CA1277268C - Immobilized acylase, process for its preparation and its use - Google Patents
Immobilized acylase, process for its preparation and its useInfo
- Publication number
- CA1277268C CA1277268C CA000464046A CA464046A CA1277268C CA 1277268 C CA1277268 C CA 1277268C CA 000464046 A CA000464046 A CA 000464046A CA 464046 A CA464046 A CA 464046A CA 1277268 C CA1277268 C CA 1277268C
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- Prior art keywords
- penicillin
- acylase
- enzyme
- immobilized
- phenol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
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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
-
- 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
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
- C12P41/007—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures by reactions involving acyl derivatives of racemic amines
-
- 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
-
- 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
- C12N11/098—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer formed in the presence of the enzymes or microbial cells
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
-
- 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
- C12P37/00—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin
- C12P37/06—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin by desacylation of the substituent in the 6 position
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
ABSTRACT
Macroporous anion exchange resins based on phenol/formaldehyde and carrying essentially secondary amino groups are suitable as carriers for the immobilization of acylases, the enzyme and carrier being bound together by means of a diisocyanate. The immobilized systems have a high enzyme activity and cleave even sterically hindered N-acyl-D,L-aminoacids stereospecifically.
Macroporous anion exchange resins based on phenol/formaldehyde and carrying essentially secondary amino groups are suitable as carriers for the immobilization of acylases, the enzyme and carrier being bound together by means of a diisocyanate. The immobilized systems have a high enzyme activity and cleave even sterically hindered N-acyl-D,L-aminoacids stereospecifically.
Description
~2t7~S~
The immobilization of acylases has already been described on many occasions: British Patent Specification 1,369,462 describes how N-acyl-D,L-aminoacids can be reacted with acylases, the L-aminoacid reacting selectively. It is mentioned that the acylase can also be adsorbed on an inert material such as cellulose, sand or aluminum oxide and can be crosslinked with a difunctional reagent such as glutaric dialdehyde. German Offenlegungsschrift 2,732,301 desc:ribes the .immobilization of proteins, special reference being made to the enzyme penicillin acylase. According to German Patent Specification 3,048,612, a penicillin-G acylase fixed in this way can be used to resolve the racemate D,~-2-amino-4-methylphosphinobutyric acid.
European Patent Application A1-43169 relates to enzyme preparations with inulinase activity. In a preferred embodiment, the enzyme is bound by reaction with glutaric dialdehyde to a macroporous resin with amino and/or hydroxy groups. A
phenol/formaldehyde resin with hydroxy groups is listed as an example and it is mentioned that a phenol/formaldehyde resin with amino groups can also be used. It is further mentioned that, in addition to dialdehydes, it is also possible to use dicarboxylic acids or their anhydrides or diisocyanates and difunctional reagents for binding the enzyme to a carrier.
In European Patent Application Al-43169, the phenol-formaldehyde resin carrier with amino groups is characterised by the trademark RDUOLITE A-7. According to European Patent Application A2-37667, this is a macroporous anion exchange resin based on phenol/formaldehyde and having a particle diameter of 250 - 840 ~m, a specific surface area of 31 m2/g, a total volume `~
- , .
. ~ ' ., ~ ' .
~' :
. . . .
.. .,~ . .- , . - .
6~3 of 0.52 cm3/g of macropores having a pore diameter of 10-200 nm, and an anion exchange capacity of 7.5 meq/g corresponding to primary and secondary amino groups. This product is sold by Diamond Shamrock. Granular or bead~shaped anion exchange resins based on phenol-formaldehyde, whose size is preferably in the range of 250 ~m to 1.4 mm, are described as being generally suitable.
It has now been found that anion exchan~e resins based on phenol-formaldehyde and preferably carrying secondary amino groups are particularly suitable for the fixakion of acylases, the enzyme being bound to the carrier with the aid of a diisocyanate. A particular advantage is that no catalyst i~
required for binding the en2yme to the carrier, in contrast, for example, to the methods mentioned in European Patent Application ~1-52365.
The invention relates to a penicillin-G-acylase immobili~ed on a macroporous phenol/formaldehyde anion exchange resin carrying carrier essentially 6econdary amino groups and having a pore diameter of 10-200 nm and a particle diameter of 250-840 ~m, the penicillin-G-acylase and the carrier being bound together by means of an alkylene diisocyanate having up to 10 carbon atoms in the alkylene chain; a process for the preparation of an immobilized penicillia-G-acylase; and, a process for cleaving amide bonds using the penicillin-G-acylase.
Possible diisocyanates are aromatic, cycloaliphatic and especially aliphatic diisocyanates such as, for example, those described in European Patent Application Al-52365. Commercially available aliphatic diisocyanates such as 2,2,4- and 2,4,4-trimethylhexame~hylene 1,6-dlisocyanates, and especially hexamethylene 1,6-diisocyanate, are preferred.
Possible acylase6 are the known products used for the cleavage of penicillin and the resolution of N-acylaminoacid racemates, such as the acylases clescribed in British Patent Specification 1,369,462, German Offenlegun~sschriften 2,352,579, 2,446,320 or ~,939,269 and especially German Patent Specification 3,048/612.
For the coupling of the enzyme to the ion exchanger, the latter is first converted to the free base form, appropriately by suspension in a strong aqueous base such as sodium hydroxide solution. After washing until the washings are neutral, and drying, the anhydrous resin is taken up in an aprotic solvent for the diisocyanate, the diisocyanate is added as such or in solution, and the whole is thoroughly mixed. After separation from the reaction solution and washing with the solvent, the reaction product can be used directly for the reàction with the enzyme. However, it can also be isolated and dried and can be kept for a relatively long period in the absence of moisture.
The resin pretreated in this way can b~ reacted directly with an aqueous solution of the enzyme, the reaction conditions depending on the properties of the enzyme. In general, the pretreated resin is stirred with the aqueous enzyme solution for a sufficient time at room temperature and the product is filtered off and washed, for example with saturated or physiological saline solution and then with water. The washed immobilized enzyme system can be used directly.
.
:.
..
' ' . ' ' :
~.~772~i8 Compared with an immobilized system obtained according to German Offenlegungsschrift 2,732,301, a penicillin-G acylase from E. coli, immobilized according to the invention, has a greatly increased activity, as shown, for example, by the fact that even a sterically hindered substrate such as N-acyl-D,L-tert.-leucine is cleaved quantitatively and stereospecifically.
The invention therefore also relates to the immobilized enzyme systems whenever prepared according to the processes of the invention and the use of the immobilized enzyme systems for the cleavage of amide bonds, especially for the resolution of N-acylamino-acid racemates.
Preferred embodiments of the invention are illus-trated in greater detail in the examples which follow.
Unless stated otherwise, percentages are by weight.
Example 1 The commercially available, macroporous anion exchange resin DUOLITE A-7, based on phenol/formaldehyde and carrying essentially secondary amino groups, is taken up in 2 N sodium hydroxide solution for conversion to the free base form, stirred and washed with deionized water until the washings are neutral. After drying in a vacuum cabinet (40 C, 50 mbar, 24 hours), 100 g of the an-hydrous resin are taken up in 200 ml of anhydrous methylene chloride and treated with 10 g of hexamethylene 1,6-diisocyanate, and the mixture is stirred for 2 hours at room temperature in the absence of moisture. The resin is then filtered off and washed thoroughly with methylene chloride. The resin is freed of methylene chloride in the absence of moist~re and sto ed i the absence of .
6~
moisture until it is needed.
Example 2 The resin obtained according to Example 1 i8 introduced into 200 ml of an aqueous solution of 10 g of penicillin-G acylase (E.C. No. 3.5.1.11, from E. coli ATCC 11 105) and stirred for 2 hours at room temperature. The product is filt~red off and washed first with saturated sodium chloride solution and then - with deionized water.
Example 3 The specific activity of the immobilized penicillin-G
amidase system obtained according to Example 2 is determined by comparison with the standard substrate penicillin-G in the following manner:
1 g of the potassium salt of penicillin-G is dissolved in 40 ml of water and treated with lQ0 mg of the immobilized system at 37C, with stirring. By keeping the pH constant at 7.B0, the quantity of penicillin-G converted to 6-aminopenicillanic acid and phenylacetic acid can be read off directly from the alkali consumption by titration with 0.1 ~ sodium hydroxide solution.
After a reaction time of 30 minut~s, 3.6 ml of 0.1 N sodium hydroxide solution have been consumed, corresponding to 360 ~mol of penicillin-G. From this, the specific activity of the immobilized system is calculated to be 120 U/g.
Example 4 10 g (33-4 mmol) of N phenacetyl-D,L-2 amino-4-methylphosphinobutyric acid are dissolved in 40 ml of water. The solution, which has a pH of 7.8, is treated at 37C with one gram of the immobilized penicillin-G amidase system according to . ~.
.' . ~ .
.
, . .
~L~%6~3 Example 2, with stirring, and the pH is kept constant at 7.8 by the addition of 0.1 N sodium hydroxide solution. The reaction has ended after 5 to 6 hours. Working-up in the customary manner (German Patent Specification 3,048l612) gives 3.5 g (16 mmol, corresponding to 96% of theory) of L~2-amino-4-methyl-phosphinobutyric acid of melting point 199-201C, [a]2=24 (c =
1, in 1 N hydrochloric acid).
Example 5 100 g (0.4 mol) of N-phenacetyl-D,L-tert.-leucine are dissolved in 400 ml of 1 N sodium hydroxide solutionl the pH is adjusted to 7.8 and the volume is made up to a total of 1000 ml.
This reaction solution is thermostated at 37C and treated with 100 g of ~enicillin-G amida~e immobilized according to Example 2, with stirring. By titration with 0.05 N sodium hydroxide solution, the pH is kept constant during the hydrolysis reaction and the progress of the reaction is recorded at the same time.
After 3 to 4 hours, no further sodium hydroxide solution is consumed. After the enzyme material has been filtered off and the filtrate acidified to pH 3 with concentrated sulfuric acid, phenylacetic acid and N-phenacetyl-D-tert.-leucine crystallize out. The phenylacetic acid can be separated from the crop of crystals by extraction with ethyl acetate/hexane, after which 45 g (0.18 mol = 90 ~ of theory) of N-phenacetyl-D-tert.-leucine of melting point 163 to 164C, [a]2D0 = 14.2 (c = 1, in methanol), remain.
23 g (0.18 mol = 90 % of theory) of L-tert.-leucine of melting point above 250C, [a]D = -9.4~ (c = 1, in water) are isolated from the concentrated mother liquor by extraction with ethanol/water.
A . 1 ' . ', ' ~
The immobilization of acylases has already been described on many occasions: British Patent Specification 1,369,462 describes how N-acyl-D,L-aminoacids can be reacted with acylases, the L-aminoacid reacting selectively. It is mentioned that the acylase can also be adsorbed on an inert material such as cellulose, sand or aluminum oxide and can be crosslinked with a difunctional reagent such as glutaric dialdehyde. German Offenlegungsschrift 2,732,301 desc:ribes the .immobilization of proteins, special reference being made to the enzyme penicillin acylase. According to German Patent Specification 3,048,612, a penicillin-G acylase fixed in this way can be used to resolve the racemate D,~-2-amino-4-methylphosphinobutyric acid.
European Patent Application A1-43169 relates to enzyme preparations with inulinase activity. In a preferred embodiment, the enzyme is bound by reaction with glutaric dialdehyde to a macroporous resin with amino and/or hydroxy groups. A
phenol/formaldehyde resin with hydroxy groups is listed as an example and it is mentioned that a phenol/formaldehyde resin with amino groups can also be used. It is further mentioned that, in addition to dialdehydes, it is also possible to use dicarboxylic acids or their anhydrides or diisocyanates and difunctional reagents for binding the enzyme to a carrier.
In European Patent Application Al-43169, the phenol-formaldehyde resin carrier with amino groups is characterised by the trademark RDUOLITE A-7. According to European Patent Application A2-37667, this is a macroporous anion exchange resin based on phenol/formaldehyde and having a particle diameter of 250 - 840 ~m, a specific surface area of 31 m2/g, a total volume `~
- , .
. ~ ' ., ~ ' .
~' :
. . . .
.. .,~ . .- , . - .
6~3 of 0.52 cm3/g of macropores having a pore diameter of 10-200 nm, and an anion exchange capacity of 7.5 meq/g corresponding to primary and secondary amino groups. This product is sold by Diamond Shamrock. Granular or bead~shaped anion exchange resins based on phenol-formaldehyde, whose size is preferably in the range of 250 ~m to 1.4 mm, are described as being generally suitable.
It has now been found that anion exchan~e resins based on phenol-formaldehyde and preferably carrying secondary amino groups are particularly suitable for the fixakion of acylases, the enzyme being bound to the carrier with the aid of a diisocyanate. A particular advantage is that no catalyst i~
required for binding the en2yme to the carrier, in contrast, for example, to the methods mentioned in European Patent Application ~1-52365.
The invention relates to a penicillin-G-acylase immobili~ed on a macroporous phenol/formaldehyde anion exchange resin carrying carrier essentially 6econdary amino groups and having a pore diameter of 10-200 nm and a particle diameter of 250-840 ~m, the penicillin-G-acylase and the carrier being bound together by means of an alkylene diisocyanate having up to 10 carbon atoms in the alkylene chain; a process for the preparation of an immobilized penicillia-G-acylase; and, a process for cleaving amide bonds using the penicillin-G-acylase.
Possible diisocyanates are aromatic, cycloaliphatic and especially aliphatic diisocyanates such as, for example, those described in European Patent Application Al-52365. Commercially available aliphatic diisocyanates such as 2,2,4- and 2,4,4-trimethylhexame~hylene 1,6-dlisocyanates, and especially hexamethylene 1,6-diisocyanate, are preferred.
Possible acylase6 are the known products used for the cleavage of penicillin and the resolution of N-acylaminoacid racemates, such as the acylases clescribed in British Patent Specification 1,369,462, German Offenlegun~sschriften 2,352,579, 2,446,320 or ~,939,269 and especially German Patent Specification 3,048/612.
For the coupling of the enzyme to the ion exchanger, the latter is first converted to the free base form, appropriately by suspension in a strong aqueous base such as sodium hydroxide solution. After washing until the washings are neutral, and drying, the anhydrous resin is taken up in an aprotic solvent for the diisocyanate, the diisocyanate is added as such or in solution, and the whole is thoroughly mixed. After separation from the reaction solution and washing with the solvent, the reaction product can be used directly for the reàction with the enzyme. However, it can also be isolated and dried and can be kept for a relatively long period in the absence of moisture.
The resin pretreated in this way can b~ reacted directly with an aqueous solution of the enzyme, the reaction conditions depending on the properties of the enzyme. In general, the pretreated resin is stirred with the aqueous enzyme solution for a sufficient time at room temperature and the product is filtered off and washed, for example with saturated or physiological saline solution and then with water. The washed immobilized enzyme system can be used directly.
.
:.
..
' ' . ' ' :
~.~772~i8 Compared with an immobilized system obtained according to German Offenlegungsschrift 2,732,301, a penicillin-G acylase from E. coli, immobilized according to the invention, has a greatly increased activity, as shown, for example, by the fact that even a sterically hindered substrate such as N-acyl-D,L-tert.-leucine is cleaved quantitatively and stereospecifically.
The invention therefore also relates to the immobilized enzyme systems whenever prepared according to the processes of the invention and the use of the immobilized enzyme systems for the cleavage of amide bonds, especially for the resolution of N-acylamino-acid racemates.
Preferred embodiments of the invention are illus-trated in greater detail in the examples which follow.
Unless stated otherwise, percentages are by weight.
Example 1 The commercially available, macroporous anion exchange resin DUOLITE A-7, based on phenol/formaldehyde and carrying essentially secondary amino groups, is taken up in 2 N sodium hydroxide solution for conversion to the free base form, stirred and washed with deionized water until the washings are neutral. After drying in a vacuum cabinet (40 C, 50 mbar, 24 hours), 100 g of the an-hydrous resin are taken up in 200 ml of anhydrous methylene chloride and treated with 10 g of hexamethylene 1,6-diisocyanate, and the mixture is stirred for 2 hours at room temperature in the absence of moisture. The resin is then filtered off and washed thoroughly with methylene chloride. The resin is freed of methylene chloride in the absence of moist~re and sto ed i the absence of .
6~
moisture until it is needed.
Example 2 The resin obtained according to Example 1 i8 introduced into 200 ml of an aqueous solution of 10 g of penicillin-G acylase (E.C. No. 3.5.1.11, from E. coli ATCC 11 105) and stirred for 2 hours at room temperature. The product is filt~red off and washed first with saturated sodium chloride solution and then - with deionized water.
Example 3 The specific activity of the immobilized penicillin-G
amidase system obtained according to Example 2 is determined by comparison with the standard substrate penicillin-G in the following manner:
1 g of the potassium salt of penicillin-G is dissolved in 40 ml of water and treated with lQ0 mg of the immobilized system at 37C, with stirring. By keeping the pH constant at 7.B0, the quantity of penicillin-G converted to 6-aminopenicillanic acid and phenylacetic acid can be read off directly from the alkali consumption by titration with 0.1 ~ sodium hydroxide solution.
After a reaction time of 30 minut~s, 3.6 ml of 0.1 N sodium hydroxide solution have been consumed, corresponding to 360 ~mol of penicillin-G. From this, the specific activity of the immobilized system is calculated to be 120 U/g.
Example 4 10 g (33-4 mmol) of N phenacetyl-D,L-2 amino-4-methylphosphinobutyric acid are dissolved in 40 ml of water. The solution, which has a pH of 7.8, is treated at 37C with one gram of the immobilized penicillin-G amidase system according to . ~.
.' . ~ .
.
, . .
~L~%6~3 Example 2, with stirring, and the pH is kept constant at 7.8 by the addition of 0.1 N sodium hydroxide solution. The reaction has ended after 5 to 6 hours. Working-up in the customary manner (German Patent Specification 3,048l612) gives 3.5 g (16 mmol, corresponding to 96% of theory) of L~2-amino-4-methyl-phosphinobutyric acid of melting point 199-201C, [a]2=24 (c =
1, in 1 N hydrochloric acid).
Example 5 100 g (0.4 mol) of N-phenacetyl-D,L-tert.-leucine are dissolved in 400 ml of 1 N sodium hydroxide solutionl the pH is adjusted to 7.8 and the volume is made up to a total of 1000 ml.
This reaction solution is thermostated at 37C and treated with 100 g of ~enicillin-G amida~e immobilized according to Example 2, with stirring. By titration with 0.05 N sodium hydroxide solution, the pH is kept constant during the hydrolysis reaction and the progress of the reaction is recorded at the same time.
After 3 to 4 hours, no further sodium hydroxide solution is consumed. After the enzyme material has been filtered off and the filtrate acidified to pH 3 with concentrated sulfuric acid, phenylacetic acid and N-phenacetyl-D-tert.-leucine crystallize out. The phenylacetic acid can be separated from the crop of crystals by extraction with ethyl acetate/hexane, after which 45 g (0.18 mol = 90 ~ of theory) of N-phenacetyl-D-tert.-leucine of melting point 163 to 164C, [a]2D0 = 14.2 (c = 1, in methanol), remain.
23 g (0.18 mol = 90 % of theory) of L-tert.-leucine of melting point above 250C, [a]D = -9.4~ (c = 1, in water) are isolated from the concentrated mother liquor by extraction with ethanol/water.
A . 1 ' . ', ' ~
Claims (4)
1. A penicillin-G-acylase immobilized on a macroporous phenol/formaldehyde anion exchange resin carrier carrying essentially secondary amino groups and having a pore diameter of 10-200 nm and a particle diameter of 250-840 µm, the penicillin-G-acylase and the carrier being bound together by means of an alkylene diisocyanatle having up to 10 carbon atoms in the alkylene chain.
2. A process for the preparation of an immobilized penicillin-G-acylase which comprises reacting a macroporous phenol/formaldehyde anion exchange resin carrier carrying essentially secondary amino groups and having a pore diameter of 10 to 200 nm and a particle diameter of 250 to 840 µm with an alkylene diisocyanate having up to 10 carbon atoms in the alkylene chain and subsequently reacting said pretreated phenol/formaldehyde anion exchange resin with said penicillin-G-acylase.
3. The process as claimed in claim 2, wherein the resin carrier in the free base form is reacted with the alkylene diisocyanate and the reaction product is reacted in an aqueous medium with a solution of the enzyme.
4. A process for cleaving amide bonds which comprises reacting N-acyl-D-L-amino acid with a penicillin-G-acylase enzyme as claimed in claim 1.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3334846.4 | 1983-09-27 | ||
| DE19833334846 DE3334846A1 (en) | 1983-09-27 | 1983-09-27 | IMMOBILIZED ACYLASE, METHOD FOR THEIR PRODUCTION AND THEIR USE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1277268C true CA1277268C (en) | 1990-12-04 |
Family
ID=6210135
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000464046A Expired - Fee Related CA1277268C (en) | 1983-09-27 | 1984-09-26 | Immobilized acylase, process for its preparation and its use |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0141223B1 (en) |
| JP (1) | JPS6094086A (en) |
| AT (1) | ATE30335T1 (en) |
| AU (1) | AU565703B2 (en) |
| CA (1) | CA1277268C (en) |
| DE (2) | DE3334846A1 (en) |
| DK (1) | DK460984A (en) |
| IL (1) | IL73059A (en) |
| ZA (1) | ZA847562B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3334848A1 (en) * | 1983-09-27 | 1985-04-04 | Hoechst Ag, 6230 Frankfurt | METHOD FOR PRODUCING THE OPTICAL ANTIPODES OF TERT.-LEUCIN |
| DE3435156A1 (en) * | 1984-09-25 | 1986-04-03 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD FOR PRODUCING THE STEREOISOMERS OF 1-AMINO-ALKYLPHOSPHONIC ACIDS OR 1-AMINOALKYLPHOSPHINIC ACIDS |
| DE4039415A1 (en) | 1990-02-03 | 1991-08-08 | Boehringer Mannheim Gmbh | Recombinant proteins without N-terminal methionine gp. - which are G-CSF derivs. useful to treat cancer and in bone marrow transplants, etc. |
| JPH05237146A (en) * | 1992-02-27 | 1993-09-17 | Yamaguchi Midori | Diaper |
| DE19529211C2 (en) * | 1995-08-09 | 1999-01-14 | Degussa | Process for the preparation of (R) tertiary leucine |
| NL1009814C2 (en) * | 1998-08-06 | 2000-02-08 | Dsm Nv | Production of N-acylated amino nitriles comprises reacting an amino nitrile with an optionally alpha-substituted phenylacetic or phenoxyacetic acid in the presence of a penicillin G or V acylase |
| JP2004344240A (en) * | 2003-05-20 | 2004-12-09 | Takasago Internatl Corp | Deodorizing method |
| CN102533888B (en) * | 2010-12-29 | 2015-07-15 | 浙江九洲药物科技有限公司 | Continuous enzymatic method for producing L-tert-leucine |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1400468A (en) * | 1972-07-22 | 1975-07-16 | Beecham Group Ltd | Enzyme preparation and use thereof |
| JPS5362890A (en) * | 1976-11-11 | 1978-06-05 | Sumitomo Chem Co Ltd | Preparation of immobilized enzyme |
| JPS56140890A (en) * | 1980-04-04 | 1981-11-04 | Sumitomo Chem Co Ltd | Immobilized lactase and its preparation |
| JPS5736986A (en) * | 1980-08-13 | 1982-02-27 | Tanabe Seiyaku Co Ltd | Immobilized aminoacylase agent and its preparation |
| SE450834B (en) * | 1980-11-19 | 1987-08-03 | Biocompat Inc | SET TO BIND A PROTEIN TO A CARRIER CONTAINING HYDROXYL GROUPS |
-
1983
- 1983-09-27 DE DE19833334846 patent/DE3334846A1/en not_active Withdrawn
-
1984
- 1984-09-20 DE DE8484111208T patent/DE3466893D1/en not_active Expired
- 1984-09-20 AT AT84111208T patent/ATE30335T1/en not_active IP Right Cessation
- 1984-09-20 EP EP84111208A patent/EP0141223B1/en not_active Expired
- 1984-09-25 IL IL73059A patent/IL73059A/en unknown
- 1984-09-26 ZA ZA847562A patent/ZA847562B/en unknown
- 1984-09-26 AU AU33557/84A patent/AU565703B2/en not_active Ceased
- 1984-09-26 CA CA000464046A patent/CA1277268C/en not_active Expired - Fee Related
- 1984-09-26 DK DK460984A patent/DK460984A/en not_active Application Discontinuation
- 1984-09-26 JP JP59199689A patent/JPS6094086A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| DK460984D0 (en) | 1984-09-26 |
| DK460984A (en) | 1985-03-28 |
| JPS6094086A (en) | 1985-05-27 |
| EP0141223A1 (en) | 1985-05-15 |
| AU3355784A (en) | 1985-04-04 |
| ATE30335T1 (en) | 1987-11-15 |
| DE3334846A1 (en) | 1985-04-04 |
| ZA847562B (en) | 1985-05-29 |
| AU565703B2 (en) | 1987-09-24 |
| DE3466893D1 (en) | 1987-11-26 |
| IL73059A0 (en) | 1984-12-31 |
| EP0141223B1 (en) | 1987-10-21 |
| IL73059A (en) | 1988-11-30 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKLA | Lapsed |