WO2022253282A1 - 一种糖基转移酶及其应用 - Google Patents

一种糖基转移酶及其应用 Download PDF

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WO2022253282A1
WO2022253282A1 PCT/CN2022/096683 CN2022096683W WO2022253282A1 WO 2022253282 A1 WO2022253282 A1 WO 2022253282A1 CN 2022096683 W CN2022096683 W CN 2022096683W WO 2022253282 A1 WO2022253282 A1 WO 2022253282A1
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seq
glycosyltransferase
rebaudioside
reaction
amino acid
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PCT/CN2022/096683
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French (fr)
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吴燕
田振华
王舒
郑孝富
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弈柯莱生物科技(上海)股份有限公司
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Priority claimed from CN202110610098.2A external-priority patent/CN115418358B/zh
Priority claimed from CN202111602506.6A external-priority patent/CN116334162A/zh
Application filed by 弈柯莱生物科技(上海)股份有限公司 filed Critical 弈柯莱生物科技(上海)股份有限公司
Priority to EP22815324.3A priority Critical patent/EP4349989A1/en
Publication of WO2022253282A1 publication Critical patent/WO2022253282A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries

Definitions

  • the invention relates to a glycosyltransferase and its application in the glycosylation reaction of steviol glycosides.
  • Steviol glycosides (Steviol glycosides, also known as steviol glycosides) is a natural sweetener extracted from the leaves of the Compositae herb stevia rebaudiana. It is a mixture of various glycosides. Different steviol glycosides have great differences in taste quality. Steviol glycosides are pure natural (from the pure natural plant stevia), high sweetness (250-450 times that of sucrose), low calorie (only 1/300 of white sugar), and economical to use (the cost is only one-third of sucrose ), good stability (heat resistance, acid resistance, alkali resistance, not easy to decompose), high safety (no toxic side effects), and other potential curative effect.
  • steviol glycoside compounds have a common aglycone: steviol (Steviol), the difference lies in the number and type of sugar groups connected at the C-13 and C-19 positions, mainly including stevioside (Stevioside), rebaudioside A (Rebaudioside A, Reb A), rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside I, dulcoside, steviolbioside and other glycosides .
  • Stevia leaves are capable of accumulating as much as 10-20% (dry weight basis) steviol glycosides.
  • rebaudioside A The major glycosides found in Stevia leaves are rebaudioside A (2-10%), stevioside (2-10%) and rebaudioside C (1-2%).
  • Other glycosides such as rebaudiosides B, D, E, F and I, steviolbioside and rubusoside, were found at much lower levels (approximately 0-0.2%).
  • rebaudioside A has the highest sweetness, and the sweetness is similar to sucrose. It is a new type of natural sweetener with high sweetness, low calorie, easy to dissolve, heat resistance and stability. Its content or purity is also a measure of the quality of stevioside main indicators.
  • steviol glycoside is a high-intensity sweetener, it has the shortcoming of post-bitterness and astringency, which severely limits its application in food, beverages and other fields that require high taste.
  • the essential cause of the bitter taste of steviol glycosides is its internal molecular structure. The more sugar groups connected to the R 1 and R 2 groups in steviol glycosides, the better the taste.
  • steviosides are found to be 110-270 times sweeter than sucrose and 150-320 times sweeter than rebaudioside A, however, even in a highly purified state, steviosides still have undesirable taste attributes such as bitterness, sweet aftertaste , licorice flavor, etc.
  • Rebaudioside D is the steviol glycoside with the most application potential. Compared with other steviol glycosides, its sweetness is high, about 300-350 times that of sucrose, and the sweetness is pure, and the taste is closer to sucrose, without bitterness and Licorice has a peculiar smell and good stability, and is an ideal natural high-intensity sweetener product.
  • the content of rebaudioside D in stevia leaves is very small (less than 5%).
  • the production of rebaudioside D by extraction requires a large amount of stevia raw materials.
  • the process of enriching rebaudioside D is cumbersome. It needs to go through the column for many times, desalting, decolorization, recrystallization, and produces a large amount of waste water in the production process. The production cost is relatively high, and it is not suitable for industrialized large-scale production.
  • UDP-glucose UDP-glucosyltransferase
  • UGT UDP-glucosyltransferase
  • Rebaudioside M (RebM) has better taste properties, but its content of dry weight of leaves is less than 0.1%, resulting in high isolation cost and high price.
  • the biocatalytic method to obtain high concentration of rebaudioside M has attracted the attention of scholars. It is currently reported that the recombinase derived from Stevia rebaudiana can catalyze rebaudioside D to rebaudioside M, but the yield is low.
  • rebaudioside D as a substrate, rebaudioside M can be obtained through the catalytic method of microbial enzyme production. Compared with the traditional extraction method, this method not only improves the production process, but also reduces the pollution to the environment and improves the yield of the target product. Yield of Rebaudioside M.
  • Glucosyltransferase is an enzyme that only transfers glucose groups in an enzymatic reaction.
  • the mechanism of action of this enzyme is to catalyze the transfer of glucose residues from sugar group donors to sugar group acceptor molecules, thereby regulating the activity of acceptor molecules.
  • Nucleoside refers to glycosylamines comprising a nucleobase (ie, a nitrogenous base) and a 5-carbon sugar (eg, ribose or deoxyribose).
  • nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine.
  • Nucleoside diphosphate refers to a glycosylamine comprising a nucleobase (ie, nitrogenous base), a 5-carbon sugar (eg, ribose or deoxyribose), and a diphosphate (ie, pyrophosphate) moiety.
  • Nucleoside diphosphate may be abbreviated as "NDP”.
  • Non-limiting examples of nucleoside diphosphates include cytidine diphosphate (CDP), uridine diphosphate (UDP), adenosine diphosphate (ADP), guanosine diphosphate (GDP), thymidine diphosphate (TDP) and Inosine diphosphate (IDP).
  • UDP-glucosyltransferase is a kind of glucosyltransferase, which uses UDP-glucose as a glycosyl donor and exists in almost all organisms.
  • UDP-glucose is the abbreviation of uridine diphosphate glucose, also referred to as UDP-glucose or UDPG. It is a vitamin composed of uridine diphosphate and glucose. It can be regarded as "active glucose” and is widely distributed. In the cells of plants, animals and microorganisms, it is the most common sugar-based donor in the synthesis of sucrose, starch, glycogen and other oligosaccharides and polysaccharides.
  • ADP-glucose, TDP-glucose, dTDP-glucose (deoxythymidine diphosphate glucose), CDP-glucose, IDP-glucose or GDP-glucose are often used as sugar donor compounds. These glycosyl donors are expensive and unfavorable for industrial production.
  • Sucrose synthase (SUS, also referred to as SuSy/SS, etc., EC2.4.1.13), which reversibly catalyzes the chemical reaction NDP-glucose + D-fructose to NDP and sucrose, belongs to the glycosyltransferase-4 subfamily It was first identified in the germ of wheat (Triticum aestivum). Each protein exists as a tetramer. The molecular weight of each subunit is about 90kDa. The gene size is generally 5.9kb, and the cDNA size is about 2.7kb. The full length of the encoded amino acid sequence is approximately 800 amino acid residues (Ross and Davies 1992). The affinity of sucrose synthase to NDP is different, and the order of affinity is UDP>ADP>dTDP>CDP>GDP.
  • CN110914445A screened the mutant of glucosyltransferase UGT76G1 that catalyzes the preparation of RI by using RA as raw material, and the screening conditions were pH7.0, MgCl2 10.3mM, and temperature 35°C.
  • CN106795523A reports sweetness data of RI and compositions containing RI.
  • the aforementioned prior art only discloses glucosyltransferases or mutants thereof having the activity of catalyzing the synthesis of RA from RI, but these enzymes have low activity and are not suitable for industrial production.
  • UDP-glucosyltransferase is increasingly used in the field of biocatalytic preparation of steviol glycosides.
  • UDP-glucosyltransferases There are many kinds of UDP-glucosyltransferases.
  • Most of the enzymes used in the field of bio-enzyme preparation of steviol glycosides are wild enzymes derived from plant cells. This wild enzyme often has the disadvantages of low enzyme activity and poor stability. This leads to higher costs for preparing steviol glycosides in industrialized large-scale production. Therefore, it is necessary to modify UDP-glucosyltransferase to obtain a modified enzyme with higher enzyme activity and better stability, so as to better serve industrial production.
  • the technical problem to be solved by the present invention is that when the existing UDP-glucosyltransferase is applied to the biocatalytic preparation of steviol glycosides, the enzyme activity is low, the stability is poor, and the conversion rate is not high when used to catalyze steviol glycosides. Therefore, the present invention provides A glycosyltransferase (ie, UDP-glucosyltransferase) and its use in the preparation of steviol glycosides.
  • a glycosyltransferase ie, UDP-glucosyltransferase
  • Glycosyltransferase (GT) of the present invention has high enzyme activity and good stability; wherein, sucrose and UDP (and/or ADP) generate UDPG (and/or ADPG) under SUS catalysis, thereby realizing UDPG (and or ADPG) ) regeneration, which solves the expensive problem of glycosyl donor UDPG (and/or ADPG); it is used to prepare steviol glycosides (such as rebaudioside A, rebaudioside D, rebaudioside M or rebaudioside Compared with the glycosyltransferase parent, diglycoside I) has a significant improvement in catalytic activity, and the conversion rate is significantly improved, thereby reducing the cost of the reaction and providing more options for optimizing process conditions for large-scale industrial production. , conducive to industrial production.
  • sucrose and UDP (and/or ADP) generate UDPG (and/or ADPG) under SUS catalysis, thereby realizing UDPG (and or ADPG)
  • the present invention provides a glycosyltransferase, the amino acid sequence of the glycosyltransferase is as shown in SEQ ID NO: 112 or an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 112 Show.
  • amino acid sequence having at least 99% sequence identity with SEQ ID NO: 112 is an amino acid residue at a residue position comprising one or more of the following residues compared with SEQ ID NO: 112 difference:
  • amino acid sequence of the glycosyltransferase may further comprise an amino acid residue difference selected from the following residue positions: K347P.
  • the amino acid sequence of the glycosyltransferase comprises an amino acid residue difference at one of the following residue positions compared with SEQ ID NO: 112: V14I, E99L, T254G, L257A, Q451E , Q265E, P271A, R333K, R12Q, A118S, E418D or S455R.
  • the amino acid sequence of the glycosyltransferase comprises amino acid residue differences selected from the following two residue positions compared with SEQ ID NO: 112: Q265E and P271A.
  • the amino acid sequence of the glycosyltransferase comprises amino acid residue differences selected from the following two residue positions compared with SEQ ID NO: 112: R333K and K347P.
  • the nucleotide sequence encoding the glycosyltransferase can be selected from the following sequences: SEQ ID NO:41, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO: 94. SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO: 104, SEQ ID NO: 107 and SEQ ID NO: 108.
  • the amino acid sequence of the glycosyltransferase may additionally have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1- 8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20 Differences in amino acid residues; differences in these residues include substitutions by conservative amino acid residues. Usually these acquisitions do not affect the enzymatic activity of the glycosyltransferase.
  • the present invention provides an isolated nucleic acid encoding the above-mentioned glycosyltransferase.
  • the nucleotide sequence of the nucleic acid can be selected from the following sequences: SEQ ID NO: 41, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 95, SEQ ID NO: ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO :107 and SEQ ID NO:108.
  • the present invention provides a recombinant expression vector comprising the above-mentioned nucleic acid.
  • the present invention provides a transformant, which is a host cell comprising the above-mentioned nucleic acid or the above-mentioned recombinant expression vector.
  • the host cell can be conventional in the art, preferably Escherichia coli (Escherichia coli).
  • the present invention provides a method for preparing the above-mentioned glycosyltransferase, the method comprising culturing the above-mentioned transformant under conditions suitable for expressing the above-mentioned glycosyltransferase.
  • the following steps can be adopted: (1) inoculate the transformant containing the glycosyltransferase into an antibiotic-containing medium such as LB medium and shake it to obtain a seed solution; (2) Transfer the seed solution in (1) to a medium containing antibiotics such as TB medium for shaking culture; (3) add IPTG to the medium in (2) to induce overnight, and collect the thalline after centrifugation; (4) Wash and resuspend the bacterial cells collected in (3), crush and centrifuge to obtain the crude enzyme solution containing the glycosyltransferase.
  • the present invention provides a composition comprising the above-mentioned glycosyltransferase.
  • the composition may, for example, be in the form of an enzyme preparation.
  • the concentration of the stevioside is 1-150g/L, preferably 100g/L.
  • sucrose synthase also known as sucrose synthase, Sucrose synthase, SuSy or SUS for short
  • sucrose synthase catalyzes reversible glucose transfer reaction
  • sucrose and UDP catalyzes sucrose and UDP to produce UDP-glucose by sucrose synthase
  • UDP-sugar donors for UDP-glycosyltransferases effectively Provides UDP-sugar donors for UDP-glycosyltransferases.
  • a UDP cycle is generated to realize the regeneration of UDP-glucose, avoiding the direct use of expensive UDP-glucose, and reducing production costs.
  • the pH of the reaction solvent for the reaction is 5-8, preferably 6.
  • the present invention provides a method for preparing rebaudioside D or rebaudioside M, which includes the step of preparing rebaudioside A according to the above-mentioned preparation method.
  • the method includes providing a stevioside substrate, a glycosyl donor and a glycosyltransferase as described above, under conditions that produce rebaudioside D or rebaudioside M
  • the stevioside substrate, glycosyl donor and glycosyltransferase were reacted as described above.
  • the present invention provides a method for preparing rebaudioside I, which involves reacting rebaudioside A with a glycosyl donor in the presence of a glycosyltransferase as described above mentioned.
  • the glycosyl donor is UDP-glucose and/or ADP-glucose.
  • the concentration of the rebaudioside A is preferably 1-150g/L, such as 100g/L, 50g/L, 80g/L, 120g/L.
  • the mass ratio between the glycosyltransferase cell and rebaudioside A is preferably 1:(3-10), such as 3:20, 1:5, 1:8.
  • the pH of the reaction solvent for the reaction is preferably 5-8, more preferably 5.5-6.
  • the temperature of the reaction system of the reaction is preferably 20-90°C, such as 60°C, 30°C, 50°C, 70°C.
  • the reaction time of the reaction is preferably 5-30h, such as 24h, 10h, 15h, 20h, 28h.
  • the glycosyl donor is preferably prepared by UDP and/or ADP in the presence of sucrose and sucrose synthase.
  • the concentration of the sucrose is preferably 100-300g/L, such as 200g/L, 150g/L, 250g/L.
  • the pH is preferably controlled by a buffer solution, such as a phosphate buffer solution.
  • the rotational speed of the reaction is preferably 500-1000 rpm, such as 600 rpm, 800 rpm.
  • the present invention provides a use of the above-mentioned glycosyltransferase in the preparation of steviol glycosides;
  • the steviol glycosides are preferably rebaudioside A, rebaudioside D, rebaudioside M or rebaudioside I.
  • the invention provides a kind of catalytic enzyme composition, it comprises glycosyltransferase and sucrose synthase, described glycosyltransferase is as above-mentioned, and the sequence of described sucrose synthase is as SEQ ID NO: 32.
  • the mass ratio of the glycosyltransferase and sucrose synthase is preferably (2-10):1, such as 5:1, 3:1, 8:1.
  • the present invention provides an application of the catalytic enzyme composition as described above in the preparation of rebaudioside A, rebaudioside D, rebaudioside M or rebaudioside I.
  • the steviol glycosides can also be called steviol glycosides, and its structural formula and the types of compounds included can be specifically referred to in the background technology section of the present invention.
  • the glycosyltransferase of the present invention has high enzyme activity and good stability; when it is used to prepare steviol glycosides (such as rebaudioside A, rebaudioside D, rebaudioside M or rebaudioside I)
  • steviol glycosides such as rebaudioside A, rebaudioside D, rebaudioside M or rebaudioside I
  • the catalytic activity has been significantly improved, and the conversion rate has been significantly improved, which solves the problem that the glycosyl donor UDPG (and/or ADPG) is expensive, thereby reducing the cost of the reaction.
  • the realization of large-scale industrial production provides more options for optimizing process conditions, which is beneficial to industrial production.
  • Fig. 1 shows a schematic diagram of the routes for preparing rebaudioside A, rebaudioside D and rebaudioside M from stevioside in Examples 1-7 of the present invention.
  • Fig. 2 shows the synthesis of rebaudioside A by the enzyme-catalyzed reaction system in Examples 1-7 of the present invention, realizing the cycle of UDPG in the biocatalytic reaction.
  • Fig. 3 is the spectrum of the substrate stevioside used in the second round of screening in Examples 1-7, the retention time of stevioside is 12.76min.
  • Fig. 4 is the spectrum of the product Rebaudioside A reference substance in Examples 1-7, the retention time is 12.38min.
  • Fig. 5 is an HPLC chart of the catalytic synthesis of RA activity of Enz.11 in Table 10.
  • Fig. 6 is the HPLC chart of the catalytic synthesis of RA activity of Enz.24 in Table 10.
  • Fig. 7 is a synthetic route for preparing rebaudioside A and rebaudioside I from stevioside.
  • Fig. 8 is the spectrum of the rebaudioside A reference substance using the HPLC detection method in Examples 8-11.
  • Fig. 10 is the HPLC pattern of reaction 8 hours in embodiment 11.
  • Fig. 11 is the HPLC profile of reaction 24 hours in embodiment 11.
  • KOD Mix enzyme was purchased from TOYOBO CO., LTD.
  • DpnI enzyme was purchased from Yingwei Jieji (Shanghai) Trading Co., Ltd.
  • E.coli Trans10 competent cells were purchased from Beijing Dingguochangsheng Biotechnology Co., Ltd.
  • E.coli BL21 (DE3) Competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.
  • Sucrose was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.
  • reaction substrates used in the first round of screening in Examples 1-7 and RA60 (stevioside, wherein the RA content is 60% and the stevioside content is about 30%) in Examples 8-11 were purchased from Chenguang Biology, product specification TSG90/RA60.
  • the reaction substrate stevioside used in the second round of screening was purchased from Bid Pharmaceuticals (purity 95%).
  • Reb A and Reb I reference substances were purchased from McLean.
  • Time(min) A% B% 0.00 90 10 15.00 60 40 20.00 0 100 24.00 0 100 24.10 90 10 32.00 90 10
  • Examples 1 to 7 mainly use the UDP-glucosyltransferase parent Enz.1 as a template, and in the first round of screening, the glycosyltransferase mutant Enz.11 with a mutation at position 308 to N was selected, and Enz.11 was used as a template to carry out In the second round of screening, UDP-glucosyltransferases with double or triple mutations were screened out.
  • ⁇ -1,3-glycosyltransferase ( ⁇ -1,3-GTase) gene of Enz.1 shown in SEQ ID NO:1 which has been connected to the pET28a plasmid vector to obtain
  • the recombinant plasmid pET28a-GT010 was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. (698 Xiangmin Road, Songjiang District, Shanghai).
  • the amino acid sequence of Enz.1 is shown in SEQ ID NO:2.
  • the PCR amplification reaction system is:
  • the PCR product was digested with DpnI and then gel-run and gel-recovered to obtain the target DNA fragment. Ligated by two-fragment homologous recombinase (Exnase II) of Novizyme. After ligation, it was transformed into E.coli Trans10 competent cells, spread on LB medium containing 50 ⁇ g/mL kanamycin, and cultured overnight at 37°C; cultured at pick point and sequenced to obtain a recombinant plasmid containing the mutant gene.
  • the above-mentioned recombinant plasmids with correct sequencing were transformed into host E. coli BL21 (DE3) competent cells to obtain genetically engineered strains containing point mutations. Pick a single colony and inoculate it into 5ml LB liquid medium containing 50 ⁇ g/ml kanamycin, and culture with shaking at 37°C for 4h.
  • sucrose synthase (SUS) gene whose numbering is Enz.2 shown in SEQ ID NO:31 is fully synthesized, and the gene has been connected to the pET28a plasmid vector to obtain the recombinant plasmid pET28a-SUS.
  • the gene synthesis company is Sangon Bioengineering (Shanghai) Co., Ltd. (698 Xiangmin Road, Songjiang District, Shanghai).
  • the amino acid sequence of sucrose synthase is shown in SEQ ID NO:32.
  • the plasmid pET28a-SUS was transformed into the host E.coli BL21(DE3) competent cells to obtain the Enz.2 genetically engineered strain. Pick a single colony and inoculate it into 5ml LB liquid medium containing 50 ⁇ g/ml kanamycin, and culture with shaking at 37°C for 4h. Transfer to 50ml of fresh TB liquid medium containing 50 ⁇ g/ml kanamycin according to 2v/v% inoculation amount, shake culture at 37°C until OD600 reaches 0.6-0.8, add IPTG to its final concentration of 0.1mM , 25 °C induction culture 20h.
  • the culture solution was centrifuged at 10,000 rpm for 10 min (for Examples 4-7) or 4,000 rpm for 20 min (for Examples 8-11), the supernatant was discarded, and the bacteria were collected. Store at -20°C for later use.
  • Examples 4-7 prepare 50mM phosphate buffer solution (PBS) at pH6.0, suspend the Enz.2 bacteria sludge obtained above according to (M/V) 1:5, and then homogenize to obtain the crude enzyme liquid, The crude enzyme solution was centrifuged, and the supernatant was taken to obtain a crude enzyme solution of sucrose synthase SUS (enzyme number Enz.2, amino acid sequence shown in SEQ ID NO:32).
  • PBS phosphate buffer solution
  • M/V phosphate buffer solution
  • the phosphate buffer solution (PBS) of preparation 50mM pH6.0, above-mentioned obtained bacterium slime is suspended according to 1:10 (M/V, g/mL), afterwards, carry out high-pressure homogenization (550Mbar, 1.5min) and then centrifuged at 12000rpm for 2min to obtain the sucrose synthase reaction enzyme solution.
  • PBS phosphate buffer solution
  • Example 2 and Example 3 were incubated at a constant temperature of 80° C. for 20 minutes, and centrifuged to obtain the supernatant to obtain the UDP-glucosyltransferase mutant reaction enzyme solution and the sucrose synthase reaction enzyme solution respectively.
  • Boil and UnBoil Two reaction conditions, Boil and UnBoil, are used for re-screening.
  • the Boil re-screening reaction conditions are the same as the primary screening reaction conditions.
  • UnBoil refers to the reaction without heating, and the rest of the reaction conditions are the same as the Boil reaction conditions.
  • Rescreening result is as shown in table 7 (the content of Reb A in the corresponding reaction solution of % numerical value in the table).
  • the gene encoding the glycosyltransferase ( ⁇ -1,3-GT enzyme) Enz.11 obtained in the first round was connected to the vector pET28a to obtain the pET28a-Enz.11 recombinant plasmid.
  • pET28a-Enz.11 as a template, the expression
  • the primer sequence shown in 8 was used for PCR amplification with KOD enzyme to obtain the gene fragment and vector fragment of the target mutant Enz.16-Enz.34.
  • the gene fragment and vector fragment of the target mutant Enz.35 were amplified by PCR.
  • the PCR product was digested with DpnI and then run and recovered from the gel.
  • the two-fragment homologous recombinase of Novizyme was used for ligation. After ligation was completed, it was transformed into E.coli Trans10 competent cells, spread on LB medium containing 50 ⁇ g/mL kanamycin, and cultured at 37°C overnight; cultured and sequenced to obtain recombinant plasmids containing mutant genes.
  • the recombinant plasmids sequenced correctly in Example 5 were transformed into host E. coli BL21 (DE3) competent cells to obtain genetically engineered strains containing point mutations. Pick a single colony and inoculate it into 5ml LB liquid medium containing 50 ⁇ g/ml kanamycin, and culture with shaking at 37°C for 4h. Transfer to 50ml of fresh TB liquid medium containing 50 ⁇ g/ml kanamycin according to 2v/v% inoculation amount, shake culture at 37°C until OD600 reaches 0.6-0.8, add IPTG to its final concentration of 0.1mM , 25 °C induction culture 20h. After the cultivation, the culture solution was centrifuged at 4000 rpm for 20 minutes, the supernatant was discarded, and the bacteria (ie, the sludge) were collected. Store at -20°C for later use.
  • Example 1 the fully synthesized ⁇ -1,3-glycosyltransferase ( ⁇ -1,3-GTase) enzyme gene shown in SEQ ID NO:1 as Enz.1 was connected to pET28a On the plasmid vector, the recombinant plasmid pET28a-Enz.1 was obtained, and the gene synthesis company was Sangon Bioengineering (Shanghai) Co., Ltd. (698 Xiangmin Road, Songjiang District, Shanghai). The amino acid sequence of Enz.1 is shown in SEQ ID NO:2. The recombinant plasmid was transformed into host Escherichia coli BL21 (DE3) competent cells to obtain an engineering strain containing transaminase Enz.1 gene.
  • the gene of transaminase Enz.2.2 ⁇ Enz.2.6 obtained by performing site-directed mutation according to the Enz.1 gene in Table 12, and Enz.16, Enz.19, Enz.23, Enz.25, Enz.27, Enz .31, the genes of Enz.32 and Enz.35, the restriction sites NdeI, HindIII, and the vector pET28a were connected to obtain transaminases Enz.2.2 ⁇ Enz.2.6, and Enz.16, Enz.19, Enz.23, Each recombinant plasmid of the gene of Enz.25, Enz.27, Enz.31, Enz.32 and Enz.35. Each recombinant plasmid was transformed into host Escherichia coli BL21 (DE3) competent cells respectively, and the engineering strains containing transaminase genes in Table 12 were obtained.
  • Enzyme number RI%(RA ⁇ RI) Enz.1 2.886 Enz.2.2 0.555 Enz.2.3 1.705 Enz.2.4 1.324 Enz.2.5 0.886 Enz.2.6 1.427 Enz.16 4.139 Enz.19 4.014 Enz.23 3.628 Enz.25 3.910 Enz.27 4.235 Enz.31 4.646 Enz.32 3.546 Enz.35 1.939

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Abstract

一种糖基转移酶及其应用，所述糖基转移酶的氨基酸序列如SEQ ID NO:112或与SEQ ID NO:112具有至少99％序列同一性的氨基酸序列所示。本发明的糖基转移酶的酶活高、稳定性好；将其用于制备甜菊糖苷时与糖基转移酶亲本相比，在催化活性方面有了明显的提高，转化率显著提升，解决了糖基供体UDPG(和/或ADPG)价格昂贵的问题，从而降低了反应的成本，利于工业化生产。

Description

一种糖基转移酶及其应用
本申请要求申请日为2021/6/1的中国专利申请2021106100982、申请日为2021/12/24的中国专利申请2021116025066的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一种糖基转移酶及其在甜菊糖苷的糖基化反应中的应用。
背景技术
甜菊糖苷(Steviol glycosides，又称甜菊醇糖苷)是从菊科草本植物甜叶菊叶中提取的天然甜味剂，是多种糖苷的混和物，不同甜菊糖苷在味质上存在较大的差异。甜菊糖苷具有纯天然(来自纯天然植物甜叶菊)、高甜度(蔗糖的250～450倍)、低热量(仅为白糖的1/300)、使用经济(成本仅为蔗糖的三分之一)、稳定性好(耐热、耐酸、耐碱，不易出现分解现象)、安全性高(无毒副作用)等优点，以及抗高血糖、抗高血压、抗炎症、抗肿瘤、抗腹泻等潜在疗效。
甜菊糖苷(甜菊糖苷类化合物)的结构式如下：
Figure PCTCN2022096683-appb-000001
序号 化合物 R 1 R 2
1 甜菊醇 H H
2 甜菊醇单糖苷 H β-Glc
3 甜菊醇双糖苷 H β-Glc-β-Glc(2-1)
4 甜茶苷 β-Glc β-Glc
5 甜菊苷(STV) β-Glc β-Glc-β-Glc(2-1)
6 莱鲍迪苷A(RA) β-Glc β-Glc-β-Glc(2-1)-β-Glc(3-1)
7 莱鲍迪苷B(RB) H β-Glc-β-Glc(2-1)-β-Glc(3-1)
8 莱鲍迪苷C(RC) β-Glc β-Glc-α-Rha(2-1)-β-Glc(3-1)
9 莱鲍迪苷D(RD) β-Glc-β-Glc(2-1) β-Glc-β-Glc(2-1)-β-Glc(3-1)
10 莱鲍迪苷E(RE) β-Glc-β-Glc(2-1) β-Glc-β-Glc(2-1)
11 莱鲍迪苷F(RF) β-Glc β-Glc-α-Xly(2-1)-β-Glc(3-1)
12 莱鲍迪苷M(RM) β-Glc-β-Glc(2-1)-β-Glc(3-1) β-Glc-β-Glc(2-1)-β-Glc(3-1)
13 杜可尔苷A β-Glc β-Glc-α-Rha(2-1)
14 莱鲍迪苷I(RI) β-Glc-β-Glc(3-1) β-Glc-β-Glc(2-1)-β-Glc(3-1)
上述甜菊糖苷类化合物，具有共同的糖苷配基：甜菊醇(Steviol)，区别在于C-13和C-19位置连接的糖基的数量和类型，主要包括甜菊苷(Stevioside)、莱鲍迪苷A(Rebaudioside A，Reb A)、莱鲍迪苷B、莱鲍迪苷C、莱鲍迪苷D、莱鲍迪苷E、莱鲍迪苷I、杜克苷、甜菊双糖苷等多种糖苷。甜菊的叶子能够累积多达10-20％(基于干重)甜菊糖苷。甜菊叶子中发现的主要糖苷是莱鲍迪苷A(2-10％)、甜菊苷(2-10％)和莱鲍迪苷C(1-2％)。其他糖苷，如莱鲍迪苷B、D、E、F和I，甜菊双糖苷和甜茶苷，以低得多的水平被发现(大约0-0.2％)。其中莱鲍迪苷A甜度最高，甜味也与蔗糖相近，是一种高甜度、低热量、易溶解、耐热、稳定的新型天然甜味剂，其含量或纯度也是衡量甜菊糖质量的主要指标。另外，莱鲍迪苷A也是合成莱鲍迪苷D(Rebaudioside D，RebD，RD)、莱鲍迪苷E(Rebaudioside E，RebE，RE)、莱鲍迪苷M(Rebaudioside M，RebM，RM)、莱鲍迪苷I(Rebaudioside I，RebI，RI)等甜菊糖类化合物的重要中间体。
虽然甜菊糖苷是一种高倍甜味剂，但存在后苦涩味这一缺点，严重限制了其在食品、饮料等对口感要求较高的领域中的应用。而引起甜菊糖苷后苦涩味的本质原因是其内在分子结构引起的，甜菊糖苷中的R 1和R 2基团上连接糖基数量越多口感越好。通常，发现甜菊苷比蔗糖甜110-270倍，莱鲍迪苷A为150至320倍，然而，即使在高度纯化的状态下，甜菊糖苷仍然具有不合需要的味道属性，如苦味、甜的余味、甘草味等。
莱鲍迪苷D是其中最有应用潜力的甜菊糖苷，与其它甜菊糖苷相比，其甜度高，约为蔗糖的300-350倍，且甜味纯正，口感也更接近蔗糖，没有苦味和甘草异味，稳定性好，是一种理想的天然高倍甜味剂产品。甜叶菊叶子中莱鲍迪苷D的含量极少(少于5％)，采用提取法生产莱鲍迪苷D需要大量的甜叶菊原料，另外富集莱鲍迪苷D的工艺繁琐，提取后需要多次过柱和脱盐、脱色、重结晶，并在生产过程中产生大量的废水，其生产成本较高，不适合工业化大生产。
目前生物酶法合成莱鲍迪苷D的方法需要外加昂贵的UDP-葡萄糖为底物之一，通过UDP-葡萄糖基转移酶(UDP-glucosyltransferase，简称UGT)的作用，并且以甜菊苷或莱鲍迪苷A为底物，催化生成莱鲍迪苷D。但由于UDP-葡萄糖极高的售价，几乎完全限制了工业化制备莱鲍迪苷D的可行性，经济性较差、缺乏市场竞争力。
莱鲍迪苷M(Rebaudioside M，RebM)具有更好的口感特性，但其占叶子干重的含量小于0.1％，导致分离成本高、价格昂贵。生物催化法获得高浓度的莱鲍迪苷M已引起了学者的关注。目前报道，来源甜叶菊的重组酶能催化莱鲍迪苷D生成莱鲍迪苷M，但产量较低。以莱鲍迪苷D为底物，通过微生物产酶催化法可获得莱鲍迪苷M，该方法较传统的提取法，不仅改善了生产流程，并且降低了对环境的污染，提高了目的产物莱鲍迪苷M的产率。但目前以生物酶催化法主要存在以下几个问题：(1)以生物酶催化莱鲍迪苷D生产莱鲍迪苷M的成本较高，并且酶催化产率有待进一步优化；(2)用于催化的糖基转移酶不易与产物分离并回收利用，且易失活；(3)天然植物中莱鲍迪苷A含量很高，而莱鲍迪苷D含量非常低，以低成本由莱鲍迪苷A直接转化为莱鲍迪苷D也是亟待解决的难题。
葡萄糖基转移酶是在酶反应中只转移葡萄糖基的酶，该酶的作用机理是催化糖基供体的葡萄糖残基转移到糖基受体分子上，从而调节受体分子的活性。
“核苷(nucleoside)”指包含核碱基(即含氮碱基)和5-碳糖(例如核糖或脱氧核糖)的糖基胺(glycosylamines)。核苷的非限制性实例包括胞苷(cytidine)、尿苷(uridine)、腺苷(adenosine)、鸟苷(guanosine)、胸苷(thymidine)和肌苷(inosine)。
“核苷二磷酸(nucleoside diphosphate)”指包含核碱基(即含氮碱基)、5-碳糖(例如核糖或脱氧核糖)和二磷酸(即焦磷酸)部分的糖基胺。“核苷二磷酸”可缩写为“NDP”。核苷二磷酸的非限制性实例包括胞苷二磷酸(CDP)、尿苷二磷酸(UDP)、腺苷二磷酸(ADP)、鸟苷二磷酸(GDP)、胸苷二磷酸(TDP)和肌苷二磷酸(IDP)。
UDP-葡萄糖基转移酶是葡萄糖基转移酶中的一种，以UDP-葡萄糖作为糖基供体，几乎存在于所有有机体中。UDP-葡萄糖是二磷酸尿苷葡糖(uridine diphosphate glucose)的简称，又简称为UDP-葡糖或者UDPG，是由尿苷二磷酸和葡萄糖组成的维生素，可看作“活性葡萄糖”，广泛分布于植物、动物和微生物的细胞内，在蔗糖、淀粉、糖原及其他寡糖和多糖合成中作葡萄糖基的供体，是最常见的一种糖基供体。除UDP-葡萄糖外，ADP-葡萄糖、TDP-葡萄糖、dTDP-葡萄糖(deoxythymidine diphosphate glucose)、CDP-葡萄糖、IDP-葡萄糖或GDP-葡萄糖等也常作为糖供体化合物。这些糖基供体价格昂贵，不利于工业化生产。
蔗糖合成酶(sucrose synthase，SUS，也简称SuSy/SS等，EC2.4.1.13)，其可逆地催化化学反应NDP-葡萄糖+D-果糖到NDP和蔗糖，属于糖基转移酶-4亚家族在小麦(Triticum aestivum)胚芽中首次被鉴定。每个蛋白以四聚体的形式存在。每个亚基分子量在90kDa左右。基因大小一般为5.9kb，cDNA大小为2.7kb左右。编码氨基酸序列全 长约800个氨基酸残基(Ross和Davies 1992)。蔗糖合成酶对NDP亲和力不同，亲和力大小依次为UDP>ADP>dTDP>CDP>GDP。
Ohta et al.(J.Appl.Glycosci.,57，199～209(2010))首次报道了从甜叶菊中分离到莱鲍迪苷I(Rebaudioside I，Reb I,RI)。Indra Prakash等(Molecules 2014,19,17345-17355)首次报道了以RA为原料，使用葡萄糖基转移酶UGT76G1的突变体UGT76G1-R11-F12制备RI的生物合成方法，该方法使用UDP-葡萄糖为糖基供体，在pH7.5、MgCl 2的条件下30℃摇瓶反应，RI收率为22.5％。Indra Prakash为发明人的专利申请CN106795523A中公布了葡萄糖基转移酶UGT76G1的GenBank登录号AAR06912.1，并公开了DNA序列。CN110914445A筛选了催化以RA为原料制备RI的葡萄糖基转移酶UGT76G1的突变体，筛选条件为pH7.0、MgCl 2 10.3mM、温度35℃。CN106795523A报道了RI的甜度数据和含RI的组合物。上述现有技术中仅公开了具有催化RA合成RI活性的葡萄糖基转移酶或其突变体，但是这些酶的活性较低，不适合工业化生产。
如今，随着天然甜味剂甜菊糖的广泛应用，以及生物催化技术的日益发展，UDP-葡萄糖基转移酶被越来越多地应用在甜菊糖苷的生物催化制备的领域中来。UDP-葡萄糖基转移酶的种类很多，目前甜菊糖苷的生物酶法制备领域中使用的酶多为来源于植物细胞中的野生酶，这种野生酶往往存在酶活低、稳定性差等缺点，从而导致应用于工业化大生产制备甜菊糖苷的成本较高。因此，有必要对UDP-葡萄糖基转移酶进行改造，从而获得酶活更高、稳定性更好的改造酶，以便更好地服务于工业化大生产。
发明内容
本发明所要解决的技术问题是现有的UDP-葡萄糖基转移酶被应用于甜菊糖苷的生物催化制备时酶活低、稳定性差因而用于催化甜菊糖苷时转化率不高等缺陷，因此本发明提供一种糖基转移酶(即，UDP-葡萄糖基转移酶)以及其在制备甜菊糖苷中的应用。本发明的糖基转移酶(GT)的酶活高、稳定性好；其中，蔗糖和UDP(和/或ADP)在SUS催化下生成UDPG(和/或ADPG)，从而实现UDPG(和或ADPG)再生，解决了糖基供体UDPG(和/或ADPG)价格昂贵的问题；将其用于制备甜菊糖苷(例如莱鲍迪苷A、莱鲍迪苷D、莱鲍迪苷M或莱鲍迪苷I)时与糖基转移酶亲本相比，在催化活性方面有了明显的提高，转化率显著提升，从而降低了反应的成本，为实现大规模工业化生产提供更多工艺条件优化的选择，利于工业化生产。
为了解决上述技术问题，本发明提供了一种糖基转移酶，所述糖基转移酶的氨基酸序列如SEQ ID NO:112或与SEQ ID NO:112具有至少99％序列同一性的氨基酸序列所 示。
较佳地，所述“与SEQ ID NO:112具有至少99％序列同一性的氨基酸序列”为与SEQ ID NO:112相比包含选自以下一个或多个的残基位置处的氨基酸残基差异：
V14I；
E99L；
T254G；
L257A；
Q451E；
Q265E；
P271A；
R333K；
R12Q；
A118S；
E418D；
S455R。
更佳地，在上述基础上，所述糖基转移酶的氨基酸序列与SEQ ID NO:112相比还可进一步包含选自以下残基位置处的氨基酸残基差异：K347P。
在某一较佳实施例中，所述糖基转移酶的氨基酸序列与SEQ ID NO:112相比包含选自以下一个残基位置处的氨基酸残基差异：V14I、E99L、T254G、L257A、Q451E、Q265E、P271A、R333K、R12Q、A118S、E418D或S455R。
在某一较佳实施例中，所述糖基转移酶的氨基酸序列与SEQ ID NO:112相比包含选自以下两个残基位置处的氨基酸残基差异：Q265E和P271A。
在某一较佳实施例中，所述糖基转移酶的氨基酸序列与SEQ ID NO:112相比包含选自以下两个残基位置处的氨基酸残基差异：R333K和K347P。
在某一较佳实施例中，编码所述糖基转移酶的核苷酸序列可选自以下的序列：SEQ ID NO:41、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:107和SEQ ID NO:108。
在一些实施方案中，糖基转移酶的氨基酸序列可在上述的特定位置以外的位置另外具有1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、1-11、1-12、1-13、1-14、1-15、1-16、1-17、1-18、1-19、1-20个氨基酸残基的差异；这些残基的差异包括以保守氨基酸残 基取代。通常这些取得不影响所述糖基转移酶的酶活。
为了解决上述技术问题，本发明提供了一种分离的核酸，所述核酸编码如上所述的糖基转移酶。
较佳地，所述核酸的核苷酸序列可选自以下的序列：SEQ ID NO:41、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:107和SEQ ID NO:108。
为了解决上述技术问题，本发明提供了一种重组表达载体，其包含如上所述的核酸。
为了解决上述技术问题，本发明提供了一种转化体，其为包含如上所述的核酸或如上所述的重组表达载体的宿主细胞。
所述宿主细胞可为本领域常规，较佳地为埃希氏大肠杆菌(Escherichia coli)。
为了解决上述技术问题，本发明提供了一种制备如上所述的糖基转移酶的方法，所述方法包括在适于表达所述糖基转移酶的条件下培养如上所述的转化体。
所述转化体表达糖基转移酶后，可采用本领域常规技术手段进行提取，例如可制备粗酶液，粗酶液制备后可进行常规的浓缩、置换，也可将粗酶液进一步经离子交换层析、亲和层析、疏水层析和分子筛层析等纯化步骤中的一种或多种以提纯所述糖基转移酶。在某一较佳实施例中，可采用以下步骤：(1)将含所述糖基转移酶的转化体接种至含抗生素的培养基例如LB培养基中振荡培养，得种子液；(2)将(1)中的种子液转接至含抗生素的培养基例如TB培养基中振荡培养；(3)向(2)中的培养基中加入IPTG诱导过夜，离心后收集菌体；(4)洗涤并重悬(3)中收集的菌体，破碎后离心，即得含所述糖基转移酶的粗酶液。
为了解决上述技术问题，本发明提供了一种组合物，其包含如上所述的糖基转移酶。所述组合物例如可以酶制剂的形式存在。
为了解决上述技术问题，本发明提供了一种用于底物的糖基化的方法，所述方法包括提供至少一种底物、如上所述的糖基转移酶，并在使得所述底物被糖基化以产生至少一种糖基化产物的条件下使所述底物与所述糖基转移酶接触；所述底物优选包括至少一种甜菊糖苷例如甜菊苷、莱鲍迪苷A或莱鲍迪苷D等。
为了解决上述技术问题，本发明提供了一种莱鲍迪苷A的制备方法，所述制备方法包括以下步骤：在如上所述的糖基转移酶的存在下，将甜菊苷(Stevioside)和糖基供体进行反应(例如在使得甜菊苷被糖基化以产生莱鲍迪苷A的条件下)，即得莱鲍迪苷A。
较佳地，所述糖基转移酶以糖基转移酶菌泥的形式存在。
较佳地，所述甜菊苷的浓度1-150g/L，优选100g/L。
较佳地，所述糖基供体与甜菊苷的摩尔比为1:1～5:1例如2.5:1。
较佳地，所述糖基供体为UDP-葡萄糖。在某一较佳实施例中，使用的糖基供体优选通过蔗糖和UDP在蔗糖合成酶的存在下制得，所述蔗糖的浓度优选为100-300g/L例如200g/L，所述UDP的浓度优选为0.05-0.2g/L例如0.1g/L。合酶(例如蔗糖合酶或海藻糖合酶)通常以相反的方向作用，以从核苷二磷酸和葡萄糖供体(例如蔗糖、海藻糖或淀粉)形成核苷二磷酸葡萄糖化合物。本发明中，所述的蔗糖合成酶(又称为蔗糖合酶，Sucrose synthase，简称为SuSy或SUS)催化可逆的葡萄糖基转移反应，通过蔗糖合成酶催化蔗糖和UDP产生UDP-葡萄糖，有效地为UDP-糖基转移酶提供了UDP-糖供体。在蔗糖合成酶和UDP-糖基转移酶构建的反应体系中，产生UDP循环，实现UDP-葡萄糖的再生，避免直接使用昂贵的UDP-葡萄糖，降低了生产成本。
较佳地，所述反应的反应溶剂的pH为5-8，优选6。
较佳地，所述反应时的转速为500-1000rpm，优选600rpm。
较佳地，所述反应的反应体系的温度为20-90℃，优选60℃。
为了解决上述技术问题，本发明提供了一种莱鲍迪苷D或莱鲍迪苷M的制备方法，其包括根据如上所述的制备方法制备莱鲍迪苷A的步骤。
在某一较佳实施例中，所述方法包括提供甜菊苷底物、糖基供体和如前所述的糖基转移酶，在使得产生莱鲍迪苷D或莱鲍迪苷M的条件下将甜菊苷底物、糖基供体和如前所述的糖基转移酶反应。
为了解决上述技术问题，本发明提供了一种莱鲍迪苷I的制备方法，在糖基转移酶的存在下将莱鲍迪苷A和糖基供体进行反应，所述糖基转移酶如上所述。
所述制备方法中，所述糖基转移酶以糖基转移酶菌体、粗酶液、纯酶、纯酶液或固定化酶的形式存在。
所述制备方法中，所述糖基供体为UDP-葡萄糖和/或ADP-葡萄糖。
所述制备方法中，所述莱鲍迪苷A的浓度较佳地为1-150g/L，例如100g/L、50g/L、80g/L、120g/L。
所述制备方法中，所述糖基转移酶菌体与莱鲍迪苷A的质量比较佳地为1:(3-10)，例如3:20、1:5、1:8。
所述制备方法中，所述反应的反应溶剂的pH优选为5-8，更优选5.5-6。
所述制备方法中，所述反应的反应体系的温度优选为20-90℃，例如60℃、30℃、50℃、70℃。
所述制备方法中，所述反应的反应时间优选为5-30h，例如24h、10h、15h、20h、28h。
所述制备方法中，所述糖基供体较佳地通过UDP和/或ADP在蔗糖和蔗糖合成酶的存在下制得。
所述蔗糖的浓度较佳地为100-300g/L，例如200g/L、150g/L、250g/L。
所述UDP或所述ADP的浓度较佳地为0.05-0.2g/L，例如0.1g/L。
所述制备方法中，所述pH较佳地由缓冲溶液控制，所述缓冲溶液例如为磷酸缓冲溶液。
所述制备方法中，所述反应的转速较佳地为500-1000rpm，例如600rpm、800rpm。
为了解决上述技术问题，本发明提供了一种如上所述的糖基转移酶在制备甜菊糖苷中的用途；所述甜菊糖苷优选为莱鲍迪苷A、莱鲍迪苷D、莱鲍迪苷M或莱鲍迪苷I。
为了解决上述技术问题，本发明提供了一种催化酶组合物，其包括糖基转移酶与蔗糖合成酶，所述糖基转移酶如上所述，所述蔗糖合成酶的序列如SEQ ID NO:32所示。
所述催化酶组合物中，所述糖基转移酶与蔗糖合成酶的质量比较佳地为(2-10):1，例如5:1、3:1、8:1。
为了解决上述技术问题，本发明提供了一种如上所述的催化酶组合物在制备莱鲍迪苷A、莱鲍迪苷D、莱鲍迪苷M或莱鲍迪苷I中的应用。
本发明中，所述的甜菊糖苷(Steviol glycosides)又可称为甜菊醇糖苷，其结构式和所包含的化合物种类具体可参见本发明的背景技术部分。
本发明的积极进步效果在于：
本发明的糖基转移酶的酶活高、稳定性好；将其用于制备甜菊糖苷(例如莱鲍迪苷A、莱鲍迪苷D、莱鲍迪苷M或莱鲍迪苷I)时与糖基转移酶亲本相比，在催化活性方面有了明显的提高，转化率显著提升，解决了糖基供体UDPG(和/或ADPG)价格昂贵的问题，从而降低了反应的成本，为实现大规模工业化生产提供更多工艺条件优化的选择，利于工业化生产。
附图说明
图1显示了本发明实施例1～7中由甜菊苷制备莱鲍迪苷A、莱鲍迪苷D、莱鲍迪苷M的路线示意图。
图2显示了本发明的实施例1～7中的酶催化反应体系合成莱鲍迪苷A，实现UDPG在生物催化反应中的循环。
图3为实施例1～7中第二轮筛选时所用底物甜菊苷的图谱，甜菊苷的保留时间为12.76min。
图4为实施例1～7中产物莱鲍迪苷A对照品的图谱，保留时间12.38min。
图5是表10中复筛Enz.11催化合成RA活性的HPLC图。
图6是表10中复筛Enz.24催化合成RA活性的HPLC图。
图7为由甜菊苷制备莱鲍迪苷A、莱鲍迪苷I的合成路线。
图8为使用实施例8～11中的HPLC检测方法，莱鲍迪苷A对照品的图谱。
图9为使用实施例8～11中的HPLC检测方法，莱鲍迪苷I对照品的图谱。
图10为实施例11中反应8小时的HPLC图谱。
图11为实施例11中反应24小时的HPLC图谱。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
本发明中的实验方法如无特别说明均为常规方法，基因克隆操作具体可参考J.萨姆布鲁克等编的《分子克隆实验指南》。
本发明中的氨基酸简写符号如无特殊说明均为本领域常规，具体简写符号对应的氨基酸如表1所示。
表1
Figure PCTCN2022096683-appb-000002
Figure PCTCN2022096683-appb-000003
所述氨基酸对应的密码子也为本领域常规，具体氨基酸与密码子的对应关系如表2所示。
表2
Figure PCTCN2022096683-appb-000004
KOD Mix酶购自TOYOBO CO.，LTD.，DpnI酶购买自英潍捷基(上海)贸易有限公司；E.coli Trans10感受态细胞购买自北京鼎国昌盛生物技术有限责任公司，E.coli BL21(DE3)感受态细胞购买自北京鼎国昌盛生物技术有限责任公司。蔗糖购自生工生物工程(上海)股份有限公司。实施例1～7中第一轮筛选所用反应底物和实施例8～11的RA60(甜菊苷，其中RA含量60％，甜菊苷含量约30％)购自晨光生物，产品规格TSG90/RA60。第二轮筛选所用反应底物甜菊苷购自毕得医药(纯度95％)。Reb A和Reb I对照品购自麦克林。
实施例1～7中转化率HPLC检测方法：色谱柱：Agilent 5 TC-C18(2)(250×4.6mm)。流动相：0.1％FA水溶液为流动相A，0.1％FA乙腈溶液为流动相B，按下表3进行梯度洗脱。检测波长：210nm；流速：1ml/min；进样体积：20μl；柱温：40℃。甜菊苷出峰时间：12.76min；Reb A出峰时间：12.38min。
表3
时间(分钟) 流动相A％ 流动相B％
0.00 70 30
15.00 60 40
20.00 30 70
25.00 30 70
25.10 70 30
32.00 70 30
实施例8～11中转化率HPLC检测方法：色谱柱：ZORBAXEclipse plus C18(4.6mm*150mm，3.5um)。流动相：0.1％TFA水溶液为流动相A，0.1％TFA乙腈溶液为流动相B，按下表4进行梯度洗脱。检测波长：210nm；流速：1ml/min；进样体积：20μL；柱温：35℃。如图8所示，Reb A出峰时间：14.504min；如图9所示，Reb I出峰时间：14.216min。
表4
Time(min) A％ B％
0.00 90 10
15.00 60 40
20.00 0 100
24.00 0 100
24.10 90 10
32.00 90 10
实施例1～7主要以UDP-葡萄糖基转移酶亲本Enz.1为模板，第一轮筛选出了308位点突变为N的糖基转移酶突变体Enz.11，以Enz.11为模板进行第二轮筛选，筛选出来了双位点或三位点突变的UDP-葡萄糖基转移酶。
实施例1 第一轮GT010-308突变体文库的构建
全合成SEQ ID NO:1所示的编号为Enz.1的β-1,3-糖基转移酶(β-1,3-GT酶)酶基因，该基因已连接在pET28a质粒载体上，得到重组质粒pET28a-GT010，基因合成公司为生工生物工程(上海)股份有限公司(上海市松江区香闵路698号)。Enz.1的氨基酸序列如SEQ ID NO:2所示。
以pET28a-GT010质粒为模板，采用表5所示的引物序列，分别以GT010-L308X-F/ET-R和ET-F/GT010-L308X-R为引物(其中X为：A、D、E、G、H、I、K、M、N、P、S、V、W)，采用KOD酶进行PCR扩增目标DNA片段和载体片段。
表5
Figure PCTCN2022096683-appb-000005
Figure PCTCN2022096683-appb-000006
PCR扩增反应体系为：
试剂 用量(μL)
KOD Mix酶 25
引物F 2
引物R 2
模板 1
去离子水 20
扩增程序如下：
Figure PCTCN2022096683-appb-000007
对PCR产物进行DpnI消化并进行跑胶及胶回收得到目标DNA片段。通过诺唯赞的 两片段同源重组酶(Exnase II)连接。连接后转化至E.coli Trans10感受态细胞，涂布在含有50μg/mL卡纳霉素的LB培养基，37℃培养过夜；挑点培养并进行测序，得到含有突变体基因的重组质粒。
实施例2 UDP-葡萄糖基转移酶突变体的制备
1.进行突变载体的蛋白表达：
将测序正确的上述重组质粒转化至宿主E.coli BL21(DE3)感受态细胞，得到含有点突变的基因工程菌株。挑单菌落接种至含50μg/ml卡那霉素的5ml LB液体培养基中，37℃震荡培养4h。按2v/v％接种量转接至50ml同样含50μg/ml卡那霉素的新鲜TB液体培养基中，37℃震荡培养至OD 600达到0.6-0.8时，加入IPTG(异丙基-β-D-硫代半乳糖苷，Isopropylβ-D-thiogalactoside)至其终浓度为0.1mM，25℃诱导培养20h。培养结束后，将培养液10000rpm离心10min，弃上清液，收集菌体(即，菌泥)。-20℃保存备用。
2.粗酶液的获取：
配制50mM pH6.0的磷酸缓冲液(PBS)，将上述所得菌泥按照(M/V)1:5进行悬浮，之后，进行均质获得粗酶液，粗酶液经离心，取上清获得UDP-葡萄糖基转移酶突变体的粗酶液。
实施例3 蔗糖合成酶SUS的制备
全合成SEQ ID NO:31所示的编号为Enz.2的蔗糖合成酶(SUS)基因，该基因已连接在pET28a质粒载体上得到重组质粒pET28a-SUS。基因合成公司为生工生物工程(上海)股份有限公司(上海市松江区香闵路698号)。蔗糖合酶的氨基酸序列如SEQ ID NO:32所示。
将质粒pET28a-SUS转化至宿主E.coli BL21(DE3)感受态细胞，得到Enz.2基因工程菌株。挑单菌落接种至含50μg/ml卡那霉素的5ml LB液体培养基中，37℃震荡培养4h。按2v/v％接种量转接至50ml同样含50μg/ml卡那霉素的新鲜TB液体培养基中，37℃震荡培养至OD 600达到0.6-0.8时，加入IPTG至其终浓度为0.1mM，25℃诱导培养20h。培养结束后，将培养液10000rpm离心10min(用于实施例4～7)或4000rpm离心20min(用于实施例8～11)，弃上清液，收集菌体。-20℃保存备用。
对于实施例4～7，配制50mM pH6.0的磷酸缓冲液(PBS)，将上述所得Enz.2菌泥按照(M/V)1:5进行悬浮，之后，进行均质获得粗酶液，粗酶液经离心，取上清获得蔗 糖合成酶SUS(酶编号Enz.2,氨基酸序列如SEQ ID NO:32所示)的粗酶液。
对于实施例8～11，配制50mM pH6.0的磷酸缓冲液(PBS)，将上述所得菌泥按照1:10(M/V、g/mL)进行悬浮，之后，进行高压均质(550Mbar、1.5min)后经12000rpm离心2min即获得蔗糖合成酶反应酶液。
实施例4 第一轮突变体的筛选
将实施例2和实施例3中得到的粗酶液分别进行80℃恒温孵育20min，离心取上清即分别获得UDP-葡萄糖基转移酶突变体反应酶液和蔗糖合成酶反应酶液。
以RA60为底物，1mL反应体系中，加入UDP-葡萄糖基转移酶突变体的反应酶液150μL，RA60终浓度为100g/L，UDP终浓度为0.1g/L，蔗糖终浓度为200g/L，蔗糖合成酶反应酶液30μL，最后加入50mM pH6.0磷酸缓冲液至终体积1mL。将配制好的反应体系置于金属浴中，60℃，600rpm下反应60min，反应液稀释50倍，进行HPLC分析Reb A的浓度(详见表6的Reb A％)。(蔗糖合成酶是用于将蔗糖上葡萄糖基转移至UDP上合成UDPG)。初筛结果如表6所示。
表6
酶编号 突变位点 Reb A％ DNA SEQ ID NO:
Enz.1 / 93.787 2
Enz.3 L308A 87.771％ 33
Enz.4 L308D 72.570％ 34
Enz.5 L308E 74.487％ 35
Enz.6 L308G 81.027％ 36
Enz.7 L308H 89.885％ 37
Enz.8 L308I 83.863％ 38
Enz.9 L308K 75.395％ 39
Enz.10 L308M 84.221％ 40
Enz.11 L308N 96.157％ 41
Enz.12 L308P 75.643％ 42
Enz.13 L308S 72.799％ 43
Enz.14 L308V 87.985％ 44
Enz.15 L308W 91.806％ 45
由表6中的初筛结果可知：相对于Enz.1,Enz.11催化效果较好，所得Reb A的产率较高。
2.复筛
采用Boil和UnBoil两种反应条件进行复筛，Boil复筛反应条件和初筛反应条件相同，UnBoil指不加热条件下反应，其余反应条件和Boil反应条件相同。复筛结果如表7所示(表中％数值对应反应液中Reb A的含量)。
表7
酶编号 Enz.3 Enz.7 Enz.1 Enz.11 Enz.14 Enz.15
Boil 84.212％ 85.939％ 92.314％ 92.100％ 83.793％ 87.168％
UnBoil 79.577％ 82.266％ 84.522％ 83.475％ 80.686％ 81.180％
由表7中的复筛结果可知：相对于Enz.1，Enz.11效果最好，其反应效果与Enz.1相当。
实施例5 第二轮突变体文库的构建
将第一轮得到的编码糖基转移酶(β-1,3-GT酶)Enz.11的基因连接载体pET28a，得到pET28a-Enz.11重组质粒，以pET28a-Enz.11为模板，采用表8所示的引物序列，采用KOD酶进行PCR扩增，获得目标突变体Enz.16-Enz.34的基因片段和载体片段。
以pET28a-Enz.34质粒为模板，GT029-L378G-F/Km-R和Km-F/GT029-L378G-R为引物序列，PCR扩增目标突变体Enz.35的基因片段和载体片段。
表8
Figure PCTCN2022096683-appb-000008
Figure PCTCN2022096683-appb-000009
Figure PCTCN2022096683-appb-000010
PCR扩增反应体系为表9-1所示：
表9-1
试剂 用量(μL)
KOD Mix 25
引物F 2
引物R 2
模板 1
去离子水 20
扩增程序如下表9-2所示：
表9-2
Figure PCTCN2022096683-appb-000011
对PCR产物进行DpnI消化并进行跑胶及胶回收。采用诺唯赞两片段同源重组酶进行连接。连接完成转化至E.coli Trans10感受态细胞，涂布在含有50μg/mL卡纳霉素的LB培养基，37℃培养过夜；挑点培养并进行测序，得到含有突变体基因的重组质粒。
实施例6 UDP-葡萄糖基转移酶突变体的制备
1.进行突变载体的蛋白表达：
将实施例5中测序正确的重组质粒转化至宿主E.coli BL21(DE3)感受态细胞，得到含有点突变的基因工程菌株。挑单菌落接种至含50μg/ml卡那霉素的5ml LB液体培养基中，37℃震荡培养4h。按2v/v％接种量转接至50ml同样含50μg/ml卡那霉素的新鲜TB液体培养基中，37℃震荡培养至OD 600达到0.6-0.8时，加入IPTG至其终浓度为0.1mM，25℃诱导培养20h。培养结束后，将培养液4000rpm离心20min，弃上清液，收集菌体(即菌泥)。-20℃保存备用。
2.粗酶液的获取：
配制50mM pH6.0的磷酸缓冲液(PBS)，将上述所得菌泥按照(M/V)1:10进行悬浮，之后，进行均质获得粗酶液，粗酶液经离心，取上清获得UDP-葡萄糖基转移酶突变体的粗酶液。-4℃保存备用。
实施例7 第二轮突变体的筛选
1.初筛
将实施例6和实施例2中得到的粗酶液分别进行80℃恒温孵育15min，离心取上清即分别获得UDP-葡萄糖基转移酶反应酶液和蔗糖合成酶反应酶液。
以甜菊苷(甜菊苷含量95％，毕得医药)为底物，1mL反应体系中，加入UDP-葡萄糖基转移酶突变体的反应酶液150μL，甜菊苷终浓度为100g/L，UDP终浓度为0.1g/L，蔗糖终浓度为200g/L，蔗糖合成酶30μL，最后加入50mM pH6.0磷酸缓冲液至终体积1mL。将配制好的反应体系置于金属浴中，60℃，600rpm下反应60min，稀释50倍，进行HPLC分析Reb A的浓度。以Enz.1和Enz.11作为双对照对20个突变体进行筛选。初筛结果如表10所示。
表10
Figure PCTCN2022096683-appb-000012
Figure PCTCN2022096683-appb-000013
表中，“/”表示同时存在不同位点的突变。
由表10中的初筛结果可知：Enz.17、Enz.18、Enz.21、Enz.22、Enz.24、Enz.26、Enz.27、Enz.28、Enz.31、Enz.32均优于对照10％以上，选择这些突变体进行复筛。此外，前述反应是将粗酶液进行80℃恒温孵育15min，离心取上清获得反应酶液再进行的反应，可以看出酶的稳定性很好。
2.复筛
复筛反应条件和初筛反应条件相同。复筛结果如表11所示。
表11
酶 Reb A％
Enz.1 47.066
Enz.11 61.639
Enz.17 73.639
Enz.18 72.940
Enz.21 66.063
Enz.22 74.680
Enz.24 76.903
Enz.26 72.557
Enz.27 63.514
Enz.28 61.659
Enz.31 66.672
Enz.32 59.815
由表11中的复筛结果确认Enz.17、Enz.18、Enz.21、Enz.22、Enz.24、Enz.26、Enz.27、Enz.28、Enz.31、Enz.32均优于亲本对照10％以上。与UDP-葡萄糖基转移酶亲本Enz.1相比，上述所得UDP-葡萄糖基转移酶突变体在催化活性方面有了明显的提高。
图1显示了本发明的实施例中由甜菊苷制备莱鲍迪苷A、莱鲍迪苷D、莱鲍迪苷M的路线示意图；图2显示了本发明的实施例中的酶催化反应体系合成莱鲍迪苷A，实现UDPG在生物催化反应中的循环。图3为第二轮筛选时所用底物甜菊苷的图谱，保留时间12.76min。图4为产物莱鲍迪苷A对照品的图谱，保留时间12.38min。图5是表11中复筛Enz.11催化合成RA活性的HPLC图；图6是表11中复筛Enz.24催化合成RA活性的HPLC图。
实施例8～12的路线示意图如图7所示。
实施例8 第一轮β-1,3-糖基转移酶突变体文库的构建
如实施例1，将全合成的如SEQ ID NO:1所示的编号为Enz.1的β-1,3-糖基转移酶(β-1,3-GT酶)酶基因，连接在pET28a质粒载体上，得到重组质粒pET28a-Enz.1，基因合成公司为生工生物工程(上海)股份有限公司(上海市松江区香闵路698号)。Enz.1的氨基酸序列如SEQ ID NO:2所示。重组质粒转化至宿主大肠杆菌BL21(DE3)感受态细胞，得到含有转氨酶Enz.1基因的工程菌株。
同样地，将表12中根据Enz.1基因进行定点突变得到的转氨酶Enz.2.2～Enz.2.6的基因，以及Enz.16、Enz.19、Enz.23、Enz.25、Enz.27、Enz.31、Enz.32和Enz.35的 基因，酶切位点NdeI、HindIII，连接载体pET28a，获得分别含有转氨酶Enz.2.2～Enz.2.6，以及Enz.16、Enz.19、Enz.23、Enz.25、Enz.27、Enz.31、Enz.32和Enz.35的基因的各重组质粒。各重组质粒分别转化至宿主大肠杆菌BL21(DE3)感受态细胞，得到表12含有转氨酶基因的工程菌株。
表12
Figure PCTCN2022096683-appb-000014
实施例9 β-1,3-糖基转移酶突变体的制备
进行突变载体的蛋白表达：
分别将实施例8中菌株挑单菌落接种至含50μg/mL卡那霉素的5ml LB液体培养基中，37℃震荡培养4h。按2％(v/v)接种量转接至50ml同样含50μg/mL卡那霉素的新鲜TB液体培养基中，37℃震荡培养至OD 600达到0.8左右时，加入IPTG(异丙基-β-D-硫代半乳糖苷，Isopropylβ-D-thiogalactoside)至其终浓度为0.1mM，25℃诱导培养20h。培养结束后，将培养液4000rpm离心20min，弃上清液，收集菌体。-20℃保存备用。
实施例10 β-1,3-糖基转移酶突变体的筛选
反应酶液的获取：
配制50mM pH 6.0的磷酸缓冲液(PBS)，分别将上述实施例6和9所得菌体按照1:10(M/V、g/mL)进行悬浮，之后使用高压均质机进行均质(550Mbar均质1.5min)；将均质后的酶液分别进行12000rpm离心2min即获得各β-1,3-糖基转移酶反应酶液。
1mL反应体系中，加入β-1,3-糖基转移酶的反应酶液150μL，RA60终浓度为50g/L，ADP终浓度为0.1g/L，蔗糖终浓度为200g/L，实施例3制得的蔗糖合成酶反应酶液30μL，最后加入50mM pH 6.0磷酸缓冲液至终体积1mL。将配制好的反应体系置于金属 浴中，60℃，600rpm下反应30min，取10μL反应液加入990μL的pH 2～3的盐酸中，涡旋，13000rpm离心10min，上清进行HPLC分析Reb I的浓度。使用HPLC检测方法获得的实验结果如表13所示。
表13
酶编号 RI％(RA→RI)
Enz.1 2.886
Enz.2.2 0.555
Enz.2.3 1.705
Enz.2.4 1.324
Enz.2.5 0.886
Enz.2.6 1.427
Enz.16 4.139
Enz.19 4.014
Enz.23 3.628
Enz.25 3.910
Enz.27 4.235
Enz.31 4.646
Enz.32 3.546
Enz.35 1.939
由表13中的初筛结果可知：Enz.31的酶活最高，因此后续选择Enz.31进行实验。
实施例11 酶Enz.31催化合成RI
1mL反应体系中，加入Enz.31的反应酶液150μL，蔗糖合成酶反应酶液30μL，RA60终浓度为100g/L，蔗糖终浓度为200g/L，ADP终浓度为0.1g/L，最后加入PBS(50mM,pH 5.5)至终体积1mL。将配制好的反应体系置于金属浴中，分别在60℃，600rpm下反应，分别在1h、2h、3h、4h、5h、6h、7h、8h、24h取10μL反应液加入990μL的pH2～3的盐酸中，涡旋，13000rpm离心10min，上清进行HPLC分析。使用HPLC检测方法获得的实验结果如表14所示。
表14
时间/h 1 2 3 4 5 6 7 8 24
RI％ 17.62 26.18 39.47 46.92 55.86 61.70 67.71 71.36 97.81
表14中的反应结果显示：反应8h后，RI的峰面积比例达71％，反应24h后，RI的峰面积比例已达97％，反应基本完全。
图10是表14中反应8小时实验结果的HPLC图。图11是表14中反应24小时实验结果的HPLC图。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (18)

  1. 一种糖基转移酶，其特征在于，所述糖基转移酶的氨基酸序列如SEQ ID NO:112或与SEQ ID NO:112具有至少99％序列同一性的氨基酸序列所示。
  2. 如权利要求1所述的糖基转移酶，其特征在于，所述与SEQ ID NO:112具有至少99％序列同一性的氨基酸序列为与SEQ ID NO:112相比包含选自以下一个或多个的残基位置处的氨基酸残基差异：
    V14I；
    E99L；
    T254G；
    L257A；
    Q451E；
    Q265E；
    P271A；
    R333K；
    R12Q；
    A118S；
    E418D；
    S455R；
    较佳地，所述糖基转移酶的氨基酸序列与SEQ ID NO:112相比还包含选自以下残基位置处的氨基酸残基差异：K347P。
  3. 如权利要求2所述的糖基转移酶，其特征在于，所述糖基转移酶的氨基酸序列与SEQ ID NO:112相比包含选自以下一个残基位置处的氨基酸残基差异：V14I、E99L、T254G、L257A、Q451E、Q265E、P271A、R333K、R12Q、A118S、E418D或S455R；
    或，所述糖基转移酶的氨基酸序列与SEQ ID NO:112相比包含选自以下残基位置处的氨基酸残基差异：Q265E和P271A；
    或，所述糖基转移酶的氨基酸序列与SEQ ID NO:112相比包含选自以下残基位置处的氨基酸残基差异：R333K和K347P。
  4. 如权利要求1～3任一项所述的糖基转移酶，其特征在于，编码所述糖基转移酶的核苷酸序列选自以下的序列：SEQ ID NO:41、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO: 107和SEQ ID NO:108。
  5. 一种分离的核酸，其特征在于，所述核酸编码如权利要求1～4任一项所述的糖基转移酶；
    较佳地，所述核酸的核苷酸序列选自以下的序列：SEQ ID NO:41、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:107、SEQ ID NO:108。
  6. 一种重组表达载体，其包含如权利要求5所述的核酸。
  7. 一种转化体，其为包含如权利要求5所述的核酸或如权利要求6所述的重组表达载体的宿主细胞；较佳地，所述宿主细胞为埃希氏大肠杆菌(Escherichia coli)。
  8. 一种制备如权利要求1～4任一项所述的糖基转移酶的方法，其特征在于，所述方法包括在适于表达所述糖基转移酶的条件下培养如权利要求7所述的转化体。
  9. 一种组合物，其包含如权利要求1～4任一项所述的糖基转移酶。
  10. 一种用于底物的糖基化的方法，所述方法包括提供至少一种底物、如权利要求1～4任一项所述的糖基转移酶，并在使得所述底物被糖基化以产生至少一种糖基化产物的条件下使所述底物与所述糖基转移酶接触。
  11. 一种莱鲍迪苷A的制备方法，其特征在于，所述制备方法包括以下步骤：在如权利要求1～4任一项所述的糖基转移酶的存在下，将甜菊苷和糖基供体进行反应，即得莱鲍迪苷A；
    较佳地：
    所述糖基转移酶以糖基转移酶菌泥的形式存在；
    和/或，所述甜菊苷的浓度1-150g/L，优选100g/L；
    和/或，所述糖基供体与甜菊苷的摩尔比为1:1～5:1；
    和/或，所述糖基供体为UDP-葡萄糖；优选通过蔗糖和UDP在蔗糖合成酶的存在下制得，所述蔗糖的浓度优选为100-300g/L例如200g/L，所述UDP的浓度优选为0.05-0.2g/L例如0.1g/L；
    和/或，所述反应的反应溶剂的pH为5-8，优选6；
    和/或，所述反应时的转速为500-1000rpm，优选600rpm；
    和/或，所述反应的反应体系的温度为20-90℃，优选60℃。
  12. 一种莱鲍迪苷D或莱鲍迪苷M的制备方法，其特征在于，其包括根据如权利要求11所述的制备方法制备莱鲍迪苷A的步骤。
  13. 一种莱鲍迪苷I的制备方法，在糖基转移酶的存在下将莱鲍迪苷A和糖基供体进行反应，其特征在于，所述糖基转移酶如权利要求1～4任一项所述，所述制备方法满足以下条件中的一种或多种：
    所述糖基转移酶以糖基转移酶菌体、粗酶液、纯酶、纯酶液或固定化酶的形式存在；
    所述糖基供体为UDP-葡萄糖和/或ADP-葡萄糖；
    所述莱鲍迪苷A的浓度为1-150g/L，优选100g/L；
    所述糖基转移酶菌体与莱鲍迪苷A的质量比为1:(3-10)，优选3:20；
    所述反应的反应溶剂的pH为5-8，优选5.5-6；
    所述反应的反应体系的温度为20-90℃，优选60℃；
    所述反应的反应时间为5-30h，优选24h。
  14. 如权利要求13所述的制备方法，其特征在于，所述糖基供体通过UDP和/或ADP在蔗糖和蔗糖合成酶的存在下制得；
    和/或，所述蔗糖的浓度为100-300g/L、优选200g/L，所述UDP或所述ADP的浓度为0.05-0.2g/L、优选0.1g/L；
    和/或，所述pH由缓冲溶液控制，所述缓冲溶液优选磷酸缓冲溶液；
    和/或，所述反应的转速为500-1000rpm，优选600rpm。
  15. 一种如权利要求1～4任一项所述的糖基转移酶在制备甜菊糖苷中的用途；所述甜菊糖苷优选为莱鲍迪苷A、莱鲍迪苷D、莱鲍迪苷M或莱鲍迪苷I。
  16. 一种催化酶组合物，其包括糖基转移酶与蔗糖合成酶，所述糖基转移酶如权利要求1～4任一项所述，所述蔗糖合成酶的序列如SEQ ID NO:32所示。
  17. 如权利要求16所述的催化酶组合物，其特征在于，所述糖基转移酶与蔗糖合成酶的质量比为(2-10):1、优选5:1。
  18. 如权利要求16或17所述的催化酶组合物在制备莱鲍迪苷A、莱鲍迪苷D、莱鲍迪苷M或莱鲍迪苷I中的应用。
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