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WO2023030065A1 - Glycosyltransférase et son application - Google Patents

Glycosyltransférase et son application Download PDF

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Publication number
WO2023030065A1
WO2023030065A1 PCT/CN2022/113874 CN2022113874W WO2023030065A1 WO 2023030065 A1 WO2023030065 A1 WO 2023030065A1 CN 2022113874 W CN2022113874 W CN 2022113874W WO 2023030065 A1 WO2023030065 A1 WO 2023030065A1
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amino acid
glycosyltransferase
rebaudioside
enz
reaction
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Chinese (zh)
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吴燕
田振华
郑孝富
王舒
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Abiochem Biotechnology Co Ltd
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Abiochem Biotechnology Co Ltd
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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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
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
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    • 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
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

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 D, Reb D), rebaudioside E, dulcoside, and steviolbioside glycosides.
  • Stevia leaves are capable of accumulating as much as 10-20% (dry weight basis) steviol glycosides.
  • glycosides found in Stevia leaves are rebaudioside A (2-10%), stevioside (2-10%) and rebaudioside C (1-2%).
  • Other glycosides such as rebaudiosides B, D, E and F, steviolbioside and rubusoside, were found at much lower levels (approximately 0-0.2%).
  • 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.
  • UDP-glucosyltransferase is a kind of glucosyltransferase, which uses UDP-glucose as a glycosyl donor and exists in almost all organisms.
  • glucosyltransferase is more and more used in the field of biocatalytic preparation of steviol glycosides.
  • the enzymes used in the field of biological enzymatic preparation of steviol glycosides often have disadvantages such as low enzyme activity and poor stability, which lead to high costs for the preparation of steviosides in large-scale industrial production. Therefore, it is necessary to modify the glucosyltransferase to obtain a modified enzyme with higher enzyme activity and better stability, so as to better serve industrial production.
  • the technical problem to be solved by the present invention is that when the existing 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 and its application in the preparation of steviol glycosides.
  • Glycosyltransferase (GT) of the present invention has high enzymatic activity and good stability; Compared with the transferase parent, the catalytic activity has been significantly improved, and the conversion rate has been significantly improved, thereby reducing the cost of the reaction and facilitating industrial production.
  • the first aspect of the present invention provides a glycosyltransferase, the glycosyltransferase comprises amino acid residues selected from one or more of the following residue positions compared with SEQ ID NO: 2 difference:
  • the 14th amino acid is I;
  • the 189th amino acid is L;
  • the 257th amino acid is A, C, L, M, S or V;
  • the 265th amino acid is E or A;
  • the 273rd amino acid is G;
  • the 302nd amino acid is G;
  • the 324th amino acid is G;
  • the 347th amino acid is G;
  • the 451st amino acid is E;
  • the 455th amino acid is D or C;
  • the difference can be obtained by mutation on the amino acid sequence shown in SEQ ID NO: 2, or on the basis of other amino acid sequences, as long as the final mutation result is the same as that shown in SEQ ID NO: If the amino acid sequence shown in 2 has the above-mentioned difference, it also falls within the protection scope of the present invention.
  • amino acid residue difference of the glycosyltransferase compared with SEQ ID NO: 2 is selected from the following groups:
  • the 265th amino acid is E; or,
  • the 265th amino acid is E, and the 451st amino acid is E;
  • amino acid at position 14 is I, amino acid at position 257 is A and amino acid at position 451 is E; or
  • amino acid at position 257 is A
  • amino acid at position 451 is E
  • amino acid at position 189 is L; or,
  • amino acid at position 257 is A
  • amino acid at position 451 is E
  • amino acid at position 273 is G
  • amino acid at position 257 is A
  • amino acid at position 451 is E
  • amino acid at position 302 is G
  • the 257th amino acid is L, and the 451st amino acid is E; or
  • the 257th amino acid is M, and the 451st amino acid is E; or
  • the 257th amino acid is S, and the 451st amino acid is E; or
  • the 257th amino acid is V, and the 451st amino acid is E; or
  • the 257th amino acid is A, the 451st amino acid is E, and the 265th amino acid is A;
  • amino acid at position 257 is A
  • amino acid at position 451 is E
  • amino acid at position 189 is L
  • amino acid at position 273 is G
  • amino acid at position 257 is A
  • amino acid at position 451 is E
  • amino acid at position 189 is L
  • amino acid at position 324 is G
  • amino acid at position 257 is A
  • amino acid at position 451 is E
  • amino acid at position 189 is L
  • amino acid at position 347 is G
  • the 257th amino acid is A
  • the 451st amino acid is E
  • the 189th amino acid is L
  • the 455th amino acid is D or C.
  • the second aspect of the present invention provides an isolated nucleic acid encoding the glycosyltransferase described in the first aspect of the present invention.
  • the third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid described in the second aspect of the present invention.
  • the fourth aspect of the present invention provides a transformant, which is a host cell comprising the nucleic acid according to the second aspect of the present invention or the recombinant expression vector according to the third aspect of the present invention.
  • the host cell can be conventional in the art, preferably Escherichia coli (Escherichia coli) such as E.coli BL21 (DE3).
  • Escherichia coli Escherichia coli
  • E.coli BL21 E.coli BL21 (DE3).
  • the fifth aspect of the present invention provides a method for preparing the glycosyltransferase described in the first aspect of the present invention, the method comprising culturing such as The transformant described in the fourth aspect of the present invention.
  • the transformant expresses the glycosyltransferase
  • it can be extracted by conventional technical means in the art, for example, a crude enzyme solution can be prepared, and after the crude enzyme solution is prepared, conventional concentration and replacement can be carried out, or the crude enzyme solution can be further subjected to ion
  • One or more of purification steps such as exchange chromatography, affinity chromatography, hydrophobic chromatography and molecular sieve chromatography are used to purify the 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 sixth aspect of the present invention provides a composition comprising the glycosyltransferase as described in the first aspect of the present invention.
  • the seventh aspect of the present invention provides a method for glycosylation of a substrate, the method comprising providing at least one substrate, the glycosyltransferase as described in the first aspect of the present invention, and contacting the substrate with the glycosyltransferase under conditions such that the substrate is glycosylated to produce at least one glycosylated product.
  • the eighth aspect of the present invention provides a method for preparing rebaudioside A, the preparation method comprising the following steps: in the presence of the glycosyltransferase as described in the first aspect of the present invention, the Rebaudioside A is obtained by reacting stevioside with a glycosyl donor.
  • the glycosyltransferase exists in the form of glycosyltransferase cells, crude enzyme solution, pure enzyme, pure enzyme solution or immobilized enzyme.
  • the concentration of the stevioside is 1-150g/L, preferably 100g/L.
  • the mass ratio of the glycosyltransferase cell to stevioside is 1:(3-10), preferably 3:20.
  • the glycosyl donor is UDP-glucose and/or ADP-glucose.
  • sucrose and sucrose synthase Preferably, produced 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.
  • the concentration of said UDP or said ADP is preferably 0.05-0.2 g/L such as 0.1 g/L.
  • the reaction solvent of the reaction has a pH of 5-8, preferably 6.
  • the pH is controlled by a buffer solution, preferably a phosphate buffer solution.
  • the rotation speed during the reaction is 500-1000 rpm, preferably 600 rpm.
  • the temperature of the reaction system of the reaction is 20-90°C, preferably 60°C.
  • the ninth aspect of the present invention provides a method for preparing rebaudioside D, which includes the step of preparing rebaudioside A according to the preparation method described in the eighth aspect of the present invention.
  • ⁇ -1,2-glycosyltransferase is also used.
  • the tenth aspect of the present invention provides a method for preparing rebaudioside M, which includes the step of preparing rebaudioside A according to the preparation method described in the eighth aspect of the present invention.
  • 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 eleventh aspect of the present invention provides a use of the glycosyltransferase described in the first aspect of the present invention in the preparation of steviol glycosides.
  • the steviol glycoside is preferably rebaudioside A, rebaudioside D or rebaudioside M.
  • Glycosyltransferase in the present invention includes NDP-glycosyltransferase, including but not limited to UDP-glucose-dependent glycosyltransferase (UDP-glycosyltransferase; UGT), ADP-glucose-dependent glycosyltransferase (ADP-glycosyltransferase; AGT), CDP-glucose-dependent glycosyltransferase (CDP-glycosyltransferase; CGT), GDP-glucose-dependent glycosyltransferase (GDP-glycosyltransferase; GGT) , TDP-glucose-dependent glycosyltransferase (TDP-glycosyltransferase; TGT) and IDP-glucose-dependent glycosyltransferase (IDP-glycosyltransferase; IGT).
  • sucrose synthase of the present invention refers to sucrose synthase (EC 2.4.1.1.13, SUS) also referred to as SuSy/SS etc.
  • Glycosyltransferase (GT) of the present invention has high enzymatic activity and good stability; Compared with the transferase parent, the catalytic activity has been significantly improved, and the conversion rate has been significantly improved, thereby reducing the cost of the reaction and facilitating industrial production.
  • the present invention combines glycosyltransferase (GT) and sucrose synthase to catalyze the synthesis of RA, RD and RM to realize a cascade reaction.
  • Sucrose and UDP can be used to realize UDPG regeneration, and sucrose and ADP can also be used to realize ADPG regeneration. It solves the problem of high prices of glycosyl donors UDPG and ADPG, and also provides multiple options for substrates, providing more options for optimizing process conditions for further large-scale industrial production, which is more conducive to large-scale industrialization.
  • Fig. 1 shows a schematic diagram of the route for preparing rebaudioside A, rebaudioside D and rebaudioside M from stevioside in an embodiment of the present invention.
  • Figure 2 shows the retention time of stevioside and rebaudioside A reference substances using HPLC detection method 1; the retention time of stevioside is 12.761min, and the retention time of rebaudioside A is 12.377min.
  • FIG. 3 is the spectrum of the Rebaudioside A reference substance using HPLC detection method 2, the retention time is 14.186min.
  • Figure 4 is the spectrum of the Rebaudioside D reference substance using HPLC detection method 2, the retention time is 11.821min.
  • Figure 5 is the spectrum of the Rebaudioside M reference substance using HPLC detection method 2, the retention time is 12.316min.
  • FIG. 6 is a graph showing the activity of Enz.7 in Table 6 to catalyze the synthesis of RA.
  • Fig. 7 is a map of Enz.10 catalytic synthesis of RA activity in Table 8.
  • Fig. 8 is a map of Enz.45 catalytic synthesis of RA activity under the condition that ADP is nucleoside diphosphate in Table 10.
  • Fig. 9 is a map of Enz.45 catalytic synthesis RM activity under the condition that UDP is nucleoside diphosphate in Table 11.
  • codons corresponding to the amino acids are also conventional in the art, and the corresponding relationship between specific amino acids and codons is shown in Table 2.
  • 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 Biotech.
  • the reaction substrate stevioside used in the first round, the second round and the third round of screening was purchased from Pide Pharmaceuticals (purity 95%), and the reaction substrate RA60 used in the synthesis of RM was purchased from Chenguang Biology (the content of RA was 60%, the content of stevioside About 30%, product specification TSG90/RA60).
  • Sucrose was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.
  • the Reb A control substance was purchased from McLean.
  • Reb D and Reb M reference substances were purchased from Qingdao Siyuan Stevia International Trade Co., Ltd.
  • HPLC detection method 1 Chromatographic column: Agilent 5TC-C18(2) (250 ⁇ 4.6mm). Mobile phase: 0.1% TFA aqueous solution was mobile phase A, 0.1% TFA acetonitrile solution was mobile phase B, and gradient elution was carried out in the following table 3. Detection wavelength: 210nm; flow rate: 1ml/min; injection volume: 20 ⁇ l; column temperature: 40°C. As shown in Figure 2, the retention time of stevioside is 12.76min, and that of Reb A is 12.38min.
  • HPLC detection method 2 Chromatographic column: ZORBAX Eclipse plus C18 (4.6mm*150mm, 3.5um). Mobile phase: 0.1% TFA aqueous solution was mobile phase A, 0.1% TFA acetonitrile solution was mobile phase B, and gradient elution was carried out in the following table 4. Detection wavelength: 210nm; flow rate: 1ml/min; injection volume: 20 ⁇ l; column temperature: 35°C. As shown in Figure 3, the peaking time of Reb A: 14.186min; as shown in Figure 4, the peaking time of Reb D: 11.821min; as shown in Figure 5, the peaking time of Reb M: 12.316min.
  • 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
  • Enz.1 Fully synthesize the ⁇ -1,3-glycosyltransferase ( ⁇ -1,3-GTase) enzyme gene numbered Enz.1 as shown in SEQ ID NO:1, which has been connected to the pET28a 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 pET28a-Enz.1 plasmid was used as a template, the primer sequences shown in Table 5 were used, and KOD enzyme was used for PCR amplification to obtain gene fragments and vector fragments of target mutants Enz.2-Enz.8.
  • the PCR amplification reaction system is:
  • the amplification procedure is as follows:
  • the PCR product was digested with DpnI and then gel-run and gel-recovered to obtain the target DNA fragment.
  • the two-fragment homologous recombinase (Exnase II, 5X CE II) of Novazin was connected to the pET28a plasmid vector to obtain the recombinant plasmids pET28a-Enz.2 ⁇ pET28a-Enz.8 of each mutant.
  • transform into E.coli Trans10 competent cells spread in LB medium containing 50 ⁇ g/mL kanamycin, and culture overnight at 37°C; pick a single colony into LB test tube (Km resistance), and culture for 8-10h , extract plasmids for sequencing.
  • 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 number is Enz.47 shown in SEQ ID NO:49 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 plasmid pET28a-SUS was transformed into host E.coli BL21(DE3) competent cells to obtain an engineering strain containing the Enz.47 gene. 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 10,000 rpm for 10 min, the supernatant was discarded, and the bacteria were collected. Store at -20°C for later use.
  • Example 4 The first round of screening of ⁇ -1,3-glycosyltransferase mutants
  • the final concentration of stevioside 95% stevioside content, Bi De Pharmaceutical
  • the final concentration of UDP is 0.1g/L
  • the final concentration of sucrose is 200g/L
  • the sucrose synthase reaction enzyme solution is 30 ⁇ L
  • 50mM pH6.0 phosphate buffer is added to the final volume of 1mL.
  • Enzyme number Discontinuity a RA% Enz.1 / / 47.826 Enz.2 V14I ATC 39.214 Enz.3 E99L CTAs 41.663 Enz.4 L257A GCG 43.981 Enz.5 Q451E GAG 39.01 Enz.6 Q265E GAA 50.444 Enz.7 L257A-Q451E GCG-GAG 52.81 Enz.8 Q265E-Q451E GAA-GAG 52.16
  • the gene encoding Enz.7 obtained in the first round was connected to the vector pET28a to obtain the pET28a-Enz.7 recombinant plasmid, using pET28a-Enz.7 as a template, using the primer sequences shown in Table 7, and using KOD enzyme for PCR amplification , to obtain gene fragments and vector fragments of target mutants Enz.9-16, Enz.18-Enz.35.
  • NNK is conventional in the field, that is, N represents A, T, G or C; K represents G or T.
  • the PCR amplification reaction system is:
  • the PCR amplification procedure is as follows:
  • the PCR product was digested with DpnI and then run and recovered from the gel. Novizym two-fragment homologous recombinase (Exnase II, 5X CE II) was used for ligation. After the connection is completed, transform into E.coli Trans10 competent cells, spread in LB medium containing 50 ⁇ g/mL kanamycin, and culture overnight at 37°C; pick a single colony into the LB test tube (Km resistance), and culture for 8-10 hours, Plasmids were extracted for sequencing identification.
  • 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 also containing 50 ⁇ g/ml kanamycin according to 2% (v/v) inoculum amount, shake culture at 37°C until OD600 reaches 0.6-0.8, then add IPTG to its final concentration 0.1mM, induced at 25°C for 20h. After the cultivation, the culture solution was centrifuged at 4000 rpm for 20 min, the supernatant was discarded, and the bacteria were collected. Store at -20°C for later use.
  • the collected bacteria were suspended in PBS (50mM, pH 6.0) at a ratio of 1:10 (M/V, g/mL), and then homogenized using a high-pressure homogenizer (550Mbar homogenization for 1.5min); after homogenization, The ⁇ -1,3-glycosyltransferase enzyme solution was treated at 80°C for 15 minutes, and centrifuged at 12000rpm for 2 minutes to obtain the reaction enzyme solution. Store at -4°C for later use.
  • stevioside (95% stevioside content, Bid Pharmaceuticals) as the substrate, add 150 ⁇ L of reaction enzyme solution of ⁇ -1,3-glycosyltransferase mutant to 1 mL reaction system, and the final concentration of stevioside is 100 g/L , the final concentration of UDP is 0.1g/L, the final concentration of sucrose is 200g/L, 30 ⁇ L of sucrose synthase, and finally 50mM pH6.0 phosphate buffer is added to the final volume of 1mL.
  • the prepared reaction system was placed in a metal bath, reacted at 60 °C and 600 rpm for 60 min, diluted 100 times, and analyzed the concentration of Reb A by HPLC.
  • the experimental results obtained using HPLC detection method 1 are shown in Table 8.
  • Enz.18 ⁇ Enz.29 were obtained by NNK of GT001-257-F/R
  • Enz.30-Enz.35 were obtained by NNK of GT001-265-F/R.
  • the gene encoding Enz.10 obtained in the second round was connected to the vector pET28a to obtain the pET28a-Enz.10 recombinant plasmid, using pET28a-Enz.10 as a template, using the primer sequences shown in Table 9, and using KOD enzyme for PCR amplification Target DNA fragments and vector fragments.
  • the PCR amplification reaction system is:
  • the PCR amplification procedure is as follows:
  • the PCR product was digested with DpnI and then run and recovered from the gel.
  • the two fragments of Novizyme homologous recombination enzyme (Exnase II, 5X CE II) were used to connect to the pET28a plasmid vector to obtain recombinant plasmids pET28a-Enz.36 ⁇ pET28a-Enz.45.
  • transform into E.coli Trans10 competent cells spread on LB medium containing 50 ⁇ g/mL kanamycin, and culture overnight at 37°C; pick a single colony into LB test tube (Km resistance), and culture for 8-10 hours , extract plasmids for sequencing.
  • Example 8 The recombinant plasmids sequenced correctly in Example 8 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 fresh TB liquid culture medium containing 50 ⁇ g/ml kanamycin according to 2% (v/v) inoculum amount, shake culture at 37°C until OD600 reaches about 0.8, add IPTG to its final concentration of 0.1mM, induced culture at 25°C for 20h. After the cultivation, the culture solution was centrifuged at 4000 rpm for 20 min, the supernatant was discarded, and the bacteria were collected. Store at -20°C for later use.
  • the collected bacteria were suspended in PBS (50mM, pH 6.0) at a ratio of 1:10 (M/V, g/mL), and then homogenized using a high-pressure homogenizer (550Mbar homogenization for 1.5min); after homogenization, The ⁇ -1,3-GT enzyme solution was treated at 80°C for 15 minutes, and centrifuged at 12000 rpm for 2 minutes to obtain the reaction enzyme solution. Store at -4°C for later use.
  • stevioside (95% stevioside content, Bid Pharmaceuticals) as the substrate, add 150 ⁇ L of reaction enzyme solution of ⁇ -1,3-glycosyltransferase mutant to 1 mL reaction system, and the final concentration of stevioside is 100 g/L , the final concentration of UDP or ADP is 0.1g/L, the final concentration of sucrose is 200g/L, 30 ⁇ L of sucrose synthase, and finally add 50mM pH6.0 phosphate buffer to a final volume of 1mL.
  • Embodiment 11 Preparation of ⁇ -1,2-glycosyltransferase
  • ⁇ -1,2-glycosyltransferase (enzyme number Enz.17) shown in the nucleotide sequence SEQ ID NO:51, a set of ⁇ -1,2-glycosyltransferase genes is synthesized from the whole gene , the gene has been connected to the pET28a plasmid vector to obtain the recombinant plasmid pET28a-Enz.17.
  • Gene synthesis company Sangon Bioengineering (Shanghai) Co., Ltd.
  • the plasmid pET28a-Enz.17 was transformed into host E.coli BL21(DE3) competent cells to obtain engineering strains containing ⁇ -1,2-glycosyltransferase gene.
  • the engineering bacteria containing the ⁇ -1,2-glycosyltransferase gene are activated by streaking on the plate, pick a single colony and inoculate it into 5ml LB liquid medium containing 50 ⁇ g/ml kanamycin, and culture it with shaking at 37°C for 12h .
  • Transfer to 50ml of fresh LB liquid medium also containing 50 ⁇ g/ml kanamycin according to 2v/v% inoculum amount shake culture at 37°C until OD600 reaches 0.6-0.8, add IPTG to its final concentration of 0.1mM, Induction culture was carried out at 24°C for 22 hours. After the cultivation, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected and stored in a -20°C ultra-low temperature refrigerator until use.
  • amino acid sequence of the ⁇ -1,2-glycosyltransferase prepared in this embodiment is shown in SEQ ID NO:52.
  • Reb A 60 (the content of Reb A is 60%) as the substrate, add 150 ⁇ L of reaction enzyme solution of ⁇ -1,3-glycosyltransferase mutant to 1mL reaction system, ⁇ -1,2-glycosyltransferase
  • the reaction enzyme solution is 120 ⁇ L
  • the final concentration of RA60 is 100g/L
  • the final concentration of UDP or ADP is 0.1g/L
  • the final concentration of sucrose is 200g/L
  • the sucrose synthase reaction enzyme solution is 30 ⁇ L
  • 50mM pH6.0 phosphate buffer is added to a final volume of 1 mL.

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Abstract

L'invention concerne une glycosyltransférase et une application de celle-ci. Par comparaison avec SEQ ID NO : 2, la glycosyltransférase contient des différences dans un ou plusieurs résidus d'acides aminés choisis parmi les positions de résidus suivantes : un acide aminé en position 14 étant I ; un acide aminé en position 189 étant L ; un acide aminé en position 257 étant A, C, L, M, S ou V ; un acide aminé en position 265 étant E ou A ; un acide aminé en position 273 étant G ; un acide aminé en position 302 étant G ; un acide aminé en position 324 étant G ; un acide aminé en position 347 étant G ; un acide aminé en position 451 étant E ; et un acide aminé en position 455 étant D ou C. La glycosyltransférase possède une activité non inférieure à celle de la glycosyltransférase possédant la séquence d'acides aminés représentée dans SEQ ID NO : 2. Lorsqu'elle est utilisée pour préparer des glycosides de stéviol, par comparaison avec une glycosyltransférase mère, la présente glycosyltransférase possède une activité catalytique améliorée et un taux de conversion accru. Elle résout le problème des prix élevés des donneurs de glycosyle UDPG et ADPG, et offre une variété de possibilités de sélection de substrat.
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