WO2023030065A1 - Glycosyltransferase and application thereof - Google Patents

Glycosyltransferase and application thereof Download PDF

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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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弈柯莱生物科技(上海)股份有限公司
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    • C12N15/09—Recombinant DNA-technology
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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
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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
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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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    • C12R2001/01—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

A glycosyltransferase and an application thereof. Compared with SEQ ID NO:2, the glycosyltransferase contains differences in one or more amino acid residues selected from the following residue positions: an amino acid at position 14 being I; an amino acid at position 189 being L; an amino acid at position 257 being A, C, L, M, S, or V; an amino acid at position 265 being E or A; an amino acid at position 273 being G; an amino acid at position 302 being G; an amino acid at position 324 being G; an amino acid at position 347 being G; an amino acid at position 451 being E; and an amino acid at position 455 being D or C. The glycosyltransferase has an activity not lower than that of the glycosyltransferase having the amino acid sequence shown in SEQ ID NO:2. When used to prepare steviol glycosides, compared with a glycosyltransferase parent, the present glycosyltransferase has improved catalytic activity and an increased conversion rate. The problem of high prices of the glycosyl donors UDPG and ADPG is solved, and a variety of substrate selection possibilities are provided.

Description

一种糖基转移酶及其应用A kind of glycosyltransferase and its application
本申请要求申请日为2021/8/30的中国专利申请202111006803.4的优先权。本申请引用上述中国专利申请的全文。This application claims the priority of the Chinese patent application 202111006803.4 with a filing date of 2021/8/30. This application cites the full text of the above-mentioned Chinese patent application.
技术领域technical field
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。The invention relates to a glycosyltransferase and its application in the glycosylation reaction of steviol glycosides.
背景技术Background technique
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。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.
甜菊糖苷(甜菊糖苷类化合物)的结构式如下：The structural formula of steviol glycosides (steviol glycosides) is as follows:
Figure PCTCN2022113874-appb-000001
Figure PCTCN2022113874-appb-000001
序号serial number 化合物compound R 1 R 1 R 2 R 2
11 甜菊醇steviol Hh Hh
22 甜菊醇单糖苷steviol monoglycoside Hh β-Glcβ-Glc
33 甜菊醇双糖苷steviol diglucoside Hh β-Glc-β-Glc(2-1)β-Glc-β-Glc(2-1)
44 甜茶苷rubusoside β-Glcβ-Glc β-Glcβ-Glc
55 甜菊苷(STV)Stevioside (STV) β-Glcβ-Glc β-Glc-β-Glc(2-1)β-Glc-β-Glc(2-1)
66 莱鲍迪苷A(RA)Rebaudioside A (RA) β-Glcβ-Glc β-Glc-β-Glc(2-1)-β-Glc(3-1)β-Glc-β-Glc(2-1)-β-Glc(3-1)
77 莱鲍迪苷B(RB)Rebaudioside B (RB) Hh β-Glc-β-Glc(2-1)-β-Glc(3-1)β-Glc-β-Glc(2-1)-β-Glc(3-1)
88 莱鲍迪苷C(RC)Rebaudioside C (RC) β-Glcβ-Glc β-Glc-α-Rha(2-1)-β-Glc(3-1)β-Glc-α-Rha(2-1)-β-Glc(3-1)
99 莱鲍迪苷D(RD)Rebaudioside D(RD) β-Glc-β-Glc(2-1)β-Glc-β-Glc(2-1) β-Glc-β-Glc(2-1)-β-Glc(3-1)β-Glc-β-Glc(2-1)-β-Glc(3-1)
1010 莱鲍迪苷E(RE)Rebaudioside E (RE) β-Glc-β-Glc(2-1)β-Glc-β-Glc(2-1) β-Glc-β-Glc(2-1)β-Glc-β-Glc(2-1)
1111 莱鲍迪苷F(RF)Rebaudioside F(RF) β-Glcβ-Glc β-Glc-α-Xly(2-1)-β-Glc(3-1)β-Glc-α-Xly(2-1)-β-Glc(3-1)
1212 莱鲍迪苷M(RM)Rebaudioside M(RM) β-Glc-β-Glc(2-1)-β-Glc(3-1)β-Glc-β-Glc(2-1)-β-Glc(3-1) β-Glc-β-Glc(2-1)-β-Glc(3-1)β-Glc-β-Glc(2-1)-β-Glc(3-1)
1313 杜可尔苷ADucorside A β-Glcβ-Glc β-Glc-α-Rha(2-1)β-Glc-α-Rha(2-1)
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(Rebaudioside D，Reb D)、莱鲍迪苷E、杜克苷、甜菊双糖苷等八种糖苷。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和F，甜菊双糖苷和甜茶苷，以低得多的水平被发现(大约0-0.2％)。The above-mentioned 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. The major glycosides found in Stevia leaves are rebaudioside A (2-10%), stevioside (2-10%) and rebaudioside C (1-2%). Other glycosides, such as rebaudiosides B, D, E and F, steviolbioside and rubusoside, were found at much lower levels (approximately 0-0.2%).
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。 Although 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. Typically, 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.
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。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. In addition, 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.
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。The current bioenzymatic synthesis of rebaudioside D requires the addition of expensive UDP-glucose as one of the substrates, through the action of UDP-glucosyltransferase (UDP-glucosyltransferase, referred to as UGT), and the addition of stevioside or rebaudioside Diglycoside A is used as a substrate to catalyze the production of rebaudioside D. However, due to the extremely high price of UDP-glucose, the feasibility of industrialized preparation of rebaudioside D is almost completely restricted, and the economy is poor and lacks market competitiveness.
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。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. Using 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. However, there are mainly the following problems with the biological enzyme catalysis method at present: (1) the cost of producing rebaudioside M with biological enzyme catalyzed rebaudioside D is relatively high, and the enzymatic catalysis yield needs to be further optimized; Because the catalyzed glycosyltransferase is not easy to separate and recycle from the product, and is easy to be inactivated; (3) the content of rebaudioside A in natural plants is very high, while the content of rebaudioside D is very low, so it can be produced by laibaudioside at low cost The direct conversion of baudioside A to rebaudioside D is also an urgent problem to be solved.
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。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。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.
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。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.
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，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。目前甜菊糖苷的生物酶法制备领域中使用的酶往往存在酶活低、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。Nowadays, with the wide application of natural sweetener stevioside and the increasing development of biocatalytic technology, glucosyltransferase is more and more used in the field of biocatalytic preparation of steviol glycosides. At present, 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.
发明内容Contents of the invention
本发明所要解决的技术问题是现有的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；将其用于制备甜菊糖苷(例如莱鲍迪苷A、莱鲍迪苷D或莱鲍迪苷M)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，利于工业化生产。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.
为了解决上述技术问题，本发明第一方面提供一种糖基转移酶，所述糖基转移酶与SEQ ID NO:2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：In order to solve the above-mentioned technical problems, 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:
第14位氨基酸为I；The 14th amino acid is I;
第189位氨基酸为L；The 189th amino acid is L;
第257位氨基酸为A、C、L、M、S或V；The 257th amino acid is A, C, L, M, S or V;
第265位氨基酸为E或A；The 265th amino acid is E or A;
第273位氨基酸为G；The 273rd amino acid is G;
第302位氨基酸为G；The 302nd amino acid is G;
第324位氨基酸为G；The 324th amino acid is G;
第347位氨基酸为G；The 347th amino acid is G;
第451位氨基酸为E；The 451st amino acid is E;
第455位氨基酸为D或C；The 455th amino acid is D or C;
并具有不低于如SEQ ID NO:2的氨基酸序列所示的糖基转移酶活性。And have not less than the glycosyltransferase activity shown in the amino acid sequence of SEQ ID NO:2.
本发明中，所述差异可以是在如SEQ ID NO:2所示的氨基酸序列上进行突变获得，也可以是以其它氨基酸序列为基础进行突变，只要其最终突变的结果与如SEQ ID NO:2所示的氨基酸序列相比具有上述差异，则同样落入本发明保护的范围。In the present invention, 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.
较佳地，所述糖基转移酶与SEQ ID NO:2相比的氨基酸残基差异选自以下组：Preferably, the amino acid residue difference of the glycosyltransferase compared with SEQ ID NO: 2 is selected from the following groups:
(1)第265位氨基酸为E；或，(1) The 265th amino acid is E; or,
第257位氨基酸为A，第451位氨基酸为E；或，A at amino acid 257 and E at amino acid 451; or,
第265位氨基酸为E，第451位氨基酸为E；The 265th amino acid is E, and the 451st amino acid is E;
(2)第14位氨基酸为I，第257位氨基酸为A且第451位氨基酸为E；或(2) amino acid at position 14 is I, amino acid at position 257 is A and amino acid at position 451 is E; or
第257位氨基酸为A、第451位氨基酸为E且第189位氨基酸为L；或，amino acid at position 257 is A, amino acid at position 451 is E, and amino acid at position 189 is L; or,
第257位氨基酸为A、第451位氨基酸为E且第273位氨基酸为G；或，amino acid at position 257 is A, amino acid at position 451 is E, and amino acid at position 273 is G; or,
第257位氨基酸为A、第451位氨基酸为E且第302位氨基酸为G；或amino acid at position 257 is A, amino acid at position 451 is E, and amino acid at position 302 is G; or
第257位氨基酸为C、第451位氨基酸为E；或C at amino acid 257 and E at amino acid 451; or
第257位氨基酸为L、第451位氨基酸为E；或The 257th amino acid is L, and the 451st amino acid is E; or
第257位氨基酸为M、第451位氨基酸为E；或The 257th amino acid is M, and the 451st amino acid is E; or
第257位氨基酸为S、第451位氨基酸为E；或The 257th amino acid is S, and the 451st amino acid is E; or
第257位氨基酸为V、第451位氨基酸为E；或The 257th amino acid is V, and the 451st amino acid is E; or
第257位氨基酸为A、第451位氨基酸为E且第265位氨基酸为A；The 257th amino acid is A, the 451st amino acid is E, and the 265th amino acid is A;
(3)第257位氨基酸为A、第451位氨基酸为E、第189位氨基酸为L且第14位氨基酸为I；或，(3) the 257th amino acid is A, the 451st amino acid is E, the 189th amino acid is L, and the 14th amino acid is I; or,
第257位氨基酸为A、第451位氨基酸为E、第189位氨基酸为L且第273位氨基酸为G；或，amino acid at position 257 is A, amino acid at position 451 is E, amino acid at position 189 is L, and amino acid at position 273 is G; or,
第257位氨基酸为A、第451位氨基酸为E、第189位氨基酸为L且第324位氨基酸为G；或，amino acid at position 257 is A, amino acid at position 451 is E, amino acid at position 189 is L, and amino acid at position 324 is G; or,
第257位氨基酸为A、第451位氨基酸为E、第189位氨基酸为L且第347位氨基酸为G；或，amino acid at position 257 is A, amino acid at position 451 is E, amino acid at position 189 is L, and amino acid at position 347 is G; or,
(3)第257位氨基酸为A、第451位氨基酸为E、第189位氨基酸为L且第455位氨基酸为D或C。(3) The 257th amino acid is A, the 451st amino acid is E, the 189th amino acid is L, and the 455th amino acid is D or C.
为了解决上述技术问题，本发明第二方面提供一种分离的核酸，所述核酸编码如本发明第一方面所述的糖基转移酶。In order to solve the above technical problems, the second aspect of the present invention provides an isolated nucleic acid encoding the glycosyltransferase described in the first aspect of the present invention.
为了解决上述技术问题，本发明第三方面提供一种重组表达载体，其包含本发明第二方面所述的核酸。In order to solve the above technical problems, 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.
为了解决上述技术问题，本发明第四方面提供一种转化体，其为包含如本发明第二方面所述的核酸或如本发明第三方面所述的重组表达载体的宿主细胞。In order to solve the above technical problems, 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.
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)例如E.coli BL21(DE3)。The host cell can be conventional in the art, preferably Escherichia coli (Escherichia coli) such as E.coli BL21 (DE3).
为了解决上述技术问题，本发明第五方面提供一种制备如本发明第一方面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本发明第四方面所述的转化体。In order to solve the above technical problems, 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.
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。After 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. In a certain preferred embodiment, 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.
为了解决上述技术问题，本发明第六方面提供一种组合物，其包含如本发明第一方面所述的糖基转移酶。In order to solve the above technical problems, the sixth aspect of the present invention provides a composition comprising the glycosyltransferase as described in the first aspect of the present invention.
为了解决上述技术问题，本发明第七方面提供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、如本发明第一方面所述的糖基转移酶，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。In order to solve the above technical problems, 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.
为了解决上述技术问题，本发明第八方面提供一种莱鲍迪苷A的制备方法，所述制备方法包括以下步骤：在如本发明第一方面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。In order to solve the above technical problems, 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.
在某一较佳实施方案中，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。In a preferred embodiment, the glycosyltransferase exists in the form of glycosyltransferase cells, crude enzyme solution, pure enzyme, pure enzyme solution or immobilized enzyme.
在某一较佳实施方案中，所述甜菊苷的浓度1-150g/L，优选100g/L。In a certain preferred embodiment, the concentration of the stevioside is 1-150g/L, preferably 100g/L.
在某一较佳实施方案中，所述糖基转移酶菌体与甜菊苷的质量比为1:(3-10)，优选 3:20。In a preferred embodiment, the mass ratio of the glycosyltransferase cell to stevioside is 1:(3-10), preferably 3:20.
在某一较佳实施方案中，所述糖基供体为UDP-葡萄糖和/或ADP-葡萄糖。In a preferred embodiment, the glycosyl donor is UDP-glucose and/or ADP-glucose.
优选地，通过UDP和/或ADP在蔗糖和蔗糖合成酶的存在下制得。Preferably, produced by UDP and/or ADP in the presence of sucrose and sucrose synthase.
所述蔗糖的浓度优选为100-300g/L例如200g/L。The concentration of the sucrose is preferably 100-300g/L such as 200g/L.
所述UDP或所述ADP的浓度优选为0.05-0.2g/L例如0.1g/L。The concentration of said UDP or said ADP is preferably 0.05-0.2 g/L such as 0.1 g/L.
在某一较佳实施方案中，所述反应的反应溶剂的pH为5-8，优选6。In a certain preferred embodiment, the reaction solvent of the reaction has a pH of 5-8, preferably 6.
在某一较佳实施方案中，所述pH由缓冲溶液控制，所述缓冲溶液优选磷酸缓冲溶液。In a preferred embodiment, the pH is controlled by a buffer solution, preferably a phosphate buffer solution.
在某一较佳实施方案中，所述反应时的转速为500-1000rpm，优选600rpm。In a certain preferred embodiment, the rotation speed during the reaction is 500-1000 rpm, preferably 600 rpm.
在某一较佳实施方案中，所述反应的反应体系的温度为20-90℃，优选60℃。In a certain preferred embodiment, the temperature of the reaction system of the reaction is 20-90°C, preferably 60°C.
为了解决上述技术问题，本发明第九方面提供一种莱鲍迪苷D的制备方法，其包括根据如本发明第八方面所述的制备方法制备莱鲍迪苷A的步骤。In order to solve the above technical problems, 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.
在制备莱鲍迪苷D时，除了使用如本发明第一方面所述的糖基转移酶之外，还使用β-1,2-糖基转移酶。When preparing rebaudioside D, in addition to using the glycosyltransferase as described in the first aspect of the present invention, β-1,2-glycosyltransferase is also used.
为了解决上述技术问题，本发明第十方面提供一种莱鲍迪苷M的制备方法，其包括根据如本发明第八方面所述的制备方法制备莱鲍迪苷A的步骤。In order to solve the above technical problems, 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.
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。In a certain preferred embodiment, 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.
为了解决上述技术问题，本发明第十一方面提供一种如本发明第一方面所述的糖基转移酶在制备甜菊糖苷中的用途。In order to solve the above technical problems, 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.
所述甜菊糖苷优选为莱鲍迪苷A、莱鲍迪苷D或莱鲍迪苷M。The steviol glycoside is preferably rebaudioside A, rebaudioside D or rebaudioside M.
本发明中“糖基转移酶”包括NDP-糖基转移酶，包括但不限于UDP-葡萄糖依赖性糖基转移酶(UDP-糖基转移酶；UGT)、ADP-葡萄糖依赖性糖基转移酶(ADP-糖基转移酶；AGT)、CDP-葡萄糖依赖性糖基转移酶(CDP-糖基转移酶；CGT)、GDP-葡萄糖依赖性糖基转移酶(GDP-糖基转移酶；GGT)、TDP-葡萄糖依赖性糖基转移酶(TDP-糖基转移酶；TGT)和IDP-葡萄糖依赖性糖基转移酶(IDP-糖基转移酶；IGT)。"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).
本发明的蔗糖合成酶指sucrose synthase(EC 2.4.1.1.13,SUS)也简称SuSy/SS等。The sucrose synthase of the present invention refers to sucrose synthase (EC 2.4.1.1.13, SUS) also referred to as SuSy/SS etc.
本发明的糖基转移酶(GT)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或莱鲍迪苷M)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，利于工业化生产。本发明联合使 用糖基转移酶(GT)与蔗糖合成酶催化合成RA、RD以及RM，实现级联反应，既可以使用蔗糖和UDP来实现UDPG再生，也可以使用蔗糖和ADP来实现ADPG再生，解决了糖基供体UDPG、ADPG价格昂贵的问题，也提供底物多种选择可能性，为进一步实现大规模工业化生产提供更多工艺条件优化的选择，更利于实现大规模工业化。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.
附图说明Description of drawings
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。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.
图2显示了使用HPLC检测方法1，甜菊苷、莱鲍迪苷A对照品的保留时间；甜菊苷的保留时间为12.761min，莱鲍迪苷A的保留时间为12.377min。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.
图3为使用HPLC检测方法2，莱鲍迪苷A对照品的图谱，保留时间为14.186min。Figure 3 is the spectrum of the Rebaudioside A reference substance using HPLC detection method 2, the retention time is 14.186min.
图4为使用HPLC检测方法2，莱鲍迪苷D对照品的图谱，保留时间为11.821min。Figure 4 is the spectrum of the Rebaudioside D reference substance using HPLC detection method 2, the retention time is 11.821min.
图5为使用HPLC检测方法2，莱鲍迪苷M对照品的图谱，保留时间为12.316min。Figure 5 is the spectrum of the Rebaudioside M reference substance using HPLC detection method 2, the retention time is 12.316min.
图6为表6中Enz.7催化合成RA活性的图谱。FIG. 6 is a graph showing the activity of Enz.7 in Table 6 to catalyze the synthesis of RA.
图7为表8中Enz.10催化合成RA活性的图谱。Fig. 7 is a map of Enz.10 catalytic synthesis of RA activity in Table 8.
图8为表10中ADP为核苷二磷酸条件下Enz.45催化合成RA活性的图谱。Fig. 8 is a map of Enz.45 catalytic synthesis of RA activity under the condition that ADP is nucleoside diphosphate in Table 10.
图9为表11中UDP为核苷二磷酸条件下Enz.45催化合成RM活性的图谱。Fig. 9 is a map of Enz.45 catalytic synthesis RM activity under the condition that UDP is nucleoside diphosphate in Table 11.
具体实施方式Detailed ways
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。The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.
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》。The experimental methods in the present invention are conventional methods unless otherwise specified. For specific gene cloning operations, please refer to "Molecular Cloning Experiment Guide" edited by J. Sambrook et al.
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。The abbreviated symbols of amino acids in the present invention are conventional in the field unless otherwise specified, and the specific amino acids corresponding to the abbreviated symbols are shown in Table 1.
表1Table 1
Figure PCTCN2022113874-appb-000002
Figure PCTCN2022113874-appb-000002
Figure PCTCN2022113874-appb-000003
Figure PCTCN2022113874-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。The 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.
表2Table 2
Figure PCTCN2022113874-appb-000004
Figure PCTCN2022113874-appb-000004
本发明的路线示意图如图1所示。The route schematic diagram of the present invention is shown in Figure 1.
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。第一轮、第二轮以及第三轮筛选所用反应底物甜菊苷购自毕得医药(纯度95％)，合成RM所用反应底物RA60购自晨光生物(其中RA含量60％，甜菊苷含量约30％，产品规格TSG90/RA60)。蔗糖购自生工生物工程(上海)股份有限公司。Reb A对照品购自麦克林。Reb D和Reb M对照品购自青岛思远甜菊国际贸易有限公司。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检测方法1：色谱柱：Agilent 5TC-C18(2)(250×4.6mm)。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3进行梯度洗脱。检测波长：210nm；流速：1ml/min；进样体积：20μl；柱温：40℃。如图2所示，甜菊苷保留时间为12.76min，Reb A保留时间为12.38min。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: 20μl; 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.
表3table 3
时间(分钟)time (minutes) 流动相A％Mobile phase A% 流动相B％Mobile phase B%
0.000.00 7070 3030
15.0015.00 6060 4040
20.0020.00 3030 7070
25.0025.00 3030 7070
25.1025.10 7070 3030
32.0032.00 7070 3030
HPLC检测方法2：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3所示，Reb A出峰时间：14.186min；如图4所示，Reb D出峰时间：11.821min；如图5所示，Reb M出峰时间：12.316min。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: 20μl; 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.
表4Table 4
Time(min)Time(min) A％A% B％B%
0.000.00 9090 1010
15.0015.00 6060 4040
20.0020.00 00 100100
24.0024.00 00 100100
24.1024.10 9090 1010
32.0032.00 9090 1010
实施例1第一轮β-1,3-糖基转移酶突变体文库的构建Example 1 Construction of the first round of β-1,3-glycosyltransferase mutant library
全合成如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，该基因已连接在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。Fully synthesize the β-1,3-glycosyltransferase (β-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.
以pET28a-Enz.1质粒为模板，采用表5所示的引物序列，采用KOD酶进行PCR扩增，获得目标突变体Enz.2～Enz.8的基因片段和载体片段。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.
表5table 5
Figure PCTCN2022113874-appb-000005
Figure PCTCN2022113874-appb-000005
PCR扩增反应体系为：The PCR amplification reaction system is:
KOD Mix：25μLKOD Mix: 25μL
ddH 2O：20μL ddHO : 20 μL
引物：2μL*2Primer: 2μL*2
模板：1μLTemplate: 1 μL
扩增程序如下：The amplification procedure is as follows:
(1)98℃3min(1) 98°C for 3 minutes
(2)98℃10s(2) 98℃10s
(3)55℃5s(3) 55℃5s
(4)68℃5s/kbp(4) 68℃5s/kbp
(5)68℃5min(5) 5min at 68°C
(6)12℃保温(6) 12°C insulation
(2)～(4)循环34次。(2)～(4) cycle 34 times.
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，5X CE II)连接至pET28a质粒载体上，得到各突变体重组质粒pET28a-Enz.2～pET28a-Enz.8。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；挑取单菌落至LB试管(Km抗性)，培养8-10h，提取质粒进行测序。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. After ligation, transform into E.coli Trans10 competent cells, spread in LB medium containing 50μg/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.
实施例2β-1,3-糖基转移酶突变体的制备Example 2 Preparation of β-1,3-glycosyltransferase mutants
1.进行突变载体的蛋白表达：1. Perform protein expression of the mutant vector:
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。 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 50μg/ml kanamycin, and culture with shaking at 37°C for 4h. According to the 2% (v/v) inoculum size, it was transferred to 50ml of fresh TB liquid medium also containing 50μg/ml kanamycin, cultured with shaking at 37°C until the OD600 reached about 0.8, and then added IPTG (isopropyl- β-D-thiogalactoside, Isopropylβ-D-thiogalactoside) to a final concentration of 0.1 mM, induced at 25°C for 20 h. 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.
2.反应酶液的获取：2. Acquisition of reaction enzyme solution:
配制50mM pH6.0的磷酸缓冲液(PBS)，将上述所得菌体按照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β-1,3-GT酶液进行80℃处理15min，12000rpm离心2min即获得反应酶液。Prepare 50mM phosphate buffer solution (PBS) with pH 6.0, suspend the bacteria obtained above at a ratio of 1:10 (M/V, g/mL), and then use a high-pressure homogenizer for homogenization (550Mbar homogenization for 1.5min) ; The homogenized β-1,3-GT enzyme solution was treated at 80°C for 15 minutes, and centrifuged at 12,000 rpm for 2 minutes to obtain the reaction enzyme solution.
实施例3蔗糖合成酶SUS的制备The preparation of embodiment 3 sucrose synthase SUS
全合成SEQ ID NO:49所示的编号为Enz.47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)。The 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).
将质粒pET28a-SUS转化至宿主E.coli BL21(DE3)感受态细胞，得到含Enz.47基因的工程菌株。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，弃上清液，收集菌体。-20℃保存备用。 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 50μg/ml kanamycin, and culture with shaking at 37°C for 4h. Transfer to 50ml of fresh TB liquid medium containing 50μg/ml kanamycin according to 2v/v% inoculation amount, shake culture at 37℃ until OD600 reaches 0.6-0.8, add IPTG to its final concentration of 0.1mM , 25 ℃ 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.
配制50mM pH6.0的磷酸缓冲液(PBS)，将上述所得Enz.47菌体按照(M/V)1:5进行悬浮，之后，进行高压均质(550～600Mbar、1min)后经12000rpm离心2min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.47,氨基酸序列如SEQ ID NO:50所示)的反应酶液。Prepare 50mM phosphate buffer solution (PBS) at pH 6.0, suspend the Enz.47 bacteria obtained above according to (M/V) 1:5, and then perform high-pressure homogenization (550-600Mbar, 1min) and centrifuge at 12000rpm The crude enzyme solution was obtained in 2 minutes, the crude enzyme solution was centrifuged, and the supernatant was taken to obtain the reaction enzyme solution of sucrose synthase SUS (enzyme number Enz.47, amino acid sequence shown in SEQ ID NO:50).
实施例4第一轮β-1,3-糖基转移酶突变体的筛选Example 4 The first round of screening of β-1,3-glycosyltransferase mutants
1mL反应体系中，加入β-1,3-糖基转移酶的反应酶液150μL，甜菊苷(甜菊苷含量95％，毕得医药)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100倍，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Reb A的浓度(详见表6的Reb A％，其代表反应液中Reb A的百分比)。使用HPLC检测方法1获得的实验结果如表6所示。In the 1mL reaction system, add 150μL of β-1,3-glycosyltransferase reaction enzyme solution, the final concentration of stevioside (95% stevioside content, Bi De Pharmaceutical) is 100g/L, and the final concentration of UDP is 0.1g/L , the final concentration of sucrose is 200g/L, the sucrose synthase reaction enzyme solution is 30μL, and finally 50mM pH6.0 phosphate buffer is added to the final volume of 1mL. Place the prepared reaction system in a metal bath, react at 60°C and 600rpm for 60min, dilute the reaction solution 100 times, take 10μL of the reaction solution and add it to 990μL of hydrochloric acid with a pH of 2~3, vortex, centrifuge at 13000rpm for 10min, and remove the supernatant. The concentration of Reb A was analyzed by HPLC (see the Reb A% of Table 6 for details, which represents the percentage of Reb A in the reaction solution). The experimental results obtained using HPLC detection method 1 are shown in Table 6.
表6Table 6
酶编号Enzyme number 突变点Discontinuity 密码子a RA％RA%
Enz.1Enz.1 // // 47.82647.826
Enz.2Enz.2 V14IV14I ATCATC 39.21439.214
Enz.3Enz.3 E99LE99L CTACTAs 41.66341.663
Enz.4Enz.4 L257AL257A GCGGCG 43.98143.981
Enz.5Enz.5 Q451EQ451E GAGGAG 39.0139.01
Enz.6Enz.6 Q265EQ265E GAAGAA 50.44450.444
Enz.7Enz.7 L257A-Q451EL257A-Q451E GCG-GAGGCG-GAG 52.8152.81
Enz.8Enz.8 Q265E-Q451EQ265E-Q451E GAA-GAGGAA-GAG 52.1652.16
由表6中的初筛结果可知：Enz.7的活性提高最大，相较于出发序列Enz.1提高幅度达10％，Enz.6、Enz.8活性均有提高，提高幅度介于5％～10％之间。后续基于Enz.7进行第二轮突变。图6是表6中Enz.7催化合成RA活性的HPLC图。From the preliminary screening results in Table 6, it can be seen that the activity of Enz.7 has the greatest improvement, compared with the starting sequence Enz.1, the improvement rate is 10%, and the activities of Enz.6 and Enz.8 are both improved, and the increase rate is between 5%. ~10%. A second round of mutations was subsequently performed based on Enz.7. Fig. 6 is the HPLC chart of the catalytic synthesis of RA activity of Enz.7 in Table 6.
实施例5第二轮β-1,3-糖基转移酶突变体文库的构建Example 5 Construction of the second round of β-1,3-glycosyltransferase mutant library
将第一轮得到的编码Enz.7的基因连接载体pET28a，得到pET28a-Enz.7重组质粒，以pET28a-Enz.7为模板，采用表7所示的引物序列，采用KOD酶进行PCR扩增，获得目标突变体Enz.9～16、Enz.18～Enz.35的基因片段和载体片段。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.
表7Table 7
Figure PCTCN2022113874-appb-000006
Figure PCTCN2022113874-appb-000006
Figure PCTCN2022113874-appb-000007
Figure PCTCN2022113874-appb-000007
其中，NNK为本领域常规，即N代表A、T、G或C；K代表G或T。Among them, NNK is conventional in the field, that is, N represents A, T, G or C; K represents G or T.
PCR扩增反应体系为：The PCR amplification reaction system is:
KOD Mix：25μL；KOD Mix: 25 μL;
ddH 2O：20μL； ddH2O : 20 μL;
引物：2μL*2；Primers: 2μL*2;
模板：1μL。Template: 1 μL.
PCR扩增程序如下：The PCR amplification procedure is as follows:
(1)98℃3min(1) 98°C for 3 minutes
(2)98℃10s(2) 98℃10s
(3)55℃5s(3) 55℃5s
(4)68℃5s/kbp(4) 68℃5s/kbp
(5)68℃5min(5) 5min at 68°C
(6)12℃保温(6) 12°C insulation
(2)～(4)循环34次。(2)～(4) cycle 34 times.
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(Exnase II，5X CE II)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；挑单菌落至LB试管(Km抗性)，培养8-10h，提取质粒进行测序鉴定。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 μg/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.
实施例6第二轮β-1,3-糖基转移酶突变体的制备Example 6 Preparation of the second round of β-1,3-glycosyltransferase mutants
1.进行突变载体的蛋白表达：1. Perform protein expression of the mutant vector:
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0.6-0.8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。-20℃保存备用。 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 50μg/ml kanamycin, and culture with shaking at 37°C for 4h. Transfer to 50ml of fresh TB liquid medium also containing 50μg/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.
2.反应酶液的获取：2. Acquisition of reaction enzyme solution:
将收集的菌体使用PBS(50mM、pH 6.0)按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β-1,3-糖基转移酶酶液进行80℃处理15min，12000rpm离心2min即获得反应酶液。-4℃保存备用。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 β-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.
实施例7第二轮突变体的筛选Example 7 The second round of screening of mutants
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β-1,3-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100倍，进行HPLC分析Reb A的浓度。使用HPLC检测方法1获得的实验结果如表8所示。Using stevioside (95% stevioside content, Bid Pharmaceuticals) as the substrate, add 150 μL of reaction enzyme solution of β-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, 30μL 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.
表8Table 8
Figure PCTCN2022113874-appb-000008
Figure PCTCN2022113874-appb-000008
Figure PCTCN2022113874-appb-000009
Figure PCTCN2022113874-appb-000009
注：Enz.18～Enz.29由GT001-257-F/R进行NNK获得，Enz.30-Enz.35由GT001-265-F/R进行NNK获得。Note: Enz.18～Enz.29 were obtained by NNK of GT001-257-F/R, and Enz.30-Enz.35 were obtained by NNK of GT001-265-F/R.
由表8中的结果可知：Enz.9、Enz.10、Enz.12、Enz.13、Enz.18、Enz.21、Enz.22、Enz.25、Enz.27、Enz.30的酶活高于Enz.1，其中Enz.10的活性最高，超出Enz.1 8％左右。图7是表8中Enz.10催化合成RA活性的HPLC图。后续在Enz.10的基础上进行新一轮突变并筛选。From the results in Table 8, it can be seen that the enzyme activity of Enz.9, Enz.10, Enz.12, Enz.13, Enz.18, Enz.21, Enz.22, Enz.25, Enz.27, Enz.30 Higher than Enz.1, of which Enz.10 has the highest activity, about 8% higher than Enz.1. Fig. 7 is the HPLC chart of the catalytic synthesis of RA activity of Enz.10 in Table 8. A new round of mutation and screening will be carried out on the basis of Enz.10.
实施例8第三轮β-1,3-糖基转移酶突变体文库的构建Example 8 Construction of the third round of β-1,3-glycosyltransferase mutant library
将第二轮得到的编码Enz.10的基因连接载体pET28a，得到pET28a-Enz.10重组质粒，以pET28a-Enz.10为模板，采用表9所示的引物序列，采用KOD酶进行PCR扩增目标DNA片段和载体片段。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.
表9Table 9
Figure PCTCN2022113874-appb-000010
Figure PCTCN2022113874-appb-000010
Figure PCTCN2022113874-appb-000011
Figure PCTCN2022113874-appb-000011
PCR扩增反应体系为：The PCR amplification reaction system is:
KOD Mix：25μL；KOD Mix: 25 μL;
ddH 2O：20μL； ddH2O : 20 μL;
引物：2μL*2；Primers: 2μL*2;
模板：1μL。Template: 1 μL.
PCR扩增程序如下：The PCR amplification procedure is as follows:
(1)98℃3min(1) 98°C for 3 minutes
(2)98℃10s(2) 98℃10s
(3)55℃5s(3) 55℃5s
(4)68℃5s/kbp(4) 68℃5s/kbp
(5)68℃5min(5) 5min at 68°C
(6)12℃保温(6) 12°C insulation
(2)～(4)循环34次。(2)～(4) cycle 34 times.
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(Exnase II，5X CE II)连接至pET28a质粒载体上，得到重组质粒pET28a-Enz.36～pET28a-Enz.45。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；挑取单菌落至LB试管(Km抗性)，培养8～10h，提取质粒进行测序。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. After ligation, transform into E.coli Trans10 competent cells, spread on LB medium containing 50 μg/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.
实施例9第三轮β-1,3-糖基转移酶突变体的制备Example 9 Preparation of the third round of β-1,3-glycosyltransferase mutants
1.进行突变载体的蛋白表达：1. Perform protein expression of the mutant vector:
将实施例8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。 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 50μg/ml kanamycin, and culture with shaking at 37°C for 4h. Transfer to 50ml fresh TB liquid culture medium containing 50μg/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.
2.反应酶液的获取：2. Acquisition of reaction enzyme solution:
将收集的菌体使用PBS(50mM、pH 6.0)按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β-1,3-GT酶液进行80℃处理15min，12000rpm离心2min即获得反应酶液。-4℃保存备用。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 β-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.
实施例10第三轮突变体的筛选Example 10 The third round of mutant screening
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β-1,3-糖基转移酶突变体的反应酶液150μL，甜菊苷终浓度为100g/L，UDP或ADP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，UDP组反应60min，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Reb A的浓度；ADP组组反应20min，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Reb A的浓度(详见表10的Reb A％)。使用HPLC检测方法1获得的实验结果如表10所示。Using stevioside (95% stevioside content, Bid Pharmaceuticals) as the substrate, add 150 μL of reaction enzyme solution of β-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, 30μL of sucrose synthase, and finally add 50mM pH6.0 phosphate buffer to a final volume of 1mL. Put the prepared reaction system in a metal bath, 60°C, 600rpm, react for 60min in the UDP group, take 10μL of the reaction solution and add it to 990μL of hydrochloric acid with a pH of 2~3, vortex, centrifuge at 13000rpm for 10min, and analyze the supernatant by HPLC Reb The concentration of A; ADP group reaction 20min, get 10 μ L reaction solution and add in the hydrochloric acid of pH2～3 of 990 μ L, vortex, 13000rpm centrifugation 10min, supernatant carries out HPLC analysis Reb A concentration (see the Reb A% of Table 10 for details) . The experimental results obtained using HPLC detection method 1 are shown in Table 10.
表10Table 10
Figure PCTCN2022113874-appb-000012
Figure PCTCN2022113874-appb-000012
由表10中的初筛结果可知：(1)UDP组：突变体Enz.36、Enz.37、Enz.41、Enz.43、Enz.44和Enz.45的酶活高于Enz.1，其中Enz.45的活性最高，超出Enz.1的酶活约12％。(2)ADP组：突变体Enz.36、Enz.37、Enz.41、Enz.43、Enz.44和Enz.45的酶活高于Enz.1，其中Enz.45的活性最高，超出Enz.1的酶活约8.8％。(3)ADP组活性高于UDP组。图8是表10中ADP为核苷二磷酸条件下Enz.45催化合成RA活性的HPLC图。From the preliminary screening results in Table 10, it can be seen that: (1) UDP group: the enzyme activities of mutants Enz.36, Enz.37, Enz.41, Enz.43, Enz.44 and Enz.45 were higher than those of Enz.1, Among them, Enz.45 has the highest activity, about 12% higher than that of Enz.1. (2) ADP group: Enzyme activity of mutants Enz.36, Enz.37, Enz.41, Enz.43, Enz.44 and Enz.45 was higher than that of Enz.1, among which Enz.45 had the highest activity, surpassing that of Enz. The enzyme activity of .1 is about 8.8%. (3) The activity of ADP group was higher than that of UDP group. Fig. 8 is an HPLC graph of Enz.45 catalytic synthesis of RA activity under the condition that ADP is nucleoside diphosphate in Table 10.
实施例11β-1,2-糖基转移酶的制备 Embodiment 11 Preparation of β-1,2-glycosyltransferase
根据如核苷酸序列SEQ ID NO:51所示的β-1,2-糖基转移酶(酶编号Enz.17)的基因，全基因合成一套β-1,2-糖基转移酶基因，该基因已连接在pET28a质粒载体上得到重组质粒pET28a-Enz.17。基因合成公司：生工生物工程(上海)股份有限公司。According to the gene of β-1,2-glycosyltransferase (enzyme number Enz.17) shown in the nucleotide sequence SEQ ID NO:51, a set of β-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.
将质粒pET28a-Enz.17转化至宿主E.coli BL21(DE3)感受态细胞，得到含有β-1,2-糖基转移酶基因的工程菌株。The plasmid pET28a-Enz.17 was transformed into host E.coli BL21(DE3) competent cells to obtain engineering strains containing β-1,2-glycosyltransferase gene.
将含有β-1,2-糖基转移酶基因的工程菌在经平皿划线活化后，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12h。按2v/v％接种量转接至50ml同样含50μg/ml卡那霉素的新鲜LB液体培养基中，37℃震荡培养至OD600达到0.6-0.8时，加入IPTG至其终浓度为0.1mM，24℃诱导培养22h。培养结束后，将培养液10000rpm离心10min，弃上清液，收集菌体，置于-20℃超低温冰箱中保存，待用。After the engineering bacteria containing the β-1,2-glycosyltransferase gene are activated by streaking on the plate, pick a single colony and inoculate it into 5ml LB liquid medium containing 50μg/ml kanamycin, and culture it with shaking at 37°C for 12h . Transfer to 50ml of fresh LB liquid medium also containing 50μg/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.
配制50mM pH6.0的磷酸缓冲液(PBS)，将收集的菌体按照(M/V)1:10进行悬浮，之后，进行高压均质(550～600Mbar、1min)，经12000rpm离心2min，取上清获得β-1,2-糖基转移酶的粗酶液。Prepare 50mM pH6.0 phosphate buffer solution (PBS), suspend the collected bacteria according to (M/V) 1:10, then perform high-pressure homogenization (550-600Mbar, 1min), centrifuge at 12000rpm for 2min, and take The crude enzyme solution of β-1,2-glycosyltransferase was obtained from the supernatant.
本实施例制备的β-1,2-糖基转移酶的氨基酸序列如SEQ ID NO:52所示。The amino acid sequence of the β-1,2-glycosyltransferase prepared in this embodiment is shown in SEQ ID NO:52.
实施例12 RM合成反应 Embodiment 12 RM synthesis reaction
以Reb A 60(Reb A含量为60％)为底物，1mL反应体系中，加入β-1,3-糖基转移酶突变体的反应酶液150μL，β-1,2-糖基转移酶的反应酶液120μL，RA60终浓度为100g/L，UDP或ADP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3.5h，取10μL反应液加入990μL pH2-3的盐酸进行涡旋涡旋，13000rpm离心10min，上清进行HPLC分析Reb A、中间产物Reb D和产物Reb M的浓度，使用HPLC检测方法2获得的实验结果如表11(使用UDP)、表12(使用ADP)所示。With Reb A 60 (the content of Reb A is 60%) as the substrate, add 150 μL of reaction enzyme solution of β-1,3-glycosyltransferase mutant to 1mL reaction system, β-1,2-glycosyltransferase The reaction enzyme solution is 120 μL, 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 30μL, and finally 50mM pH6.0 phosphate buffer is added to a final volume of 1 mL. Put the prepared reaction system in a metal bath, react at 60°C and 600rpm for 3.5h, take 10μL of the reaction solution and add 990μL of pH2-3 hydrochloric acid to vortex, centrifuge at 13000rpm for 10min, and analyze the supernatant by HPLC. The concentration of product Reb D and product Reb M, the experimental results obtained using HPLC detection method 2 are shown in Table 11 (using UDP) and Table 12 (using ADP).
表11Table 11
酶enzyme RA％RA% RD％RD% RM％RM%
Enz.36Enz.36 5.565.56 10.2310.23 84.2184.21
Enz.41Enz.41 7.497.49 10.3710.37 82.1582.15
Enz.44Enz.44 8.488.48 11.5611.56 79.9679.96
Enz.45Enz.45 7.477.47 9.859.85 82.6782.67
表12Table 12
酶enzyme RA％RA% RD％RD% RM％RM%
Enz.36Enz.36 2.212.21 3.553.55 94.2494.24
Enz.41Enz.41 1.171.17 4.064.06 94.7794.77
Enz.44Enz.44 1.561.56 3.973.97 94.4894.48
Enz.45Enz.45 1.741.74 4.254.25 94.0194.01
由表11、表12中结果可知，Enz.36、Enz.41、Enz.44、Enz.45这四个突变体均适合应用于以RA60为原料进行RM的合成，UDP条件下反应3.5h转化率可达80％以上；ADP条件下反应3.5h，RM转化率可高达94％以上。图9是表11中UDP为核苷二磷酸条件下Enz.45催化合成RM活性的HPLC图。From the results in Table 11 and Table 12, it can be seen that the four mutants Enz.36, Enz.41, Enz.44, and Enz.45 are all suitable for the synthesis of RM using RA60 as a raw material, and the reaction is 3.5h under UDP conditions. The rate can reach more than 80%; under the condition of ADP for 3.5 hours, the conversion rate of RM can be as high as 94%. Fig. 9 is an HPLC chart of Enz.45 catalytic synthesis RM activity under the condition that UDP is nucleoside diphosphate in Table 11.

Claims (11)

  1. 一种糖基转移酶，其特征在于，所述糖基转移酶与SEQ ID NO:2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：A glycosyltransferase, characterized in that, compared with SEQ ID NO: 2, the glycosyltransferase comprises amino acid residue differences selected from one or more of the following residue positions:
    第14位氨基酸为I；The 14th amino acid is I;
    第189位氨基酸为L；The 189th amino acid is L;
    第257位氨基酸为A、C、L、M、S或V；The 257th amino acid is A, C, L, M, S or V;
    第265位氨基酸为E或A；The 265th amino acid is E or A;
    第273位氨基酸为G；The 273rd amino acid is G;
    第302位氨基酸为G；The 302nd amino acid is G;
    第324位氨基酸为G；The 324th amino acid is G;
    第347位氨基酸为G；The 347th amino acid is G;
    第451位氨基酸为E；The 451st amino acid is E;
    第455位氨基酸为D或C；The 455th amino acid is D or C;
    并具有不低于如SEQ ID NO:2的氨基酸序列所示的糖基转移酶活性。And have not less than the glycosyltransferase activity shown in the amino acid sequence of SEQ ID NO:2.
  2. 如权利要求1所述的糖基转移酶，其特征在于，所述糖基转移酶与SEQ ID NO:2相比的氨基酸残基差异选自以下组：Glycosyltransferase as claimed in claim 1, is characterized in that, the amino acid residue difference of described glycosyltransferase compared with SEQ ID NO:2 is selected from following group:
    (1)第265位氨基酸为E；或，(1) The 265th amino acid is E; or,
    第257位氨基酸为A，第451位氨基酸为E；或，A at amino acid 257 and E at amino acid 451; or,
    第265位氨基酸为E，第451位氨基酸为E；The 265th amino acid is E, and the 451st amino acid is E;
    (2)第14位氨基酸为I，第257位氨基酸为A且第451位氨基酸为E；或(2) amino acid at position 14 is I, amino acid at position 257 is A and amino acid at position 451 is E; or
    第257位氨基酸为A、第451位氨基酸为E且第189位氨基酸为L；或，amino acid at position 257 is A, amino acid at position 451 is E, and amino acid at position 189 is L; or,
    第257位氨基酸为A、第451位氨基酸为E且第273位氨基酸为G；或，amino acid at position 257 is A, amino acid at position 451 is E, and amino acid at position 273 is G; or,
    第257位氨基酸为A、第451位氨基酸为E且第302位氨基酸为G；或amino acid at position 257 is A, amino acid at position 451 is E, and amino acid at position 302 is G; or
    第257位氨基酸为C、第451位氨基酸为E；或C at amino acid 257 and E at amino acid 451; or
    第257位氨基酸为L、第451位氨基酸为E；或The 257th amino acid is L, and the 451st amino acid is E; or
    第257位氨基酸为M、第451位氨基酸为E；或The 257th amino acid is M, and the 451st amino acid is E; or
    第257位氨基酸为S、第451位氨基酸为E；或The 257th amino acid is S, and the 451st amino acid is E; or
    第257位氨基酸为V、第451位氨基酸为E；或The 257th amino acid is V, and the 451st amino acid is E; or
    第257位氨基酸为A、第451位氨基酸为E且第265位氨基酸为A；The 257th amino acid is A, the 451st amino acid is E, and the 265th amino acid is A;
    (3)第257位氨基酸为A、第451位氨基酸为E、第189位氨基酸为L且第14位 氨基酸为I；或，(3) the 257th amino acid is A, the 451st amino acid is E, the 189th amino acid is L and the 14th amino acid is I; or,
    第257位氨基酸为A、第451位氨基酸为E、第189位氨基酸为L且第273位氨基酸为G；或，amino acid at position 257 is A, amino acid at position 451 is E, amino acid at position 189 is L, and amino acid at position 273 is G; or,
    第257位氨基酸为A、第451位氨基酸为E、第189位氨基酸为L且第324位氨基酸为G；或，amino acid at position 257 is A, amino acid at position 451 is E, amino acid at position 189 is L, and amino acid at position 324 is G; or,
    第257位氨基酸为A、第451位氨基酸为E、第189位氨基酸为L且第347位氨基酸为G；或，amino acid at position 257 is A, amino acid at position 451 is E, amino acid at position 189 is L, and amino acid at position 347 is G; or,
    第257位氨基酸为A、第451位氨基酸为E、第189位氨基酸为L且第455位氨基酸为D或C。Amino acid 257 is A, amino acid 451 is E, amino acid 189 is L and amino acid 455 is D or C.
  3. 一种分离的核酸，其特征在于，所述核酸编码如权利要求1或2所述的糖基转移酶。An isolated nucleic acid, characterized in that the nucleic acid encodes the glycosyltransferase according to claim 1 or 2.
  4. 一种重组表达载体，其包含如权利要求3所述的核酸。A recombinant expression vector comprising the nucleic acid according to claim 3.
  5. 一种转化体，其为包含如权利要求3所述的核酸或如权利要求4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)例如E.coli BL21(DE3)。A transformant, which is a host cell comprising the nucleic acid as claimed in claim 3 or the recombinant expression vector as claimed in claim 4; preferably, the host cell is Escherichia coli (Escherichia coli) such as E. coli BL21(DE3).
  6. 一种制备如权利要求1或2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5所述的转化体。A method for preparing the glycosyltransferase according to claim 1 or 2, wherein the method comprises culturing the transformant according to claim 5 under conditions suitable for expressing the glycosyltransferase .
  7. 一种组合物，其包含如权利要求1或2所述的糖基转移酶。A composition comprising the glycosyltransferase according to claim 1 or 2.
  8. 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或2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。A method for the glycosylation of a substrate, said method comprising providing at least one substrate, a glycosyltransferase as claimed in claim 1 or 2, and allowing said substrate to be glycosylated to The substrate is contacted with the glycosyltransferase under conditions that produce at least one glycosylation product.
  9. 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或2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；A preparation method for rebaudioside A, characterized in that the preparation method comprises the following steps: in the presence of a glycosyltransferase as claimed in claim 1 or 2, carrying out stevioside and a glycosyl donor reaction to obtain rebaudioside A;
    较佳地，所述制备方法满足以下条件中的一种或多种：Preferably, the preparation method meets one or more of the following conditions:
    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；The glycosyltransferase exists in the form of glycosyltransferase bacteria, crude enzyme solution, pure enzyme, pure enzyme solution or immobilized enzyme;
    所述甜菊苷的浓度1-150g/L，优选100g/L；The concentration of the stevioside is 1-150g/L, preferably 100g/L;
    所述糖基转移酶菌体与甜菊苷的质量比为1:(3-10)，优选3:20；The mass ratio of the glycosyltransferase thallus to stevioside is 1:(3-10), preferably 3:20;
    所述糖基供体为UDP-葡萄糖和/或ADP-葡萄糖；优选通过UDP和/或ADP在蔗糖 和蔗糖合成酶的存在下制得，所述蔗糖的浓度优选为100-300g/L例如200g/L，所述UDP或所述ADP的浓度优选为0.05-0.2g/L例如0.1g/L；The glycosyl donor is UDP-glucose and/or ADP-glucose; it is preferably prepared by UDP and/or ADP in the presence of sucrose and sucrose synthase, and the concentration of the sucrose is preferably 100-300g/L such as 200g /L, the concentration of the UDP or the ADP is preferably 0.05-0.2g/L such as 0.1g/L;
    所述反应的反应溶剂的pH为5-8，优选6；The pH of the reaction solvent of the reaction is 5-8, preferably 6;
    所述pH由缓冲溶液控制，所述缓冲溶液优选磷酸缓冲溶液；The pH is controlled by a buffer solution, preferably a phosphate buffer solution;
    所述反应的转速为500-1000rpm，优选600rpm；The rotating speed of described reaction is 500-1000rpm, preferably 600rpm;
    所述反应的反应体系的温度为20-90℃，优选60℃。The temperature of the reaction system of the reaction is 20-90°C, preferably 60°C.
  10. 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9所述的制备方法制备莱鲍迪苷A的步骤。A method for preparing rebaudioside D or rebaudioside M, characterized in that it comprises the step of preparing rebaudioside A according to the preparation method as claimed in claim 9.
  11. 一种如权利要求1或2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或莱鲍迪苷M。A use of the glycosyltransferase according to claim 1 or 2 in the preparation of steviol glycosides; said steviol glycosides are preferably rebaudioside A, rebaudioside D or rebaudioside M.
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107164435A (en) * 2017-05-27 2017-09-15 中国药科大学 A kind of Rebaudiodside A KA preparation method
CN108486160A (en) * 2018-03-13 2018-09-04 兴化格林生物制品有限公司 The method that high density fermentation synthesizes rebaudioside A
WO2019161634A1 (en) * 2018-07-19 2019-08-29 邦泰生物工程(深圳)有限公司 Preparation method for rebaudioside a, enzyme for rebaudioside a preparation, and application
US20200140836A1 (en) * 2018-05-25 2020-05-07 Bontac Bio-Engineering (Shenzhen) Co., Ltd Method and enzyme for preparation of enzyme-modified stevia sugar and use of enzyme-modified stevia sugar
CN111621456A (en) * 2020-05-25 2020-09-04 安徽金禾实业股份有限公司 Method for preparing rebaudioside A through liquid fermentation
CN112375750A (en) * 2020-12-02 2021-02-19 南京工业大学 Glycosyltransferase mutant and method for catalytically synthesizing rebaudioside A by using same
CN112805295A (en) * 2018-07-30 2021-05-14 科德克希思公司 Engineering glycosyltransferases and methods of glycosylation of steviol glycosides

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2015261617C1 (en) * 2010-06-02 2019-01-31 Minnesota Specialty Yeast Llc Recombinant production of steviol glycosides
WO2019211230A1 (en) * 2018-04-30 2019-11-07 Dsm Ip Assets B.V. Steviol glycoside transport
BR112021026306A2 (en) * 2019-06-25 2022-09-06 Manus Bio Inc URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASE ENZYME
CN115418358B (en) * 2021-06-01 2024-01-30 弈柯莱生物科技(集团)股份有限公司 Glycosyltransferase and application thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107164435A (en) * 2017-05-27 2017-09-15 中国药科大学 A kind of Rebaudiodside A KA preparation method
CN108486160A (en) * 2018-03-13 2018-09-04 兴化格林生物制品有限公司 The method that high density fermentation synthesizes rebaudioside A
US20200140836A1 (en) * 2018-05-25 2020-05-07 Bontac Bio-Engineering (Shenzhen) Co., Ltd Method and enzyme for preparation of enzyme-modified stevia sugar and use of enzyme-modified stevia sugar
WO2019161634A1 (en) * 2018-07-19 2019-08-29 邦泰生物工程(深圳)有限公司 Preparation method for rebaudioside a, enzyme for rebaudioside a preparation, and application
CN112805295A (en) * 2018-07-30 2021-05-14 科德克希思公司 Engineering glycosyltransferases and methods of glycosylation of steviol glycosides
CN111621456A (en) * 2020-05-25 2020-09-04 安徽金禾实业股份有限公司 Method for preparing rebaudioside A through liquid fermentation
CN112375750A (en) * 2020-12-02 2021-02-19 南京工业大学 Glycosyltransferase mutant and method for catalytically synthesizing rebaudioside A by using same

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