IL39063A - Water-insoluble enzyme products - Google Patents
Water-insoluble enzyme productsInfo
- Publication number
- IL39063A IL39063A IL39063A IL3906372A IL39063A IL 39063 A IL39063 A IL 39063A IL 39063 A IL39063 A IL 39063A IL 3906372 A IL3906372 A IL 3906372A IL 39063 A IL39063 A IL 39063A
- Authority
- IL
- Israel
- Prior art keywords
- enzyme
- insoluble
- product
- water
- process according
- Prior art date
Links
- 108090000790 Enzymes Proteins 0.000 title claims description 54
- 102000004190 Enzymes Human genes 0.000 title claims description 54
- 229940088598 enzyme Drugs 0.000 claims description 53
- 238000000034 method Methods 0.000 claims description 15
- 150000004676 glycans Chemical class 0.000 claims description 14
- 229920001282 polysaccharide Polymers 0.000 claims description 14
- 239000005017 polysaccharide Substances 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 125000003172 aldehyde group Chemical group 0.000 claims description 9
- 239000007853 buffer solution Substances 0.000 claims description 8
- 239000001913 cellulose Substances 0.000 claims description 7
- 229920002678 cellulose Polymers 0.000 claims description 7
- 102000004142 Trypsin Human genes 0.000 claims description 4
- 108090000631 Trypsin Proteins 0.000 claims description 4
- 239000012588 trypsin Substances 0.000 claims description 4
- 108090000526 Papain Proteins 0.000 claims description 3
- 239000004365 Protease Substances 0.000 claims description 3
- 229940055729 papain Drugs 0.000 claims description 3
- 235000019834 papain Nutrition 0.000 claims description 3
- 125000003277 amino group Chemical group 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 2
- 239000000047 product Substances 0.000 description 21
- 230000000694 effects Effects 0.000 description 9
- 239000000243 solution Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 239000000758 substrate Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 239000012429 reaction media Substances 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- FYFFGSSZFBZTAH-UHFFFAOYSA-N methylaminomethanetriol Chemical compound CNC(O)(O)O FYFFGSSZFBZTAH-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 description 1
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- WTHXTWHYLIZJBH-UHFFFAOYSA-N acetic acid;azane Chemical compound N.CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O WTHXTWHYLIZJBH-UHFFFAOYSA-N 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000004397 blinking Effects 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 229960001305 cysteine hydrochloride Drugs 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- RGHFKWPGWBFQLN-UHFFFAOYSA-M sodium;5,5-diethylpyrimidin-3-ide-2,4,6-trione Chemical compound [Na+].CCC1(CC)C([O-])=NC(=O)NC1=O RGHFKWPGWBFQLN-UHFFFAOYSA-M 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 229920003176 water-insoluble polymer Polymers 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
- C12N11/02—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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
Water-insoluble enzyme products 0*03 0Ό&3 03 'R iWK D'T3K {11313*171 Socie'te' des Produits Nestle* S.A.
This invention is concerned with the preparation o products having enzyme activity which are insoluble in aqueous media · Enzyme reactions such as enzymatic hydrolyses, are generally carried out with one or more enzymes in solution in the medium containing the substrate, the term "substrate" designating the substance subjected to the action of the enzyme. When the enzyme is dissolved in the reaction medium, its separation from the substrate is particularly difficult, and when it is desired to stop the reaction it. is generally necessary to inactivate the enzyme, as by heating, with the consequence that the enzyme is irrecoverable.
To overcome these difficulties, various process&i'-S have been proposed for preparing water-insoluble enzyme products. Using these substances enzymatic reactions may be carried out by passing the substrate over a bed of the insoluble product, or simply by adding the insoluble enzyme to a solution of the substrate and subsequent, separation of the enzyme by mechanical means such as filtration or cent.ri-fugation.
There exist, various methods for preparing insoluble enzymes, including absorption on an insoluble support, encapsulation in a polymeric matrix, cross-linking with bi-functional substances and covalent. bonding to an insoluble carrier .
Products obtained by absorption of the enzyme are however frequently relatively unstable. Enzymes encapsulated in a polymeric matrix, whilst, being fairly stable, units constituting the matrix. Insolubilisati'on by cross- blinking involves formation of a large number of bonds or bridges which generally necessitates the prior protection of the active groups of the enzyme. In addition, insolu-bilisation methods involving cross-linking do not. always afford the possibility of forming the products in shapes, such as beads or granules, allowing for easy mechanical separation.
Covalent bonding of the enzyme to a water-insoluble polymer frequently requires the formation of active sites on the support, by various reactions such as diazotisation, or use of copolymers. As the formation of bonds may however damage some of the active groups of the enzyme, it. is usually necessary first to protect these, and to remove the protecting groups after the bonding which complicates the process .
An object of the present invention is to provide in novel waterVsoluble enzyme products, and a simple process for their preparation.
The invention provides a water-insoluble enzyme product, comprising an enzyme covalently bonded to free aldehyde groups of a water-insoluble oxidised polysaccharide.
By free aldehyde groups are meant, aldehyde groups carried by the oxidised polysaccharide which are capable of forming chemical bonds with other substances.
Oxidation of polysaccharides such as cellulose leads to the formation of free aldehyde groups by the oxidation of alcohol groups carried by the polymer chain. Thus, for example, oxidation of a polysaccharide such as cellulose polymer having 10 - 12% of reducing groups, that- is the number of free aldehyde groups corresponds to 10 - 12% of the number of glucose residues in the polysaccharide.
The detection and estimation of free aldehyde groups may be conveniently effected by the method of Park and Johnson (estimation of reducing sugars) adapted for insoluble substances, as described by J. S. Thompson and G. D. Schockman in Analytical Biochemistry, 22^, 260-268 (1968) .
The process of the present invention may be applied to enzymes containing reactive groups capable of forming co-valent bonds with the free aldehyde groups of the polysaccharide. Free amino groups are one example of such reactive groups, and these may be attached to amino acid residues, a specific example being the £-amino group of lysine.
Bonding of the enzyme to the insoluble oxidised polysaccharide may for example be effected by simply mixing the enzyme with the polysaccharide for a few hours in a buffer solution, at. temperatures below the inactivation temperature of the enzyme. The insoluble product may be separated from the reaction medium mechanically, as by filtration or centrifugation, and washed before being dried (e.g. lyophilised) .
The reaction conditions are by no means critical, provided that the properties of the enzyme or polymer are not. destroyed. Hence the upper temperature limit, is the inactivation temperature of the enzyme, whereas the lower limit, is 0°C The relative proportions of the reactants may be varied within wide limits, and it is preferred to carry out. the reaction in a buffer solution at a pH of 7 not critical provided that the reactants are not' affected. The pH may thus generally lie in the range 1 to 11. Reaction times may range from a few minutes to several hours. Upon completion of the reaction the water-insoluble enzyme product, may be conveniently separated from the medium by filtration, centrifugation or decanting.
The invention is illustrated by the following examples. In these examples the enzyme activities of the products are expressed as percentages of the activity of the amount, of soluble enzyme which is equal to the amount, of insoluble enzyme recovered. If after separation of the product, the reaction medium has no enzymatic activity, the amount, of insolubilised enzyme may be calculated on the basis that the ratios amount, of insoluble enzyme recovered : amount, of enzyme used, and weight of insoluble product, recovered : total weight, of polysaccharide and enzyme used, are equal. Should the residual medium still have some enzyme activity, which means that not. all the enzyme used has been insolubilised, this calculation is not. appropriate and the amount, of enzyme insolubilised is determined experimentally, as by nitrogen analysis of the insoluble product. Example 1 g of cellulose are oxidised at. 4°C in 2 litres of 0.05M aqueous sodium periodate. Progress of the reaction is followed by titration of the periodate.
After 125 hours' reaction, when 0.5 moles of periodate have been used up per glucose unit, of the polysaccharide the oxidation is stopped by addition of ethylene glycol.
The oxidised cellulose is recovered, washed twice with dis- groups. An 0.2M buffer solution of sodium barbital * hydrochloride is prepared and its pH is adjusted to 8.0. 100 mg of oxidised cellulose are then mixed in the buffer solution with 80 mg of crystallised papain. The mixture is maintained at ambient, temperature with stirring for 3 hours and the insoluble product is recovered by centrifu- gation. The supernatant has no enzyme activity.
The insoluble product is washed 3 times with a buffer solution of pH 7.2 containing, per litre, 1.21 g cysteine hydrochloride, 13.5 g potassium phosphate and 20 ml of a 0. IN solution of the disodium salt of ethylene di¬ amine tetraacetic acid, and finally washed twice with dis¬ tilled water.
The enzyme activity of the product is obtained by determination (U.V. absorption spectrum at 405 nm) of the amount of p-nitro-aniline liberated by the enzyme from a -3 solution containing 2.10 mole of o , -benzoyl-DL-argmine 4-nit.roanilide per litre of the buffer solution used for washing, together with 5% by weight of dimethylsulphoxide .
The enzyme activity of the product, determined by this method is 85%.
Example 2 The pH of a 0.2M aqueous solution of trihydroxy- methyl-amino methane is adjusted to 8.0 with, hydrochloric acid. 0.08 moles/litre of calcium chloride are added and the solution is diluted fourfold to give concentrations of 0.05 and 0.02 mole/litre respectively of trihydroxymethyl- amino methane and calcium chloride. 200 mg of crystallised trypsin and 250 mg of oxi then mixed in 50 ml of the solution and the mixture is maintained at. 4°C with stirring for 20 hours. The insolu ble product, is then recovered by centrifugation, washed 3 times with the buffer solution and twice with distilled water and lyophilised. 190 mg of insoluble product are obtained containing 30 mg of trypsin (determined by analysi of total nitrogen, as the supernatant had some enzyme activity) .
The activity of the insoluble product determined a in Example 1 is 33%.
Claims (14)
1. A water-insoluble enzyme product, comprising an enzyme covalently bonded to free aldehyde groups of a water- insoluble oxidised polysaccharide.
2. A product according to claim 1 in which the oxidised polysaccharide is oxidised cellulose.
3. A product according to claim 1 or claim 2 in which the enzyme contains amino groups which are covalently bonded to the free aldehyde groups.
4. A product, according to any one of the preceding claims in which the enzyme is papain or trypsin.
5. An enzyme product, substantially as herein described with reference to the Examples. as claimed in Claim 1
6. A process for preparing water-insoluble enzyme products/ which comprises reacting an enzyme with a water-insoluble oxidised polysaccharide and recovering a water-insoluble enzyme product.
7. A process according to claim 6 in which the reaction is effected in a buffer solution.
8. A process according to claim 6 or claim 7 in which the reaction is effected at. a pH of 1 to 11.
9. A process according to claim 8 in which the reaction is effected at a pH of 7 to 8.
10. A process according to any one of claims 6 to 9 in which the enzyme is papain or trypsin.
11. A process according to any one of claims 6 to 10 in which the oxidised polysaccharide is oxidised cellulose.
12. A process according to any one of claims 6 to ll in which the recovered enzyme product, is dried.
13. A process for preparing a water-insoluble enzyme product, substantially as herein described wit reference to the Examples.
14. An enzyme product, obtained by a process according to any one of claims 6 to 13. for the Ap licants
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH468771A CH536860A (en) | 1971-03-31 | 1971-03-31 | Process for preparing a product endowed with enzymatic activity, insoluble in aqueous medium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| IL39063A0 IL39063A0 (en) | 1972-05-30 |
| IL39063A true IL39063A (en) | 1974-11-29 |
Family
ID=4280751
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IL39063A IL39063A (en) | 1971-03-31 | 1972-03-23 | Water-insoluble enzyme products |
Country Status (10)
| Country | Link |
|---|---|
| JP (1) | JPS5622515B1 (en) |
| AT (1) | AT314451B (en) |
| BE (1) | BE780939A (en) |
| CA (1) | CA976098A (en) |
| CH (1) | CH536860A (en) |
| DE (1) | DE2215160C3 (en) |
| DK (1) | DK129460B (en) |
| FR (1) | FR2132080B1 (en) |
| GB (1) | GB1349498A (en) |
| IL (1) | IL39063A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS54113492A (en) | 1978-02-24 | 1979-09-05 | Sanyo Chem Ind Ltd | Preparation of glucoprotein derivative |
| CS206823B1 (en) * | 1979-04-28 | 1981-07-31 | Karel Filka | Sorbents for saccharides,glycoproteins and polymers,containing saccharides and method of their manufacture |
| DE3033029A1 (en) * | 1979-09-28 | 1981-04-23 | Vsesojuznyj kardiologičeskij naučnyj centr Akademii medicinskich Nauk SSSR,, Moskva | Thrombolytic urokinase water-soluble deriv. - obtd. by reaction of urokinase with aldehyde dextran |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DD83154A1 (en) * | 1970-05-22 | 1971-07-12 | Process for the preparation of water-insoluble proteins, proteids and peptides |
-
1971
- 1971-03-31 CH CH468771A patent/CH536860A/en not_active IP Right Cessation
-
1972
- 1972-03-20 BE BE780939A patent/BE780939A/en not_active IP Right Cessation
- 1972-03-23 IL IL39063A patent/IL39063A/en unknown
- 1972-03-24 GB GB1383872A patent/GB1349498A/en not_active Expired
- 1972-03-27 CA CA138,232A patent/CA976098A/en not_active Expired
- 1972-03-27 FR FR7210644A patent/FR2132080B1/fr not_active Expired
- 1972-03-28 DE DE2215160A patent/DE2215160C3/en not_active Expired
- 1972-03-28 DK DK151072AA patent/DK129460B/en not_active IP Right Cessation
- 1972-03-30 AT AT279172A patent/AT314451B/en not_active IP Right Cessation
- 1972-03-31 JP JP3182172A patent/JPS5622515B1/ja active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| DE2215160B2 (en) | 1979-08-09 |
| DE2215160C3 (en) | 1980-05-08 |
| JPS5622515B1 (en) | 1981-05-26 |
| IL39063A0 (en) | 1972-05-30 |
| GB1349498A (en) | 1974-04-03 |
| FR2132080B1 (en) | 1976-06-11 |
| AT314451B (en) | 1974-04-10 |
| BE780939A (en) | 1972-09-20 |
| DE2215160A1 (en) | 1972-10-12 |
| DK129460B (en) | 1974-10-14 |
| DK129460C (en) | 1975-04-01 |
| CH536860A (en) | 1973-05-15 |
| CA976098A (en) | 1975-10-14 |
| FR2132080A1 (en) | 1972-11-17 |
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