CA1052305A - Insolubilized proteins on polymeric condensation products - Google Patents
Insolubilized proteins on polymeric condensation productsInfo
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- CA1052305A CA1052305A CA231,082A CA231082A CA1052305A CA 1052305 A CA1052305 A CA 1052305A CA 231082 A CA231082 A CA 231082A CA 1052305 A CA1052305 A CA 1052305A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
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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
- 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
- C12N11/091—Phenol resins; Amino resins
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G12/00—Condensation polymers of aldehydes or ketones with only compounds containing hydrogen attached to nitrogen
- C08G12/02—Condensation polymers of aldehydes or ketones with only compounds containing hydrogen attached to nitrogen of aldehydes
- C08G12/04—Condensation polymers of aldehydes or ketones with only compounds containing hydrogen attached to nitrogen of aldehydes with acyclic or carbocyclic compounds
- C08G12/06—Amines
- C08G12/08—Amines aromatic
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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
- 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
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/087—Acrylic polymers
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- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Compositions Of Macromolecular Compounds (AREA)
- Polymers With Sulfur, Phosphorus Or Metals In The Main Chain (AREA)
- Phenolic Resins Or Amino Resins (AREA)
- Macromolecular Compounds Obtained By Forming Nitrogen-Containing Linkages In General (AREA)
- Peptides Or Proteins (AREA)
Abstract
ABSTRACT
This invention relates to a process for the fixing or insolu-bilising of proteins, which process comprises bonding the protein to a polymeric condensation product obtained by reacting a carbocyclic, aromatic polyamine with an aliphatic dialdehyde or acrolein, other than the 1,3-phenylenediamine glutardialdehyde condensation product.
This invention relates to a process for the fixing or insolu-bilising of proteins, which process comprises bonding the protein to a polymeric condensation product obtained by reacting a carbocyclic, aromatic polyamine with an aliphatic dialdehyde or acrolein, other than the 1,3-phenylenediamine glutardialdehyde condensation product.
Description
ifA~3~
The present invention is concerned with the fixing or insolu-bilising of proteins.
An object of the Canadian Patent Appllcation No. 192,733 is the manufackure of the novel condensation product obtained by reacting 1,3-phenylenediamine with glutardialdehyde, which is distinguished by having a large bonding capacity for proteins.
It has now been found in accordance with the present invention that a series of further structurally related amines may be reacted with the same or with structurally similar dialdehydes, or also with acrolein, in the same manner to give condensation products which are likewise suitable in an outstanding manner for the same purpose~j namely for the fixing or insolubilising of proteins.
Accordingly, the present invention provides a process for the fixing or insolubilising of proteins, which process comprises bonding the protein to a polymeric condensation product obtained by reacting a carbocyclic, aromatic polyamine with an aliphatic dialdehyde or acro-~ lein, other than the 1,3-phenylenediamine glutardialdehyde condensation ; product.
The polyamine-aldehyde resins can be made reacting a carbocyclic aromatic polymaine with an aliphatic dialdehyde or acrolein to give a condensation product suitable for the fixing or insolubilising of proteins ,:
~s~s ~the manufacture of the 1,3-phenylenediamine glutardialdehyde condensation product being excluded).
Carbocyclic, aromatic polyamines which can be used in the for-mation of the condensation product include diamines or triamines, that is mononuclear compounds such as phenylenediamines (e.g. p-phenylenediamine) and phenylenetriamines ~e.g. 2,4,6-triaminophenol, 2,~,6-triaminotoluene), polynuclear compounds such as biphenyldiamines (e.g. ben~idine) or poly-nuclear compounds with aliphatic bridges, such as diaminodiphenylalkanes ~e.g. diaminodiphenylmethanes).
Apart from amino groups, the aromatic nuclei can also carry other substituents such as halogen atoms ~e.g. chlorine), lower alkyl groups ~e.g. methyl, ethyl), lower alkoxy groups ~e.g. methoxy, ethoxy) or hydroxy, carboxy, mercapto, lower alkylthio or sulphonyl groups.
Finally, mixtures of different polyamines can also be employed as reaction components in the manufacture of the condensation products in accordance with the invention.
Preferred polyamine reaction components are banzidine and 2,4, 6-triaminotoluene.
~xamples of aliphatic dialdehydes which can be used in the for-mation of the condensation product include glutardialdehyde, succinic acid dialdehyde, glyoxal, etc. Glutardialdehyde is preferably used.
. . , ~
~/~S'~3~5 ~:`
~hether or not the condensation products manufactured according to the process of the present invention are suitable for the fixing or insolubilising of enzymes can be experimentally determined in a simple manner by treating the condensation product with an enzyme, preferably amyloglucosidase~ and measuring the activity of the fixed ~nzyme. The suitabili~y is verified if this activity comes to at least 50 to 100 units per gram of ; condensation product carrier material.
The following test procedure can be used for this purpose:
A chromatography column (1.5 x 15 cm~ is filled with 1 g of the condensation product carrier materialO A solution of 1 g of amyloglucosidase (50 units/mg) in 100 ml of 16 mmol acetate buffer (pH 4.8) is passed through the column at rdom temperature and with a flow rate of 20 ml per hour. As already mentioned, ; the fixe~ enzyme should have an activity of at least 50 to 100 units ~ of carrier material.
The activity is measured according to Zulkoski by way of the glucose liberated from soluble starch. The liberated glucose is determined enzymatically by means of the glucose-oxidase/peroxidase test [Bergmeyer~ Methoden der enzymatischen Analyse, Volume I, (19~0) page 41~]. [1 ~lit = that amoun~ of enzyme which liberates 1 ~mol of glucose/min from soluble starch at a reaction temperature of 60 C and at pH 4.8].
Alternatively, the suitability is verified if the condensation product carrier material fixes at least 10 mg of a protein (e.g. beef serum albumin) per gram of carrier. The --'1--~z~os amount of fixed protein can be determined by hydrolysing the carrier-fixed protein with 6-N hydrochloric acid and subsequently determining the amount of free amino acids with the aid of any practicable analysis method for amino acids.
Proteins which can be bonded to the condessation products in ac-cordance with the present invention include polypeptides, antigens, anti-bodies, protein inhibitors and~ especially~ enzymes~ The enzymes can be of vegetahle, animal or microbial origin. The enzymes may be hydrolases (peptidases, proteinases, desaminases, carbohydrases, esterases~ nucleases), lyases or desmolases (hydrolyases, decarboxylases, aldolases), transferases, iso~erases, oxidoreductases and ligases. Examples of enzymes from which insoluble enzyme preparations provided by this invention can be n~nufactured are alcoholdehydrogenase, naringinase, hesperidinase~-glucosidase, ~-amyl-la~ invertase, amyloglucosidase, urease, trypsin, ficin, papain, bromelin, subt;lopeptidase, rennin, glucoseisomerase, glucoseoxidase, peroxidase, catalase, acylase, cytochrome, ribonuclease, phosphodiesterase and adenyldeaminase.
According to ~Comprehensive Biochemistryl~ by Florkin ~. and Stotz E. H. Volume 13, third edition, Elsevier Publishing Company, the enzymes have the following enzyme numbers:
alcoholdehydrogenase 1 ol~
~-glucosidase 3.~.1.21 -amylase 3.2.1.2 invertase 3.2.1~26 amyloglucosidase 3.2.1.33 urease 3.5.1.5.
t~ypsin 3.4.4.4 ficin 3.4.4.12 papain 3.4~4.10 ~, ', ' ! ~
~05'~3~5 bromelin 3.4.4.24 subtilopeptidase 3.4.4~16 rennin 3.4.4 3 glucoseisomerase 5.3~1 5 glucoseoxidase 1.1.3 4 peroxidase 1.11.l.7 catalase 1.11.1.6 acylase 3~1.1.6 cytochrome 1.9.3.1 ribonuclease 2.7.7.16 phosphodiesterase 3.1.4.1 adenyldeaminase 3.5.4.2 Naringinase and hesperidinase ha~e no numbers because they are enzyme mixtures of ~-glucosidase and rhamnosidase~
The molar ratio of the polyamine and aldehyde reaction partners expediently lies at between l:l and l:10, a ratiolof about 1:3 being pre-ferred.
The reaction can be carried out in a manner known per se; for example~ in aqueous solution, preferably with the addition of an acid such
The present invention is concerned with the fixing or insolu-bilising of proteins.
An object of the Canadian Patent Appllcation No. 192,733 is the manufackure of the novel condensation product obtained by reacting 1,3-phenylenediamine with glutardialdehyde, which is distinguished by having a large bonding capacity for proteins.
It has now been found in accordance with the present invention that a series of further structurally related amines may be reacted with the same or with structurally similar dialdehydes, or also with acrolein, in the same manner to give condensation products which are likewise suitable in an outstanding manner for the same purpose~j namely for the fixing or insolubilising of proteins.
Accordingly, the present invention provides a process for the fixing or insolubilising of proteins, which process comprises bonding the protein to a polymeric condensation product obtained by reacting a carbocyclic, aromatic polyamine with an aliphatic dialdehyde or acro-~ lein, other than the 1,3-phenylenediamine glutardialdehyde condensation ; product.
The polyamine-aldehyde resins can be made reacting a carbocyclic aromatic polymaine with an aliphatic dialdehyde or acrolein to give a condensation product suitable for the fixing or insolubilising of proteins ,:
~s~s ~the manufacture of the 1,3-phenylenediamine glutardialdehyde condensation product being excluded).
Carbocyclic, aromatic polyamines which can be used in the for-mation of the condensation product include diamines or triamines, that is mononuclear compounds such as phenylenediamines (e.g. p-phenylenediamine) and phenylenetriamines ~e.g. 2,4,6-triaminophenol, 2,~,6-triaminotoluene), polynuclear compounds such as biphenyldiamines (e.g. ben~idine) or poly-nuclear compounds with aliphatic bridges, such as diaminodiphenylalkanes ~e.g. diaminodiphenylmethanes).
Apart from amino groups, the aromatic nuclei can also carry other substituents such as halogen atoms ~e.g. chlorine), lower alkyl groups ~e.g. methyl, ethyl), lower alkoxy groups ~e.g. methoxy, ethoxy) or hydroxy, carboxy, mercapto, lower alkylthio or sulphonyl groups.
Finally, mixtures of different polyamines can also be employed as reaction components in the manufacture of the condensation products in accordance with the invention.
Preferred polyamine reaction components are banzidine and 2,4, 6-triaminotoluene.
~xamples of aliphatic dialdehydes which can be used in the for-mation of the condensation product include glutardialdehyde, succinic acid dialdehyde, glyoxal, etc. Glutardialdehyde is preferably used.
. . , ~
~/~S'~3~5 ~:`
~hether or not the condensation products manufactured according to the process of the present invention are suitable for the fixing or insolubilising of enzymes can be experimentally determined in a simple manner by treating the condensation product with an enzyme, preferably amyloglucosidase~ and measuring the activity of the fixed ~nzyme. The suitabili~y is verified if this activity comes to at least 50 to 100 units per gram of ; condensation product carrier material.
The following test procedure can be used for this purpose:
A chromatography column (1.5 x 15 cm~ is filled with 1 g of the condensation product carrier materialO A solution of 1 g of amyloglucosidase (50 units/mg) in 100 ml of 16 mmol acetate buffer (pH 4.8) is passed through the column at rdom temperature and with a flow rate of 20 ml per hour. As already mentioned, ; the fixe~ enzyme should have an activity of at least 50 to 100 units ~ of carrier material.
The activity is measured according to Zulkoski by way of the glucose liberated from soluble starch. The liberated glucose is determined enzymatically by means of the glucose-oxidase/peroxidase test [Bergmeyer~ Methoden der enzymatischen Analyse, Volume I, (19~0) page 41~]. [1 ~lit = that amoun~ of enzyme which liberates 1 ~mol of glucose/min from soluble starch at a reaction temperature of 60 C and at pH 4.8].
Alternatively, the suitability is verified if the condensation product carrier material fixes at least 10 mg of a protein (e.g. beef serum albumin) per gram of carrier. The --'1--~z~os amount of fixed protein can be determined by hydrolysing the carrier-fixed protein with 6-N hydrochloric acid and subsequently determining the amount of free amino acids with the aid of any practicable analysis method for amino acids.
Proteins which can be bonded to the condessation products in ac-cordance with the present invention include polypeptides, antigens, anti-bodies, protein inhibitors and~ especially~ enzymes~ The enzymes can be of vegetahle, animal or microbial origin. The enzymes may be hydrolases (peptidases, proteinases, desaminases, carbohydrases, esterases~ nucleases), lyases or desmolases (hydrolyases, decarboxylases, aldolases), transferases, iso~erases, oxidoreductases and ligases. Examples of enzymes from which insoluble enzyme preparations provided by this invention can be n~nufactured are alcoholdehydrogenase, naringinase, hesperidinase~-glucosidase, ~-amyl-la~ invertase, amyloglucosidase, urease, trypsin, ficin, papain, bromelin, subt;lopeptidase, rennin, glucoseisomerase, glucoseoxidase, peroxidase, catalase, acylase, cytochrome, ribonuclease, phosphodiesterase and adenyldeaminase.
According to ~Comprehensive Biochemistryl~ by Florkin ~. and Stotz E. H. Volume 13, third edition, Elsevier Publishing Company, the enzymes have the following enzyme numbers:
alcoholdehydrogenase 1 ol~
~-glucosidase 3.~.1.21 -amylase 3.2.1.2 invertase 3.2.1~26 amyloglucosidase 3.2.1.33 urease 3.5.1.5.
t~ypsin 3.4.4.4 ficin 3.4.4.12 papain 3.4~4.10 ~, ', ' ! ~
~05'~3~5 bromelin 3.4.4.24 subtilopeptidase 3.4.4~16 rennin 3.4.4 3 glucoseisomerase 5.3~1 5 glucoseoxidase 1.1.3 4 peroxidase 1.11.l.7 catalase 1.11.1.6 acylase 3~1.1.6 cytochrome 1.9.3.1 ribonuclease 2.7.7.16 phosphodiesterase 3.1.4.1 adenyldeaminase 3.5.4.2 Naringinase and hesperidinase ha~e no numbers because they are enzyme mixtures of ~-glucosidase and rhamnosidase~
The molar ratio of the polyamine and aldehyde reaction partners expediently lies at between l:l and l:10, a ratiolof about 1:3 being pre-ferred.
The reaction can be carried out in a manner known per se; for example~ in aqueous solution, preferably with the addition of an acid such
2~ as a mineral acid (e.g. hydrochloric acid). In a particular embodiment, the reaction is carried out in the presence of an inert, fine-grained, preferably inorganic, - 5a-~5'~3~5 especially silicate-contain~g~ juuant.~ p~e~fo~-u~ch adjuvants are silica gel, pumice-stone, diatomaceous earth (kieselguhr)~ bentonite, wollastonite, porous glass and also metal oxides such as aluminium oxide or hydroxylapatite. It is preferred to use silica gel ~e.gO with a particle si7~e of 0.05-0.2 mm, 70-325 mesh) or pumice-stone (e.g. with a particle siæe of 0.05-10 mm). The presence of such an adjuvant gives rise to a homogeneous particle formation in the reaction andg as a result~ an improved sedimentation is achieved. The reaction in the presence of an adjuvant is expediently carr~d out by initially bringing the particles into contact with one of th~ two reaction compo~ènts and then adding the second component with simultaneous or subsequent slight acidification.
The reaction can be carried out in a homogeneous phase or, preferably~ in a two-phase system with the addition of an acid such as a mineral acid ~e.g. sulphuric acid, phosphoric acid or, preferably, hydrochlori~ acid) or an organic acid (e.g. a carboxylic acid such as acetic acid) with vigorous stirring or shaking. The use of a two-phase system promotes the formation ~0 of spherical~ substantially homogeneous particles which are easy to filter and which sediment well, these particles being especially well suited as a carrier materialO
Inert, water-immiscible organic solvents (e.g. di~hcloro-methane, chloroform, carbon tetrachloride~ benzene~ toluene, ethyl acetate~ dioxane, carbon disulphide3 are suitable as the second phaseO Chlaroform is preferably used as the inertg water-immiscible solvent, but acetone is also suitable for this purpose.
~SZ3~
The reaction temperature is not critical, it can lie, for example, at between 0C and 50C, preferably at room temperature (i.eO at about 18-22 C.). In accordance with the process provided by this invention there is obtained a conden-sation product carrier material which, although already very active, can be still further activated by diazotisation. The diazotisation can be carried out in a manner known per se by treatment with nitrite and acid.
In order to fix the protein to the carrier material, the latter is treated with an aqueous s~-lution, preferably a buffered solution~ of the protein (1-50 mg/ml) at a temperature of 0 -30 C~ preferably 4 C to room temperature. This treatment can be carried out while stirring or shaking. Because of the high activity of the carrier material, the fixing of the proteins can also be advantageously carried out by simply filtering the protein solution through a carrier layer, preferably a column filled with the carrier material~ as is common, for example, ; in column chromatography. Thus, for example, culture f~ltrates of microorganisms which contain proteins or enzymes can be allowed to run directly through a column of carrier material, the proteins or enzymes being selectively fixed to the carrier.
The column is expediently rinsed with a buffer solution and 1-M potassium chloride solution in order to remove the non-fixed proteins.
The proteins fixed to the carrier material are generally very stable and very high specific activities (unit/g) are achieved in the case of in enzymesD Loadings of, for example, 1:3-10 parts by weight of protein/carrier material are achieved.
The reaction can be carried out in a homogeneous phase or, preferably~ in a two-phase system with the addition of an acid such as a mineral acid ~e.g. sulphuric acid, phosphoric acid or, preferably, hydrochlori~ acid) or an organic acid (e.g. a carboxylic acid such as acetic acid) with vigorous stirring or shaking. The use of a two-phase system promotes the formation ~0 of spherical~ substantially homogeneous particles which are easy to filter and which sediment well, these particles being especially well suited as a carrier materialO
Inert, water-immiscible organic solvents (e.g. di~hcloro-methane, chloroform, carbon tetrachloride~ benzene~ toluene, ethyl acetate~ dioxane, carbon disulphide3 are suitable as the second phaseO Chlaroform is preferably used as the inertg water-immiscible solvent, but acetone is also suitable for this purpose.
~SZ3~
The reaction temperature is not critical, it can lie, for example, at between 0C and 50C, preferably at room temperature (i.eO at about 18-22 C.). In accordance with the process provided by this invention there is obtained a conden-sation product carrier material which, although already very active, can be still further activated by diazotisation. The diazotisation can be carried out in a manner known per se by treatment with nitrite and acid.
In order to fix the protein to the carrier material, the latter is treated with an aqueous s~-lution, preferably a buffered solution~ of the protein (1-50 mg/ml) at a temperature of 0 -30 C~ preferably 4 C to room temperature. This treatment can be carried out while stirring or shaking. Because of the high activity of the carrier material, the fixing of the proteins can also be advantageously carried out by simply filtering the protein solution through a carrier layer, preferably a column filled with the carrier material~ as is common, for example, ; in column chromatography. Thus, for example, culture f~ltrates of microorganisms which contain proteins or enzymes can be allowed to run directly through a column of carrier material, the proteins or enzymes being selectively fixed to the carrier.
The column is expediently rinsed with a buffer solution and 1-M potassium chloride solution in order to remove the non-fixed proteins.
The proteins fixed to the carrier material are generally very stable and very high specific activities (unit/g) are achieved in the case of in enzymesD Loadings of, for example, 1:3-10 parts by weight of protein/carrier material are achieved.
3~
The carrier-bonded proteins provided by this inven~ion, especially enzymes, can be used in a manner known per se; for example, for analytical or preparative purposes or in food technology such as in the manufacture of glucose from starch ~see3 or example., Scientific American 224~ (No. 3) 26-33 ( 1971); Angew. Chemie 849 (8) 319-268 ( 1972); Chemiker Zeitung 96, (11), 595-602 ~1972); ~ & EN, ~15.2.71), 86-87].
In this important large-scale process, amyloglucosidase fixed to the condensation product provided by the present invention can be utilised for the manufacture of glucose from the "glucose syrup" (prehydrolysed starch). The condensation products can then be transferred~ for example, onto columns (fixed-bed process). A further application results in the enzymatic degradation of lactose in milk products by means of lactases (e.g. I'sweets" from whey).
The following Examples illustrate the present invenkion.
The quantitative data of the carrier material obtained or used is given with respect to dry weight.
xample 1 To a solution or suspension of 5 g of the polyamine in 200 ml of chloroform, there are added portionwise with vigorous s~irring, firstly 20 g of aldehyde (80 ml of a 25% solution ~
water) and, after a further 5 minutes, 20 ml of 7-N hydrochloric acid. The reaction mixture solidifies. It is treated with 300 ml of water and shaken for 5 minutes until it again becomes liquid. The polymeric particles are left to stand for 1 hour with occasional stirring. After vacuum filtration of the mixture over a Bùchner funnel, the polymeric part:icles are 30~i washed with three 200 ml portions of acetone and then with 0.1-~ sodium hydroxide solution. The residual chloroform is removed by washing with acetone. The thus obtained carrier particles are spherical, homogeneous and capable of good filtration; they are stored in water or in d~llute sodium hydroxide. They can be employed for enzyme fixing~9 optionally after diazotisation.
Example 2 A chromatography column (1.5 x 15 cm) is filled with 1 g of carrier material and washed with the buffer solution used in the following enzyme fixing. The buffered enzyme solution (1-100 mg of protein per ml of buffer) is passed through the column with a flow rate of 20 ml/hour.
The coupling capacity is exhausted ~eh protein can be detected in the eluate, which can be detected, for exa~ple by measurement of the absorption spectrum at 280 m,u. The non-fixed enzyme is removed by washing the column with 1-M
potassium chloride and the buffer solution. There then follows an activity determination of an aliquot of the enzyme-fixed material with the respective substrate under given reaction conditions. Then an aliquot of the enzyme solution is washsd with waterg dried and the dried material determined analogously.
The number of units of enzyme activity/g of carrier material can be determined in this manner.
The condensation products manufactured according to Example 1 and their corresponding activities determined according to Example 2 are compiled in the following Table:
~ ~5~3~5 _ ~ _ _ . .
~ O
h ~ ~ >
~ ' _ _ _ __ . _ _ ~ .
~9'~ '7 _10 -
The carrier-bonded proteins provided by this inven~ion, especially enzymes, can be used in a manner known per se; for example, for analytical or preparative purposes or in food technology such as in the manufacture of glucose from starch ~see3 or example., Scientific American 224~ (No. 3) 26-33 ( 1971); Angew. Chemie 849 (8) 319-268 ( 1972); Chemiker Zeitung 96, (11), 595-602 ~1972); ~ & EN, ~15.2.71), 86-87].
In this important large-scale process, amyloglucosidase fixed to the condensation product provided by the present invention can be utilised for the manufacture of glucose from the "glucose syrup" (prehydrolysed starch). The condensation products can then be transferred~ for example, onto columns (fixed-bed process). A further application results in the enzymatic degradation of lactose in milk products by means of lactases (e.g. I'sweets" from whey).
The following Examples illustrate the present invenkion.
The quantitative data of the carrier material obtained or used is given with respect to dry weight.
xample 1 To a solution or suspension of 5 g of the polyamine in 200 ml of chloroform, there are added portionwise with vigorous s~irring, firstly 20 g of aldehyde (80 ml of a 25% solution ~
water) and, after a further 5 minutes, 20 ml of 7-N hydrochloric acid. The reaction mixture solidifies. It is treated with 300 ml of water and shaken for 5 minutes until it again becomes liquid. The polymeric particles are left to stand for 1 hour with occasional stirring. After vacuum filtration of the mixture over a Bùchner funnel, the polymeric part:icles are 30~i washed with three 200 ml portions of acetone and then with 0.1-~ sodium hydroxide solution. The residual chloroform is removed by washing with acetone. The thus obtained carrier particles are spherical, homogeneous and capable of good filtration; they are stored in water or in d~llute sodium hydroxide. They can be employed for enzyme fixing~9 optionally after diazotisation.
Example 2 A chromatography column (1.5 x 15 cm) is filled with 1 g of carrier material and washed with the buffer solution used in the following enzyme fixing. The buffered enzyme solution (1-100 mg of protein per ml of buffer) is passed through the column with a flow rate of 20 ml/hour.
The coupling capacity is exhausted ~eh protein can be detected in the eluate, which can be detected, for exa~ple by measurement of the absorption spectrum at 280 m,u. The non-fixed enzyme is removed by washing the column with 1-M
potassium chloride and the buffer solution. There then follows an activity determination of an aliquot of the enzyme-fixed material with the respective substrate under given reaction conditions. Then an aliquot of the enzyme solution is washsd with waterg dried and the dried material determined analogously.
The number of units of enzyme activity/g of carrier material can be determined in this manner.
The condensation products manufactured according to Example 1 and their corresponding activities determined according to Example 2 are compiled in the following Table:
~ ~5~3~5 _ ~ _ _ . .
~ O
h ~ ~ >
~ ' _ _ _ __ . _ _ ~ .
~9'~ '7 _10 -
Claims (25)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the fixing or insolubilising of proteins, which process comprises bonding the protein to a polymeric condensation product obtained by reacting a carbocyclic, aromatic polyamine with an aliphatic dialdehyde or acrolein, other than the 1,3-phenylenediamine glutardialdehyde condensation product.
2. A process according to claim 1, wherein in the condensation product a diamine is used as the carbooyclic, aromatic polyamine.
3. A process according to claim 1 or claim 2, wherein in the condensa-tion product glutardialdehyde is used as the dialdehyde.
4. A process according to claim 1, wherein the condensation product is obtained by reaction of benzidine with glutardialdehyde.
5. A process according to claim 1, wherein the condensation product is obtained by reaction of 2,4,6-triaminotoluene with glutardialdehyde.
6. A process according to claim 1, wherein the condensation product is obtained by reaction of 1 mole of polyamine with 1-10 moles of aldehyde.
7. A process according to claim 1, wherein the reaction to obtain the condensation product is carried out in the presence of a fine-grained, solid, inert adjuvant.
8. A process according to claim 7, wherein the adjuvant is a silicate or a silicate-containing material.
9. A process according to claim 8, wherein silica gel is used as the adjuvant.
10. A process according to claim 1, wherein the reaction to obtain the condensation product is carried out in a two-phase system.
11. A process according to cliam 10, wherein the two-phase system con-sists of an aqueous phase and a water-immiscible organic phase.
12. A process according to claim 11, wherein the water-immiscible organ-ic phase is chloroform.
13. A process according to claim 1, wherein the condensation product is diazotised.
14. A process according to claim 1 wherein the protein is an enzyme.
15. A process according to claim 14, wherein the bonding is carried out by passing a solution of the protein through a column containing the conden-sation product.
16. A protein bound to a condensation product and obtained according to the process claimed in claim 1, 2 or 3.
17. A protein bound to a condensation product and obtained according to the process claimed in claim 4, 5 or 6.
18. A protein bound to a condensation product and obtained according to the process claimed in claim 7, 8 or 9.
19. A protein bound to a condensation product and obtained according to the process claimed in claim 10, 11 or 12.
20. A protein bound to a condensation product and obtained according to the process claimed in claim 13.
21. An enzyme bound to a condensation product and obtained according to the process claimed in claim 1, 2 or 3.
22. An enzyme bound to a condensation product and obtained according to the process claimed in claim 4, 5 or 6.
23. An enzyme bound to a condensation product and obtained according to the process claimed in claim 7, 8 or 9.
24. An enzyme bound to a condensation product and obtained according to the process claimed in claim 10, 11 or 12.
25. An enzyme bound to a condensation product and obtained according to the process claimed in claim 13.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK45874AA DK138750B (en) | 1973-02-22 | 1974-01-29 | Polycondensation product for binding proteins or enzymes. |
| CH941574A CH592694A5 (en) | 1974-07-09 | 1974-07-09 | Condensn prods of aromatic polyamines with dialdehydes or acrolein |
| DK446374AA DK141533B (en) | 1974-01-29 | 1974-08-21 | Polycondensation product for binding proteins or enzymes. |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1052305A true CA1052305A (en) | 1979-04-10 |
Family
ID=27176221
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA231,082A Expired CA1052305A (en) | 1974-01-29 | 1975-07-08 | Insolubilized proteins on polymeric condensation products |
Country Status (9)
| Country | Link |
|---|---|
| JP (1) | JPS5133185A (en) |
| BE (1) | BE831168R (en) |
| CA (1) | CA1052305A (en) |
| DE (1) | DE2529604A1 (en) |
| DK (1) | DK141533B (en) |
| FR (1) | FR2277841A2 (en) |
| GB (1) | GB1512066A (en) |
| NL (1) | NL7508189A (en) |
| SE (1) | SE7410542L (en) |
Families Citing this family (32)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PT64790B (en) * | 1975-02-18 | 1977-07-07 | Uop Inc | Immobilized enzyme conjugates |
| JPS52116131A (en) * | 1976-03-26 | 1977-09-29 | Toshiba Corp | Microprogram instruction control unit |
| DE2708018A1 (en) * | 1977-02-24 | 1978-09-07 | Boehringer Mannheim Gmbh | BIOLOGICALLY ACTIVE PROTEIN FIXED TO POLYAMIDE AND THE PROCESS FOR ITS PRODUCTION |
| JPS59232064A (en) * | 1983-06-14 | 1984-12-26 | Nippon Sanso Kk | Preparation of seasoning |
| JPS60176563A (en) * | 1984-02-22 | 1985-09-10 | San Ei Chem Ind Ltd | Production of oleophilic food dye |
| KR20240056621A (en) | 2005-07-26 | 2024-04-30 | 크나우프 인설레이션, 인크. | Binders and materials made therewith |
| BRPI0721232B1 (en) | 2007-01-25 | 2023-01-24 | Knauf Insulation Limited | COMPOSITE WOOD PLATE |
| US9828287B2 (en) | 2007-01-25 | 2017-11-28 | Knauf Insulation, Inc. | Binders and materials made therewith |
| CN101668713B (en) | 2007-01-25 | 2012-11-07 | 可耐福保温材料有限公司 | Mineral fibre board |
| EP2137223B1 (en) | 2007-04-13 | 2019-02-27 | Knauf Insulation GmbH | Composite maillard-resole binders |
| GB0715100D0 (en) | 2007-08-03 | 2007-09-12 | Knauf Insulation Ltd | Binders |
| CA2753945C (en) * | 2009-03-04 | 2015-06-16 | Adrien Pierre Cote | Electronic devices comprising structured organic films |
| WO2011015946A2 (en) | 2009-08-07 | 2011-02-10 | Knauf Insulation | Molasses binder |
| WO2011102330A1 (en) * | 2010-02-18 | 2011-08-25 | 綜研化学株式会社 | Novel polyazomethine |
| WO2011138459A1 (en) | 2010-05-07 | 2011-11-10 | Knauf Insulation | Carbohydrate binders and materials made therewith |
| EP3922655A1 (en) * | 2010-05-07 | 2021-12-15 | Knauf Insulation | Carbohydrate polyamine binders and materials made therewith |
| CA2801546C (en) | 2010-06-07 | 2018-07-10 | Knauf Insulation | Fiber products having temperature control additives |
| US9567425B2 (en) * | 2010-06-15 | 2017-02-14 | Xerox Corporation | Periodic structured organic films |
| CA2834816C (en) | 2011-05-07 | 2020-05-12 | Knauf Insulation | Liquid high solids binder composition |
| FR2982267A1 (en) * | 2011-11-07 | 2013-05-10 | Knauf Insulation | Binder, useful e.g. for consolidating loosely assembled matter e.g. fibers, plastics and rubbers, fabricating articles, and for ceiling tiles or office panels, comprises a polymeric product of a carbohydrate reactant and a nucleophile |
| FR2982268A1 (en) * | 2011-11-07 | 2013-05-10 | Knauf Insulation | Binder, useful e.g. for consolidating loosely assembled matter e.g. fibers, plastics and rubbers, fabricating articles, and for ceiling tiles or office panels, comprises a polymeric product of a carbohydrate reactant and a polyamine |
| GB201206193D0 (en) | 2012-04-05 | 2012-05-23 | Knauf Insulation Ltd | Binders and associated products |
| GB201214734D0 (en) | 2012-08-17 | 2012-10-03 | Knauf Insulation Ltd | Wood board and process for its production |
| WO2014086777A2 (en) | 2012-12-05 | 2014-06-12 | Knauf Insulation | Binder |
| MX2016010192A (en) | 2014-02-07 | 2017-01-09 | Knauf Insulation Inc | Uncured articles with improved shelf-life. |
| GB201408909D0 (en) | 2014-05-20 | 2014-07-02 | Knauf Insulation Ltd | Binders |
| GB201517867D0 (en) | 2015-10-09 | 2015-11-25 | Knauf Insulation Ltd | Wood particle boards |
| GB201610063D0 (en) | 2016-06-09 | 2016-07-27 | Knauf Insulation Ltd | Binders |
| GB201701569D0 (en) | 2017-01-31 | 2017-03-15 | Knauf Insulation Ltd | Improved binder compositions and uses thereof |
| GB201804907D0 (en) | 2018-03-27 | 2018-05-09 | Knauf Insulation Ltd | Composite products |
| GB201804908D0 (en) | 2018-03-27 | 2018-05-09 | Knauf Insulation Ltd | Binder compositions and uses thereof |
| CN115557601A (en) * | 2022-11-08 | 2023-01-03 | 成都理工大学 | Biomass microsphere, preparation method and application thereof, bioreactor and underground well |
-
1974
- 1974-08-19 SE SE7410542A patent/SE7410542L/en unknown
- 1974-08-21 DK DK446374AA patent/DK141533B/en unknown
-
1975
- 1975-07-02 DE DE19752529604 patent/DE2529604A1/en not_active Ceased
- 1975-07-08 CA CA231,082A patent/CA1052305A/en not_active Expired
- 1975-07-08 JP JP50083898A patent/JPS5133185A/ja active Pending
- 1975-07-08 FR FR7521397A patent/FR2277841A2/en active Granted
- 1975-07-09 NL NL7508189A patent/NL7508189A/en not_active Application Discontinuation
- 1975-07-09 BE BE158126A patent/BE831168R/en active
- 1975-07-09 GB GB28888/75A patent/GB1512066A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| BE831168R (en) | 1976-01-09 |
| GB1512066A (en) | 1978-05-24 |
| JPS5133185A (en) | 1976-03-22 |
| NL7508189A (en) | 1976-01-13 |
| DK446374A (en) | 1976-01-10 |
| FR2277841A2 (en) | 1976-02-06 |
| DK141533B (en) | 1980-04-14 |
| DK141533C (en) | 1980-09-15 |
| FR2277841B2 (en) | 1981-08-07 |
| SE7410542L (en) | 1976-01-12 |
| DE2529604A1 (en) | 1976-01-29 |
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