CN113699132A - Application of cyclodextrin glucosyltransferase from bacillus circulans and glycosylation method of ginsenoside compound - Google Patents

Abstract

The invention relates to the fields of bioengineering and medicine, and particularly discloses cyclodextrin glucosyltransferase from bacillus circulans and application of a coding gene thereof. The enzyme is a CGTase with the highest substrate heterozygosity, can be used for obtaining glycosylation products of more ginsenoside compounds, can enrich a ginsenoside compound library, and lays a foundation for further expanding application of the ginsenoside compounds, discovering new pharmaceutically active compounds and application.

Description

Application of cyclodextrin glucosyltransferase from bacillus circulans and glycosylation method of ginsenoside compound

Technical Field

The invention relates to the fields of bioengineering and medicine, and particularly discloses cyclodextrin glucosyltransferase from bacillus circulans and application of a coding gene thereof to catalyzing glycosylation of ginsenoside compounds.

Background

The ginsenoside is mainly derived from important secondary metabolites of various rare medicinal plants such as Araliaceae ginseng (Panax ginseng), American ginseng (American ginseng), pseudo-ginseng (Panax notogeng) and the like, is a main active component for exerting the drug effect of the medicinal plants, has wide application prospect in the fields of medicine, health products, nutriment and cosmetic development, and more new medicinal preparations containing the ginsenoside and health products are developed and put on the market. With the intensive research on the pharmacological activity of ginsenoside, the ginsenoside is found to have the pharmacological activities of resisting tumors, protecting cardiac muscle, protecting nervous system and the like.

Cyclodextrin glucosyltransferases (CGTases, EC 2.4.1.19), which catalyze the cleavage and cyclization of alpha-1, 4 bonds in starch or polysaccharides to form cyclodextrin CDs, catalyze four different reaction types, including cyclization, coupling, disproportionation and hydrolysis. Wherein the specific cyclization reaction of CGTase is mainly applied to the preparation of cyclodextrin.

Numerous studies have shown that glycosylation of natural products can effectively improve the original properties of natural products, including increased water solubility, improved mouthfeel, increased stability, etc. The invention utilizes the transglycosylation function of CGTase and uses ginsenoside as a substrate to obtain a transglycosylation compound with enhanced pharmacological activity, increased water solubility or new medicinal value and other transglycosylation compounds, thereby enlarging the application range of the ginsenoside or increasing the economic benefit of the ginsenoside.

CGTase derived from Bacillus circulans catalyzes various ginsenosides and generates glycosylation in different degrees, which is the CGTase with the highest substrate heterozygosity found at present, and the glycosylation product enriches a ginsenoside compound library, thus laying a foundation for further expanding application of the ginsenosides.

Disclosure of Invention

The invention aims to provide a cyclodextrin glucosyltransferase of bacillus circulans and application of a coding gene thereof.

The invention also provides a glycosylation method of the ginsenoside compound.

The technical scheme of the invention is as follows: the cyclodextrin glucosyltransferase or the gene thereof from the bacillus circulans can be applied to catalyzing glycosylation reaction of ginsenoside compounds.

Specifically, the cyclodextrin glucosyltransferase derived from bacillus circulans contains an amino acid sequence shown as SEQ ID No. 1. Preferably, the amino acid sequence of the cyclodextrin glucosyltransferase derived from bacillus circulans is shown as SEQ ID No. 1.

The cyclodextrin glucosyltransferase gene derived from bacillus circulans encodes an amino acid sequence shown in SEQ ID No. 1.

Preferably, the nucleotide sequence of the cyclodextrin glucosyltransferase gene derived from bacillus circulans is shown as SEQ ID No. 2.

The recombinant vector or the host cell expressing the amino acid sequence shown in SEQ ID No.1 can be used for catalyzing glycosylation of ginsenoside compounds.

The recombinant vector or host cell containing the nucleotide sequence shown in SEQ ID No.2 can be used for catalyzing glycosylation of ginsenoside compounds.

A method for glycosylation of ginsenoside compounds comprises using ginsenoside compounds and dextrin as raw materials, and using cyclodextrin glucosyltransferase from Bacillus circulans, and recombinant vector or host cell capable of expressing cyclodextrin glucosyltransferase from Bacillus circulans as catalyst.

The ginsenoside compounds include notoginsenoside R1, ginsenoside Rd, ginsenoside Rh1, ginsenoside Rh2, ginsenoside Rg3, ginsenoside Rg11, notoginsenoside R2, ginsenoside Re, ginsenoside Ck, ginsenoside F2 and ginsenoside F1.

The glycosylation reaction of ginsenoside compounds increases the amount of glucose to 1-6, preferably 1-4.

The invention catalyzes a plurality of ginsenosides to generate glycosylation with different degrees by cyclodextrin glucosyltransferase CGTase from Bacillus circulans, the enzyme is CGTase with the highest substrate heterogeneity found at present, can be used for obtaining glycosylation products of a plurality of types of ginsenosides, can enrich the ginsenosides compound library, and lays a foundation for further expanding application of the ginsenosides compounds, finding new pharmaceutically active compounds and application, therefore, the invention has wide application prospect and market value.

Drawings

FIG. 1 is a PCR gel electrophoresis chart of the colonies obtained in example 2

FIG. 2 is an SDS-PAGE pattern of BcGTase protein of example 3

FIG. 3 is LC-MS diagram of example 4 showing the reaction product of glycosylation of notoginsenoside R1

FIG. 4 is the LC-MS diagram of example 4 in catalyzing ginsenoside Rd glycosylation reaction product

FIG. 5 is the LC-MS diagram of the glycosylation reaction product of ginsenoside Rh1 catalyzed by example 4

FIG. 6 is LC-MS diagram of example 4 showing the reaction product of glycosylation of ginsenoside Rh2

FIG. 7 is a LC-MS diagram of example 4 in catalyzing ginsenoside Rg3 glycosylation reaction products

FIG. 8 is the LC-MS diagram of the product of the glycosylation reaction of ginsenoside R1 catalyzed by example 4

FIG. 9 is LC-MS diagram of example 4 showing the reaction product of glycosylation of notoginsenoside R2

FIG. 10 is the LC-MS diagram of the product of glycosylation reaction of ginsenoside Re catalyzed by example 4

FIG. 11 is the LC-MS diagram of the product of glycosylation reaction of ginsenoside Ck catalyzed in example 4

FIG. 12 is the LC-MS diagram of the product of the glycosylation reaction of ginsenoside F2 catalyzed by example 4

FIG. 13 is LC-MS diagram of example 4 showing the reaction product of ginsenoside F1 glycosylation

Detailed Description

Example 1 Bacillus circulans (Bacillus circulans) CGTase Gene Synthesis

The Bacillus circulans gene sequence (GenBack: AF302787) was downloaded by NCBI and selected as pET32(a) as vector, Hind III and Not-I as polyclonal insertion sites, and gene synthesis was performed by GeneWIZ, Shanghai, Inc.

The Bc-CGTase gene sequence is shown as SEQ ID No.2:

atgaaaagatttatgaaactaacagccgtatggacactctggttatccctcacgctgggcctcttgagcccggtccacgcagccccggatacctcggtatccaacaagcagaatttcagcacggatgtcatatatcagatcttcaccgaccggttctcggacggcaatccggccaacaatccgaccggcgcggcatttgacggatcatgtacgaatcttcgcttatactgcggcggcgactggcaaggcatcatcaacaaaatcaacgacggttatttgaccggcatgggcattacggccatctggatttcacagcctgtcgagaatatctacagcgtgatcaactactccggcgtccataatacggcttatcacggctactgggcgcgggacttcaagaagaccaatccggcctacggaacgatgcaggacttcaaaaacctgatcgacaccgcgcatgcgcataacataaaagtcatcatcgactttgcaccgaaccatacatctccggcttcttcggatgatccttcctttgcagagaacggccgcttgtacgataacggcaacctgctcggcggatacaccaacgatacccaaaatctgttccaccattatggcggcacggatttctccaccattgagaacggcatttataaaaacctgtacgatctggctgacctgaatcataacaacagcagcgtcgatgtgtatctgaaggatgccatcaaaatgtggctcgacctcggggttgacggcattcgcgtggacgcggtcaagcatatgccattcggctggcagaagagctttatgtccaccattaacaactacaagccggtcttcaccttcggcgaatggttccttggcgtcaatgagattagtccggaataccatcaattcgctaacgagtccgggatgagcctgctcgatttccgctttgcccagaaggcccggcaagtgttcagggacaacaccgacaatatgtacggcctgaaagcgatgctggagggctctgaagtagactatgcccaggtgaatgaccaggtgaccttcatcgacaatcatgacatggagcgtttccacaccagcaatggcgacagacggaagctggagcaggcgctggcctttaccctgacttcacgcggtgtgcctgccatctattacggcagcgagcagtatatgtctggcgggaatgatccggacaaccgtgctcggattccttccttctccacgacgacgaccgcatatcaagtcatccaaaagctcgctccgctccgcaaatccaacccggccatcgcttacggttccacacaggagcgctggatcaacaacgatgtgatcatctatgaacgcaaattcggcaataacgtggccgttgttgccattaaccgcaatatgaacacaccggcttcgattaccggccttgtcacttccctcccgcagggcagctataacgatgtgctcggcggaattctgaacggcaatacgctaaccgtgggtgctggcggtgcagcttccaactttactttggctcctggcggcactgctgtatggcagtacacaaccgatgccacagctccgatcatcggcaatgtcggcccgatgatggccaagccaggggtcacgattacgattgacggccgcggcttcggctccggcaagggaacggtttacttcggtacaacggcagtcactggcgcggacatcgtagcttgggaagatacacaaatccaggtgaaaatccctgcggtccctggcggcatctatgatatcagagttgccaacgcagccggagcagccagcaacatctacgacaatttcgaggtgctgaccggagaccaggtcaccgttcggttcgtaatcaacaatgccacaacggcgctgggacagaatgtgttcctcacgggcaatgtcagcgagctgggcaactgggatccgaacaacgcgatcggcccgatgtataatcaggtcgtctaccaatacccgacttggtattatgatgtcagcgttccggcaggccaaacgattgaatttaaattcctgaaaaagcaaggctccaccgtcacatgggaaggcggcgcgaatcgcaccttcaccaccccaaccagcggcacggcaacgatgaatgtgaactggcagccttaa

the Bc-CGTase amino acid sequence is shown as SEQ ID No.1:

MKRFMKLTAVWTLWLSLTLGLLSPVHAAPDTSVSNKQNFSTDVIYQIFTDRFSDGNPANNPTGAAFDGSCTNLRLYCGGDWQGIINKINDGYLTGMGITAIWISQPVENIYSVINYSGVHNTAYHGYWARDFKKTNPAYGTMQDFKNLIDTAHAHNIKVIIDFAPNHTSPASSDDPSFAENGRLYDNGNLLGGYTNDTQNLFHHYGGTDFSTIENGIYKNLYDLADLNHNNSSVDVYLKDAIKMWLDLGVDGIRVDAVKHMPFGWQKSFMSTINNYKPVFTFGEWFLGVNEISPEYHQFANESGMSLLDFRFAQKARQVFRDNTDNMYGLKAMLEGSEVDYAQVNDQVTFIDNHDMERFHTSNGDRRKLEQALAFTLTSRGVPAIYYGSEQYMSGGNDPDNRARIPSFSTTTTAYQVIQKLAPLRKSNPAIAYGSTQERWINNDVIIYERKFGNNVAVVAINRNMNTPASITGLVTSLPQGSYNDVLGGILNGNTLTVGAGGAASNFTLAPGGTAVWQYTTDATAPIIGNVGPMMAKPGVTITIDGRGFGSGKGTVYFGTTAVTGADIVAWEDTQIQVKIPAVPGGIYDIRVANAAGAASNIYDNFEVLTGDQVTVRFVINNATTALGQNVFLTGNVSELGNWDPNNAIGPMYNQVVYQYPTWYYDVSVPAGQTIEFKFLKKQGSTVTWEGGANRTFTTPTSGTATMNVNWQP

EXAMPLE 2 transformation of E.coli with vector

Experimental procedures reference kit (Vital organism TOP10 chemical ly component Cell)

(1) Taking out the competent cells from a refrigerator at the temperature of-80 ℃, unfreezing at room temperature, and then rapidly putting on ice;

(2) add ligation product 5. mu.L into 50. mu.L of competent cells (in clean bench);

(3) standing on ice for 30min, thermally shocking at 42 deg.C for 90s, and immediately standing on ice for 5 min;

(4) adding 500 μ L of LB culture solution without antibiotics at 37 deg.C and shaking at 200rpm for about 1 hr;

(5) centrifuging at room temperature and 5000rpm for 1min, reserving 100 μ L of supernatant, and blowing and resuspending;

(6) coating the suspension on an LB solid culture medium containing 100mg/L Amp +, and inversely placing the suspension in a constant-temperature incubator at 37 ℃ for culture for 12-16 h;

(7) selecting 8 monoclonal colonies, inoculating the colonies in 300 mu L LB liquid medium containing 100mg/L Amp +, and shake culturing at 37 ℃ and 200rpm for more than 3 h;

(8) as shown in FIG. 1, the monoclonal positive bacteria were detected, and the target band appeared at the corresponding position and had a length of 2142 b.

a. The reaction solution preparation is shown in Table 1, wherein the upstream primer uses a universal primer, and the downstream primer uses a gene primer; PCR reaction procedures table 2.

TABLE 1 PCR reaction solution formulation

TABLE 2 PCR reaction procedure

The PCR product was detected by 0.8% agarose gel electrophoresis under the following conditions: 150V, 15 min; the colony PCR gel electrophoresis is shown in FIG. 1, and has a length of 2142 b.

mu.L of the bacterial suspension (pET32a + BcCGTase) positive for colony PCR detection was added to 5mL of LB (Amp +100mg/L) medium and cultured overnight in a shaker at 37 ℃ and 200 rpm. The thalli is cracked, the supernatant is obtained by centrifugation, the supernatant is recovered by hanging a column, washed and centrifuged, and eluted to obtain a Plasmid solution (the Plasmid extraction step refers to a Kit Easypure Plasmid MiniPrep Kit full-type gold EM101), BL21 is transformed (the method is as same as Trans1-T1 competence), and the mixture is coated on a plate and cultured in a constant temperature incubator at 37 ℃ for overnight. And (4) selecting spots, checking bacteria (the method is as same as the method of Trans1-T1 competence), and reserving a positive clone strain.

EXAMPLE 3 protein Induction expression and purification

(1) Protein induced expression

a. 10. mu.L of each of the bacterial solutions pET32a-BcCGTase-BL21 and pET32a + -BL21 (as a negative control) was added to 0.5mL of LB medium (containing Ka +100mg/L), cultured overnight, and activated twice.

b. mu.L of the resulting suspension was added to 5mL of LB medium (containing Amp +100mg/L) and cultured at 37 ℃ at 200rpm for about 6 hours. Adding 1mL of bacterial liquid into 200mL of LB (containing Amp +100mg/L) culture medium at 200rpm and 37 ℃ to culture until the OD600 value reaches 0.4-0.6;

c. adding isopropyl-beta-D-thiogalactoside (IPTG) to make the final concentration 0.2mmol/L, and culturing overnight (24h) at 80rpm and 37 ℃;

d.45, centrifuging at 000rpm for 15min to enrich the thallus, collecting the thallus in 50mL centrifuge tubes in batches, resuspending in 20mL PBS buffer (pH 7.4), re-centrifuging, and removing the supernatant; the cells were resuspended in 20mL of PBS buffer (pH 7.4).

e.200w (grade changing rod 3, power 35%), 2s ultrasound, 4s interval, ultrasonic crushing on ice for 15min, wherein the temperature of the bacterial liquid is not more than 4 ℃;

f. the ultrasonication solution was centrifuged at 12,000rpm at 4 ℃ for 15min, and the supernatant, i.e., the crude protein solution, was collected. (in preliminary experiments, 1mL of sonicate was centrifuged at 4 ℃ for 15min at 12,000rpm, and the supernatant, i.e., crude protein solution, was collected and the pellet was resuspended in an equal volume of 1 XPBS buffer.)

(2) Protein purification and concentration

The respective solutions required for purification were prepared as indicated using a Bio-Scale Mini profinity IMAC Cartridges purification column, adding ultrapure water to 1000mL, KOH or H₃PO₄Adjusting pH to 7.4, filtering with 0.22 μm microporous membrane, and standing at 4 deg.C.

TABLE 3 buffer solution formulation

a. Passing the obtained supernatant crude protein solution and each buffer solution through a 0.22 mu m microporous filter membrane;

b. the column was equilibrated with wash buffer 1 at 5 column volumes (5mL), rinsed slowly at 2 mL/min;

c. sampling the crude protein liquid at 2 mL/min;

d. washing the column by using wash buffer 1 and wash buffer 2 with 6 times of column volume in sequence, wherein the flow rate is 2 mL/min;

e. eluting the sample by using an elution buffer with 10 times of column volume, collecting the purified fraction of the target protein (pre-experiment: 1mL of eluent is respectively collected by using 1.5mL centrifuge tubes and is sequentially numbered), and slowly eluting at 2 mL/min;

f. after elution, the column was slowly rinsed with 2mL/min using wash buffer 1 at 5 column volumes;

g. the protein sample is concentrated by using millipore molecular sieve concentration column (50kDa) and the aim of desalting is fulfilled,

the method comprises the following steps:

centrifuging at 1.4 deg.C to less than 5000 Xg, and concentrating to less than 0.5-1 mL.

2. 3mL PBS buffer was added and centrifuged to less than 500. mu.L.

3. The concentrated protease was placed on ice for subsequent experiments.

Note: to avoid protein degradation, each of the above steps was performed on ice.

(3) SDS-PAGE electrophoretic analysis

mu.L of each of the supernatants obtained in the above experiment was added with 4. mu.L of protein loading buffer (6 XProtein loading buffer), mixed well and boiled in boiling water for 8min (heat denaturation at 12,000rpm for 2 min), and the supernatant was subjected to SDS-PAGE analysis.

a. 10% SDS-PAGE gel was prepared, and the formulations of the separation gel and the concentration gel are shown in the table, in which the amounts of the two gels are shown. Adding the separating glue into a glue making tank, coagulating for about 30min, adding the concentrated glue, and rapidly inserting into a comb. Immediately after setting, the product was used or wrapped with a wet absorbent paper and stored at 4 ℃.

TABLE 4 formulations of the separation and concentration gums

Composition (I)	Separating glue	Composition (I)	Concentrated glue
				Mini-Q H2O	5.0mL	Mini-Q H2O	3.7mL
30％Acr-Bis(29:1)	4.3mL	30％Acr-Bis(29:1)	0.67mL
				1M Tris·HCl(pH＝8.8)	3.5mL	1M Tris·HCl(pH＝6.8)	0.5mL
10％SDS	0.1mL	10％SDS	0.04mL
				10％AP	0.1mL	10％AP	0.04mL
TEMED	0.004mL	TEMED	0.004mL

b. And 8 mu.L of the denatured sample supernatant was uniformly added to the lane of the concentrated gel, and the electrophoresis program was performed using a Bio-Rad electrophoresis apparatus: 80V 20min 120V 60 min.

c. After the reaction is finished, taking out the gel, carefully cutting off the concentrated gel, placing the separated gel in a glass plate added with Coomassie brilliant blue fast dye solution, shaking for about 10-30min by a side shaking table, taking out the gel and placing the gel in clear water, heating the gel for 30s by a medium fire in a microwave oven, and decoloring the gel repeatedly until protein bands are obviously visible.

The molecular weight of the BcGTase protein is predicted to be 79 kDa. The results are shown in FIG. 2, M: marker; 1: no-load supernatant; 2: no-load precipitation; 3: and (5) purifying the protein. FIG. 2 shows that the difference between the empty and the supernatant is not significant, but there is a significant band at the corresponding position after purification, and the expression level of the enzyme may not be too high.

Example 4 in vitro enzyme functional validation

In vitro enzyme function validation experiments were as follows: the in vitro recombinant BcCGTase crude enzyme obtained in the embodiment, a substrate ginsenoside compound, dextrin and Tris-HCL buffer solution form a reaction system, and the reaction system is placed in a constant temperature mixer for reaction at 50 ℃ and 300rpm for 24 hours. The reaction system is shown in Table 5:

TABLE 5 in vitro recombinant enzyme activity reaction System

Components	Sample addition amount
		20mM Tris-Hcl(pH＝9.0)	400μL
Crude enzyme	50μL(10μg)
		Dextrin (Dextrin)	1mg
Ginsenoside substrate (1mg/ml)	50μL

The reaction was completed, boiled in boiling water for five minutes, extracted with ethyl acetate and evaporated to dryness, dissolved in 200. mu.L of methanol, centrifuged at 12000rpm for five minutes, and the supernatant was extracted for LC-MS detection. The blank solution was not added with enzyme (CK), and the rest of the procedure was the same as above; the Standard (Standard) solution was loaded at a concentration of 0.25 mg/ml.

Chromatographic conditions are as follows: waters acquitt UPLCT3 chromatography column (2.1 x 100 mm); the mobile phase A is 0.01 percent of formic acid water solution, and the mobile phase B is 0.01 percent of formic acid acetonitrile; gradient elution procedure, 0-7 min (2-20% B), 7-11 min (20-22% B), 11-20 min (22-60% B), 20-25 min (60-65% B), 25-28 min (65% B), 28-30 min (65-90% B), 30-33 min (95% B), 33-38 min (2% B); the flow rate is 0.4 ml/min; the sample volume is 1 mu L; the column temperature was 35 ℃.

Mass spectrum conditions: electrospray ion source (ESI), adopt negative ion mode scanning, capillary voltage is 2500V, and taper hole voltage is 30V, desolventizing gas is nitrogen gas, the gas flow: 600L/h, the desolvation temperature of 450 ℃, the ion source temperature of 150 ℃, the scanning range of m/z 50-1800Da, the scanning time of 0.35s and the collision gas of argon. Mass spectrum data acquisition and processing software MassLynx V4.1 workstation. The collision energy is 6eV in the low energy scanning and 15-30 eV in the high energy scanning. The instrument was calibrated using sodium formate solution, using leucine-enkephalin as external standard, and mass calibration (200ng/ml) was performed during data acquisition with a calibration flow rate of 5 μ L/min under positive and negative ions.

The results of using notoginsenoside R1, notoginsenoside R2, ginsenoside F1, ginsenoside F2, ginsenoside Rh1, ginsenoside Rh2, ginsenoside Rg1, ginsenoside Rg3, ginsenoside Re, ginsenoside Rd and ginsenoside Ck as substrates are as follows:

(1) the products of adding 1, 2, 3, 2, and 3 glucoses appear in the LC-MS diagram from right to left in sequence by catalyzing Notoginsenoside R1 (Notogenoside R1), as shown in FIG. 3. CK is blank control.

(2) Catalytic ginsenoside Rd (ginsenoside Rd), and a product obtained by adding 1 glucose appears on an LC-MS diagram, as shown in figure 4.

(3) Catalyzing Ginsenoside Rh1(Ginsenoside Rh1), products of adding 1, 2, 3 and 4 glucose appear from right to left on an LC-MS diagram in sequence, as shown in figure 5.

(4) Catalyzing Ginsenoside Rh2(Ginsenoside Rh2), a product of adding 1 glucose appears on an LC-MS diagram, as shown in figure 6.

(5) Catalyzing Ginsenoside Rg3(Ginsenoside Rg3), products of adding 1 and 2 glucose appear in turn from right to left on LC-MS diagram, as shown in figure 7.

(6) Catalyzing Ginsenoside Rg11(Ginsenoside Rg11), products of adding 1, 2, 1 and 3 glucose appear in turn from right to left on LC-MS diagram, as shown in figure 8.

(7) The products of adding 1 and 2 glucose appear on LC-MS diagram from right to left in sequence by catalyzing Notoginsenoside R2 (Notogenoside R2), as shown in FIG. 9.

(8) Catalyzing ginsenoside Re (ginsenoside Re), and products of adding 1 and 2 glucose appear in sequence from right to left on an LC-MS diagram, as shown in figure 10.

(9) Catalytic ginsenoside Ck (ginsenoside Ck), and products of adding 1, 2 and 3 glucose appear in sequence from right to left on an LC-MS diagram, as shown in FIG. 11.

(10) Catalyzing Ginsenoside F2(Ginsenoside F2), products of adding 1, 2 glucose appear in sequence from right to left on the LC-MS diagram as shown in fig. 12.

(11) Catalyzing Ginsenoside F1(Ginsenoside F1), products of adding 1, 2, 1, 3, 4 glucose appear in sequence from right to left on LC-MS diagram, as shown in fig. 13.

The LC-MS detection result shows that CGTase enzyme derived from Bacillus circulans has obvious transglycosylation effect on notoginsenoside R1, notoginsenoside R2, ginsenoside F1, ginsenoside F2, ginsenoside Rh1, ginsenoside Rh2, ginsenoside Rg1, ginsenoside Rg3, ginsenoside Re, ginsenoside Rd and ginsenoside Ck.

The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. The invention belongs to the technical field of the invention, and a plurality of equivalent substitutions or obvious modifications can be made without departing from the concept of the invention, and the invention has the same or similar performance or use and shall belong to the protection scope of the invention.

Sequence listing

<110> Shanghai medical university

<120> application of cyclodextrin glucosyltransferase from bacillus circulans and glycosylation method of ginsenoside compound

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 713

<212> PRT

<213> Bacillus circulans

<400> 1

Met Lys Arg Phe Met Lys Leu Thr Ala Val Trp Thr Leu Trp Leu Ser

1 5 10 15

Leu Thr Leu Gly Leu Leu Ser Pro Val His Ala Ala Pro Asp Thr Ser

20 25 30

Val Ser Asn Lys Gln Asn Phe Ser Thr Asp Val Ile Tyr Gln Ile Phe

35 40 45

Thr Asp Arg Phe Ser Asp Gly Asn Pro Ala Asn Asn Pro Thr Gly Ala

50 55 60

Ala Phe Asp Gly Ser Cys Thr Asn Leu Arg Leu Tyr Cys Gly Gly Asp

65 70 75 80

Trp Gln Gly Ile Ile Asn Lys Ile Asn Asp Gly Tyr Leu Thr Gly Met

85 90 95

Gly Ile Thr Ala Ile Trp Ile Ser Gln Pro Val Glu Asn Ile Tyr Ser

100 105 110

Val Ile Asn Tyr Ser Gly Val His Asn Thr Ala Tyr His Gly Tyr Trp

115 120 125

Ala Arg Asp Phe Lys Lys Thr Asn Pro Ala Tyr Gly Thr Met Gln Asp

130 135 140

Phe Lys Asn Leu Ile Asp Thr Ala His Ala His Asn Ile Lys Val Ile

145 150 155 160

Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala Ser Ser Asp Asp Pro

165 170 175

Ser Phe Ala Glu Asn Gly Arg Leu Tyr Asp Asn Gly Asn Leu Leu Gly

180 185 190

Gly Tyr Thr Asn Asp Thr Gln Asn Leu Phe His His Tyr Gly Gly Thr

195 200 205

Asp Phe Ser Thr Ile Glu Asn Gly Ile Tyr Lys Asn Leu Tyr Asp Leu

210 215 220

Ala Asp Leu Asn His Asn Asn Ser Ser Val Asp Val Tyr Leu Lys Asp

225 230 235 240

Ala Ile Lys Met Trp Leu Asp Leu Gly Val Asp Gly Ile Arg Val Asp

245 250 255

Ala Val Lys His Met Pro Phe Gly Trp Gln Lys Ser Phe Met Ser Thr

260 265 270

Ile Asn Asn Tyr Lys Pro Val Phe Thr Phe Gly Glu Trp Phe Leu Gly

275 280 285

Val Asn Glu Ile Ser Pro Glu Tyr His Gln Phe Ala Asn Glu Ser Gly

290 295 300

Met Ser Leu Leu Asp Phe Arg Phe Ala Gln Lys Ala Arg Gln Val Phe

305 310 315 320

Arg Asp Asn Thr Asp Asn Met Tyr Gly Leu Lys Ala Met Leu Glu Gly

325 330 335

Ser Glu Val Asp Tyr Ala Gln Val Asn Asp Gln Val Thr Phe Ile Asp

340 345 350

Asn His Asp Met Glu Arg Phe His Thr Ser Asn Gly Asp Arg Arg Lys

355 360 365

Leu Glu Gln Ala Leu Ala Phe Thr Leu Thr Ser Arg Gly Val Pro Ala

370 375 380

Ile Tyr Tyr Gly Ser Glu Gln Tyr Met Ser Gly Gly Asn Asp Pro Asp

385 390 395 400

Asn Arg Ala Arg Ile Pro Ser Phe Ser Thr Thr Thr Thr Ala Tyr Gln

405 410 415

Val Ile Gln Lys Leu Ala Pro Leu Arg Lys Ser Asn Pro Ala Ile Ala

420 425 430

Tyr Gly Ser Thr Gln Glu Arg Trp Ile Asn Asn Asp Val Ile Ile Tyr

435 440 445

Glu Arg Lys Phe Gly Asn Asn Val Ala Val Val Ala Ile Asn Arg Asn

450 455 460

Met Asn Thr Pro Ala Ser Ile Thr Gly Leu Val Thr Ser Leu Pro Gln

465 470 475 480

Gly Ser Tyr Asn Asp Val Leu Gly Gly Ile Leu Asn Gly Asn Thr Leu

485 490 495

Thr Val Gly Ala Gly Gly Ala Ala Ser Asn Phe Thr Leu Ala Pro Gly

500 505 510

Gly Thr Ala Val Trp Gln Tyr Thr Thr Asp Ala Thr Ala Pro Ile Ile

515 520 525

Gly Asn Val Gly Pro Met Met Ala Lys Pro Gly Val Thr Ile Thr Ile

530 535 540

Asp Gly Arg Gly Phe Gly Ser Gly Lys Gly Thr Val Tyr Phe Gly Thr

545 550 555 560

Thr Ala Val Thr Gly Ala Asp Ile Val Ala Trp Glu Asp Thr Gln Ile

565 570 575

Gln Val Lys Ile Pro Ala Val Pro Gly Gly Ile Tyr Asp Ile Arg Val

580 585 590

Ala Asn Ala Ala Gly Ala Ala Ser Asn Ile Tyr Asp Asn Phe Glu Val

595 600 605

Leu Thr Gly Asp Gln Val Thr Val Arg Phe Val Ile Asn Asn Ala Thr

610 615 620

Thr Ala Leu Gly Gln Asn Val Phe Leu Thr Gly Asn Val Ser Glu Leu

625 630 635 640

Gly Asn Trp Asp Pro Asn Asn Ala Ile Gly Pro Met Tyr Asn Gln Val

645 650 655

Val Tyr Gln Tyr Pro Thr Trp Tyr Tyr Asp Val Ser Val Pro Ala Gly

660 665 670

Gln Thr Ile Glu Phe Lys Phe Leu Lys Lys Gln Gly Ser Thr Val Thr

675 680 685

Trp Glu Gly Gly Ala Asn Arg Thr Phe Thr Thr Pro Thr Ser Gly Thr

690 695 700

Ala Thr Met Asn Val Asn Trp Gln Pro

705 710

<210> 2

<211> 2142

<212> DNA

<213> Bacillus circulans

<400> 2

atgaaaagat ttatgaaact aacagccgta tggacactct ggttatccct cacgctgggc 60

ctcttgagcc cggtccacgc agccccggat acctcggtat ccaacaagca gaatttcagc 120

acggatgtca tatatcagat cttcaccgac cggttctcgg acggcaatcc ggccaacaat 180

ccgaccggcg cggcatttga cggatcatgt acgaatcttc gcttatactg cggcggcgac 240

tggcaaggca tcatcaacaa aatcaacgac ggttatttga ccggcatggg cattacggcc 300

atctggattt cacagcctgt cgagaatatc tacagcgtga tcaactactc cggcgtccat 360

aatacggctt atcacggcta ctgggcgcgg gacttcaaga agaccaatcc ggcctacgga 420

acgatgcagg acttcaaaaa cctgatcgac accgcgcatg cgcataacat aaaagtcatc 480

atcgactttg caccgaacca tacatctccg gcttcttcgg atgatccttc ctttgcagag 540

aacggccgct tgtacgataa cggcaacctg ctcggcggat acaccaacga tacccaaaat 600

ctgttccacc attatggcgg cacggatttc tccaccattg agaacggcat ttataaaaac 660

ctgtacgatc tggctgacct gaatcataac aacagcagcg tcgatgtgta tctgaaggat 720

gccatcaaaa tgtggctcga cctcggggtt gacggcattc gcgtggacgc ggtcaagcat 780

atgccattcg gctggcagaa gagctttatg tccaccatta acaactacaa gccggtcttc 840

accttcggcg aatggttcct tggcgtcaat gagattagtc cggaatacca tcaattcgct 900

aacgagtccg ggatgagcct gctcgatttc cgctttgccc agaaggcccg gcaagtgttc 960

agggacaaca ccgacaatat gtacggcctg aaagcgatgc tggagggctc tgaagtagac 1020

tatgcccagg tgaatgacca ggtgaccttc atcgacaatc atgacatgga gcgtttccac 1080

accagcaatg gcgacagacg gaagctggag caggcgctgg cctttaccct gacttcacgc 1140

ggtgtgcctg ccatctatta cggcagcgag cagtatatgt ctggcgggaa tgatccggac 1200

aaccgtgctc ggattccttc cttctccacg acgacgaccg catatcaagt catccaaaag 1260

ctcgctccgc tccgcaaatc caacccggcc atcgcttacg gttccacaca ggagcgctgg 1320

atcaacaacg atgtgatcat ctatgaacgc aaattcggca ataacgtggc cgttgttgcc 1380

attaaccgca atatgaacac accggcttcg attaccggcc ttgtcacttc cctcccgcag 1440

ggcagctata acgatgtgct cggcggaatt ctgaacggca atacgctaac cgtgggtgct 1500

ggcggtgcag cttccaactt tactttggct cctggcggca ctgctgtatg gcagtacaca 1560

accgatgcca cagctccgat catcggcaat gtcggcccga tgatggccaa gccaggggtc 1620

acgattacga ttgacggccg cggcttcggc tccggcaagg gaacggttta cttcggtaca 1680

acggcagtca ctggcgcgga catcgtagct tgggaagata cacaaatcca ggtgaaaatc 1740

cctgcggtcc ctggcggcat ctatgatatc agagttgcca acgcagccgg agcagccagc 1800

aacatctacg acaatttcga ggtgctgacc ggagaccagg tcaccgttcg gttcgtaatc 1860

aacaatgcca caacggcgct gggacagaat gtgttcctca cgggcaatgt cagcgagctg 1920

ggcaactggg atccgaacaa cgcgatcggc ccgatgtata atcaggtcgt ctaccaatac 1980

ccgacttggt attatgatgt cagcgttccg gcaggccaaa cgattgaatt taaattcctg 2040

aaaaagcaag gctccaccgt cacatgggaa ggcggcgcga atcgcacctt caccacccca 2100

accagcggca cggcaacgat gaatgtgaac tggcagcctt aa 2142

Claims (10)

1. Application of cyclodextrin glucosyltransferase or its gene derived from Bacillus circulans in catalyzing glycosylation reaction of ginsenoside compounds.

2. The use according to claim 1, wherein the cyclodextrin glycosyltransferase derived from Bacillus circulans comprises the amino acid sequence shown in SEQ ID No. 1.

3. The use according to claim 1, wherein the amino acid sequence of the cyclodextrin glucosyltransferase from bacillus circulans is shown in SEQ ID No. 1.

4. The use according to claim 1, wherein the cyclodextrin glucosyltransferase gene from bacillus circulans encodes the amino acid sequence shown in SEQ ID No. 1.

5. The use according to claim 1, wherein the nucleotide sequence of the cyclodextrin glucosyltransferase gene from bacillus circulans is shown in SEQ ID No. 2.

6. The recombinant vector or host cell expressing the amino acid sequence shown in SEQ ID No.1 is used for catalyzing the glycosylation reaction of the ginsenoside compounds.

7. The recombinant vector or host cell containing the nucleotide sequence shown in SEQ ID No.2 is used for catalyzing the glycosylation reaction of the ginsenoside compounds.

8. A method for glycosylation of ginsenoside compounds is characterized in that the ginsenoside compounds and dextrin are used as raw materials, and cyclodextrin glucosyltransferase from bacillus circulans, a recombinant vector or host cell capable of expressing the cyclodextrin glucosyltransferase from bacillus circulans are used as catalysts.

9. The method of claim 8, wherein the ginsenoside compounds comprise notoginsenoside R1, ginsenoside Rd, ginsenoside Rh1, ginsenoside Rh2, ginsenoside Rg3, ginsenoside Rg11, notoginsenoside R2, ginsenoside Re, ginsenoside Ck, ginsenoside F2 and ginsenoside F1.

10. The method of claim 8, wherein the amount of glucose added is 1-6.

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