties:
(1) Maximum enzyme activity was reached at 50℃and pH 8;
(2) Under the condition of the existence of a substrate, the enzyme activity below 35 ℃ can retain more than 95 percent of the enzyme activity in 2 hours;
(3) In the presence of a substrate, the enzyme activity of pH 7-9 can be maintained for more than 75% in 2 hours;
(4) Metal ions (EDTA, zn) are additionally added 2+ ,Mg 2+ ,Ca 2+ ,Ni 2+ ,Mn 2+ ,Cu 2+ And Ba 2+ )，
The enzyme activity all shows a decreasing trend, zn 2+ ,Ni 2+ And Cu 2+ Directly inactivating the enzyme;
the kinetic parameters Km for the substrates rubusoside, stevioside, rebaudioside a, rebaudioside E and D were 1.2mM, 2.1mM, 2.4mM, 2.3mM and 6.9mM, respectively.
In the glycosylation reaction, the crude enzyme solution is subjected to bioconversion in a buffer solution with pH of 6-8, the reaction temperature is 20-50 ℃, the reaction time is 1-36h, the initial reaction concentration of a substrate (rubusoside, stevioside and rebaudioside A, E, D, M) is 1-50g/L, the mass ratio of sucrose to rubusoside is 1-15, the total protein concentration of the crude enzyme solution is 2-10mg/mL, and the crude enzyme activity ratio of sucrose synthase to glycosyltransferase is 1-20. Rubusoside is used as a substrate, lbUGT is used for catalyzing for 36 hours, the rubusoside conversion rate is 80%, the rubus 1G yield is 43%, and the rubus 2G yield is 25%; stevioside is used as a substrate, lbUGT is used for catalyzing for 36 hours, the stevioside conversion rate is 87%, the St1G yield is 48%, and the St2G yield is 29%; rebaudioside a is a substrate, catalyzed by LbUGT for 36h, rebaudioside a conversion is 90%, RA1G yield is 49%, RM2 yield is 42%; rebaudioside E is a substrate, catalyzed by LbUGT for 36h, rebaudioside E conversion is 73%, st2G yield is 67%; rebaudioside D, lbUGT catalyzes for 36h with a rebaudioside D conversion of 65% and RM2 yield of 58%; rebaudioside M was the substrate and catalyzed by LbUGT for 36h with a rebaudioside M conversion of 93% and RM1G yield of 87%. Glycosyltransferase LbUGT preferentially catalyzes rebaudiosides a and M to produce RA1G, RM2 and RM1G.
Glycosyltransferase NsUGT from fructus Myrtilli (Nyssa sinensis), the amino acid sequence is shown in SEQ ID NO. 5, and the nucleotide sequence is shown in SEQ ID NO. 6;
the glycosyltransferase NsUGT has the following enzymatic properties:
(1) Maximum enzyme activity was reached at 50℃and pH 8;
(2) Under the condition of the existence of a substrate, the enzyme activity below 35 ℃ can retain more than 95 percent of the enzyme activity in 2 hours;
(3) In the presence of a substrate, the enzyme activity of pH 7-9 can be maintained for more than 75% in 2 hours;
(4) Metal ions (EDTA, zn) are additionally added 2+ ,Mg 2+ ,Ca 2+ ,Ni 2+ ,Mn 2+ ,Cu 2+ And Ba 2+ ) The enzyme activity all shows a decreasing trend, zn 2+ ,Ni 2+ And Cu 2+ Directly inactivating the enzyme;
(5) The kinetic parameters Km for the substrates rubusoside, stevioside, rebaudioside a, rebaudioside E and D were 3.8mM, 1.1mM, 4.6mM, 2.1mM and 6.5mM, respectively.
In the glycosylation reaction, the crude enzyme solution is subjected to bioconversion in a buffer solution with pH of 6-8, the reaction temperature is 20-50 ℃, the reaction time is 1-36h, the initial reaction concentration of a substrate (rubusoside, stevioside and rebaudioside A, E, D, M) is 1-50g/L, the mass ratio of sucrose to rubusoside is 1-15, the total protein concentration of the crude enzyme solution is 2-10mg/mL, and the crude enzyme activity ratio of sucrose synthase to glycosyltransferase is 1-20. Rubusoside is used as a substrate, the NSUGT is catalyzed for 36 hours, the rubusoside conversion rate is 75%, the rubus 1G yield is 43%, and the rubus 2G yield is 19%; stevioside is used as a substrate, nsUGT is used for catalyzing for 36 hours, the stevioside conversion rate is 100%, the St1G yield is 54%, and the St2G yield is 43%; rebaudioside a is a substrate, nsUGT catalyzes for 36h, rebaudioside a conversion is 73%, RA1G yield is 34%, RM2 yield is 22%; rebaudioside E is a substrate, nsUGT catalyzes for 36h, rebaudioside E conversion is 100%, st2G yield is 95%; rebaudioside D is a substrate, nsUGT catalyzes for 36h, rebaudioside D conversion is 75%, RM2 yield is 67%; rebaudioside M was the substrate, nsUGT catalyzed for 36h, rebaudioside M conversion was 72%, RM1G yield was 67%. Glycosyltransferase NsUGT preferentially catalyzes stevioside and rebaudioside E to produce St1G and St2G.
A glycosyltransferase NtUGT derived from tobacco (Nicotiana tomentosiformis) has an amino acid sequence shown in SEQ ID NO. 7 and a nucleotide sequence shown in SEQ ID NO. 8.
The glycosyltransferase NtUGT has the following enzymatic properties:
(1) Maximum enzyme activity was reached at 55℃and pH 8;
(2) Under the condition of the existence of a substrate, the enzyme activity below 35 ℃ can retain more than 95 percent of the enzyme activity in 2 hours;
(3) In the presence of a substrate, the enzyme activity of pH 7-9 can be maintained for more than 80% in 2 hours;
(4) Metal ions (EDTA, zn) are additionally added 2+ ,Mg 2+ ,Ca 2+ ,Ni 2+ ,Mn 2+ ,Cu 2+ And Ba 2+ ) The enzyme activity all shows a decreasing trend, zn 2+ ,Ni 2+ And Cu 2+ Directly inactivating the enzyme;
(5) The kinetic parameters Km for the substrates rubusoside, stevioside, rebaudioside a, rebaudioside E and D were 1.9mM, 4.5mM, 3.8mM, 2.7mM and 2.6mM, respectively.
In the glycosylation reaction, the crude enzyme solution is subjected to bioconversion in a buffer solution with pH of 6-8, the reaction temperature is 20-50 ℃, the reaction time is 1-36h, the initial reaction concentration of a substrate (rubusoside, stevioside and rebaudioside A, E, D, M) is 1-50g/L, the mass ratio of sucrose to rubusoside is 1-15, the total protein concentration of the crude enzyme solution is 2-10mg/mL, and the crude enzyme activity ratio of sucrose synthase to glycosyltransferase is 1-20. Rubusoside is used as a substrate, ntUGT is catalyzed for 36 hours, the rubusoside conversion rate is 70%, the Rub1G yield is 41%, and the Rub2G yield is 17%; stevioside is used as a substrate, ntUGT is used for catalyzing for 36 hours, the stevioside conversion rate is 83%, the St1G yield is 51%, and the St2G yield is 28%; rebaudioside a is a substrate, the NtUGT catalyzes for 36 hours, the rebaudioside a conversion is 67%, the RA1G yield is 41%, the RM2 yield is 13%; rebaudioside E is the substrate, ntUGT catalyzes for 36h, rebaudioside E conversion is 66%, st2G yield is 62%; rebaudioside D was the substrate, catalyzed for 36h at a glycosyltransferase of NtUGT, rebaudioside D conversion 100%, RM2 yield 96%; rebaudioside M was the substrate, ntUGT catalyzed for 36h, rebaudioside M conversion 86%, RM1G yield 73%. Glycosyltransferase NtUGT preferentially catalyzes rebaudioside D to produce RM2.
The application of the glycosyltransferase (VvUGT, lbUGT, nsUGT, ntUGT) from the sources of the fruit tree, the medlar, the grape or the tobacco in catalyzing glycosylation reaction. In the glycosylation reaction, the glycosyltransferase glycosylates stevioside compounds, and beta-glucoside is added to C-6 'and C-2' of C19 connected with the first glycosyl. When the C-6 'is not connected with the glycoside, the beta-glucoside is preferentially added at the C-6' position; when the C-6 'has been linked to the glycoside, β -glucoside is added at the C-2' position.
The glycosylation reaction can be widely applied to catalyzing the glycosylation reaction of stevioside compounds. The steviol glycoside compound is one or more of the following groups: steviol glycoside compounds present in natural plants, extracted steviol glycoside compounds and synthetic steviol glycoside compounds.
The steviol glycoside compounds include, but are not limited to, rubusoside, stevioside, rebaudioside A, rebaudioside B, rebaudioside D, rebaudioside E, and rebaudioside M.
A recombinant vector comprising at least one nucleotide sequence of the above glycosyltransferase.
A recombinant cell comprising the recombinant vector or the nucleotide sequence of the glycosyltransferase can be expressed to obtain the glycosyltransferase.
A method for producing glycosyltransferase by fermenting the recombinant cell.
Cloning glycosyltransferase genes, constructing an expression vector, transferring the expression vector into host bacteria to obtain engineering bacteria of glycosyltransferase, fermenting the constructed engineering bacteria to produce enzyme, and performing ultrasonic crushing or high-pressure crushing on the bacterial cells obtained by fermentation to obtain crude enzyme liquid of glycosyltransferase. The host bacteria include, but are not limited to, E.coli, saccharomyces cerevisiae, pichia pastoris, corynebacterium glutamicum, and the like.
The glycosyltransferase can be widely applied to stevioside compound glycosylation reaction, such as rubusoside, stevioside, rebaudioside A, rebaudioside B, rebaudioside D, rebaudioside E, rebaudioside M and the like.
Use of glycosyltransferase (VvUGT, lbUGT, nsUGT, ntUGT) derived from a fruit tree of the blue, fruit of the Chinese wolfberry, grape or tobacco source to glycosylate steviol glycosides, comprising the steps of:
(1) Constructing engineering bacteria: cloning glycosyltransferase genes and sucrose synthase genes, constructing vectors for co-expression or separate expression of double enzymes, and transferring the vectors into host bacteria to obtain engineering bacteria of UDP-glycosyltransferase and/or sucrose synthase;
(2) Preparation of the glycosylation product: fermenting the constructed engineering bacteria to produce enzyme, and performing ultrasonic crushing or high-pressure crushing on the thallus obtained by fermentation to obtain crude enzyme liquid of glycosyltransferase and sucrose synthase;
(3) Carrying out glycosylation reaction on the crude enzyme solution, a glycosylation substrate and sucrose in a conversion system; the glycosyltransferase catalyzes a substrate to carry out a conversion reaction to prepare a glycosylation product; sucrose synthase catalyzes the production of UDP-glucose as a glycosyl donor from UDP produced by this process.
The host bacteria include, but are not limited to, E.coli, saccharomyces cerevisiae, pichia pastoris, bacillus subtilis, corynebacterium glutamicum, and the like.
Further improved, the engineering bacteria are escherichia coli, saccharomyces cerevisiae, pichia pastoris, bacillus subtilis, corynebacterium glutamicum and the like which are introduced with UDP-glycosyltransferase genes (VvUGT, lbUGT, nsUGT or NtUGT) and sucrose synthase genes StSUS.
The initial reaction concentration of the glycosylation substrate is 1-1000g/L, the mass ratio of sucrose to the glycosylation substrate is 1-200, and the total protein concentration of the crude enzyme solution prepared by the engineering bacteria which co-express or respectively express the two enzymes in a reaction system is 2-10mg/mL. The ratio of crude enzyme activity of sucrose synthase to glycosyltransferase is 1-20.
In another improvement, the transformation reaction adopts an aqueous phase system or a two-phase system, and the crude enzyme solution is subjected to biological transformation in a buffer solution with pH of 6-8.
The aqueous phase system is in a buffer solution with pH of 6-8, and the buffer solution comprises potassium phosphate, sodium phosphate and HEPES buffer solution.
The biphasic system is a biphasic system in which an organic phase, for example DMSO, is mixed with a buffer, which is phosphate buffer (pH 6-8).
In another improvement, the reaction temperature of the conversion reaction is 20-50 ℃ and the reaction time is 1-96h.
The rubusoside is used as a substrate to synthesize a first glycosylation product Rub1G and a second glycosylation product Rub2G:
the conversion reaction adopts a water phase system, the crude enzyme solution is subjected to biological conversion in a buffer solution with pH value of 6-8, the reaction temperature is 20-50 ℃, the reaction time is 1-36h, the initial reaction concentration of a substrate is 1-20g/L, the mass ratio of sucrose to rubusoside is 1-15, the total protein concentration of the crude enzyme solution is 2-10mg/mL, and the crude enzyme activity ratio of sucrose synthase to glycosyltransferase is 1-20.
Further, the crude enzyme solution is subjected to biotransformation in a buffer solution with pH of 7.2, the reaction temperature is 37 ℃, the reaction time is 1-40h, the initial reaction concentration of the substrate is 10g/L, the mass ratio of sucrose to rubusoside is 5, and the total protein concentration of the crude enzyme solution is 10mg/mL. Catalyzing for 36h when glycosyltransferase is VvUGT, wherein the rubusoside conversion rate is 100%, the Rub1G yield is 45%, and the Rub2G yield is 40%; catalyzing for 36h when the glycosyltransferase is LbUGT, wherein the rubusoside conversion rate is 80%, the Rub1G yield is 43%, and the Rub2G yield is 25%; catalyzing for 36h when glycosyltransferase is NsUGT, wherein the rubusoside conversion rate is 75%, the Rub1G yield is 43%, and the Rub2G yield is 19%; the catalyst was used for 36h when the glycosyltransferase was NtUGT, the rubusoside conversion was 70%, the Rub1G yield was 41% and the Rub2G yield was 17%. In the production of rubusoside as a substrate and a product Rub1G, rub G, the yield of VvUGT is highest.
Stevioside is used as a substrate to synthesize a first glycosylation product St1G and a second glycosylation product St2G:
the conversion reaction adopts a water phase system, the crude enzyme solution is subjected to biological conversion in a buffer solution with pH value of 6-8, the reaction temperature is 20-50 ℃, the reaction time is 1-36h, the initial reaction concentration of a substrate is 1-20g/L, the mass ratio of sucrose to stevioside is 1-15, the total protein concentration of the crude enzyme solution is 2-10mg/mL, and the crude enzyme activity ratio of sucrose synthase to glycosyltransferase is 1-20.
Further, the crude enzyme solution is subjected to biotransformation in a buffer solution with pH of 7.2, the reaction temperature is 37 ℃, the reaction time is 1-40h, the initial reaction concentration of the substrate is 10g/L, the mass ratio of sucrose to stevioside is 5, and the total protein concentration of the crude enzyme solution is 10mg/mL. Catalyzing for 36h when the glycosyltransferase is VvUGT, wherein the stevioside rate is 80%, the St1G yield is 35%, and the St2G yield is 27%; catalyzing for 36h when the glycosyltransferase is LbUGT, wherein the stevioside conversion rate is 87%, the St1G yield is 48%, and the St2G yield is 29%; catalyzing for 36h when the glycosyltransferase is NsUGT, wherein the stevioside conversion rate is 100%, the St1G yield is 54%, and the St2G yield is 43%; catalytic 36h at a glycosyltransferase of NtUGT, stevioside conversion of 83%, st1G yield of 51% and St2G yield of 28%. The yield of NsUGT is highest in the production of the product St1G, st G by taking stevioside as a substrate.
Rebaudioside a is a substrate to synthesize a first glycosylation product RA1G and a second glycosylation product RM2:
the transformation reaction adopts a water phase system, the crude enzyme solution is subjected to biological transformation in a buffer solution with pH of 6-8, the reaction temperature is 20-50 ℃, the reaction time is 1-36h, the initial reaction concentration of a substrate is 1-20g/L, the mass ratio of sucrose to rebaudioside A is 1-50, the total protein concentration of the crude enzyme solution is 2-10mg/mL, and the crude enzyme activity ratio of sucrose synthase to glycosyltransferase is 1-20.
Further, the crude enzyme solution is subjected to bioconversion in a buffer solution with pH of 7.2, the reaction temperature is 37 ℃, the reaction time is 1-40h, the initial reaction concentration of a substrate is 10g/L, the mass ratio of sucrose to rebaudioside A is 5, and the total protein concentration of the crude enzyme solution is 10mg/mL. Catalyzing for 36h when the glycosyltransferase is VvUGT, wherein the rebaudioside A conversion rate is 60%, the RA1G yield is 30%, and the RM2 yield is 18%; catalyzing for 36h when the glycosyltransferase is LbUGT, wherein the rebaudioside A conversion rate is 90%, the RA1G yield is 49%, and the RM2 yield is 42%; catalyzing for 36h when the glycosyltransferase is NsUGT, wherein the rebaudioside A conversion rate is 73%, the RA1G yield is 34%, and the RM2 yield is 22%; the catalyst was used for 36h when the glycosyltransferase was NtUGT, the rebaudioside A conversion was 67%, the RA1G yield was 41% and the RM2 yield was 13%. Of the production of the product RA1G, RM2 with rebaudioside a as a substrate, lbUGT yields are highest.
Rebaudioside E is a substrate to synthesize St2G:
the transformation reaction adopts a water phase system, the crude enzyme solution is subjected to biological transformation in a buffer solution with pH of 6-8, the reaction temperature is 20-50 ℃, the reaction time is 1-36h, the initial reaction concentration of a substrate is 1-20g/L, the mass ratio of sucrose to rebaudioside E is 1-50, the total protein concentration of the crude enzyme solution is 2-10mg/mL, and the crude enzyme activity ratio of sucrose synthase to glycosyltransferase is 1-20.
Further, the crude enzyme solution is subjected to bioconversion in a buffer solution with pH of 7.2, the reaction temperature is 37 ℃, the reaction time is 1-40h, the initial reaction concentration of a substrate is 10g/L, the mass ratio of sucrose to rebaudioside E is 5, and the total protein concentration of the crude enzyme solution is 10mg/mL. Catalyzing for 36h when the glycosyltransferase is VvUGT, wherein the rebaudioside E conversion rate is 20%, and the St2G yield is 13%; catalyzing for 36h when the glycosyltransferase is LbUGT, wherein the rebaudioside E conversion rate is 73%, and the St2G yield is 67%; catalyzing for 36h when the glycosyltransferase is NsUGT, wherein the rebaudioside E conversion rate is 100%, and the St2G yield is 95%; the catalyst was used for 36h when the glycosyltransferase was NtUGT, the rebaudioside E conversion was 66% and the St2G yield was 62%. Among the production of the product St2G with rebaudioside E as a substrate, the NSUGT yield is highest.
Rebaudioside D is a substrate to synthesize RM2:
the transformation reaction adopts a biphasic (aqueous phase and organic phase) system, crude enzyme solution is subjected to biological transformation in a buffer solution with pH of 6-8, the reaction temperature is 20-50 ℃, the reaction time is 1-40h, the initial reaction concentration of a substrate is 1-20g/L, the mass ratio of sucrose to rebaudioside D is 1-50, the total protein concentration of the crude enzyme solution is 2-10mg/mL, and the crude enzyme activity ratio of sucrose synthase to glycosyltransferase is 1-20.
Further, the crude enzyme solution is subjected to bioconversion in a buffer solution with pH of 7.2, the reaction temperature is 37 ℃, the reaction time is 1-36h, the initial reaction concentration of a substrate is 5g/L, the mass ratio of sucrose to rebaudioside D is 5, and the total protein concentration of the crude enzyme solution is 10mg/mL. Catalyzing for 36h when the glycosyltransferase is VvUGT, wherein the rebaudioside D conversion rate is 0%, and the RM2 yield is 0%; catalyzing for 36h when the glycosyltransferase is LbUGT, wherein the rebaudioside D conversion rate is 65%, and the RM2 yield is 58%; catalyzing for 36h when the glycosyltransferase is NsUGT, wherein the rebaudioside D conversion rate is 75%, and the RM2 yield is 67%; the catalyst was used for 36h when the glycosyltransferase was NtUGT, the rebaudioside D conversion was 100%, and the RM2 yield was 96%. Among the production of the product St2G with rebaudioside D as a substrate, the NtUGT yield is highest.
Rebaudioside M is a substrate to synthesize RM1G:
the transformation reaction adopts a water phase system, the crude enzyme solution is subjected to biological transformation in a buffer solution with pH of 6-8, the reaction temperature is 20-50 ℃, the reaction time is 1-36h, the initial reaction concentration of a substrate is 1-20g/L, the mass ratio of sucrose to rebaudioside M is 1-50, the total protein concentration of the crude enzyme solution is 2-10mg/mL, and the crude enzyme activity ratio of sucrose synthase to glycosyltransferase is 1-20.
Further, the crude enzyme solution is subjected to bioconversion in a buffer solution with pH of 7.2, the reaction temperature is 37 ℃, the reaction time is 1-40h, the initial reaction concentration of a substrate is 5g/L, the mass ratio of sucrose to rebaudioside M is 5, and the total protein concentration of the crude enzyme solution is 10mg/mL. Catalyzing for 36h when the glycosyltransferase is VvUGT, wherein the rebaudioside M conversion rate is 0%, and the RM1G yield is 0%; catalyzing for 36h when the glycosyltransferase is LbUGT, wherein the rebaudioside M conversion rate is 93%, and the RM1G yield is 87%; catalyzing for 36h when the glycosyltransferase is NsUGT, wherein the rebaudioside M conversion rate is 72%, and the RM1G yield is 67%; the catalyst was used for 36h when the glycosyltransferase was NtUGT, the rebaudioside M conversion was 86%, and the RM1G yield was 73%. In the production of the product RM1G with rebaudioside M as a substrate, the LbUGT yield is highest.
The glycosylated steviol glycoside derivative is purified and the structure is identified by one-and two-dimensional NMR. It was determined that Rub1G was the first glycosyl linkage 1,6-O- β -D-glucoside attached at C19 of rubusoside and Rub2G was the first glycosyl linkage 1,2-O- β -D-glucoside attached at C19 of Rub 1G; st1G is the first glycosyl linkage 1,6-O- β -D-glucoside attached at C19 of stevioside, st2G is the first glycosyl linkage 1,2-O- β -D-glucoside attached at C19 of St 1G; RA1G is the first glycosyl linkage 1,6-O- β -D-glucoside attached at C19 of rebaudioside A, RM2 is the first glycosyl linkage 1,2-O- β -D-glucoside attached at C19 of Rub 1G; st2G is the first glucosyl linkage 1, 6-O-beta-D-glucoside attached at the C19 of RE; RM2 is the first glucosyl linkage 1, 6-O-beta-D-glucoside attached at the C19 of RD; RM1G is the first glucosyl group attached to 1, 6-O-. Beta. -D-glucoside at the C19 linkage of rebaudioside M.
When the stevioside compound is rubusoside, the first glycosylation product is Rub1G, and the second glycosylation product is Rub2G, and the chemical formula is shown as follows:
When the stevioside compound is stevioside, the first glycosylation product is St1G, and the second glycosylation product is St2G, and the chemical formula is as follows:
when the stevioside compound is rebaudioside a, the first glycosylation product is RA1G and the second glycosylation product is RM2.
When the steviol glycoside is rebaudioside E, the first glycosylation product is St2G.
When the steviol glycoside compound is rebaudioside D, the first glycosylation product is RM2.
When the stevioside compound is rebaudioside M, the first glycosylation product is RM1G, the chemical formula is as follows:
the derivative of the glycosylated stevioside compound has high sweetness and good taste, and can be used as a potential high-quality sweetener.
And evaluating the sweetness of the derivative of the glycosylated stevioside compound and the sweetness and taste of the corresponding product.
Sensory evaluation was performed on purified Rub1G, rub1G, st1G, st G, RA1G, RM and RM1G and substrates (rubusoside, stevioside, rebaudioside a, rebaudioside D, rebaudioside E, rebaudioside M) with rubusoside as control. The purified derivatives and their substrates were evaluated for sweetness and mouthfeel by multiple volunteers. Sweetness evaluation: the samples to be evaluated are diluted by 1 to 1000X and are compared with 1 to 50 percent of sucrose aqueous solution for evaluation. Taste evaluation, relative taste grade was evaluated with rubusoside as control.
Further, sweetness assessments 20 volunteers, including 10 females and 10 males, assessed taste quality and sweetness intensity. Rub1G, rub1G, st1G, st2G, RA1G, RM2, RM1G, rubusoside, stevioside, rebaudioside A, rebaudioside D, rebaudioside E, and rebaudioside M were dissolved in water, respectively, to give a 5% (w/v) solution, which was then diluted 60-510×, with a 30×interval. Volunteers tasted each sample and compared to 5% sucrose to estimate sweetness. The taste evaluation included "excellent (+4), fairly good (+3), better (+2), slightly better (+1), the same (0) or worse (-1)" 6 grades with the taste quality of rubusoside as rated "0" relative to rubusoside. Sweetness evaluation, namely, the glycosylated derivative and the corresponding substrate are subjected to sweetness increase by 10-20%, and the taste is improved by 1-2 grades.
The glycosylated stevioside derivatives have strong stability.
Evaluation of stability of glycosylated steviol glycoside derivatives under powder storage, acidic aqueous solution and heat treatment. And (5) storing the powder, and evaluating the residual amount of the sample for 1-36 months. Preparing aqueous solution of ph1-10, evaluating for 1-10 days, and evaluating the residual amount of the sample. Heating in 80-98deg.C water bath for 10-90min, and calculating the residual amount of sample.
Further, the purified powder is preserved in a cool and ventilated place for 12 months, and the residual amount of the sample is more than 98%. Incubation in aqueous ph2-7 for 7 days, the residual sample amounts were all between 97-99%. Heat treating in water bath at 95deg.C for 30min, and the residual amount is 95-98%.
UDP-glycosyltransferases typically immobilise on glycosylation to catalyze some type of glycosidic linkage, such as UGT76G1 catalyzing a 1, 3-beta-D-glycidic glycosidic linkage, and UGTSL2 catalyzing a 1, 2-beta-D-glycidic glycosidic linkage. However, both catalyze not only the C19 glycosylation but also the C13 glycosylation, reducing the efficiency of extending the glycoside chain at the C19 position. In addition, UGTSL2 catalyzes 1, 2-beta-D-glycidic glycosidic bond of stevioside compound and also accompanies by-product of 1, 6-beta-D-glycidic glycosidic bond, which results in reduced product purity, increased difficulty in extracting product and complicated process. The 4 UDP-glycosyltransferases of the invention not only specifically catalyze the glycosylation of C19, but also catalyze the glycosylation of 1, 6-beta-D-glycidic glycosidic bond or 1, 2-beta-D-glycidic glycosidic bond without producing other glycosidic bond products, and only enter the glycosylation of 1, 2-beta-D-glycidic glycosidic bond after the substrate is glycosylated with all 1, 6-beta-D-glycidic glycosidic bonds in the first glucosyl group connected by C19. The glycosylation method effectively improves the glycosylation efficiency of C19, ensures the purity of the product, and simplifies the process of product purification.
The beneficial effects are that:
the glycosyltransferase (VvUGT, lbUGT, nsUGT, ntUGT) from the blue fruit tree, the medlar, the grape or the tobacco is obtained by a gene digging technology, the 4 enzymes have natural high stability and affinity for stevioside compounds, C19 can be sequentially glycosylated to connect C-6 'and C-2' of the first glycosyl, and beta-glucoside is added.
The invention constructs glycosyltransferase from blue fruit tree, matrimony vine, grape or tobacco with sucrose synthase by using genetic engineering technology to obtain co-expression high-yield recombinant strain, obtains cheap and easily available high-activity biological enzyme suitable for industrialization, and synthesizes a large amount of glycosylation products by enzyme method.
Compared with other methods for glycosylating stevioside compounds, the project relates to the rare 1, 6-O-beta-D-glycosidic reaction catalyzed by glycosyltransferase at C19, and byproducts are almost absent; in the case of the 1, 6-O-beta-D-glycoside already present, there are few side reactions in the C19 which continue to catalyze the 1, 2-O-beta-D-glycoside reaction. The enzyme has high-efficiency conversion capability on stevioside compounds, especially stevioside and rebaudioside M. Glycosyltransferases derived from blue fruit trees, medlar, grapes or tobacco are also focused on glycosylation at the C19 position, so that the bitter taste of stevioside compounds is greatly eliminated, and the development of high-quality novel stevioside compound sweeteners is facilitated.
Drawings
FIG. 1 SDS-PAGE analysis of crude VvUGT, lbUGT, nsUGT and NtUGT extracts. (M: protein Marker,1:VvUGT supernatant, 2:VvUGT pellet, 3:LbUGT supernatant, 4:LbUGT pellet, 5:NsUGT supernatant, 6:NsUGT pellet, 7:NtUGT supernatant, 8:NtUGT pellet)
FIG. 2 SDS-PAGE analysis of purified VvUGT, lbUGT, nsUGT and NtUGT. (M: protein Marker,1: purified VvUGT,2: purified LbUGT,3: purified NsUGT,4: purified NtUGT)
FIG. 3 optimum temperature and temperature stability curves for VvUGT, lbUGT, nsUGT and NtUGT;
FIG. 4 optimum pH curves for VvUGT, lbUGT, nsUGT and NtUGT;
FIG. 5 temperature stability curves for VvUGT, lbUGT, nsUGT and NtUGT;
FIG. 6 pH stability curves for VvUGT, lbUGT, nsUGT and NtUGT;
FIG. 7 shows the structure of a part of glycosylated derivatives of steviol glycosides.
Detailed Description
The invention will be better understood from the following examples. However, it will be readily understood by those skilled in the art that the specific material ratios, process conditions and results thereof described in the examples are only illustrative of the present invention and should not be nor limited to the invention described in detail in the claims.
Example 1
Construction of engineering bacterium of escherichia coli for expressing glycosyltransferase VvUGT, lbUGT, nsUGT and NtUGT genes and enzyme activity determination
The glycosyltransferase VvUGT, lbUGT, nsUGT and NtUGT are obtained by using a gene mining technology, the amino acid sequence of the VvUGT is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 2; the LbUGT has an amino acid sequence shown as SEQ ID NO. 3, and a nucleotide sequence shown as SEQ ID NO. 4; the amino acid sequence of the NsUGT is shown as SEQ ID NO. 5, and the nucleotide sequence is shown as SEQ ID NO. 6; the amino acid sequence of the NtUGT is shown as SEQ ID NO. 7, and the nucleotide sequence is shown as SEQ ID NO. 8.
The two ends of glycosyltransferase VvUGT, lbUGT, nsUGT and NtUGT are added with NcoI and EcoRI restriction sites respectively, the gene fragments are inserted into the corresponding restriction sites of an expression vector pRSFDue-1 through double restriction and connection, and placed under the control of a T7 promoter to respectively construct recombinant plasmids pRSF-VvUGT, pRSF-LbUGT, pRSF-NtUGT and pRSF-NtUGT for expressing glycosyltransferase genes. The recombinant plasmids were transformed into E.coli BL21 (DE 3), and engineering bacteria E.coli BL21 (pRSF-VvUGT), E.coli BL21 (pRSF-LbUGT), E.coli BL21 (pRSF-NsUGT) and E.coli BL21 (pRSF-NtUGT) were constructed, respectively. SDSP-PAGE analysis is shown in FIG. 1.
Engineering bacteria E.coli BL21 (pRSF-VvUGT), E.coli BL21 (pRSF-LbUGT), E.coli BL21 (pRSF-NtUGT) and E.coli BL21 (pRSF-NtUGT) were picked up respectively and cultured overnight in LB medium (tryptone 10g/L, sodium chloride 10g/L, yeast extract 5 g/L) containing 50. Mu.g/mL kanamycin at 37 ℃. When the OD600 value of the culture medium reaches 0.5-0.6, the culture medium is inoculated into 100mLLB culture medium containing corresponding antibiotics according to the inoculation amount of 2 percent, 0.1mM IPTG is added after the culture medium is cooled, after 36 hours of induced expression, bacterial liquid is centrifuged at 6500rpm at 4 ℃ for 3 minutes, and the supernatant is discarded and deposited for later use. The bacterial precipitate is taken, washed twice by potassium phosphate buffer (100 mmol/L, pH 7.2), the washed precipitate is suspended in the potassium phosphate buffer, cells are subjected to ultrasonic disruption treatment (power 300W, ultrasonic treatment for 1s and intermittent treatment for 2s and total time is 10 min), the disrupted suspension is centrifuged at 8000rpm at 4 ℃ for 20min, and the supernatant is taken to be crude enzyme solution.
The enzyme activity of glycosyltransferase was measured as 3mL of a reaction system containing 50mM potassium phosphate buffer at pH 7.2, 2mM substrate (rubusoside, stevioside, rebaudioside A, rebaudioside E, rebaudioside D, rebaudioside M) and 3mM UDPG,1mg VvUGT,LbUGT,NsUGT or NtUGT crude protein, and reacted at 37 ℃. High Performance Liquid Chromatography (HPLC) detection, and enzyme activity was calculated. The specific data of enzyme activity are shown in Table 1.
Table 1.Vvugt, lbUGT, nsUGT, and NtUGT related enzyme activity data.
Example 2: protein purification of glycosyltransferase VvUGT, lbUGT, nsUGT and NtUGT
The purification step was carried out at 4 ℃. The engineering bacteria E.coli BL21 (pRSF-VvUGT), E.coli BL21 (pRSF-LbUGT), E.coli BL21 (pRSF-NsUGT) and E.coli BL21 (pRSF-NtUGT) of example 1 were individually taken for bacterial precipitation, resuspended in appropriate lysis buffers (including 500mM NaCl and 10% glycerol and 20mM sodium phosphate buffer, pH 8.0) and the cells were subsequently sonicated. The crushed suspension is centrifuged twice at 6665g for 15min each time. Filtering the supernatant with 0.2 μm pore size water-based fiber membrane, and purifying egg with 6 His tag with high affinity nickel resin FF pre-packed column (GenScript, nanjing, china) White. Eluting recombinant protein from the column with gradient-ascending imidazole gradient (eluting with imidazole concentration gradients of 40mM, 80mM, 120mM, 160mM and 200 mM), combining components with glycosyltransferase activity, and eluting with membrane of Mm Ltracel-30The mixture was concentrated in a MLtra-15 centrifugal filter (Ireland Merck Mibo Co., ltd.) while the buffer was changed to 50mM HEPES-NaOH (pH 7.0). SDS-PAGE analysis of purified target protein VvUGT, lbUGT, nsUGT and NtUGT is shown in FIG. 2.
Example 3: determination of the optimum temperature for an enzyme-catalyzed reaction
To a pH 7.250mM sodium phosphate buffer reaction system was added 3mM UDP, 2mM rubusoside, 10. Mu.g of the VvUGT, lbUGT, nsUGT or NtUGT single protein purified in example 2 to a final volume of 300. Mu.L. The enzyme activities were measured under the reaction conditions of 25, 30, 35, 40, 45, 50 and 55℃respectively. The reaction was stopped by incubating in a water bath at 95℃for 5 min.
Wherein the highest enzyme activity is set as 100%, and the relative enzyme activity values at other reaction temperatures are calculated in sequence. According to the results, optimum temperature curves (FIG. 3) were drawn, and the optimum temperatures of VvUGT, lbUGT, nsUGT and NtUGT were 45 ℃,50 ℃,50 ℃ and 55 ℃, respectively.
Example 4: determination of the optimum pH for an enzyme-catalyzed reaction
Buffers of different pH were formulated as follows:
pH 4.0-5.0：100mM disodiumhydrogen-citric acidbuffer
pH 6.0-8.0：100mM sodiumphosphatebuffer
pH 9.0-10.0：100mM glycine buffer
3mM UDP, 2mM rubusoside, 10. Mu.g of the single protein VvUGT, lbUGT, nsUGT or NtUGT purified in example 2 was added sequentially to the reaction system of pH4, 5, 6, 7, 8, 9, 10 buffer to a final volume of 300. Mu.L, reacted at 37℃and incubated at 95℃in a water bath for 5min to terminate the reaction. HPLC detection and calculation of enzyme activity.
Wherein the highest enzyme activity is set as 100%, and the relative enzyme activity values under other reaction pH values are calculated in sequence. The optimum pH was plotted according to the results (FIG. 4), and both VvUGT, lbUGT, nsUGT and NtUGT optimum pH were pH8.
Example 5: measurement of the thermal stability of an enzyme-catalyzed reaction
To the reaction system were added 3mM UDPG,2mM stevioside, pH 7.250mM sodium phosphate buffer, and 10. Mu.g of the VvUGT, lbUGT, nsUGT or NtUGT single protein purified in example 2 to a final volume of 300. Mu.L. Incubation for 2h at 30, 35, 40, 45, 50, 55 and 60 ℃ in the absence of substrate, respectively, followed by addition of substrate and reaction at 37 ℃ and incubation for 5min at 95 ℃ in a water bath was terminated. HPLC detection and calculation of enzyme activity.
The highest enzyme activity was set to 100%, and the relative enzyme activity values in other reactions were calculated in order, whereby a thermostable curve was drawn (fig. 5). Both VvUGT, lbUGT, nsUGT and NtUGT can retain more than 95% of their enzyme activity in 2 hours at temperatures below 35 ℃ in the absence of substrate.
Real-time example 6: determination of the pH stability of an enzyme-catalyzed reaction
Buffers of different pH were formulated as in example 4.
The purified VvUGT, lbUGT, nsUGT or NtUGT single proteins of example 2 were incubated at pH 4, 5, 6, 7, 8, 9, 10 for 2h, respectively. Subsequently, 3mM UDPG,2mM stevioside, and 10. Mu.g of incubated LbUGT were sequentially added to the reaction system, the reaction temperature was 37℃and the reaction volume was 300. Mu.L. The reaction was stopped by incubation in a water bath at 95℃for 5 min. HPLC detection and calculation of enzyme activity.
The highest enzyme activity was set to 100%, and the relative values of other enzyme activities were calculated in order, whereby a pH-stable curve was drawn (FIG. 6). The VvUGT, lbUGT and NsUGT still have more than 75 percent of activity, and the NtUGT still has more than 80 percent of activity after being incubated for 2 hours under the condition of pH 7-9.
Example 7: enzyme catalytic kinetic parameter determination
Different substrates (rubusoside, stevioside, rebaudioside A, E and D) are respectively prepared into different concentrations (0.1, 0.2, 0.4, 0.8, 1.2, 2.0, 3.0 and 5.0 mM), and a 1-10mM UDPG,pH 7.250mM sodium phosphate buffer reaction system and 10 mu g of the purified VvUGT, lbUGT, nsUGT or NtUGT single protein in the embodiment 2 are sequentially added into the reaction system, wherein the reaction temperature is 37 ℃, and the reaction volume is 300 mu L. Samples at each concentration were taken in 3 replicates and averaged. And drawing a standard curve by taking the standard concentration as an abscissa and the absorbance as an ordinate, and carrying out linear regression through Origin 2021 to obtain a standard equation. The enzymatic kinetics parameters of VvUGT, lbUGT, nsUGT and NtUGT-catalyzed rubusoside, stevioside, rebaudioside A, E, and D were obtained, respectively, and the detailed data are shown in table 2.
TABLE 2VvUGT, lbUGT, nsUGT and NtUGT related kinetic parameters
Example 8: divalent Metal ion influence of enzyme catalyzed reactions
Preparation of 3mM different divalent Metal ions (EDTA, zn) 2+ ,Mg 2+ ,Ca 2+ ,Ni 2+ ,Mn 2+ ,Cu 2+ And Ba 2+ ) Is a solution of (a) and (b). 3mM of different divalent metal ions, 3mM of UDPG,2mM of stevioside and 10. Mu.g of the single protein of VvUGT, lbUGT, nsUGT or NtUGT purified in example 2 are sequentially added into the reaction system, wherein the reaction temperature is 37 ℃ and the reaction volume is 300. Mu.L. The reaction was stopped by incubation in a water bath at 95℃for 5 min. HPLC detection and calculation of enzyme activity.
According to the enzyme activity detection without adding metal ions as a blank control, the enzyme activity is set to be 100%, and other enzyme activity relative values are calculated in sequence. Preliminary inferences were made that the addition of divalent metal ions reduced the enzymatic activity of VvUGT, lbUGT, nsUGT or NtUGT by 10-30%. Wherein Zn is 2+ ,Ni 2+ And Cu 2+ Directly inactivating VvUGT, lbUGT, nsUGT or NtUGT. This is similar to most GT-B folded glycosyltransferases, and is a non-metal dependent enzyme.
Example 9: construction of engineering bacterium of colibacillus expressing double enzyme genes
The recombinant plasmid pRSF-StSUS for expressing potato-derived sucrose synthase gene is constructed by adding NcoI and EcoRI cleavage sites at both ends of potato-derived sucrose synthase gene StSUS, inserting the gene fragment into the corresponding cleavage site of expression vector pRSFDue-1 by double cleavage and ligation, and placing under the control of T7 promoter.
By Nanjing Jinsri Biotechnology Co.LtdThe kit is used for cloning target glycosyltransferase genes onto pRSF-StSUS plasmid vectors between NdeI and XhoI cleavage sites to obtain construction double-enzyme recombinant expression plasmids pRSF-VvUGT-StSUS, pRSF-LbUGT-StSUS, pRSF-NsUGT-StSUS and pRSF-NtUGT-StSUS. The double-enzyme recombinant expression plasmid is transformed into E.coli BL21 (DE 3) to obtain engineering bacteria E.coli BL21 (pRSF-VvUGT-StSUS), E.coli BL21 (pRSF-LbUGT-StSUS), E.coli BL21 (pRSF-NtUGT-StSUS) and E.coli BL21 (pRSF-NtUGT-StSUS) for co-expressing double enzymes respectively.
Example 10: coli engineering bacteria fermentation enzyme producing method for expressing double enzyme genes
Engineering bacteria E.coli BL21 (pRSF-LbUGT-StSUS) containing expressed double enzyme genes constructed in example 9 were picked up and cultured overnight at 37℃with shaking in LB medium (tryptone 10g/L, sodium chloride 10g/L, yeast extract 5 g/L) containing 50. Mu.g/mL kanamycin. When the OD600 value of the culture medium reaches 0.5-0.6, the culture medium is inoculated into 100mL of LB culture medium containing corresponding antibiotics according to the inoculation amount of 2%, 0.1mM IPTG is added after cooling, after 36h of induced expression, bacterial liquid is centrifuged at 6500rpm at 4 ℃ for 3min, the supernatant is discarded, and the sediment is reserved.
The bacterial precipitate is taken, washed twice by potassium phosphate buffer (100 mmol/L, pH 7.2), the washed precipitate is suspended in the potassium phosphate buffer, cells are subjected to ultrasonic treatment (power 300W, ultrasonic treatment for 1s and intermittent treatment for 2s and total time is 10 min), the crushed suspension is centrifuged at 8000rpm at 4 ℃ for 20min, and the supernatant is taken to be crude enzyme solution.
Example 11:10G/L rubusoside as substrate to synthesize Rub1G and Rub2G
10g/L rubusoside, 50g/L sucrose, a proper amount of E.coli BL21 (pRSF-VvUGT-StSUS), E.coli BL21 (pRSF-LbUGT-StSUS) or E.coli BL21 (pRSF-NtUGT-StSUS) crude enzyme solution (10 mg/mL total protein) and pH 7.250mM sodium phosphate buffer solution described in example 10 are sequentially added into a reaction system to a final volume of 20mL, and the mixture is placed in a shaking table at 37 ℃ for stirring reaction at 200rpm for 36h after uniform mixing. Inactivating in 95deg.C water bath for 10min, centrifuging at 12,000rpm for 1min, collecting supernatant, filtering with 0.45 μm water-based filter membrane, and detecting with high performance liquid chromatography. The 36h catalytic specification data are shown in Table 3. In the production of rubusoside as a substrate and a product Rub1G, rub G, the yield of VvUGT is highest.
The chromatographic conditions for HPLC are as follows, and the detection methods for the products in the following examples are identical:
chromatographic column: agilentTC-C18 column (250 mm. Times.4.6 mm; netherlands); mobile phase a: acetonitrile + 1%o formic acid, mobile phase B: water + 1%o formic acid; flow rate: 1mL min-1; column temperature: 55 ℃; detection wavelength: 210nm, detection time: 30min. Detection conditions: 25-47% of phase A (0-15 min), 47-100% of phase A (15-20 min), 100-25% of phase A (25-30 min).
Example 12: synthesis of St1G and St2G with 10G/L stevioside as substrate
10g/L stevioside, 50g/L sucrose and a proper amount of E.coli BL21 (pRSF-VvUGT-StSUS), E.coli BL21 (pRSF-LbUGT-StSUS) or E.coli BL21 (pRSF-NtUGT-StSUS) crude enzyme solution (10 mg/mL total protein) and pH 7.250mM sodium phosphate buffer solution described in example 10 are sequentially added into a reaction system to a final volume of 20mL, and the mixture is placed in a shaking table at 37 ℃ and stirred at 200rpm for reaction for 24 hours. Inactivating in 95deg.C water bath for 10min, centrifuging at 12,000rpm for 1min, collecting supernatant, filtering with 0.45 μm water-based filter membrane, and detecting with high performance liquid chromatography. The 36h catalytic specification data are shown in Table 3. Of the production of the product St1G, st G using St as substrate, the yield of NSUGT was highest.
Example 13: synthesis of RA1G and RM2 with 10G/L rebaudioside A as substrate
10g/L rebaudioside A,50g/L sucrose and a proper amount of E.coli BL21 (pRSF-VvUGT-StSUS), E.coli BL21 (pRSF-LbUGT-StSUS), E.coli BL21 (pRSF-NsUGT-StSUS) or E.coli BL21 (pRSF-NtUGT-StSUS) crude enzyme solution (10 mg/mL total protein) and pH 7.250mM sodium phosphate buffer solution according to embodiment 10 are sequentially added into a reaction system to a final volume of 20mL, and the mixture is placed in a shaking table at 37 ℃ for stirring reaction at 200rpm for 36h. Inactivating in 95deg.C water bath for 10min, centrifuging at 12,000rpm for 1min, collecting supernatant, filtering with 0.45 μm water-based filter membrane, and detecting with high performance liquid chromatography. The 36h catalytic specification data are shown in Table 3. Of the production of the product RA1G, RM2 with rebaudioside a as a substrate, lbUGT yields are highest.
Example 14: synthesis of St2G with 10G/L rebaudioside E as substrate
10g/L rebaudioside E and 50g/L sucrose are sequentially added into a reaction system, a proper amount of E.coli BL21 (pRSF-VvUGT-StSUS) described in embodiment 10, E.coli BL21 (pRSF-LbUGT-StSUS) or E.coli BL21 (pRSF-NtUGT-StSUS) crude enzyme solution (10 mg/mL total protein) and pH 7.250mM sodium phosphate buffer solution are added to a final volume of 20mL, and the mixture is placed in a shaking table at 37 ℃ for stirring reaction at 200rpm for 36h after uniform mixing. Inactivating in 95deg.C water bath for 10min, centrifuging at 12,000rpm for 1min, collecting supernatant, filtering with 0.45 μm water-based filter membrane, and detecting with high performance liquid chromatography. The 36h catalytic specification data are shown in Table 3. Among the production of the product St2G with rebaudioside E as a substrate, the NSUGT yield is highest.
Example 15: synthesis of RM2 using 5g/L rebaudioside D as substrate
Sequentially adding 5g/L rebaudioside D,50g/L sucrose and a proper amount of E.coli BL21 (pRSF-VvUGT-StSUS), E.coli BL21 (pRSF-LbUGT-StSUS) or E.coli BL21 (pRSF-NtUGT-StSUS) crude enzyme solution (10 mg/mL total protein) and pH 7.250mM sodium phosphate buffer solution according to embodiment 10 to a final volume of 20mL, uniformly mixing, and placing in a shaking table at 37 ℃ for stirring reaction at 200rpm for 36h. Inactivating in 95deg.C water bath for 10min, centrifuging at 12,000rpm for 1min, collecting supernatant, filtering with 0.45 μm water-based filter membrane, and detecting with high performance liquid chromatography. The 36h catalytic specification data are shown in Table 3. Among the production of the product St2G with rebaudioside D as a substrate, the NtUGT yield is highest.
Example 16: synthesis of RM1G using 5G/L rebaudioside M as substrate
Sequentially adding 5g/L rebaudioside M and 50g/L sucrose into a reaction system, mixing a proper amount of E.coli BL21 (pRSF-VvUGT-StSUS) described in embodiment 10, E.coli BL21 (pRSF-LbUGT-StSUS) or E.coli BL21 (pRSF-NtUGT-StSUS) crude enzyme solution (10 mg/mL total protein) and pH 7.250mM sodium phosphate buffer to a final volume of 20mL, and placing the mixture in a shaking table at 37 ℃ for stirring reaction at 200rpm for 36h. Inactivating in 95deg.C water bath for 10min, centrifuging at 12,000rpm for 1min, collecting supernatant, filtering with 0.45 μm water-based filter membrane, and detecting with high performance liquid chromatography. The 36h catalytic specification data are shown in Table 3. In the production of the product RM1G with rebaudioside M as a substrate, the LbUGT yield is highest.
TABLE 3 conversion of steviol glycosides and yield of glycosylated derivatives thereof by 3VvUGT, lbUGT, nsUGT, ntUGT
Example 17: purification and structural characterization of glycosylated derivatives
Adsorbing the glycosylation product by macroporous resin, eluting with 30-90% ethanol water solution, concentrating by rotary steaming instrument, and crystallizing at 4deg.C to obtain Rub1G, rub1G, st1G, st G, RA1G, RM2 and RM1G with purity of above 95%. Verification of glycosidic bond species and by detection of methylation products 1 H NMR, 13 C NMR，Distortionless Enhancement Polarization Transfer(DEPT), 1 H- 1 H correlation spectroscopy(COSY), 1 H- 13 C Heteronuclear Single Quantum Coherence(HSQC), 1 H- 13 C Heteronuclear Multiple Bond Correlation(HMBC), 1 H- 1 H Nuclear Overhauser Effect Spectroscopy (NOESY) and Total Correlation Spectroscopy (TOCSY), the glycosylated derivatives were characterized structurally.
The methylation product detection results are shown in Table 4, and the partial NMR analysis data are shown in Table 5. It was determined that Rub1G was the first glycosyl linkage 1,6-O- β -D-glucoside attached at C19 of rubusoside and Rub2G was the first glycosyl linkage 1,2-O- β -D-glucoside attached at C19 of Rub 1G; st1G is the first glycosyl linkage 1,6-O- β -D-glucoside attached at C19 of stevioside, st2G is the first glycosyl linkage 1,2-O- β -D-glucoside attached at C19 of St 1G; RA1G is the first glycosyl linkage 1,6-O- β -D-glucoside attached at C19 of rebaudioside A, RM2 is the first glycosyl linkage 1,2-O- β -D-glucoside attached at C19 of Rub 1G; st2G is the first glucosyl linkage 1, 6-O-beta-D-glucoside attached at the C19 of RE; RM2 is the first glucosyl linkage 1, 6-O-beta-D-glucoside attached at the C19 of RD; RM1G is the first glucosyl group attached to 1, 6-O-. Beta. -D-glucoside at the C19 linkage of rebaudioside M. Rub1G, rub1G, st1G, st2G, RA1G, RM2 and RM1G structures are shown in the following formulas:
Glycosylated derivative of partial stevioside compound
TABLE 4 detection results of methylation products of Rub1G and Rub2G mixtures
* Relative molar amount = peak area/molecular weight;
* Relative molar ratio (%) =relative molar amount/sum of relative molar amounts of the components
TABLE 5 analysis of one-and two-dimensional NMR structural characterization data for Rub2G
Example 18: sweetness and taste identification of derivatives of LbUGT glycosylated stevioside compounds
Sensory evaluation was performed on purified Rub1G, rub1G, st1G, st G, RA1G, RM and RM1G and substrates (rubusoside, stevioside, rebaudioside a, rebaudioside D, rebaudioside E, rebaudioside M) with rubusoside as control. The taste quality and sweetness intensity were evaluated in 20 volunteers, including 10 females and 10 males. Rub1G, rub1G, st1G, st2G, RA1G, RM2, RM1G, rubusoside, stevioside, rebaudioside A, rebaudioside D, rebaudioside E, and rebaudioside M were dissolved in water, respectively, to give a 5% (w/v) solution, which was then diluted 60-510×, with a 30×interval. Volunteers tasted each sample and compared to 5% sucrose to estimate sweetness. A subjective equivalence Point (PSE) was calculated to represent the relative sweetness intensity of each sample [8]. The quality of mouthfeel relative to rubusoside was evaluated as "excellent (+4), fairly good (+3), better (+2), slightly better (+1), the same (0) or worse (-1)".
As shown in Table 6, the LbUGT glycosylated derivative has about 10-20% increased sweetness relative to its substrate, 1-2 level increased taste, greatly reduced bitter and astringent tastes, and improved quality of the sweetener.
TABLE 6 sensory evaluation of steviol glycosides and their glycosylated derivatives
a The relative sweetness was set to 1 for 5% (mass/volume) sucrose aqueous solution.
b Taste grade setting the taste grade of rubusoside to 0, the taste quality relative to rubusoside was evaluated as "excellent (+4), fairly good (+3), better (+2), slightly better (+1), the same (0) or worse (-1)".
Example 18: identification of the stability of derivatives of glycosylated steviol glycosides
The storage stability of VvUGT, lbUGT, nsUGT and NtUGT glycosylated steviol glycoside derivatives in powder form in dry and cool environment was examined. Purified Rub1G, rub1G, st1G, st G, RA1G, RM2 and RM1G were stored as powders in dry, cool environment for one year. After the storage, 1mg/mL of each aqueous solution was prepared and HPLC detection was performed.
The pH stability of derivatives of VvUGT, lbUGT, nsUGT and NtUGT glycosylated steviol glycosides at pH2.0-7.0 was examined. 1 mL of each buffer (100 mM glycine-HCl buffer, pH2.0 and 3.0;100mM disodium hydrogen citrate buffer, pH 4.0, 5.0, 6.0;100mM sodium phosphate buffer, pH 7.0) containing 1mg/mL of Rub1G, rub1G, st1G, st2G, RA1G, RM2 and RM1G, respectively, at different acidity pH were incubated for 7 days at room temperature.
Heat treatment stability of glycosylated steviol glycoside derivatives 1mL of aqueous solution containing 1mg/mL of Rub1G, rub1G, st1G, st G, RA1G, RM2 or RM1G respectively were tested in a 95 ℃ water bath for 30 min.
The results show that the derivatives of the VvUGT, lbUGT, nsUGT and NtUGT glycosylated stevioside compounds have good stability. After being stored in a powder form for one year, the residual quantity reaches more than 98 percent. The residual amounts were all 98% or more after 7 days of incubation in aqueous ph2-7, and the specific residual amount data are shown in Table 7. Heat treating in water bath at 95deg.C for 30min, and the residual amount is 95-98%. The derivatives of the glycosylated stevioside compounds can ensure structural integrity under the conditions of long-term storage, addition to beverages and heat sterilization treatment.
TABLE 7 acidic Water environmental stability of glycosylated derivatives
Sequence listing
<110> university of Nanjing Industrial science
<120> method for preparing glycosylated stevioside compound and derivative thereof by enzymatic method
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 446
<212> PRT
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
Met Asp Ala Arg Gln Ser Asp Gly Ile Ser Val Leu Met Phe Pro Trp
1 5 10 15
Leu Ala His Gly His Ile Ser Pro Phe Leu Gln Leu Ala Lys Lys Leu
20 25 30
Ser Lys Arg Asn Phe Ser Ile Tyr Phe Cys Ser Thr Pro Val Asn Leu
35 40 45
Asp Pro Ile Lys Gly Lys Leu Ser Glu Ser Tyr Ser Leu Ser Ile Gln
50 55 60
Leu Val Lys Leu His Leu Pro Ser Leu Pro Glu Leu Pro Pro Gln Tyr
65 70 75 80
His Thr Thr Asn Gly Leu Pro Pro His Leu Met Pro Thr Leu Lys Met
85 90 95
Ala Phe Asp Met Ala Ser Pro Asn Phe Ser Asn Ile Leu Lys Thr Leu
100 105 110
His Pro Asp Leu Leu Ile Tyr Asp Phe Leu Gln Pro Trp Ala Pro Ala
115 120 125
Ala Ala Ser Ser Leu Asn Ile Pro Ala Val Gln Phe Leu Ser Thr Gly
130 135 140
Ala Thr Leu Gln Ser Phe Leu Ala His Arg His Arg Lys Pro Gly Ile
145 150 155 160
Glu Phe Pro Phe Gln Glu Ile His Leu Pro Asp Tyr Glu Ile Gly Arg
165 170 175
Leu Asn Arg Phe Leu Glu Pro Ser Ala Gly Arg Ile Ser Asp Arg Asp
180 185 190
Arg Ala Asn Gln Cys Leu Glu Arg Ser Ser Arg Phe Ser Leu Ile Lys
195 200 205
Thr Phe Arg Glu Ile Glu Ala Lys Tyr Leu Asp Tyr Val Ser Asp Leu
210 215 220
Thr Lys Lys Lys Met Val Thr Val Gly Pro Leu Leu Gln Asp Pro Glu
225 230 235 240
Asp Glu Asp Glu Ala Thr Asp Ile Val Glu Trp Leu Asn Lys Lys Cys
245 250 255
Glu Ala Ser Ala Val Phe Val Ser Phe Gly Ser Glu Tyr Phe Val Ser
260 265 270
Lys Glu Glu Met Glu Glu Ile Ala Tyr Gly Leu Glu Leu Ser Asn Val
275 280 285
Asp Phe Ile Trp Val Val Arg Phe Pro Met Gly Glu Lys Ile Arg Leu
290 295 300
Glu Asp Ala Leu Pro Pro Gly Phe Leu His Arg Leu Gly Asp Arg Gly
305 310 315 320
Met Val Val Glu Gly Trp Ala Pro Gln Arg Lys Ile Leu Gly His Ser
325 330 335
Ser Ile Gly Gly Phe Val Ser His Cys Gly Trp Ser Ser Val Met Glu
340 345 350
Gly Met Lys Phe Gly Val Pro Ile Ile Ala Met Pro Met His Leu Asp
355 360 365
Gln Pro Ile Asn Ala Lys Leu Val Glu Ala Val Gly Val Gly Arg Glu
370 375 380
Val Lys Arg Asp Glu Asn Arg Lys Leu Glu Arg Glu Glu Ile Ala Lys
385 390 395 400
Val Ile Lys Glu Val Val Gly Glu Lys Asn Gly Glu Asn Val Arg Arg
405 410 415
Lys Ala Arg Glu Leu Ser Glu Thr Leu Arg Lys Lys Gly Asp Glu Glu
420 425 430
Ile Asp Val Val Val Glu Glu Leu Lys Gln Leu Cys Ser Tyr
435 440 445
<210> 2
<211> 1341
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
atggacgcta ggcaaagtga tggaatatca gtcctgatgt ttccgtggct ggcgcacggc 60
cacattagcc cgttcttgca actggcgaag aagttatcga aacgaaattt ctctatctat 120
ttctgctcca ctccggtgaa tctggacccg atcaagggta aattgtccga aagctacagc 180
ctgagcattc agctggttaa actccacctg ccttcccttc cggaactgcc accgcaatat 240
cacaccacaa atggcctgcc accgcattta atgccgacgc tgaaaatggc gtttgatatg 300
gcgtcaccga atttctccaa catcttgaag accctacacc cggacctgct tatctacgac 360
ttcctgcagc cgtgggctcc ggcggccgcg tcaagcttga acattccggc tgtgcaattt 420
ctgagcaccg gtgcgaccct gcaaagcttt ctggcacatc gccatagaaa gccgggtatt 480
gagttcccgt ttcaggagat ccaccttccg gattacgaga ttggtcgtct gaaccgtttt 540
ctggagccgt ccgctggccg tatttcggac cgtgaccgtg caaatcagtg tctggagcgc 600
agctctcgtt tcagcctgat taagacgttt cgtgaaatcg aagcgaaata cctggactat 660
gttagcgatc tgaccaaaaa gaagatggtt accgttggtc cgctgctgca agatccagaa 720
gatgaggacg aagcgaccga catcgtggag tggctaaaca aaaagtgcga agctagtgcc 780
gtttttgtgt cttttggcag cgagtacttc gtgtccaaag aagagatgga ggaaatcgcg 840
tatggactgg aattgtctaa cgtggatttc atttgggttg tccgcttccc gatgggtgag 900
aaaatccgtc tggaagatgc attgccgccg ggttttctgc accgcttggg cgaccgcggt 960
atggtggtcg aggggtgggc gccacagcgt aagattctgg gtcacagctc catcggcggt 1020
ttcgtaagcc attgcggttg gagcagcgtc atggagggta tgaaattcgg cgttccgatt 1080
atcgccatgc cgatgcattt ggaccagcct atcaacgcca agttggttga ggcggtcggc 1140
gttggtcgcg aagttaaacg cgatgaaaac cgtaaattgg agcgtgagga aatcgcaaag 1200
gtgattaaag aggttgttgg tgaaaagaac ggcgagaacg ttcgtcgtaa agcacgtgag 1260
ctgtctgaaa ccttacgtaa aaagggcgac gaggagatcg atgtggtggt ggaagagctc 1320
aagcagctgt gtagctacta a 1341
<210> 3
<211> 454
<212> PRT
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
Met Gly Thr Glu Val Thr Val His Lys Asn Thr Leu Arg Val Leu Met
1 5 10 15
Phe Pro Trp Leu Ala Tyr Gly His Ile Ser Pro Phe Leu Asn Val Ala
20 25 30
Lys Lys Leu Val Asp Arg Gly Phe Leu Ile Tyr Leu Cys Ser Thr Ala
35 40 45
Ile Asn Leu Lys Ser Thr Ile Lys Lys Ile Pro Glu Lys Tyr Ser Asp
50 55 60
Ser Ile Gln Leu Ile Glu Leu His Leu Pro Glu Leu Pro Glu Leu Pro
65 70 75 80
Pro His Tyr His Thr Thr Asn Gly Leu Pro Pro His Leu Asn His Thr
85 90 95
Leu Gln Lys Ala Leu Lys Met Ser Lys Pro Asn Phe Ser Lys Ile Leu
100 105 110
Gln Asn Leu Lys Pro Asp Leu Val Ile Tyr Asp Leu Leu Gln Gln Trp
115 120 125
Ala Glu Gly Val Ala Asn Glu Gln Asn Ile Pro Ala Val Lys Leu Leu
130 135 140
Thr Ser Gly Ala Ala Val Leu Ser Tyr Phe Phe Asn Leu Val Lys Lys
145 150 155 160
Pro Gly Val Glu Phe Pro Phe Pro Ala Ile Tyr Leu Arg Lys Asn Glu
165 170 175
Leu Glu Lys Met Ser Glu Leu Leu Ala Gln Ser Ala Lys Asp Lys Glu
180 185 190
Pro Asp Gly Val Asp Pro Phe Ala Asp Gly Asn Met Gln Val Met Leu
195 200 205
Met Ser Thr Ser Arg Ile Ile Glu Ala Lys Tyr Ile Asp Tyr Phe Ser
210 215 220
Gly Leu Ser Asn Trp Lys Val Val Pro Val Gly Pro Pro Val Gln Asp
225 230 235 240
Pro Ile Ala Asp Asp Ala Asp Glu Met Glu Leu Ile Asp Trp Leu Gly
245 250 255
Lys Lys Asp Glu Asn Ser Thr Val Phe Val Ser Phe Gly Ser Glu Tyr
260 265 270
Phe Leu Ser Lys Glu Asp Arg Glu Glu Ile Ala Phe Gly Leu Glu Leu
275 280 285
Ser Asn Val Asn Phe Ile Trp Val Ala Arg Phe Pro Lys Gly Glu Glu
290 295 300
Gln Asn Leu Glu Asp Ala Leu Pro Lys Gly Phe Leu Glu Arg Ile Gly
305 310 315 320
Asp Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln Pro Arg Ile Leu
325 330 335
Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys Gly Trp Asn Ser
340 345 350
Val Met Glu Ser Val Asp Phe Gly Val Pro Ile Ile Ala Met Pro Ile
355 360 365
His Leu Asp Gln Pro Met Asn Ala Arg Leu Ile Val Glu Leu Gly Val
370 375 380
Ala Val Glu Ile Val Arg Asp Asp Tyr Gly Lys Ile His Arg Glu Glu
385 390 395 400
Ile Ala Glu Ile Leu Lys Asp Val Ile Ala Gly Lys Ser Gly Glu Asn
405 410 415
Leu Lys Ala Lys Met Arg Asp Ile Ser Lys Asn Leu Lys Ser Ile Arg
420 425 430
Asp Glu Glu Met Asp Thr Ala Ala Glu Glu Leu Ile Gln Leu Cys Lys
435 440 445
Asn Ser Pro Lys Leu Lys
450
<210> 4
<211> 1365
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
atgggcaccg aagtgaccgt tcataaaaac accctgcgcg tgctgatgtt tccgtggctg 60
gcgtacggtc atattagccc gtttttgaac gtggccaaga agctggtcga ccgcggtttt 120
ctgatctatc tgtgctcaac cgcgattaat ctgaagagca ccattaaaaa gatcccggaa 180
aaatacagcg atagcattca gttgattgag ttgcacttgc cggagctgcc ggagttgccg 240
ccacactacc ataccaccaa tggtctccca ccacacctta accacactct gcaaaaggct 300
ctgaaaatga gcaagccgaa tttcagcaag atcctgcaaa acttgaagcc ggatctcgtc 360
atctatgatc tgctgcagca gtgggcagaa ggtgtcgcta atgaacaaaa catcccggcg 420
gttaaattgc taacgagcgg cgcggcagtg ctctcctatt tcttcaatct ggttaaaaaa 480
ccgggtgtgg aatttccgtt tccggcgatt tacctgcgca aaaacgaact agagaagatg 540
tctgagttgc tggcgcagtc tgcaaaagac aaagaaccag atggtgttga cccgttcgcc 600
gatggtaata tgcaggttat gctgatgtcg accagccgta ttatcgaggc gaagtacatt 660
gattacttca gcggtctgtc taactggaaa gttgttccgg tgggtccgcc cgtgcaggac 720
ccgattgcgg atgatgcgga cgagatggaa ttgatcgact ggttgggcaa gaaggacgag 780
aacagcacgg tttttgttag ctttggttcg gagtatttcc tgagcaaaga ggacagagaa 840
gaaatcgcgt ttggcttgga actttccaac gttaatttca tctgggttgc gcgttttccg 900
aaaggcgagg agcaaaacct ggaggacgcg ctgcctaagg gattcctgga gcgcatcggc 960
gatcgtggcc gtgttctgga taaattcgca ccgcagccgc gtatcctgaa tcacccgagc 1020
acgggtggtt tcatcagcca ttgtggttgg aacagcgtga tggaaagcgt ggacttcggc 1080
gtaccgatca tcgccatgcc gattcacctg gaccagccta tgaacgcccg tctgatcgta 1140
gagctcggcg tggcggtgga gattgtgcgt gacgactacg gcaaaatcca tcgtgaagag 1200
atcgcggaga tcctgaagga cgttattgct ggcaagagcg gtgaaaactt gaaagcgaaa 1260
atgcgtgata tttccaagaa cctgaaatcc attcgcgacg aagaaatgga taccgctgct 1320
gaagagctga ttcaactgtg caaaaactcg ccgaagctga agtaa 1365
<210> 5
<211> 481
<212> PRT
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 5
Met Glu Ser Ile Pro Pro Pro Ser Met Thr Asn Asp Gly Ser Lys Leu
1 5 10 15
His Ile Val Ile Phe Pro Trp Leu Ala Phe Gly His Met Ile Pro Phe
20 25 30
Leu Glu Val Ala Lys Leu Ile Ala Gln Lys Gly His Lys Val Ser Phe
35 40 45
Val Ser Ser Pro Arg Asn Ile Asp Arg Leu Pro Lys Leu Pro Pro Asn
50 55 60
Leu Thr Pro Leu Leu Asn Phe Val Lys Phe Pro Leu Pro Thr Met Asp
65 70 75 80
Asn Leu Pro Glu Asn Ala Glu Ala Thr Thr Asp Leu Pro Tyr His Glu
85 90 95
Val Lys Tyr Leu Lys Ile Ala Tyr Asp Leu Leu Gln Gln Pro Met Thr
100 105 110
Gln Phe Leu Gln Ser Ser Ala Pro Asp Trp Val Ile Tyr Asp Phe Ala
115 120 125
Ser Tyr Trp Leu Gly Pro Ile Ala Ala Glu Leu Gly Ile Ser Ser Leu
130 135 140
Tyr Phe Cys Ile Phe Ile Ala Ala Ala Met Cys Tyr Cys Gly Pro Val
145 150 155 160
Ser Ser Leu Met Gly Thr Gln Asp Ala Arg Ser Lys Pro Glu Asp Phe
165 170 175
Thr Val Pro Pro Lys Trp Ile Pro Phe Lys Ser Thr Val Ala Phe Arg
180 185 190
Leu Phe Glu Ile Leu Arg Asn Leu Asp Asp Val Thr Gly Asp Asp Glu
195 200 205
Ser Ile Pro Val Thr Tyr Arg Ser Ala Ala Ser Ile Gln Gly Cys Asp
210 215 220
Ala Val Ala Leu Arg Ser Cys Tyr Glu Phe Glu Pro Glu Trp Leu Lys
225 230 235 240
Leu Leu Glu Glu Ile Tyr Gln Lys Pro Val Ile Pro Val Gly Gln Leu
245 250 255
Pro Thr Thr Leu Tyr Asn Ala Cys Tyr Asp Asp Glu Asn Asp Glu Trp
260 265 270
Lys Gly Met Lys Gln Trp Leu Asp Lys Gln Val Lys Ala Ser Val Val
275 280 285
Tyr Val Ala Phe Gly Ser Glu Ala Lys Pro Ser Gln Ala Glu Leu Thr
290 295 300
Glu Ile Ala Leu Gly Leu Glu Leu Ser Gly Leu Pro Phe Phe Trp Ile
305 310 315 320
Leu Arg Glu Arg Arg Gly Leu Ala Asp Thr Glu Leu Ile Glu Leu Pro
325 330 335
Asp Gly Phe Glu Glu Arg Thr Lys Gly Arg Gly Met Val Trp Thr Ser
340 345 350
Trp Ala Pro Gln Leu Lys Ile Leu Ser His Asn Ser Val Gly Gly Phe
355 360 365
Leu Thr His Ser Gly Trp Thr Ser Val Val Glu Ala Leu Gln Phe Glu
370 375 380
Arg Ala Leu Ile Leu Leu Thr Phe Leu Ala Asp Gln Gly Leu Asn Ala
385 390 395 400
Arg Val Leu Glu Glu Lys Lys Met Gly Tyr Ser Ile Pro Arg Asp Glu
405 410 415
Arg Asp Gly Ser Phe Thr Arg Asp Ser Val Ala Asp Ser Leu Arg Met
420 425 430
Val Met Val Glu Glu Lys Gly Lys Ile Tyr Arg Asp Lys Ala Lys Glu
435 440 445
Met Ser Lys Leu Phe Gly Asp Arg Asp Lys Gln Asp Arg Tyr Thr Asn
450 455 460
Asn Leu Leu Asp Tyr Leu Gln Thr His Arg Gln Arg Val Lys Lys Gly
465 470 475 480
Gln
<210> 6
<211> 1446
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 6
atggaatcaa taccaccccc tagtatgaca aacgacggca gcaagttgca cattgttatc 60
tttccgtggc tggcgtttgg ccacatgatt ccgtttctgg aggtggccaa attaatcgcg 120
cagaaaggcc acaaggtgag ctttgtttcg tcgccgcgta atattgatcg cttgccgaaa 180
ctgccaccga atctgacgcc cctgcttaac tttgttaagt tcccgctgcc gacgatggat 240
aatttgccgg aaaacgctga ggctaccacc gatttgccgt atcatgaagt aaaatacctg 300
aagattgcgt atgacctgtt gcaacagccg atgacccagt ttctgcaatc ctccgctccg 360
gactgggtta tttacgactt cgcgtcctat tggttaggtc caatcgcggc cgagctgggt 420
attagctctc tgtacttctg cattttcatc gcggcggcca tgtgctactg cggtccggtg 480
agctcgctga tgggcaccca agatgcccgt tccaagccgg aagatttcac cgttccgcca 540
aaatggatcc cgtttaaatc gaccgtagcg tttcgtctgt tcgaaattct gcgcaacctc 600
gacgacgtta ccggtgatga cgagagcatc ccggttacct atcgttctgc cgcgtctatt 660
cagggttgcg atgcagtcgc tctgcgtagc tgttacgaat ttgagccgga atggctgaag 720
ctgctggaag agatctacca gaaaccggtc atcccggtgg gtcaactgcc gacgaccctg 780
tacaatgcat gttacgacga cgagaacgat gagtggaaag ggatgaaaca atggctggac 840
aaacaggtta aggcatccgt cgtgtatgtg gcgttcggct ctgaagcgaa accgagccag 900
gcagagttga ccgaaatcgc tctgggcttg gagctgagcg gtctgccatt cttctggatt 960
ttgagagaac gccgtggctt ggctgacacc gagttgattg aattacctga tggtttcgag 1020
gaaagaacaa aaggccgtgg tatggtgtgg acgagctggg caccgcagct gaagatcctg 1080
agccataaca gcgttggtgg tttccttact cacagcggct ggaccagcgt ggtggaagcg 1140
ctgcaattcg agcgtgcact gatcctcctc acgtttttgg cggaccaggg tttgaatgcg 1200
cgtgttctgg aggagaaaaa aatgggttat agcatcccgc gcgacgagcg cgacggaagt 1260
tttacccgtg actccgttgc ggattcactg cgcatggtca tggtggaaga aaagggtaaa 1320
atctaccgcg acaaggcgaa agaaatgagc aagcttttcg gcgatcggga taagcaagat 1380
cgttatacca acaacctgct ggattatctg caaacccatc gtcagcgtgt gaagaagggt 1440
cagtaa 1446
<210> 7
<211> 454
<212> PRT
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 7
Met Asp Thr Glu His Thr Lys Leu Lys Ala Phe Leu Phe Pro Trp Leu
1 5 10 15
Ala Tyr Gly His Ile Ser Pro Phe Leu Glu Leu Ala Lys Lys Leu Ser
20 25 30
Asp Arg Gly Phe Leu Ile Glu Leu Cys Ser Thr Pro Ile Asn Leu Ser
35 40 45
Phe Ile Gln Lys Arg Ile Pro Gln Ser Tyr Ser Ser Thr Ile Gln Leu
50 55 60
Val Glu Leu Ile Leu Pro Glu Phe Pro Gln Leu Pro Pro His Tyr His
65 70 75 80
Thr Thr Asn Gly Leu Pro Leu His Leu Asn Ser Thr Leu His Lys Ala
85 90 95
Leu Lys Leu Ala Lys Pro Asn Leu Phe Thr Ile Leu Lys Thr Arg Lys
100 105 110
Pro Asp Leu Ile Ile Tyr Asp Val Met Gln Leu Trp Thr Phe Gly Val
115 120 125
Ala Ser Ser Leu Asn Ile Pro Ser Ala Arg Phe Phe Thr Ser Gly Ala
130 135 140
Ala Met Cys Ser Tyr Phe Val His Leu Tyr Lys Asn Leu Glu Val Glu
145 150 155 160
Tyr Pro Phe Pro Ala Leu His Leu Tyr Asp Tyr Glu Ile Glu Arg Ala
165 170 175
Arg Lys Leu Val Gln Arg Asn Asp Lys Glu Asn Glu Asn Lys Asp Asp
180 185 190
Gln Pro Glu Glu Glu Met Pro Pro Gln Glu Gly Ile Met Leu Ile Ser
195 200 205
Thr Ser Arg Glu Leu Glu Gly Lys Tyr Met Asn Tyr Leu Ala Glu Ile
210 215 220
Ile Glu Thr Arg Ile Phe Pro Ile Gly Thr Leu Val Gln Asp Pro Val
225 230 235 240
Ala Ser Gly Asp Glu Asn Met Asn Ile Met Gln Trp Leu Asp Ser Lys
245 250 255
Lys Glu Leu Ser Thr Val Phe Val Ser Phe Gly Ser Glu Tyr Phe Leu
260 265 270
Thr Lys Glu Glu Arg Glu Glu Ile Ala Leu Gly Leu Glu Leu Ser Gln
275 280 285
Val Asn Phe Ile Trp Val Val Arg Phe Pro Lys Gly Glu Lys Gln Asn
290 295 300
Leu Glu Glu Ala Leu Pro Gln Gly Phe Leu Glu Arg Val Gly Asp Arg
305 310 315 320
Gly Met Ile Val Glu Trp Ala Pro Gln Ala Lys Ile Leu Thr His Ser
325 330 335
Ser Ile Gly Gly Phe Val Ser His Cys Gly Trp Asn Ser Ile Ser Glu
340 345 350
Ser Ile Glu Phe Gly Val Pro Ile Ile Ala Ile Pro Met Gln Leu Asp
355 360 365
Gln Pro Met Asn Ala Lys Leu Leu Val Glu Ile Gly Val Ala Leu Glu
370 375 380
Val Val Arg Asp Asp Asn Gly Ser Leu His Arg Glu Glu Ile Ala Ser
385 390 395 400
Val Ile Lys Asp Val Thr Ser Gly Glu Ser Ser Glu Thr Met Arg Cys
405 410 415
Lys Val Arg Asn Leu Gly Lys Asn Leu Arg Thr Lys Ser Val Glu Asp
420 425 430
Met Asp Ala Thr Val Glu Glu Leu Arg Glu Leu Val Gly Glu Ser Arg
435 440 445
Ser Lys Asn Gly Ser Phe
450
<210> 8
<211> 1365
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 8
atggatactg aacacacaaa actaaaggct ttcttgttcc cgtggctggc gtatggtcat 60
atttccccgt ttttggagtt ggcgaagaaa ttgtctgatc gtggtttcct gatcgaactg 120
tgttcgactc cgatcaacct tagcttcatc cagaaacgta ttccgcaaag ctacagcagc 180
acgatccagc tggtggagct gatcctgccg gagttcccgc aactgccacc gcattaccat 240
accaccaacg gtctgccgct gcacctgaac agcacgctgc acaaagcgtt gaagcttgct 300
aagccgaatt tgttcaccat tctgaaaacc agaaagccgg atctgatcat ctacgacgtt 360
atgcaactgt ggacctttgg cgtcgctagt tcgctcaaca tcccgagcgc acgcttcttt 420
acctcgggcg cggcaatgtg cagctatttc gttcatctct acaaaaactt ggaagttgag 480
tacccgtttc cggcacttca cctgtacgac tacgaaatcg agcgtgcacg taaactggtg 540
cagcgtaacg ataaagagaa tgaaaacaaa gacgaccagc cagaagaaga gatgccgcct 600
caagaaggta ttatgctgat tagcacgagc cgtgagttgg agggcaaata tatgaactat 660
ttggctgaaa tcatcgaaac tcgcattttt ccgatcggca ctctggtaca ggatccggtt 720
gccagcggtg acgagaacat gaatattatg cagtggctgg acagcaaaaa ggaattatct 780
acggtttttg tgtccttcgg ctctgaatat ttcttaacca aagaggaacg tgaagagata 840
gcgctgggtc tggagttgag ccaggttaat tttatctggg ttgttcgttt cccgaaaggc 900
gaaaaacaga atctggaaga ggcgctgcca caaggttttt tggagcgcgt gggtgaccgc 960
ggtatgattg tggagtgggc accgcaagcg aagatcctga cccacagctc cattggcggc 1020
ttcgtgagcc actgcggttg gaacagcatc tcagaatcta ttgagtttgg cgttccgatt 1080
attgcgattc cgatgcagct ggaccaaccg atgaacgcca agttgctggt agagattggt 1140
gtggccctgg aggtcgtgcg tgatgataat ggctccctgc accgtgaaga gatcgcctcc 1200
gtgattaagg acgtgacctc tggggagagc agcgagacca tgcgttgcaa ggtgcgcaac 1260
ctgggtaaga acctgcgcac caagagcgtt gaagatatgg atgcgaccgt cgaggaactg 1320
cgtgagctgg tcggtgaatc tagatccaag aacggcagct tttaa 1365
<210> 9
<211> 2418
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 9
atggccgaac gtgtcctgac ccgtgtccat agtctgcgtg aacgtgttga tgctaccctg 60
gctgcccacc gtaatgaaat cctgctgttt ctgagtcgta ttgaaagcca cggcaaaggt 120
atcctgaaac cgcacgaact gctggcagaa tttgatgcta ttcgccagga tgacaaaaac 180
aaactgaacg aacatgcatt cgaagaactg ctgaaaagca cccaagaagc tatcgtcctg 240
ccgccgtggg tggcactggc aattcgtctg cgcccgggcg tttgggaata catccgtgtt 300
aacgtcaatg cgctggttgt ggaagaactg agtgtgccgg aatatctgca gtttaaagaa 360
gaactggtcg atggcgcgtc caacggtaat ttcgtgctgg aactggactt tgaaccgttc 420
accgcctcat ttccgaaacc gaccctgacg aaatcgattg gcaacggtgt tgaatttctg 480
aatcgtcatc tgagcgccaa aatgttccac gataaagaat ctatgacccc gctgctggaa 540
tttctgcgcg cacatcacta taaaggtaaa accatgatgc tgaacgatcg tattcagaac 600
agcaatacgc tgcaaaatgt gctgcgcaaa gcggaagaat acctgatcat gctgccgccg 660
gaaaccccgt acttcgaatt tgaacataaa ttccaggaaa ttggcctgga aaaaggctgg 720
ggtgatacgg cagaacgtgt gctggaaatg gtttgcatgc tgctggatct gctggaagct 780
ccggacagct gtaccctgga aaaatttctg ggtcgcattc cgatggtttt caacgtcgtg 840
atcctgtctc cgcacggcta ttttgcgcag gaaaatgtcc tgggttaccc ggataccggc 900
ggtcaggttg tctatattct ggaccaagtg ccggccctgg aacgtgaaat gctgaaacgc 960
atcaaagaac agggcctgga tattatcccg cgtattctga tcgtcacccg tctgctgccg 1020
gacgcagtgg gcaccacgtg cggtcaacgt attgaaaaag tgtatggcgc tgaacattca 1080
cacatcctgc gtgttccgtt tcgcaccgaa aaaggtattg tccgtaaatg gatctcgcgc 1140
tttgaagtgt ggccgtacat ggaaacgttc attgaagatg ttgcaaaaga aatctcagcg 1200
gaactgcagg ccaaaccgga cctgattatc ggcaactata gcgaaggtaa tctggcggcc 1260
tctctgctgg cccataaact gggcgtgacc caatgtacga ttgcacacgc tctggaaaaa 1320
accaaatatc cggattcgga catctactgg aaaaaattcg atgaaaaata ccatttcagc 1380
tctcagttca ccgcagatct gattgctatg aaccacacgg actttattat caccagtacg 1440
ttccaggaaa tcgcgggctc caaagatacc gtgggtcaat acgaaagtca tatggccttt 1500
acgatgccgg gcctgtatcg cgtggttcac ggtatcaacg ttttcgatcc gaaattcaac 1560
attgtctccc cgggtgcaga catcaatctg tatttttcat actcggaaac cgaaaaacgt 1620
ctgacggctt tccatccgga aatcgatgaa ctgctgtata gcgatgtgga aaacgacgaa 1680
cacctgtgcg ttctgaaaga tcgcaccaaa ccgattctgt ttacgatggc gcgtctggac 1740
cgcgttaaaa atctgaccgg cctggtcgaa tggtacgcca aaaacccgcg tctgcgcggt 1800
ctggtgaatc tggtcgtggt tggcggtgat cgtcgcaaag aatctaaaga cctggaagaa 1860
caggcggaaa tgaagaaaat gtacgaactg atcgaaaccc ataacctgaa tggccagttc 1920
cgttggatca gttcccaaat gaaccgtgtt cgcaatggcg aactgtatcg ctacatcgca 1980
gatacgaaag gtgcttttgt ccagccggcg ttttacgaag ccttcggcct gaccgtcgtg 2040
gaagcgatga cgtgcggtct gccgaccttc gcaacgaatc atggcggccc ggcagaaatt 2100
atcgttcacg gcaaaagtgg ttttcatatt gatccgtatc acggcgaaca ggcagctgat 2160
ctgctggccg actttttcga aaaatgtaaa aaagacccgt cacattggga aaccatttcg 2220
atgggcggtc tgaaacgcat cgaagaaaaa tatacctggc aaatttacag cgaatctctg 2280
ctgacgctgg cggccgtgta cggtttctgg aaacacgttt ctaaactgga tcgtctggaa 2340
attcgtcgct atctggaaat gttttatgcg ctgaaatacc gcaaaatggc ggaagccgtg 2400
ccgctggcag ctgaataa 2418

Claims (4)

1. A method for enzymatic preparation of glycosylated steviol glycoside compounds, characterized in that in the presence of glycosyltransferase the glycosyl group of the glycosyl donor is transferred to the C-6 'of the C19 linked first glycosyl group of steviol glycoside compounds and/or the glycosyl group of the glycosyl donor is transferred to the C2' of the C19 linked first glycosyl group of steviol glycoside compounds;
the glycosyltransferase is derived from grapeVitis vinifera) A kind of electronic deviceVvUGT or derived from fructus LyciiLycium barbarum) A kind of electronic deviceLbUGT or from blue fruit treeNyssa sinensis) A kind of electronic deviceNsUGT or derived from tobaccoNicotiana tomentosiformis) A kind of electronic deviceNtUGT；
VvThe UGT amino acid sequence is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 2; LbThe UGT amino acid sequence is shown as SEQ ID NO. 3, and the nucleotide sequence is shown as SEQ ID NO. 4;Nsthe UGT amino acid sequence is shown as SEQ ID NO. 5, and the nucleotide sequence is shown as SEQ ID NO. 6;Ntthe UGT amino acid sequence is shown as SEQ ID NO. 7, and the nucleotide sequence is shown as SEQ ID NO. 8;
glycosyltransferaseVvThe UGT has substrate of rubusoside, stevioside, rebaudioside A or rebaudioside E;
glycosyltransferaseLbThe UGT has substrate of rubusoside, stevioside, rebaudioside A, rebaudioside E or rebaudioside D;
glycosyltransferaseNsThe UGT has substrate of rubusoside, stevioside, rebaudioside A, rebaudioside E or rebaudioside D;
glycosyltransferaseNtThe UGT has substrate of rubusoside and steviosideRebaudioside a, rebaudioside E or rebaudioside D.
2. The method for preparing glycosylated steviol glycoside compounds according to claim 1, characterized by comprising the following steps:
constructing engineering bacteria: cloning glycosyltransferase genes and sucrose synthase genes, constructing vectors for co-expression or separate expression of double enzymes, and transferring the vectors into host bacteria to obtain engineering bacteria of UDP-glycosyltransferase and/or sucrose synthase;
preparation of the glycosylation product: fermenting the constructed engineering bacteria to produce enzyme, and performing ultrasonic crushing or high-pressure crushing on the thallus obtained by fermentation to obtain crude enzyme liquid of glycosyltransferase and sucrose synthase;
Carrying out glycosylation reaction on the crude enzyme solution, a glycosylation substrate and sucrose in a conversion system; the glycosyltransferase catalyzes a substrate to carry out a conversion reaction to prepare a glycosylation product; sucrose synthase catalyzes the production of UDP-glucose as a glycosyl donor from UDP produced by this process.
3. The method for preparing glycosylated stevioside compounds by using the enzymatic method according to claim 1, wherein the glycosylation reaction is carried out on glycosyltransferase crude enzyme liquid, stevioside compounds and glycosyl donors in a conversion system, the adding amount of glycosyltransferase is 0.1-5 g/L, the adding amount of stevioside compounds is 0.1-1000 g/L, the adding amount of glycosyl donors is 0.1-3000 g/L, the reaction temperature is 20-80 ℃, the reaction time is 2-96 h, and the reaction pH is 5-10.
4. The method of enzymatic preparation of glycosylated steviol glycosides according to claim 1, wherein the steviol glycosides include, but are not limited to, rubusoside, stevioside, rebaudioside a, rebaudioside B, rebaudioside D, rebaudioside E, rebaudioside M.
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Publication number Priority date Publication date Assignee Title
CN105408339A (en) * 2013-05-28 2016-03-16 谱赛科有限责任公司 High-purity steviol glycosides
CN112135528A (en) * 2018-05-08 2020-12-25 伊比西(北京)植物药物技术有限公司 Composition of steviol glycoside and/or polysaccharide derivative thereof
CN113508124A (en) * 2019-02-15 2021-10-15 谱赛科美国股份有限公司 High purity steviol glycosides
WO2021232750A1 (en) * 2020-05-19 2021-11-25 Epc Natural Products Co., Ltd. Sweetener and flavor compositions

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Publication number Priority date Publication date Assignee Title
CN105408339A (en) * 2013-05-28 2016-03-16 谱赛科有限责任公司 High-purity steviol glycosides
CN112135528A (en) * 2018-05-08 2020-12-25 伊比西(北京)植物药物技术有限公司 Composition of steviol glycoside and/or polysaccharide derivative thereof
CN113508124A (en) * 2019-02-15 2021-10-15 谱赛科美国股份有限公司 High purity steviol glycosides
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hypothetical protein F0562_003078 [Nyssa sinensis];Yang,X. et al.;GenBank: KAA8546691.1;全文 *
Roach,M.J. et al..Beta-D-glucosyl crocetin beta-1,6-glucosyltransferase [Vitis vinifera]. GenBank: RVW36893.1.2019,全文. *
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