CN113980931B - Application of glucuronic acid hydrolase and mutant thereof in preparation of oleanolic acid-beta-D-glucopyranosyl ester - Google Patents
Application of glucuronic acid hydrolase and mutant thereof in preparation of oleanolic acid-beta-D-glucopyranosyl ester Download PDFInfo
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- CN113980931B CN113980931B CN202111382752.5A CN202111382752A CN113980931B CN 113980931 B CN113980931 B CN 113980931B CN 202111382752 A CN202111382752 A CN 202111382752A CN 113980931 B CN113980931 B CN 113980931B
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- glycoside
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- 229940097043 glucuronic acid Drugs 0.000 title claims abstract description 60
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 title claims abstract description 51
- KMKFOIBUKYMVRJ-YHFBEQRYSA-N Oleanolic acid 28-O-beta-D-glucopyranoside Chemical compound O=C([C@]12CCC(C[C@H]1C=1[C@@]([C@@]3(CC[C@H]4C(C)(C)[C@@H](O)CC[C@]4(C)[C@H]3CC=1)C)(C)CC2)(C)C)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O KMKFOIBUKYMVRJ-YHFBEQRYSA-N 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title abstract description 10
- MPDGHEJMBKOTSU-YKLVYJNSSA-N 18beta-glycyrrhetic acid Chemical compound C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C(O)=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](O)C1(C)C MPDGHEJMBKOTSU-YKLVYJNSSA-N 0.000 claims abstract description 89
- MPDGHEJMBKOTSU-UHFFFAOYSA-N Glycyrrhetinsaeure Natural products C12C(=O)C=C3C4CC(C)(C(O)=O)CCC4(C)CCC3(C)C1(C)CCC1C2(C)CCC(O)C1(C)C MPDGHEJMBKOTSU-UHFFFAOYSA-N 0.000 claims abstract description 47
- 229960003720 enoxolone Drugs 0.000 claims abstract description 47
- 229930182490 saponin Natural products 0.000 claims abstract description 45
- 150000007949 saponins Chemical class 0.000 claims abstract description 45
- 238000006243 chemical reaction Methods 0.000 claims abstract description 36
- 150000001875 compounds Chemical class 0.000 claims abstract description 36
- JKLISIRFYWXLQG-UHFFFAOYSA-N Epioleonolsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4CCC3C21C JKLISIRFYWXLQG-UHFFFAOYSA-N 0.000 claims abstract description 34
- YBRJHZPWOMJYKQ-UHFFFAOYSA-N Oleanolic acid Natural products CC1(C)CC2C3=CCC4C5(C)CCC(O)C(C)(C)C5CCC4(C)C3(C)CCC2(C1)C(=O)O YBRJHZPWOMJYKQ-UHFFFAOYSA-N 0.000 claims abstract description 34
- MIJYXULNPSFWEK-UHFFFAOYSA-N Oleanolinsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C MIJYXULNPSFWEK-UHFFFAOYSA-N 0.000 claims abstract description 34
- 229940100243 oleanolic acid Drugs 0.000 claims abstract description 34
- HZLWUYJLOIAQFC-UHFFFAOYSA-N prosapogenin PS-A Natural products C12CC(C)(C)CCC2(C(O)=O)CCC(C2(CCC3C4(C)C)C)(C)C1=CCC2C3(C)CCC4OC1OCC(O)C(O)C1O HZLWUYJLOIAQFC-UHFFFAOYSA-N 0.000 claims abstract description 34
- -1 glycoside compounds Chemical class 0.000 claims abstract description 33
- 241000168720 Panax japonicus Species 0.000 claims abstract description 32
- 235000003174 Panax japonicus Nutrition 0.000 claims abstract description 32
- MIJYXULNPSFWEK-GTOFXWBISA-N 3beta-hydroxyolean-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C MIJYXULNPSFWEK-GTOFXWBISA-N 0.000 claims abstract description 29
- LPLVUJXQOOQHMX-UHFFFAOYSA-N glycyrrhetinic acid glycoside Natural products C1CC(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2=O)C(O)=O)C)(C)CC2)(C)C2C(C)(C)C1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O LPLVUJXQOOQHMX-UHFFFAOYSA-N 0.000 claims abstract description 26
- 229960004949 glycyrrhizic acid Drugs 0.000 claims abstract description 26
- UYRUBYNTXSDKQT-UHFFFAOYSA-N glycyrrhizic acid Natural products CC1(C)C(CCC2(C)C1CCC3(C)C2C(=O)C=C4C5CC(C)(CCC5(C)CCC34C)C(=O)O)OC6OC(C(O)C(O)C6OC7OC(O)C(O)C(O)C7C(=O)O)C(=O)O UYRUBYNTXSDKQT-UHFFFAOYSA-N 0.000 claims abstract description 26
- 235000019410 glycyrrhizin Nutrition 0.000 claims abstract description 26
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 claims abstract description 26
- VTAJIXDZFCRWBR-UHFFFAOYSA-N Licoricesaponin B2 Natural products C1C(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2)C(O)=O)C)(C)CC2)(C)C2C(C)(C)CC1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O VTAJIXDZFCRWBR-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000001685 glycyrrhizic acid Substances 0.000 claims abstract description 24
- 239000001397 quillaja saponaria molina bark Substances 0.000 claims abstract description 24
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 claims abstract description 15
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 claims abstract description 15
- 150000008134 glucuronides Chemical group 0.000 claims abstract description 15
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 claims abstract description 15
- RMIVRCBSQPCSCQ-BDANYOJNSA-N methyl (2s,4as,6ar,6as,6br,8ar,10s,12as,14br)-10-hydroxy-2,4a,6a,6b,9,9,12a-heptamethyl-13-oxo-3,4,5,6,6a,7,8,8a,10,11,12,14b-dodecahydro-1h-picene-2-carboxylate Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C)CC[C@@](C(=O)OC)(C)C[C@H]5C4=CC(=O)[C@@H]3[C@]21C RMIVRCBSQPCSCQ-BDANYOJNSA-N 0.000 claims abstract description 15
- OFLDMHFCMNKHGN-UHFFFAOYSA-N calendulaglycoside e Chemical compound C12CC(C)(C)CCC2(C(O)=O)CCC(C2(CCC3C4(C)C)C)(C)C1=CCC2C3(C)CCC4OC(C(C1O)O)OC(C(O)=O)C1OC1OC(CO)C(O)C(O)C1O OFLDMHFCMNKHGN-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229930182470 glycoside Natural products 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 11
- 230000003301 hydrolyzing effect Effects 0.000 claims abstract description 10
- CAKDFKUXFMLCAR-UIOGXPPZSA-L (2s,3s,4s,5r,6r)-6-[(2s,3r,4s,5s,6s)-2-[[(3s,4ar,6ar,6bs,8as,11s,12ar,14ar,14bs)-11-methoxycarbonyl-4,4,6a,6b,8a,11,14b-heptamethyl-14-oxo-2,3,4a,5,6,7,8,9,10,12,12a,14a-dodecahydro-1h-picen-3-yl]oxy]-6-carboxylato-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydr Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@H]1CC[C@]2(C)[C@H]3C(=O)C=C4[C@@]([C@@]3(CC[C@H]2C1(C)C)C)(C)CC[C@@]1(C)CC[C@](C[C@H]14)(C)C(=O)OC)C([O-])=O)[C@@H]1O[C@H](C([O-])=O)[C@@H](O)[C@H](O)[C@H]1O CAKDFKUXFMLCAR-UIOGXPPZSA-L 0.000 claims abstract description 7
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- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 claims description 12
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- AJBZENLMTKDAEK-UHFFFAOYSA-N 3a,5a,5b,8,8,11a-hexamethyl-1-prop-1-en-2-yl-1,2,3,4,5,6,7,7a,9,10,11,11b,12,13,13a,13b-hexadecahydrocyclopenta[a]chrysene-4,9-diol Chemical compound CC12CCC(O)C(C)(C)C1CCC(C1(C)CC3O)(C)C2CCC1C1C3(C)CCC1C(=C)C AJBZENLMTKDAEK-UHFFFAOYSA-N 0.000 claims description 6
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- ILRKKHJEINIICQ-OOFFSTKBSA-N Monoammonium glycyrrhizinate Chemical compound N.O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@H]1CC[C@]2(C)[C@H]3C(=O)C=C4[C@@H]5C[C@](C)(CC[C@@]5(CC[C@@]4(C)[C@]3(C)CC[C@H]2C1(C)C)C)C(O)=O)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O ILRKKHJEINIICQ-OOFFSTKBSA-N 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 2
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 claims description 2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01031—Beta-glucuronidase (3.2.1.31)
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Abstract
The invention discloses application of glucuronic acid hydrolase and a mutant thereof in catalyzing and hydrolyzing glycoside compounds containing terminal glucuronide structures to obtain corresponding aglycone compounds or corresponding deglycosylated compounds. The research result of the invention shows that the glucuronic acid hydrolase and the mutant thereof can be used for efficiently hydrolyzing calendula glycoside E, panax japonicus saponin IVa, glycyrrhizic acid, methyl glycyrrhizate and the like to correspondingly generate oleanolic acid, oleanolic acid-beta-D-glucopyranosyl ester, glycyrrhetinic acid, methyl glycyrrhetinate, mono glucuronic acid glycyrrhetinic acid methyl ester and the like. The invention expands the application of glucuronic acid hydrolase and provides a high-efficiency bioconversion method for preparing the characteristic product. The conversion conditions related by the invention are mainly temperature and reaction time, and the conversion reaction is carried out at room temperature or under the condition of temperature control. The method has the characteristics of simple enzyme preparation mode, simple reaction system, mild reaction condition, controllable reaction degree and the like, and is suitable for industrial preparation.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of glucuronic acid hydrolase and mutant thereof in the production of oleanolic acid, oleanolic acid-beta-D-glucopyranosyl ester, glycyrrhetinic acid, methyl glycyrrhetinate, mono glucuronic acid glycyrrhetinic acid and mono glucuronic acid methyl glycyrrhetinate.
Background
Triterpene saponins are the main pharmacodynamic active substances of many traditional Chinese medicines, and can be mainly divided into tetracyclic triterpene and pentacyclic triterpene. The oleanolic acid beta-D-glucopyranosyl ester belongs to a pentacyclic triterpene compound, and is a glycosyl ester compound formed by dehydration and condensation of C-28 carboxyl of oleanolic acid and C-1 hydroxyl of glucose. The content of the extract in medicinal plants is very rare, the extract is difficult to directly extract from the plants, and no proper preparation method exists, so that pharmacological research and application development of the extract are limited. In addition, oleanolic acid has strong pharmacological activities such as antibiosis, antidiabetic, antioxidation, anti-tumor, antiatherosclerotic and the like. In recent years, due to the low permeability and solubility of oleanolic acid, the oleanolic acid cannot be extracted from natural plants with high efficiency, so that the purification cost is high, and the development of the dosage forms is difficult. Therefore, the design of a proper preparation mode of the oleanolic acid has important guiding significance for the development and industrial application of the pharmaceutical dosage forms. With the continuous development of modern biotechnology such as enzyme engineering and bioconversion, a powerful tool is provided for efficiently preparing specific products. Through investigation, the panax japonicus saponin IVa is the oleanolic acid serving as a structural parent nucleus, and a part of sub-glucuronic acid and a part of sub-glucose are respectively combined at the C-3 position and the C-28 position of the oleanolic acid through glycosidic bonds, so that the panax japonicus saponin IVa can be used as a candidate substrate for converting and generating the oleanolic acid beta-D-glucopyranosyl ester. The calendula glycoside E is formed by connecting C-3 hydroxyl of oleanolic acid and one molecule of glucuronic acid through glycosidic bond, and can be used as a precursor substrate for converting and generating oleanolic acid. Therefore, the target products such as oleanolic acid and oleanolic acid beta-D-glucopyranosyl ester can be obtained efficiently by means of biotransformation by using these precursor substances or plant extracts containing the precursor substances.
The invention discloses application of glucuronic acid hydrolase. Molecular cloning, recombinant protein expression and enzyme activity detection are carried out on glucuronic acid hydrolase. Through substrate screening, the hydrolase is found to have the effect of efficiently converting calendula glycoside E into oleanolic acid and converting panax japonicus saponin IVa into oleanolic acid beta-D-glucopyranosyl ester; and also has the effects of converting glycyrrhizic acid into mono-glucuronic acid glycyrrhetinic acid or glycyrrhetinic acid, and hydrolyzing methyl glycyrrhetinate into mono-glucuronic acid glycyrrhetinic acid methyl ester or glycyrrhetinic acid methyl ester. The invention expands the application of glucuronic acid hydrolase and provides a high-efficiency bioconversion method for preparing the characteristic product.
Disclosure of Invention
The invention aims to provide application of glucuronic acid hydrolase and mutant thereof in catalyzing and hydrolyzing glycoside compounds containing terminal glucuronide structures to obtain corresponding aglycone compounds or corresponding deglycosylated compounds.
The aim of the invention can be achieved by the following technical scheme:
has the sequence shown in SEQ NO:2 or a mutant of the glucuronic acid hydrolase in catalyzing and hydrolyzing a glycoside compound containing a terminal glucuronide structure to obtain a corresponding aglycone compound or a corresponding deglycosylated compound.
As a preferred technical scheme: the glucoside compound containing the terminal glucuronide structure is any one of calendoside E, panax japonicus saponin IVa, glycyrrhizic acid and methyl glycyrrhizate, or a medicinal material extract containing the compound; the corresponding aglycone compound or the corresponding deglycosylated compound obtained by the conversion is oleanolic acid, oleanolic acid-beta-D-glucopyranosyl ester, glycyrrhetinic acid, methyl glycyrrhetinate, mono glucuronic acid glycyrrhetinic acid or mono glucuronic acid glycyrrhetinic acid methyl ester.
Further preferred is: the reaction of catalytic hydrolysis of a glycoside compound containing a terminal glucuronide structure to obtain a corresponding aglycone compound or a corresponding deglycosylated compound is any one of (1) to (4):
(1) The glucuronic acid hydrolase or the mutant of the glucuronic acid hydrolase catalyzes and hydrolyzes calendula glycoside E or extract containing the calendula glycoside E in pH buffer solution to obtain oleanolic acid;
(2) The glucuronic acid hydrolase or the mutant of the glucuronic acid hydrolase catalyzes and hydrolyzes the panax japonicus saponin IVa or the extract containing the panax japonicus saponin IVa in a pH buffer solution to obtain oleanolic acid-beta-D-glucopyranosyl ester;
(3) The glucuronic acid hydrolase or the mutant of the glucuronic acid hydrolase catalyzes and hydrolyzes glycyrrhizic acid, monoammonium glycyrrhizinate or licorice extract containing glycyrrhizic acid in pH buffer solution to obtain mono-glucuronic acid glycyrrhetinic acid or glycyrrhetinic acid;
(4) The glucuronic acid hydrolase or the mutant of the glucuronic acid hydrolase catalyzes and hydrolyzes methyl glycyrrhiza extract containing methyl glycyrrhiza in pH buffer solution to obtain methyl glycyrrhiza or methyl glycyrrhiza.
Further preferred is: the extract containing calendula extract E is total saponins of rhizoma Panacis Japonici extract, total saponins of Aralia elata extract, aralia elata extract or total saponins of Aralia taibaiensis extract;
the extract containing the panax japonicus saponin IVa is total saponins extract of panax japonicus, total saponins extract of aralia elata, extract of aralia elata or total saponins extract of aralia taibaiensis.
Further preferred is: the pH buffer solution is at least one of phosphate buffer solution, citric acid-disodium hydrogen phosphate buffer solution and Tris-HCl buffer solution, and the pH value is between 4.0 and 10.0; the reaction temperature of the catalytic hydrolysis is 4-65 ℃.
A method for the catalytic hydrolysis of a glycoside compound comprising a terminal glucuronide structure to give the corresponding aglycone compound or the corresponding deglycosylated compound, using a glycoside having the structure as shown in SEQ NO:2 or a mutant of the glucuronic acid hydrolase catalyzes the hydrolysis of a glycoside compound containing a terminal glucuronide structure to obtain a corresponding aglycone compound or a corresponding deglycosylated compound. Preferably: the glucoside compound containing the terminal glucuronide structure is any one of calendoside E, panax japonicus saponin IVa, glycyrrhizic acid and methyl glycyrrhizate, or a medicinal material extract containing the compound; the corresponding aglycone compound or the corresponding deglycosylated compound obtained by the conversion is oleanolic acid, oleanolic acid-beta-D-glucopyranosyl ester, glycyrrhetinic acid, methyl glycyrrhetinate, mono glucuronic acid glycyrrhetinic acid or mono glucuronic acid glycyrrhetinic acid methyl ester.
The glucuronic acid hydrolase is a glucuronic acid hydrolase derived from escherichia coli, and the base sequence for encoding the protein can be obtained through NCBI and other databases, and the accession number is CP084529.1. The amino acid sequence is shown in SEQ NO:2. Modifying the amino acid sequence of the glucuronic acid hydrolase by molecular means to obtain the glucuronic acid hydrolase mutant, wherein the glucuronic acid hydrolase mutant is at least one of (1) to (11):
(1) Setting SEQ NO:2, and mutating the 81 st glycine and the 126 th lysine of the amino acid sequence shown in the formula 2 into arginine;
(2) Setting SEQ NO:2, the 126 th lysine and the 137 th glutamic acid of the amino acid sequence shown in the formula 2 are mutated into arginine;
(3) Setting SEQ NO:2, the 137 th glutamic acid and 203 th aspartic acid of the amino acid sequence shown in the formula 2 are mutated into arginine;
(4) Setting SEQ NO:2, the 81 st glycine, the 126 th lysine and the 137 th glutamic acid of the amino acid sequence shown in the formula 2 are mutated into arginine;
(5) Setting SEQ NO:2, the 81 st glycine, the 126 th lysine and the 151 st aspartic acid of the amino acid sequence shown in the formula 2 are mutated into arginine;
(6) Setting SEQ NO:2, the 126 th lysine, the 151 th aspartic acid and the 203 th aspartic acid of the amino acid sequence shown in the formula 2 are mutated into arginine;
(7) Setting SEQ NO:2, the 137 th glutamic acid, the 151 th aspartic acid and the 203 th aspartic acid of the amino acid sequence shown in the formula 2 are mutated into arginine;
(8) Setting SEQ NO:2, the 81 st glycine, the 126 th lysine, the 137 th glutamic acid and the 151 th aspartic acid of the amino acid sequence shown in the formula 2 are mutated into arginine;
(9) Setting SEQ NO:2, the 81 st glycine, the 126 th lysine, the 137 th glutamic acid and the 203 th aspartic acid of the amino acid sequence shown in the formula 2 are mutated into arginine;
(10) Setting SEQ NO:2, the 81 st glycine, the 126 th lysine, the 151 st aspartic acid and the 203 st aspartic acid of the amino acid sequence shown in the formula 2 are mutated into arginine;
(11) Setting SEQ NO:2, glycine 81, lysine 126, glutamic acid 137, aspartic acid 151 and aspartic acid 203 of the amino acid sequence shown in the formula 2 are mutated into arginine.
The invention inserts the coding gene of the protein or the mutant strain thereof into a prokaryotic expression plasmid, and transfers the recombinant plasmid into an escherichia coli expression strain to obtain a recombinant escherichia coli strain for expressing the recombinant protein. Because the hydrolase is derived from escherichia coli and is completely matched with an escherichia coli expression system, a large amount of purer protease liquid can be quickly obtained by constructing a recombinant expression system through plasmids, and the purification step of recombinant protein is omitted. The crude enzyme solution can be directly used for the conversion reaction.
The glucuronic acid hydrolase realizes gene cloning and recombination by related operation of molecular biology, and then constructs recombinant bacteria. Culturing the recombinant strain, carrying out protein induced expression, collecting fermentation liquor, centrifugally collecting thalli, centrifugally collecting supernatant or freeze-drying supernatant after the thalli are crushed, and the like.
The crude protease solution obtained by the method is directly applied to catalytic hydrolysis of glycoside compounds containing a terminal glucuronide structure, such as calendula glycoside E, panax japonicus saponin IVa, glycyrrhizic acid and the like, or medicinal material extracts containing the compounds, and the corresponding aglycone compounds or corresponding deglycosylated compounds are obtained through conversion. The conversion system consists of only crude enzyme solution-substrate-pH buffer solution or crude enzyme solution-substrate.
Studies show that the glucuronic acid recombinase and the mutant thereof derived from escherichia coli can be used for preparing compounds such as oleanolic acid, oleanolic acid-beta-D-glucopyranosyl ester, glycyrrhetinic acid, mono-glucuronic acid glycyrrhetinic acid, methyl glycyrrhetinate and the like through conversion. The method comprises the following steps:
(1) The substrate for converting oleanolic acid can be calendula extract or medicinal extract containing calendula extract, such as rhizoma Panacis Japonici total saponin, rhizoma Panacis Japonici crude extract, chinese Aralia total saponin, aralia crude extract, etc.
(2) The substrate for converting oleanolic acid-beta-D-glucopyranosyl ester can be ginsenoside IVa or medicinal extract containing ginsenoside IVa such as total saponins of Panax japonicus, crude extract of Panax japonicus, total saponins of Aralia elata, crude extract of Aralia elata, etc.
(3) The substrate for converting glycyrrhetinic acid and mono glucuronic acid glycyrrhetinic acid can be glycyrrhizic acid, monoammonium glycyrrhizinate or glycyrrhiza extract containing glycyrrhizic acid.
(4) The substrate for conversion of methyl glycyrrhetinate and methyl glycyrrhetinate monoglucuronate can be methyl glycyrrhizate or Glycyrrhrizae radix extract containing methyl glycyrrhizate.
The invention can purposefully control the conversion products, such as the intermediate of glycyrrhetinic acid, which is needed to prepare glycyrrhetinic acid through hydrolysis of glycyrrhizic acid, by controlling the reaction time. Under the optimized feeding proportion and reaction time, the content of the glycyrrhetinic acid monoglucuronide in the reaction product can reach a ratio of 80% or more. The reaction time is prolonged under the same reaction feeding proportion condition, or the feeding ratio of enzyme is increased under the same reaction time, so that all glycyrrhizic acid and mono glucuronic acid glycyrrhetinic acid can be converted into glycyrrhetinic acid, and the conversion ratio of glycyrrhetinic acid can reach 90% or more.
The invention has the beneficial effects that:
experiments prove that the recombinant expressed glucuronic acid hydrolase crude extract can be used for efficiently hydrolyzing calendered glycoside E, panax japonicus saponin IVa, glycyrrhizic acid, methyl glycyrrhetate and the like to correspondingly generate oleanolic acid, oleanolic acid-beta-D-glucopyranosyl ester, glycyrrhetinic acid, methyl glycyrrhetinate, mono-glucuronic acid glycyrrhetinic acid methyl glycyrrhetinate. The invention expands the application of glucuronic acid hydrolase and provides a high-efficiency bioconversion method for preparing the characteristic product. The conversion conditions related by the invention are mainly temperature and reaction time, and the conversion reaction is carried out at room temperature or under the condition of temperature control. The method has the characteristics of simple enzyme preparation mode, simple reaction system, mild reaction condition, controllable reaction degree and the like, and is suitable for industrial preparation.
Drawings
FIG. 1 shows SDS-PAGE of crude enzyme of Gus-GH2 in example 2.
FIG. 2 shows the results of the hydrolysis of calendula E from recombinant Gus-GH2 to oleanolic acid in the extract of Panax japonicus of example 3.
FIG. 3 shows the results of the hydrolysis of the recombinant Gus-GH2 of the panax japonicus total saponins IVa to oleanolic acid-. Beta. -D-glucopyranosyl ester in example 7.
FIG. 4 shows the results of the hydrolysis of glycyrrhizic acid in glycyrrhizin extract by recombinant Gus-GH2 to obtain glycyrrhetinic acid in example 8.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
EXAMPLE 1 cloning of E.coli-derived glucuronic acid hydrolase and Gene encoding same
Taking the genome of escherichia coli BL21 as a template, and designing and synthesizing corresponding primers; carrying out molecular cloning, constructing genetic engineering bacteria and expressing GUS genes. The cells were directly used as a Polymerase Chain Reaction (PCR) amplification template, and amplified using 5'-ATTCCATATGatgttacgtcctgtagaaaccccaa-3' and 5'-ATATCTCGAGtcattgtttgcctccctgctg-3' as primers, respectively. The PCR system is as follows: 2 XTaq Mixture 25. Mu.L, 1. Mu.L of each of the upstream and downstream primers (10 μm), and a very small amount of cell and ddH 2 O22. Mu.L. The PCR conditions were: first, the mixture is pre-denatured for 10m at 95 DEG Cin, then 30 cycles of 95 ℃ for 30s,58 ℃ for 30s, and 72 ℃ for 2 min; finally, the extension is carried out for 10min at 72 ℃. The PCR products are analyzed by agarose gel electrophoresis, and then the target DNA fragment is recovered by the kit. Double-digestion (37 ℃ C., 2 h) of the recovered product with pET-28a (+) vector was performed with restriction enzymes NdeI and XhoI, respectively; purifying the product by gel electrophoresis, recovering the purified enzyme-cut DNA product by a kit, connecting the product with T4 ligase at 16 ℃ for 6 hours, transforming the connected product into E.coli BL21 (DE 3) competent cells, selecting monoclonal, and carrying out PCR verification by using T7 primers to obtain recombinant bacteria BL21 (DE 3)/pET-28 a (+) -GUS. Plasmid sequencing of the extracted recombinant bacteria verifies that the gene of the PCR product is named GUS, and the nucleotide sequence of the PCR product is shown in SEQ NO:1, the sequence is consistent with the sequence of the reported E.coli glucuronic acid hydrolase in NCBI database, the cloning is correct, the protein coded by the gene is named as Gus-GH2, and the amino acid sequence of the protein is SEQ NO in a sequence table: 2. the plasmid containing the PCR product was designated pET-28a (+) -GUS, which is the sequence shown in SEQ No:1 inserted into the NdeI and XhoI double cleavage sites of pET-28a (+) vector.
EXAMPLE 2 preparation of Gus-GH2 crude enzyme solution
The BL21 (DE 3)/pET-28 a (+) -GUS single colony obtained in example 1 was inoculated into LB liquid medium containing kanamycin (final concentration: 50. Mu.g/mL), cultured at 37℃for 12 hours, 1mL of the bacterial liquid (1% by volume) was added to 100mL of fresh LB liquid medium, simultaneously kanamycin (final concentration: 50. Mu.g/mL) was added, and when the OD600 reached 0.5 by culture at 37℃was removed, placed in ice water for cooling for 10 minutes, followed by addition of IPTG at a final concentration of 0.2mM, and induced in a shaker at 16℃at 110rpm for 20 hours. The induced fermentation broth was centrifuged at 5000rpm at 4℃for 15min to collect the cells. The cells were collected by centrifugation after washing with 50mL of physiological saline 2 times. The cells were resuspended in 20mM pH7.4 sodium phosphate buffer and the cells were broken up by a high pressure homogenizer at a temperature of 4 ℃. The cells were pelleted by centrifugation at 12000rpm at 4℃for 20 min. The crude enzyme supernatant was lyophilized and stored at 4 ℃. SDS-PAGE protein electrophoresis of the Gus-GH2 crude enzyme solution is shown in FIG. 1.
EXAMPLE 3 conversion of Gus-GH2 to Panax japonicus Total saponins to Oleanolic acid
Dissolving a proper amount of total saponins extract of rhizoma Panacis Japonici containing calendula glycoside E (calendula glycoside E content is not less than 20%) in 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH of 7.0 to obtain substrate solution with final concentration of 10mg/ml, adding 5 times of crude enzyme solution prepared in example 2, reacting at 37deg.C in a shaker at 110rpm for 6 hr, adding water saturated n-butanol solution with equal volume, extracting for 3 times, mixing n-butanol parts, concentrating by rotary volatilizing instrument, evaporating to dryness, washing with proper amount of methanol, and evaporating solvent to obtain white powder, i.e. crude product containing oleanolic acid. Taking a proper amount of product powder, dissolving the product powder into a chromatographic test sample of 0.5mg/ml by methanol, and analyzing the chromatographic test sample by high performance liquid chromatography. The conditions of the high performance liquid chromatography are as follows: the liquid phase system was an Agilent 1260 single pump quaternary system, the chromatographic column used Agilent ZORBAX SB-C18 (4.6 mm. Times.250 mm 5 μm), mobile phase A was acetonitrile, phase B was 0.05% phosphoric acid, flow rate 1ml/min. The phase A increases from 23% to 40% with a gradient of 0 to 20 min; 20 to 30min, phase a increases from 40% to 75%; 30 th to 32 th min, phase A increased from 75% to 90%; phase a was maintained at 90% from 32 to 40 min. The sampling time is 40min, the column temperature is 30 ℃, the detector is DAD detection, the wavelength is 256nm, and the sample injection volume is 10 μl. Calendula glycoside E has been converted to oleanolic acid with a conversion ratio of 97.8% calculated on the molar mass of calendula glycoside E. As shown in the results of FIG. 2, calendula E in the Panax japonicus extract was hydrolyzed by recombinant Gus-GH2 to oleanolic acid under the experimental conditions shown.
EXAMPLE 4 Gus-GH2 hydrolysis of calendered E to Oleanolic acid
Dissolving a proper amount of calendoside E sample (with purity of 98%) into substrate solution with final concentration of 10mg/ml by using dimethyl sulfoxide, adding 10 times of the volume of crude enzyme solution prepared in example 2, reacting for 3 hours in a shaking table with speed of 110rpm at 37 ℃, adding an equal volume of water saturated n-butanol solution, extracting for 3 times, combining n-butanol parts, concentrating and evaporating to dryness by using a rotary volatilizing instrument, washing with proper amount of methanol, and evaporating solvent to obtain white powder, namely crude oleanolic acid. An appropriate amount of the product powder was dissolved in methanol to give a chromatographic test sample of 0.5mg/ml, and analyzed by thin layer chromatography or high performance liquid chromatography (refer to the conditions described in example 3), the conversion ratio of calendoside E to oleanolic acid was 98.6% based on the molar mass of calendoside E.
EXAMPLE 5 Gus-GH2 hydrolysis of Panax japonicus saponin IVa conversion to Oleanolic acid-beta-D-glucopyranosyl ester
Taking a proper amount of a panax japonicus saponin IVa sample (purity is 98%), dissolving the sample into a substrate solution with a final concentration of 10mg/ml by using dimethyl sulfoxide, adding 10 times of the volume of the crude enzyme solution prepared in the example 2, reacting for 5 hours in a shaking table with a speed of 110rpm at 37 ℃, adding an equal volume of water saturated n-butanol solution, extracting for 3 times, combining n-butanol parts, concentrating and evaporating to dryness by using a rotary volatilizing instrument, washing with proper amount of methanol, and evaporating the solvent to obtain white powder, namely the crude product of oleanolic acid-beta-D-glucopyranosyl ester. An appropriate amount of the product powder was dissolved in methanol to give a chromatographic test sample of 0.5mg/ml, and analyzed by high performance liquid chromatography (refer to the conditions described in example 3), and the conversion ratio of the ginsenoside IVa to oleanolic acid-beta-D-glucopyranosyl ester was 99.6% based on the molar mass of the ginsenoside IVa.
EXAMPLE 6 conversion of Gus-GH2 hydrolysis of aralia Total saponins to Oleanolic acid-beta-D-glucopyranosyl esters
Taking a proper amount of aralia elata saponin extract (containing not less than 32 percent of panax japonicus saponin IVa), dissolving the aralia elata saponin extract into a substrate solution with the final concentration of 10mg/ml by using methanol, adding 10 times of the crude enzyme solution prepared in the example 2, reacting for 6 hours in a shaking table with the speed of 110rpm at 37 ℃, adding an equal volume of water saturated n-butanol solution for extraction for 3 times, combining n-butanol parts, concentrating and evaporating the n-butanol parts by a rotary volatilizing instrument, washing the mixture with a proper amount of methanol, and evaporating the solvent to obtain white powder, namely a crude product containing oleanolic acid-beta-D-glucopyranosyl ester. An appropriate amount of product powder was taken and dissolved in methanol to give a chromatographic test sample of 0.5mg/ml, and analyzed by high performance liquid chromatography (refer to the conditions described in example 3), and the conversion ratio of the ginsenoside IVa in the fractions was 97.9% based on the molar mass of the ginsenoside IVa.
EXAMPLE 7 conversion of Gus-GH2 hydrolysis of Panax japonicus Total saponins to Oleanolic acid-beta-D-glucopyranosyl esters
Dissolving a proper amount of total saponins of panax japonicus extract (containing not less than 25% of panax japonicus IVa) into a substrate solution with a final concentration of 10mg/ml by using methanol, adding 10 times of the crude enzyme solution prepared in example 2, reacting for 6 hours in a shaking table at 37 ℃ and 110rpm, adding an equal volume of water saturated n-butanol solution for extraction for 3 times, combining n-butanol parts, concentrating and evaporating to dryness by a rotary volatilizing instrument, washing by using a proper amount of methanol, and evaporating the solvent to obtain white powder, namely a crude product containing oleanolic acid-beta-D-glucopyranosyl ester. Taking a proper amount of product powder, dissolving the product powder into a chromatographic test sample of 0.5mg/ml by methanol, and analyzing the chromatographic test sample by thin layer chromatography or high performance liquid chromatography (refer to the condition described in the example 3), wherein the conversion ratio of the ginsenoside IVa in the components is 99.0 percent calculated according to the molar mass of the ginsenoside IVa. As shown in the results of FIG. 3, under the experimental conditions, the panax japonicus saponin IVa in the total saponins of panax japonicus is hydrolyzed and converted into oleanolic acid-beta-D-glucopyranosyl ester by recombinant Gus-GH 2.
Example 8 conversion of Gus-GH2 hydrolyzed Glycyrrhiza extract to Glycyrrhetinic acid monoglucuronate
Dissolving proper amount of Glycyrrhrizae radix extract (containing glycyrrhizic acid and glycyrrhetinic acid with content of not less than 50%), 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH of 7.0 to obtain substrate solution with final concentration of 10mg/ml, adding 5 times volume of crude enzyme solution prepared in example 2, reacting at 37deg.C in a shaker at 110rpm for 1 hr, adding equal volume of water saturated n-butanol solution, extracting for 3 times, mixing n-butanol parts, concentrating and evaporating to dryness by rotary volatilizing instrument, washing with proper amount of methanol, and evaporating solvent to obtain white powder which is crude product containing glycyrrhetinic acid monoglucuronate (content of glycyrrhetinic acid monoglucuronic acid is not less than 35%). Dissolving appropriate amount of product powder with methanol to obtain 0.5mg/ml chromatographic test sample, and analyzing by thin layer chromatography or high performance liquid chromatography. The high performance liquid chromatography conditions of glycyrrhizic acid and glycyrrhetinic acid product are as follows: the liquid phase system was an Agilent 1260 single pump quaternary system using Agilent ZORBAX SB-C18 (4.6 mm 250mm 5 μm) column with mobile phase A of acetonitrile and phase B of 0.05% phosphoric acid at a flow rate of 1ml/min. Gradient 0 to 20min was used with phase a increasing from 23% to 40%; 20 to 30min, phase a increases from 40% to 75%; 30 th to 32 th min, phase A increased from 75% to 90%; phase a was maintained at 90% from 32 to 40 min. The sampling time is 40min, the column temperature is 30 ℃, the detector is DAD detection, the wavelength is 256nm, and the sample injection volume is 5 μl. The glycyrrhizic acid in the components is converted into mono-glucuronic acid glycyrrhetinic acid and glycyrrhetinic acid, the conversion ratio is 87.1% and the conversion ratio of the mono-glucuronic acid glycyrrhetinic acid is 6.7% calculated according to the mole mass of the glycyrrhizic acid. As shown in the results of FIG. 4, glycyrrhizic acid in glycyrrhizin extract was converted into glycyrrhetinic acid by hydrolysis of recombinant Gus-GH2 under the experimental conditions shown.
Example 9 conversion of Gus-GH2 to Glycyrrhetinic acid monoammonium salt hydrolysis
Dissolving proper amount of monoammonium glycyrrhizinate into substrate solution with final concentration of 10mg/ml by using dimethyl sulfoxide, adding 10 times of the volume of crude enzyme solution prepared in example 2, reacting for 6 hours in a shaking table at 37 ℃ and 110rpm, adding equal volume of water saturated n-butanol solution, extracting for 3 times, combining n-butanol parts, concentrating and evaporating to dryness by using a rotary volatilizing instrument, washing by using proper amount of methanol, and evaporating solvent to obtain white powder, namely glycyrrhetinic acid crude product. And dissolving a proper amount of product powder into a chromatographic test sample of 0.5mg/ml by using methanol, and analyzing by using high performance liquid chromatography (the analysis condition is referred to in reference example 8), wherein the glycyrrhizic acid in the components is completely converted into the glycyrrhetinic acid, and the conversion ratio is 98.7 percent according to the mole mass of the glycyrrhizic acid.
GUS sequence:
atgttacgtcctgtagaaaccccaacccgtgaaatcaaaaaactcgacggcctgtgggcattcagtctggatcgcgaaaactgtggaattgatcagcgttggtgggaaagcgcgttacaagaaagccgggcaattgctgtgccaggcagttttaacgatcagttcgccgatgcagatattcgtaattatgcgggcaacgtctggtatcagcgcgaagtctttataccgaaaggttgggcaggccagcgtatcgtgctgcgtttcgatgcggtcactcattacggcaaagtgtgggtcaataatcaggaagtgatggagcatcagggcggctatacgccatttgaagccgatgtcacgccgtatgttattgccgggaaaagtgtacgtatcaccgtttgtgtgaacaacgaactgaactggcagactatcccgccgggaatggtgattaccgacgaaaacggcaagaaaaagcagtcttacttccatgatttctttaactatgccgggatccatcgcagcgtaatgctctacaccacgccgaacacctgggtggacgatatcaccgtggtgacgcatgtcgcgcaagactgtaaccacgcgtctgttgactggcaggtggtggccaatggtgatgtcagcgttgaactgcgtgatgcggatcaacaggtggttgcaactggacaaggcactagcgggactttgcaagtggtgaatccgcacctctggcaaccgggtgaaggttatctctatgaactgtgcgtcacagccaaaagccagacagagtgtgatatctacccgcttcgcgtcggcatccggtcagtggcagtgaagggcgaacagttcctgattaaccacaaaccgttctactttactggctttggtcgtcatgaagatgcggacttgcgtggcaaaggattcgataacgtgctgatggtgcacgaccacgcattaatggactggattggggccaactcctaccgtacctcgcattacccttacgctgaagagatgctcgactgggcagatgaacatggcatcgtggtgattgatgaaactgctgctgtcggctttaacctctctttaggcattggtttcgaagcgggcaacaagccgaaagaactgtacagcgaagaggcagtcaacggggaaactcagcaagcgcacttacaggcgattaaagagctgatagcgcgtgacaaaaaccacccaagcgtggtgatgtggagtattgccaacgaaccggatacccgtccgcaaggtgcacgggaatatttcgcgccactggcggaagcaacgcgtaaactcgacccgacgcgtccgatcacctgcgtcaatgtaatgttctgcgacgctcacaccgataccatcagcgatctctttgatgtgctgtgcctgaaccgttattacggatggtatgtccaaagcggcgatttggaaacggcagagaaggtactggaaaaagaacttctggcctggcaggagaaactgcatcagccgattatcatcaccgaatacggcgtggatacgttagccgggctgcactcaatgtacaccgacatgtggagtgaagagtatcagtgtgcatggctggatatgtatcaccgcgtctttgatcgcgtcagcgccgtcgtcggtgaacaggtatggaatttcgccgattttgcgacctcgcaaggcatattgcgcgttggcggtaacaagaaagggatcttcactcgcgaccgcaaaccgaagtcggcggcttttctgctgcaaaaacgctggactggcatgaacttcggtgaaaaaccgcagcagggaggcaaacaatga
GUD protein sequence:
MLRPVETPTREIKKLDGLWAFSLDRENCGIDQRWWESALQESRAIAVPGSFNDQFADADIRNYAGNVWYQREVFIPKGWAGQRIVLRFDAVTHYGKVWVNNQEVMEHQGGYTPFEADVTPYVIAGKSVRITVCVNNELNWQTIPPGMVITDENGKKKQSYFHDFFNYAGIHRSVMLYTTPNTWVDDITVVTHVAQDCNHASVDWQVVANGDVSVELRDADQQVVATGQGTSGTLQVVNPHLWQPGEGYLYELCVTAKSQTECDIYPLRVGIRSVAVKGEQFLINHKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDWIGANSYRTSHYPYAEEMLDWADEHGIVVIDETAAVGFNLSLGIGFEAGNKPKELYSEEAVNGETQQAHLQAIKELIARDKNHPSVVMWSIANEPDTRPQGAREYFAPLAEATRKLDPTRPITCVNVMFCDAHTDTISDLFDVLCLNRYYGWYVQSGDLETAEKVLEKELLAWQEKLHQPIIITEYGVDTLAGLHSMYTDMWSEEYQCAWLDMYHRVFDRVSAVVGEQVWNFADFATSQGILRVGGNKKGIFTRDRKPKSAAFLLQKRWTGMNFGEKPQQGGKQ*
sequence listing
<110> Shanghai university of Chinese medicine
<120> application of glucuronic acid hydrolase and mutant thereof in preparation of oleanolic acid-beta-D-glucopyranosyl ester
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1812
<212> DNA
<213> Escherichia coli BL21 (Escherichia coli)
<400> 1
atgttacgtc ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg cctgtgggca 60
ttcagtctgg atcgcgaaaa ctgtggaatt gatcagcgtt ggtgggaaag cgcgttacaa 120
gaaagccggg caattgctgt gccaggcagt tttaacgatc agttcgccga tgcagatatt 180
cgtaattatg cgggcaacgt ctggtatcag cgcgaagtct ttataccgaa aggttgggca 240
ggccagcgta tcgtgctgcg tttcgatgcg gtcactcatt acggcaaagt gtgggtcaat 300
aatcaggaag tgatggagca tcagggcggc tatacgccat ttgaagccga tgtcacgccg 360
tatgttattg ccgggaaaag tgtacgtatc accgtttgtg tgaacaacga actgaactgg 420
cagactatcc cgccgggaat ggtgattacc gacgaaaacg gcaagaaaaa gcagtcttac 480
ttccatgatt tctttaacta tgccgggatc catcgcagcg taatgctcta caccacgccg 540
aacacctggg tggacgatat caccgtggtg acgcatgtcg cgcaagactg taaccacgcg 600
tctgttgact ggcaggtggt ggccaatggt gatgtcagcg ttgaactgcg tgatgcggat 660
caacaggtgg ttgcaactgg acaaggcact agcgggactt tgcaagtggt gaatccgcac 720
ctctggcaac cgggtgaagg ttatctctat gaactgtgcg tcacagccaa aagccagaca 780
gagtgtgata tctacccgct tcgcgtcggc atccggtcag tggcagtgaa gggcgaacag 840
ttcctgatta accacaaacc gttctacttt actggctttg gtcgtcatga agatgcggac 900
ttgcgtggca aaggattcga taacgtgctg atggtgcacg accacgcatt aatggactgg 960
attggggcca actcctaccg tacctcgcat tacccttacg ctgaagagat gctcgactgg 1020
gcagatgaac atggcatcgt ggtgattgat gaaactgctg ctgtcggctt taacctctct 1080
ttaggcattg gtttcgaagc gggcaacaag ccgaaagaac tgtacagcga agaggcagtc 1140
aacggggaaa ctcagcaagc gcacttacag gcgattaaag agctgatagc gcgtgacaaa 1200
aaccacccaa gcgtggtgat gtggagtatt gccaacgaac cggatacccg tccgcaaggt 1260
gcacgggaat atttcgcgcc actggcggaa gcaacgcgta aactcgaccc gacgcgtccg 1320
atcacctgcg tcaatgtaat gttctgcgac gctcacaccg ataccatcag cgatctcttt 1380
gatgtgctgt gcctgaaccg ttattacgga tggtatgtcc aaagcggcga tttggaaacg 1440
gcagagaagg tactggaaaa agaacttctg gcctggcagg agaaactgca tcagccgatt 1500
atcatcaccg aatacggcgt ggatacgtta gccgggctgc actcaatgta caccgacatg 1560
tggagtgaag agtatcagtg tgcatggctg gatatgtatc accgcgtctt tgatcgcgtc 1620
agcgccgtcg tcggtgaaca ggtatggaat ttcgccgatt ttgcgacctc gcaaggcata 1680
ttgcgcgttg gcggtaacaa gaaagggatc ttcactcgcg accgcaaacc gaagtcggcg 1740
gcttttctgc tgcaaaaacg ctggactggc atgaacttcg gtgaaaaacc gcagcaggga 1800
ggcaaacaat ga 1812
<210> 2
<211> 603
<212> PRT
<213> Escherichia coli BL21 (Escherichia coli)
<400> 2
Met Leu Arg Pro Val Glu Thr Pro Thr Arg Glu Ile Lys Lys Leu Asp
1 5 10 15
Gly Leu Trp Ala Phe Ser Leu Asp Arg Glu Asn Cys Gly Ile Asp Gln
20 25 30
Arg Trp Trp Glu Ser Ala Leu Gln Glu Ser Arg Ala Ile Ala Val Pro
35 40 45
Gly Ser Phe Asn Asp Gln Phe Ala Asp Ala Asp Ile Arg Asn Tyr Ala
50 55 60
Gly Asn Val Trp Tyr Gln Arg Glu Val Phe Ile Pro Lys Gly Trp Ala
65 70 75 80
Gly Gln Arg Ile Val Leu Arg Phe Asp Ala Val Thr His Tyr Gly Lys
85 90 95
Val Trp Val Asn Asn Gln Glu Val Met Glu His Gln Gly Gly Tyr Thr
100 105 110
Pro Phe Glu Ala Asp Val Thr Pro Tyr Val Ile Ala Gly Lys Ser Val
115 120 125
Arg Ile Thr Val Cys Val Asn Asn Glu Leu Asn Trp Gln Thr Ile Pro
130 135 140
Pro Gly Met Val Ile Thr Asp Glu Asn Gly Lys Lys Lys Gln Ser Tyr
145 150 155 160
Phe His Asp Phe Phe Asn Tyr Ala Gly Ile His Arg Ser Val Met Leu
165 170 175
Tyr Thr Thr Pro Asn Thr Trp Val Asp Asp Ile Thr Val Val Thr His
180 185 190
Val Ala Gln Asp Cys Asn His Ala Ser Val Asp Trp Gln Val Val Ala
195 200 205
Asn Gly Asp Val Ser Val Glu Leu Arg Asp Ala Asp Gln Gln Val Val
210 215 220
Ala Thr Gly Gln Gly Thr Ser Gly Thr Leu Gln Val Val Asn Pro His
225 230 235 240
Leu Trp Gln Pro Gly Glu Gly Tyr Leu Tyr Glu Leu Cys Val Thr Ala
245 250 255
Lys Ser Gln Thr Glu Cys Asp Ile Tyr Pro Leu Arg Val Gly Ile Arg
260 265 270
Ser Val Ala Val Lys Gly Glu Gln Phe Leu Ile Asn His Lys Pro Phe
275 280 285
Tyr Phe Thr Gly Phe Gly Arg His Glu Asp Ala Asp Leu Arg Gly Lys
290 295 300
Gly Phe Asp Asn Val Leu Met Val His Asp His Ala Leu Met Asp Trp
305 310 315 320
Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr Pro Tyr Ala Glu Glu
325 330 335
Met Leu Asp Trp Ala Asp Glu His Gly Ile Val Val Ile Asp Glu Thr
340 345 350
Ala Ala Val Gly Phe Asn Leu Ser Leu Gly Ile Gly Phe Glu Ala Gly
355 360 365
Asn Lys Pro Lys Glu Leu Tyr Ser Glu Glu Ala Val Asn Gly Glu Thr
370 375 380
Gln Gln Ala His Leu Gln Ala Ile Lys Glu Leu Ile Ala Arg Asp Lys
385 390 395 400
Asn His Pro Ser Val Val Met Trp Ser Ile Ala Asn Glu Pro Asp Thr
405 410 415
Arg Pro Gln Gly Ala Arg Glu Tyr Phe Ala Pro Leu Ala Glu Ala Thr
420 425 430
Arg Lys Leu Asp Pro Thr Arg Pro Ile Thr Cys Val Asn Val Met Phe
435 440 445
Cys Asp Ala His Thr Asp Thr Ile Ser Asp Leu Phe Asp Val Leu Cys
450 455 460
Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gln Ser Gly Asp Leu Glu Thr
465 470 475 480
Ala Glu Lys Val Leu Glu Lys Glu Leu Leu Ala Trp Gln Glu Lys Leu
485 490 495
His Gln Pro Ile Ile Ile Thr Glu Tyr Gly Val Asp Thr Leu Ala Gly
500 505 510
Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu Glu Tyr Gln Cys Ala
515 520 525
Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg Val Ser Ala Val Val
530 535 540
Gly Glu Gln Val Trp Asn Phe Ala Asp Phe Ala Thr Ser Gln Gly Ile
545 550 555 560
Leu Arg Val Gly Gly Asn Lys Lys Gly Ile Phe Thr Arg Asp Arg Lys
565 570 575
Pro Lys Ser Ala Ala Phe Leu Leu Gln Lys Arg Trp Thr Gly Met Asn
580 585 590
Phe Gly Glu Lys Pro Gln Gln Gly Gly Lys Gln
595 600
Claims (5)
1. Has the sequence shown in SEQ NO:2 in catalyzing and hydrolyzing a glycoside compound containing a terminal glucuronide structure to obtain a corresponding aglycone compound or a corresponding deglycosylated compound, characterized in that: the glycoside compound containing the terminal glucuronide structure is any one of calendula glycoside E, panax japonicus saponin IVa, glycyrrhizic acid and methyl glycyrrhizate, or a medicinal material extract containing the compound; the corresponding aglycone compound or the corresponding deglycosylated compound obtained by conversion is oleanolic acid, oleanolic acid-beta-D-glucopyranosyl ester, glycyrrhetinic acid, methyl glycyrrhetinate, mono glucuronic acid glycyrrhetinic acid or mono glucuronic acid methyl glycyrrhetinate.
2. The use according to claim 1, characterized in that: the reaction of catalytically hydrolyzing the glycoside compound containing the terminal glucuronide structure to obtain the corresponding aglycone compound or the corresponding deglycosylated compound is any one of (1) to (4):
(1) The glucuronic acid hydrolase catalyzes and hydrolyzes calendula glycoside E or extract containing calendula glycoside E in pH buffer solution to obtain oleanolic acid;
(2) The glucuronic acid hydrolase catalyzes and hydrolyzes the panax japonicus saponin IVa or the extract containing the panax japonicus saponin IVa in a pH buffer solution to obtain oleanolic acid-beta-D-glucopyranosyl ester;
(3) The glucuronic acid hydrolase catalyzes and hydrolyzes glycyrrhizic acid, monoammonium glycyrrhizinate or licorice extract containing glycyrrhizic acid in pH buffer solution to obtain mono-glucuronic acid glycyrrhetinic acid or glycyrrhetinic acid;
(4) The glucuronic acid hydrolase is used for catalyzing and hydrolyzing methyl glycyrrhiza extract containing methyl glycyrrhiza in pH buffer solution to obtain methyl glycyrrhiza monoglucuronate or methyl glycyrrhiza.
3. The use according to claim 2, characterized in that:
the extract containing calendula extract E is total saponins of rhizoma Panacis Japonici extract, total saponins of Aralia elata extract, aralia elata extract or total saponins of Aralia taibaiensis extract;
the extract containing the panax japonicus saponin IVa is total saponins extract of panax japonicus, total saponins extract of aralia elata, extract of aralia elata or total saponins extract of aralia taibaiensis.
4. The use according to claim 2, characterized in that: the pH buffer solution is at least one of phosphate buffer solution, citric acid-disodium hydrogen phosphate buffer solution and Tris-HCl buffer solution, and the pH value is between 4.0 and 10.0;
the reaction temperature of the catalytic hydrolysis is 4-65 ℃.
5. A method for catalytic hydrolysis of a glycoside compound having a terminal glucuronide structure to give the corresponding aglycone compound or the corresponding deglycosylated compound, characterized by: use is made of a polypeptide having the sequence as set forth in SEQ ID NO:2, catalyzing and hydrolyzing the glycoside compound containing the terminal glucuronide structure by glucuronic acid hydrolase with the amino acid sequence shown in the formula 2 to obtain a corresponding aglycone compound or a corresponding deglycosylated compound;
the glycoside compound containing the terminal glucuronide structure is any one of calendula glycoside E, panax japonicus saponin IVa, glycyrrhizic acid and methyl glycyrrhizate, or a medicinal material extract containing the compound; the corresponding aglycone compound or the corresponding deglycosylated compound obtained by conversion is oleanolic acid, oleanolic acid-beta-D-glucopyranosyl ester, glycyrrhetinic acid, methyl glycyrrhetinate, mono glucuronic acid glycyrrhetinic acid or mono glucuronic acid methyl glycyrrhetinate.
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