IL43784A - Manufacture of hesperetin dihydrochalcone glucoside by means of an enzyme system - Google Patents
Manufacture of hesperetin dihydrochalcone glucoside by means of an enzyme systemInfo
- Publication number
- IL43784A IL43784A IL43784A IL4378473A IL43784A IL 43784 A IL43784 A IL 43784A IL 43784 A IL43784 A IL 43784A IL 4378473 A IL4378473 A IL 4378473A IL 43784 A IL43784 A IL 43784A
- Authority
- IL
- Israel
- Prior art keywords
- carrier
- enzyme
- derivative
- process according
- hesperidinase
- Prior art date
Links
- 102000004190 Enzymes Human genes 0.000 title claims description 46
- 108090000790 Enzymes Proteins 0.000 title claims description 45
- 229930182478 glucoside Natural products 0.000 title claims description 15
- -1 hesperetin dihydrochalcone glucoside Chemical class 0.000 title claims description 15
- 238000004519 manufacturing process Methods 0.000 title claims description 11
- 238000000034 method Methods 0.000 claims description 29
- 230000000694 effects Effects 0.000 claims description 20
- 108010030923 hesperidinase Proteins 0.000 claims description 17
- 239000001100 (2S)-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one Substances 0.000 claims description 16
- QUQPHWDTPGMPEX-UHFFFAOYSA-N Hesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(COC4C(C(O)C(O)C(C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-UHFFFAOYSA-N 0.000 claims description 15
- QUQPHWDTPGMPEX-UTWYECKDSA-N aurantiamarin Natural products COc1ccc(cc1O)[C@H]1CC(=O)c2c(O)cc(O[C@@H]3O[C@H](CO[C@@H]4O[C@@H](C)[C@H](O)[C@@H](O)[C@H]4O)[C@@H](O)[C@H](O)[C@H]3O)cc2O1 QUQPHWDTPGMPEX-UTWYECKDSA-N 0.000 claims description 15
- APSNPMVGBGZYAJ-GLOOOPAXSA-N clematine Natural products COc1cc(ccc1O)[C@@H]2CC(=O)c3c(O)cc(O[C@@H]4O[C@H](CO[C@H]5O[C@@H](C)[C@H](O)[C@@H](O)[C@H]5O)[C@@H](O)[C@H](O)[C@H]4O)cc3O2 APSNPMVGBGZYAJ-GLOOOPAXSA-N 0.000 claims description 15
- VUYDGVRIQRPHFX-UHFFFAOYSA-N hesperidin Natural products COc1cc(ccc1O)C2CC(=O)c3c(O)cc(OC4OC(COC5OC(O)C(O)C(O)C5O)C(O)C(O)C4O)cc3O2 VUYDGVRIQRPHFX-UHFFFAOYSA-N 0.000 claims description 15
- QUQPHWDTPGMPEX-QJBIFVCTSA-N hesperidin Chemical compound C1=C(O)C(OC)=CC=C1[C@H]1OC2=CC(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO[C@H]4[C@@H]([C@H](O)[C@@H](O)[C@H](C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-QJBIFVCTSA-N 0.000 claims description 15
- 229940025878 hesperidin Drugs 0.000 claims description 15
- ARGKVCXINMKCAZ-UHFFFAOYSA-N neohesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(CO)O3)OC3C(C(O)C(O)C(C)O3)O)=CC(O)=C2C(=O)C1 ARGKVCXINMKCAZ-UHFFFAOYSA-N 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 14
- QGGZBXOADPVUPN-UHFFFAOYSA-N dihydrochalcone Chemical compound C=1C=CC=CC=1C(=O)CCC1=CC=CC=C1 QGGZBXOADPVUPN-UHFFFAOYSA-N 0.000 claims description 14
- PXLWOFBAEVGBOA-UHFFFAOYSA-N dihydrochalcone Natural products OC1C(O)C(O)C(CO)OC1C1=C(O)C=CC(C(=O)CC(O)C=2C=CC(O)=CC=2)=C1O PXLWOFBAEVGBOA-UHFFFAOYSA-N 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- 150000004984 aromatic diamines Chemical class 0.000 claims description 11
- 108010047754 beta-Glucosidase Proteins 0.000 claims description 9
- 102000006995 beta-Glucosidase Human genes 0.000 claims description 9
- 239000012876 carrier material Substances 0.000 claims description 8
- 239000005373 porous glass Substances 0.000 claims description 8
- 230000002414 glycolytic effect Effects 0.000 claims description 7
- YBRVSVVVWCFQMG-UHFFFAOYSA-N 4,4'-diaminodiphenylmethane Chemical compound C1=CC(N)=CC=C1CC1=CC=C(N)C=C1 YBRVSVVVWCFQMG-UHFFFAOYSA-N 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 150000004760 silicates Chemical class 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- DQFBYFPFKXHELB-UHFFFAOYSA-N Chalcone Natural products C=1C=CC=CC=1C(=O)C=CC1=CC=CC=C1 DQFBYFPFKXHELB-UHFFFAOYSA-N 0.000 claims 1
- 125000000751 azo group Chemical group [*]N=N[*] 0.000 claims 1
- 235000005513 chalcones Nutrition 0.000 claims 1
- AIONOLUJZLIMTK-AWEZNQCLSA-N hesperetin Chemical compound C1=C(O)C(OC)=CC=C1[C@H]1OC2=CC(O)=CC(O)=C2C(=O)C1 AIONOLUJZLIMTK-AWEZNQCLSA-N 0.000 claims 1
- 229960001587 hesperetin Drugs 0.000 claims 1
- AIONOLUJZLIMTK-UHFFFAOYSA-N hesperetin Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(O)=CC(O)=C2C(=O)C1 AIONOLUJZLIMTK-UHFFFAOYSA-N 0.000 claims 1
- 235000010209 hesperetin Nutrition 0.000 claims 1
- FTODBIPDTXRIGS-UHFFFAOYSA-N homoeriodictyol Natural products C1=C(O)C(OC)=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 FTODBIPDTXRIGS-UHFFFAOYSA-N 0.000 claims 1
- DQFBYFPFKXHELB-VAWYXSNFSA-N trans-chalcone Chemical compound C=1C=CC=CC=1C(=O)\C=C\C1=CC=CC=C1 DQFBYFPFKXHELB-VAWYXSNFSA-N 0.000 claims 1
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- 239000007979 citrate buffer Substances 0.000 description 5
- 108010001078 naringinase Proteins 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 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 description 3
- PNEYBMLMFCGWSK-UHFFFAOYSA-N Alumina Chemical compound [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 3
- 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 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 235000009508 confectionery Nutrition 0.000 description 3
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 2
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 150000008131 glucosides Chemical class 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- WZCQRUWWHSTZEM-UHFFFAOYSA-N 1,3-phenylenediamine Chemical compound NC1=CC=CC(N)=C1 WZCQRUWWHSTZEM-UHFFFAOYSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 239000000440 bentonite Substances 0.000 description 1
- 229910000278 bentonite Inorganic materials 0.000 description 1
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 1
- HFACYLZERDEVSX-UHFFFAOYSA-N benzidine Chemical group C1=CC(N)=CC=C1C1=CC=C(N)C=C1 HFACYLZERDEVSX-UHFFFAOYSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 150000004985 diamines Chemical class 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 229910044991 metal oxide Inorganic materials 0.000 description 1
- 150000004706 metal oxides Chemical class 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 239000010456 wollastonite Substances 0.000 description 1
- 229910052882 wollastonite Inorganic materials 0.000 description 1
Classifications
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/096—Polyesters; Polyamides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
- C12N11/12—Cellulose or derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Inorganic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Saccharide Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Seasonings (AREA)
Description
fhe manufacture of heeperetin dihydro- chaloon© glueoelde by means of an enzyme system eiVAUDAU & CIE SOCIETE AMOM Ref. 6550/4 The present invention is concerned with a process for the manufacture of hesperetin dihydrochalcone glucoside.
It is known that the flavourless glycoside hesperidin, which is principally present in the peel of citrus fruits, can be converted into the very sweet (and consequently usable as a sweetener) hesperetin dihydrochalcone glucoside of the formula by treatment with alkali, hydrogenation and enzymatic hydrolysi (see, for example, United States Patent Specification No. 3» 583»894-). In so doing, the hesperidin dihydrochalcone of the formula 43784/2 .is subjected to the action of the enzyme system of naringinase the β-glucosidase activity of which has been extensively destroyed, so that it predominantly cleaves only the rhamnose. The inactivation of the β-glucosidase of the enzyme system is carried out by heating an aqueous naringinase suspension to a temperature of about 60°-65°C for 30-120 minutes at a pH of 6.4-6.8. This procedure is* however, associated with a partial loss of rhamnosidase activity, the absolute magnitude of which is not reproducible.
The technique of binding enzymes to carriers £>y " means of a polyfunctional silan compound is known from DOS 1,944,418. In this known method several reaction steps are required until the stage where the protein can be bound to the carrier is reached.
A similar method is also described in U.S. Patent Specification 3,669,841.
It has now surprisingly been found in accordance with the present invention that by bonding a glycolytic enzyme or enzyme system having rhamnosidase activity, such as is exhibited, for example by naringinase and hesperidinase , to a solid activated carrier which is covered with a layer of aromatic 4-mine , the β-glucosidase activity is selectively blocked.
The present invention is based on the foregoing finding and is accordingly concerned with a process for the manufacture of hesperetin dihydrochalcone glucoside from hesperidin dihydrochalcone by means of glycolytic enzymes or enzyme systems having rhamnosidase activity, such as is exhibited by hesperidinase and naringinase, said process being carried out using a carrier-bonded enzyme or enzyme system concerned with a glycolytic enzyme or enzyme system derivative having rhanmosidase activity and inhibited β-glucosidase activity^ which has extensively lost the β-glu'cosidase activity, especially with respect to the hesperidin dihydrochalcone as the substrate, by coupling of the enzyme or enzyme system to a solid carrier material, as well as its manufacture'.
An enzyme or enzyme system which is preferred in the presen invention is hesperidinase, a commercially available product.
Suitable solid carrier materials which are used for the manufacture ofenzyme preparations in accordance with the present invention, especially hesperidinase having inhibited β--glucosidase activity, are polymers having an organic or inorganic basis. Examples of materials having an inorganic basis are natural and synthetic silicates and silicate--containing substances such as, for example, glass, sand, silica gel, kieselguhr, bentonite and wollastonite as well as metal oxides such as aluminium oxide and hydroxylapatite.
Suitable polymers having an organic basis are, for example, cellulose and polyamides. In an especially preferred · embodiment of the present invention, there can be used as the carrier material porous glass; for example, in the form in which it is commercially available, preferably with a particle size of about 80-400 mesh and an average pore diameter of about 75-2000 iL The carrier material can, however, also have another form and/or size if the conditions should require this.
The solid carrier material is expediently covered with layer of an aromatic diamine. This can advantageously be carried out by treatment with a solution of the amine, if desired with slight warming. Suitable aromatic diamines are, for example, 1,3-phenylenediamine, 4,4' -diamino-diphenyl, 4»4' -diamino-stilbene and 4,4' -diamino-diphenyl-methane as well as the correspondingly substituted compounds. 4,4'- -diamino-diphenyl-methane is the especially preferred diamine. The thus-obtained product is then diazotised in a manner known per se; for example, by reaction with a nitrite and an acid (e.g. hydrochloric acid or acetic acid), expediently at a temperature between 0°C and room temperature. In accordance with the previously described procedure, the carrier particles are given a strongly adhering covering which is activated for the bonding of the protein.
In order to bond the enzyme to the activated solid carrier, the carrier is treated with a solution of the enzyme, a ratio of protein/carrier of 1-50 mg/g being expediently maintained. There may be used as the solvent for the enzyme any solvent which is customarily used in enzyme chemistry, but buffer solutions (e.g. phosphate or citrate buffers) are preferably used. The reaction of the enzyme with the diazotised carrier material is preferably carried out in a buffer solution having a pH of between about .5 and 8.0 while shaking for about 30 minutes to 24 hours, preferably for 1-2 hours, and at a temperature between 0°C and 30°C. When hesperidinase is used, a buffer having a pH of 6.0 to 7· is especially preferred and the preferred reaction temperature is ca 4°C.
The thus-obtained enzyme which is bonded to a solid activated carrier and the β-glucosidase activity of which is selectively inhibited, is suitable for the degradation of hesperidin dihydrochalcone to hesperetin dihydrochalcone glucoside. In addition, the thus-obtained insolublised enzyme has known advantageous properties; it can be readily separated from the substrate (e.g. by filtration or by centrifuging) , it can be used several times and it is especially suitable for use in continuous processing. Finally, the enzyme preparation obtained according to the present invention has a high stability. In the case of hesperidinase bonded to porous glass, there is observed an especially good activity and stability in a continuous process in columns such as is known, for example, in chromatography.
The reaction of the hesperidin dihydrochalcone with the carrier-bonded enzyme can be carried out in a manner known per se; namely in stationary plant (e.g. in stirred reactors) and, as mentioned earlier, advantageously in continuous plant (e.g. in reaction columns). In so doing, an initial substrate concentration of about 5% is preferably used, but solutions having higher or lower concentrations can, of course, also be used. The working-up of the reaction product does not pose any problems and it can be carried out in a known manner. For example, it can be carried out simply by concentratin the reaction mixture to dryness without further purification since the aglycone which may be present in slight amounts as a byproduct is also sweet and physiologically harmless.
The following Examples illustrate the present invention: Example 1 Manufacture of the activated carrier o 1 g of porous glass (120-200 mesh, pore diameter: 2000 A) was added to a solution of 200 mg of 4,4' -diamino-diphenyl--methane in 10 ml of dry benzene. The mixture was warmed for 5 minutes on a water-bath (80°C) with occasional shaking, then evaporated under a vacuum until all the benzene had been removed. The residue was added to 20 ml of ice-cold water, treated while stirring with 300 mg of sodium nitrite and then with dilute hydrochloric acid (20 vol-%) to a pH of 1-2. The resulting mixture was then stirred for 10 minutes at 0°C, the pH adjusted to 5 hy the addition of 1-N sodium hydroxide and the supernatant decanted off. The residue was washed several times with ice-cold water, which had been brought to pH 4-5 by the addition of hydrochloric acid, until the supernatant was colourless. The thus-obtained particles were used immediately for the enzyme Bonding.
Example 2 Bonding of the enzyme 1 g of the carrier material manufactured according to the procedure described in Example 1 was added to 25 ml of an ice-cold solution of 2 mg of hesperidinase ( 5· units/mg) in 0.05-M phosphate buffer (pH 6. 5-7.8) and shaken gently at 4°C for 1 hour. The mixture was then filtered, the particles were washed three times with 0 ml of l-M potassium chloride solution each time and once with 50 nil of 0.05-M citrate buffer (pH 6. 5)1 suspended in 50 ml of 0.05-ΓΊ citrate buffer (pH 6. 5) a d then stored at °C.
Example 3 Conversion of hesperidin dihydrochalcone into hesperetin dihydrochalcone glucoside 2 g of hesperidinase (4.3 units) bonded to porous glass, manufactured according to the procedure described in Example 2 were shaken for 2 hours at 45°C with a solution of 500 mg of hesperidin dihydrochalcone in 20 ml of 0.05-M citrate buffer (pH · )· Chromatographic analysis of the resulting solution showed that, after this time, the hesperidin dihydrochalcone had been quantitatively converted into hesperetin dihydrochalcone glucoside, whereby traces of the corresponding aglycone could be detected. The carrier-bonded enzyme was filtered off, washed with 0.05-M citrate buffer (pH 6. 5) at 45°C and used immediately or stored at 4°C for future use.
The filtrate was combined with the wash-solutions and concentrated to dryness under reduced pressure. The residue was extracted three times with 50 ml of dioxane. After removal of the solvent, the extract yielded 20 mg (70% yield) of the very sweet hesperetin dihydrochalcone glucoside.
A similarly good yield of glucoside was obtained by a triple direct extraction of the reaction solution with ethyl acetate without previous concentration.
By cooling the reaction solution with ice, a crystalline separation of the glucoside is achieved. This working-up is, in fact, not quantitative, but well-suited to large batches where extraction methods are less suitable.
Example 4 Conversion of hesperidin dihydrochalcone into hesperetin dihydrochalcone glucoside 3 g of hesperidinase (8.1 units) bonded to porous glass, manufactured according to the procedure described in Example 2, were treated in a glass column (1.0 x 10 cm) with a solution of 50 ni of hesperidin dihydrochalcone in 0.05-M citrate (pH 4.5) at 45°C, a flow rate of 11 mlAour being maintained. Continuous chromatographic analysis showed the quantitative degradation of the hesperidin dihydrochalcone to hesperetin dihydrochalcone glucoside and rhamnose as well as a trace of aglycone.
Example Conversion of hesperidin dihydrochalcone into hesperetin dihydrochalcone glucoside 118 g of hesperidin dihydrochalcone were quantitatively hydrolysed in a reaction column containing 5 S of carrier--bonded hesperidinase (20 mg of enzyme) in a continuous operation over a period of 2 weeks with an initial flow rate of 20 ml hour which returned to 10 ml/hour at the end of the operation, without the activity of the enzyme being exhausted. 43784/2
Claims (22)
1. ) A process for the manufacture of hesperetin dihydro- ^ chalcone glucoaide from hesperidin dihydrochalcone by means of .a glycolytic enzyme or enzyme system having rhamnosidase activity, said process being carried out using a carrier-bonded enzyme or enzyme system having inhibited β-glucosidase activity, wherein the carrier material is covered with a layer of aromatic diamine.
2. ) A process according to claim 1, wherein a carrier-bonded hesperidinase having inhibited jS-g ucosidase activity is used.
3. ) A process according to claim 2, wherein the carrier is an activated silica-containing material.
4. ) A process according to claim 2 and claim 3, wherein the , • i carrier -consists of porous glass having a layer of a ! diazotised aromatic diamine. j
5. ) A process according to claim 4, wherein said aromatic diamine is 4, '.-diamino-diphenyl-methane .
6. ) A process for the manufacture of a glycolytic enzyme derivative or an enzyme system derivative having rhamnosidase activity and inhibited β-glucosidase activity, which process comprises treating a carrier which is covered with a layer of aromatic diamine with a solution of an enzyme or enzyme system.
7. ) A process according ta claim 6, wherein hesperidinase is used as the enzyme.
8. ) A process according to claim 6 and claim 7, wherein an activated silicate-containing material is used as the carrier.
9. ) A process according to any one of claims 6 to 8 inclusive, wherein there is used as the carrier porous glass which is covered with a layer of diazotised aromatic diamine.
10. ) A process according to claim 9» wherein 4 , 4 ' -diamino--diphenyl-methane is used as the aromatic diamine.
11. ) A process according to any one of claims 7 to 10 inclusive, wherein the reaction of the hesperidinase with the . carrier is carried out in a buffer solution having a pH of 4.5-8.0.
12. ) A process according to claim 11, wherein the pH of the buffer solution is 7.5·
13. ) A process according to claim 11 and claim 12, wherein the reaction is carried out at abour 4°C.
14. ) A process for the manufacture of hesperetin dihydro-chalcone glucoside, substantially as hereinbefore described with reference to Examples 3, 4 and 5. glycolytic
15. ) A process for the manufacture of aR^enzyme derivative or an enzyme system derivative having rhamnosidase activity and inhibited '^-glucosidase activity, substantially as hereinbefore described with reference to Examples 1 and 2.
16. ) Hesperetin dihydrochalcone glucoside, when manufactured by the process claimed in any one of claims 1 to 5 and 1 or by an obvious chemical equivalent thereof. 43784/2
17. ) A glycolytic enzyme derivative or enzyme system derivative having rhamnosidase activity and inhibited β-glucosidase activity which comprises the enzyme or enzyme system bonded to a carrier which is covered with a layer of aromatic diamine.
18. ) A derivative according to claim 17 which consists of hesperidinase bonded to a carrier.
19. ) A hesperidihase derivative according to claim 18, wherein the carrier is an activated silica-contining material.
20. ) A hesperidinase derivative according to claims 17 and 18, wherein the carrier consists of porous glass which is covered with a layer of diazotised aromatic diamine.
21. ) A hesperidinase derivative according to claim 19, wherein said aromatic diamine is 4,4'-diamino-diphenyl-methane.
22. ) A hesperidinase derivative according to any one of claims 16 to 20 inclusive, wherein the bonding of the enzyme to the carrier is effected via azo groups. For fhe Appl
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH260273A CH579098A5 (en) | 1973-02-22 | 1973-02-22 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| IL43784A0 IL43784A0 (en) | 1974-03-14 |
| IL43784A true IL43784A (en) | 1977-04-29 |
Family
ID=4237445
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IL43784A IL43784A (en) | 1973-02-22 | 1973-12-10 | Manufacture of hesperetin dihydrochalcone glucoside by means of an enzyme system |
Country Status (8)
| Country | Link |
|---|---|
| JP (1) | JPS49116295A (en) |
| CH (1) | CH579098A5 (en) |
| DE (1) | DE2402221A1 (en) |
| ES (1) | ES423449A1 (en) |
| FR (1) | FR2228069B1 (en) |
| GB (1) | GB1404306A (en) |
| IL (1) | IL43784A (en) |
| NL (1) | NL7400180A (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103819520A (en) * | 2014-02-14 | 2014-05-28 | 闻永举 | Method for preparing mono-glucoside through selective hydrolysis of flavone rutinoside or neohesperidoside |
| CN108220366A (en) * | 2018-01-23 | 2018-06-29 | 山东奔月生物科技有限公司 | Hesperetin dihydrochalcone -7-O- glucoside bioanalysis synthesis technologies |
| CN110200256A (en) * | 2019-06-28 | 2019-09-06 | 山东奔月生物科技股份有限公司 | The preparation method of new-type compound sweetener |
| CN110551011A (en) * | 2019-09-26 | 2019-12-10 | 陕西嘉禾生物科技股份有限公司 | synthesis method of hesperetin dihydrochalcone |
| CN113881659B (en) * | 2021-09-24 | 2024-02-20 | 华南理工大学 | A method for preparing hesperetin dihydrochalcone glucoside by immobilized enzyme method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CH579533A5 (en) * | 1972-07-12 | 1976-09-15 | Givaudan & Cie Sa |
-
1973
- 1973-02-22 CH CH260273A patent/CH579098A5/xx not_active IP Right Cessation
- 1973-12-10 IL IL43784A patent/IL43784A/en unknown
-
1974
- 1974-01-07 NL NL7400180A patent/NL7400180A/xx unknown
- 1974-01-17 DE DE2402221A patent/DE2402221A1/en active Pending
- 1974-02-14 JP JP49018095A patent/JPS49116295A/ja active Pending
- 1974-02-20 FR FR7405744A patent/FR2228069B1/fr not_active Expired
- 1974-02-21 GB GB793374A patent/GB1404306A/en not_active Expired
- 1974-02-21 ES ES423449A patent/ES423449A1/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| DE2402221A1 (en) | 1974-08-29 |
| FR2228069A1 (en) | 1974-11-29 |
| ES423449A1 (en) | 1976-05-16 |
| GB1404306A (en) | 1975-08-28 |
| CH579098A5 (en) | 1976-08-31 |
| JPS49116295A (en) | 1974-11-06 |
| NL7400180A (en) | 1974-08-26 |
| IL43784A0 (en) | 1974-03-14 |
| FR2228069B1 (en) | 1978-03-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4174439A (en) | Process for isolating glucopyranose compound from culture broths | |
| CA2451971C (en) | Process for producing sugar chain asparagine derivative | |
| EP1591532B1 (en) | Process for producing sugar chain asparagine derivative | |
| US7273934B2 (en) | Three-branched sugar-chain asparagine derivatives, the sugar-chain asparagines, the sugar chains, and processes for producing these | |
| HU197926B (en) | Process for recovering glycopeptide antibiotics | |
| IL43784A (en) | Manufacture of hesperetin dihydrochalcone glucoside by means of an enzyme system | |
| KR20010080330A (en) | Method for enzymatic splitting of rutinosides | |
| US5256788A (en) | Moranoline derivatives and their production and the use of moranoline and its derivatives as a stabilizing agent for enzymes | |
| RU2293571C1 (en) | Method for production of affinity adsorbent for cellulolitic enzyme fractionation | |
| KR101860796B1 (en) | Purification method for ascorbic acid glycoside | |
| JP3826426B2 (en) | Process for producing 2-chloro-4-nitrophenyl 4-O-β-D-galactopyranosyl-α-maltoside | |
| JPH0430276B2 (en) | ||
| EP0560982A1 (en) | Process for producing sugar and transfusion | |
| JP3630378B2 (en) | Method for producing galactosylglycerols | |
| JPH053280B2 (en) | ||
| JPH0675510B2 (en) | Method for producing moranolin derivative | |
| JP2690779B2 (en) | L-ascorbic acid derivative and method for producing the same | |
| JP2527064B2 (en) | Enzyme stabilization method, stabilizer and enzyme | |
| JPH09224693A (en) | Production of 1-menthyl-alpha-d-glucopyranoside by enzyme reaction | |
| CH589667A5 (en) | Hesperetin dihydrochalcone glucoside sweetener prepn. - by hesperidin dihydrochalcone degradation using carrier-bonded glycolytic enzyme system | |
| JPH06284897A (en) | Production of beta-form polyphenol glycoside | |
| JP2006022191A (en) | Three-branched sugar chain asparagine derivative, the sugar chain asparagine, the sugar chain and their preparation methods | |
| JPH09313196A (en) | Production of glycoside by immobilized enzyme | |
| JPH06141881A (en) | Production of stevia sweetener containing highly sweet saccharide added thereto by immobilized enzyme | |
| JPS6017511B2 (en) | Purification method of invertase |