KR800000587B1 - Process for immobilizing microbial cells - Google Patents

Abstract

Whole microbial cells may be immobillixed on polymers and used for their enzymic properties. Thus, glycidyl methacrylate in H2O was miced with N, N'-methylenebiacrylamide, after stirring Me methacrylate was added and soln. was held a 5≰C for 15 min. then, dimethyaminopropionitrile and NH4 persulfate were added and the soln. was held at 10≰C until polymn. began. the mixt. was stirred for 2 hr, H2O wsa added, and the polmer was sepd. A suspension of wet, fresh Aspergillus niger cells was mixed with the polmer in H2O, and the solid material was removed and washed.

Description

Non-migrating method of microbial cells

The present invention relates to polymerizable enzyme products. More specifically, the present invention relates to a method for producing non-migrated microbial cells.

Enzymes are much better than conventional catalysts for many chemical reactions due to their high specificity. Until recently, however, the use of enzymes has been very limited due to a number of factors, such as high isolation prices, a tendency to destabilize when isolated from the environment of the whole cell, and availability only in soluble form.

Non-migrating enzymes by attaching them to or incorporating them into a solid support helps overcome these difficulties and make more efficient use of the enzymes. Various physical and chemical methods used for demobilization are described in magazines such as O.R. Zaborsky's "Immobilized Enzymes" (CRC Press, 1973). Whole cell demigration was also used to overcome enzymatic isolation and stabilization issues. But so far, its use has been limited to the physical method of demigration. Typical examples are reconstituted bark collagen [Biotech. & Bioengr., XV, page 565, 1973], is a cell uptake of Streptomyces pheoclomogenes, including the active glucose isomerase, and a hydroxylase enzyme that converts Compound S to cortisol [Scientific American, Gel uptake of fungal cells, including the March 1971 issue, page 30. However, this non-migration method prevents the cells from separating from the support medium. Due to the nature of the binding force, the reaction conditions are very limited to prevent or minimize such separation and subsequent loss of enzymatic activity and possibly contamination of the process stream. Thus, a more stable cell demobilization system is an object of the present invention.

It has now been discovered that microbial cells can be effectively demobilized by chemically covalently binding the cells to the water-insoluble particulate polymer matrix. Chemical bonds may be formed with reactive monomers that have been previously formed or prior to polymerization. In addition, treating the cells with a multifunctional crosslinker prior to, during or after binding reduces the loss of enzyme from the cells.

According to the present invention, there is provided a composition comprising microbial cells covalently bonded to a water-insoluble particulate polymer matrix.

Also provided is a method of immobilizing microbial cells by reacting the microbial cells with a binding bacterium of the polymer to form a covalent bond with the water-insoluble particulate polymer matrix.

In an embodiment of the invention, the microbial cells are chemically bound to the water-insoluble particulate polymer through reactive groups on the polymer. Although chemical covalent bonding of isolated enzymes to polymers is often practiced, it is surprising that the whole cell itself, which is a source of enzymes and a size larger than the enzyme, can also be effectively demobilized by chemical covalent bonds. Such bonds are frequently generated by reacting cellular amino groups with reactive polymer substituents. In addition, other airways, such as hydroxyl and sulfohydryl, may also be used intracellularly and may play a role in the binding mechanism. This is true because, for example, amino reactive polymer substituents such as epoxide, halocarbonyl and halomethylcarbonyl groups are also reactive to hydroxyl and sulfhydryl groups such as those present in the cell.

Bonding can occur before or after polymer formation. In the former case, the cell is contacted with a polymerizable ethylene unsaturated monomer containing a reactive group, and then the cell transport monomer is polymerized or copolymerized in the presence of a crosslinking monomer and an initiator system. In the latter case, the cells are brought into direct contact with the particulate polymer comprising the reactive group.

Suitable polymerizable ethylenically unsaturated monomers and polymers derived therefrom for carrying out the present invention by pre- or post-bonding methods are those of formula (I):

In the common sense

R ₁ is hydrogen, methyl or chloro,

이며,or

Is,

R ₂ is halo, azido, 2,3-epoxypropoxy,

2,3-epithiopropoxy, N- (2,3-epoxypropyl) amino, N-[(P-diazonium chloride) phenyl] amino, acrylooxy, lower alkoxy or benzenesulfonyloxy,

X is oxygen or NR ₃ (R ₃ is hydrogen or alkyl of C ₁ -C ₆ ),

Y is alkylene of C ₂ -C ₃ ,

n is an integer of 1-2,

Z is haloacetyl, 2- (4,6-dichloro) -S-triazinyl, P-toluenesulfonyl, P- (halomethyl) benzoyl or cyano,

X 'is the same as X except X' is oxygen when Z is P-toluenesulfonyl.

Monomers and polymers comprising epoxide, halocarbonyl and halomethylcarbonyl groups are preferred for bond formation. Particularly preferred are 2,3-epoxypropyl methacrylate (glycidyl methacrylate), 2,3-epithiopropyl methacrylate, methacryloyl chloride and bromoacetyl hydroxyethyl methacrylate which are reactive monomers It is a polymer containing.

In the case of monomers and polymers containing non-reactive functional groups, such as when R ₂ is hydroxy or Z is hydrogen in the monomer of formula (I), the functional groups can be converted into reactive substituents by known methods. In this case it is sometimes necessary to use multifunctional low molecular weight reagents. Thus, when R ₂ in the formula (I) is hydroxy, the carboxyl group of the monomer or polymer can be activated by reacting with a suitable carboxyl-activating reagent as follows. Carbodiimides such as dicyclohexylcarbodiimide or ethyl morpholinocarbodiide; N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's Reagent K); Keteneimines such as pentamethylene ketene cyclohexylimine; Acetyl acid ethers such as ethoxy acetylene; Hexahalocyclotriphosphatriazine; N-hydroxyphthalimide or N-hydroxy succinimide and other reagents used to form peptide bonds, monomers or polymers when Z in the formula (I) is hydrogen The hydroxyl group of can likewise be activated by reaction with halo-Z, in particular bromo acetyl bromide, cyanogen bromide and 2-amino-4,6-dichloro-1,3,5-triazine, by standard methods.

Non-migrating processes can be applied to microbial cells such as bacteria, yeast, actinomycetes and fungi. Preferred cells include oxidoreductases, such as glucose oxidase, in which the main enzyme system does not particularly require soluble cofactors; hydrolases, such as α, β-amylase and peptidase and especially penicillin acylase; Lyases, preferably lyases such as those involved in the degradation of carbon-oxygen bonds and in particular those involved in the degradation of carbon-nitrogen bonds and aspartate ammonia lyase; And isomerases such as racemas, epimerases, cis-trans isomerases and especially glucose isomerases. Preferred bacteria include Pseudomonas, Chisantonamonas, Acetobacter, Alcaligenes, Furabobacterium, Escherichia, Aerobacter, Erwinia, Serratia proteus, Micrococcus, Sarcina, Lactobacillus, Bacillus , Nocadia, streptomyces and corynebacterium. Preferred fungi and yeast genera include Rhodotorula, Rhodosporidium, Sporobolomyces, Mucor, Fussium, Penicillium, Aspergillus, Glomerella, Lazopus, Saccharomyces, Considiobol Ruth, Visoklamis, Candida, Ketoumium, Trichoderma, Septoria, Coprinus, Neurospora, Humucola and Tricosphoron. Preferred actinomycetes are Michaelloplispora. Particularly preferred species are Escherichia coli, Bacterium cardaberis, Proteus letgeri, Rhodochola glacilis, Penicillium chrysogenium and Aspergillus niger.

The reaction of covalently binding the cell to the reactive polymer or the cell to the reactive monomer and then polymerizing or copolymerizing the monomer is carried out in an aqueous medium. The temperature upon polymerization to the polymer or monomer or after polymerization to the monomer can vary widely but the preferred range is 5-50 ° C., in particular 10-30 ° C. The time required for bonding varies depending on the system and bonding temperature but is usually 0.5-35 hours, the preferred time for binding to the polymer is 15-30 hours and the preferred time for binding to the monomer is 1-5 hours. Polymerization usually completes in 1-6 hours. The weight ratio of polymer to cell (dry) can vary widely, with a preferred ratio being 0.1-25, in particular 0.5-4.

When the cell is bound to the reactive monomer, the next aqueous addition polymerization is carried out with or without one or more comonomers in the presence of the bitubular crosslinking monomer and the initiator system. Thus, comonomers, crosslinking monomers and initiator systems commonly used in this type of polymerization and which do not disrupt the enzymatic activity of the cells can be readily used.

Examples of suitable comonomers include acrylic acid, α-chloro acrylic acid, methacrylic acid and their glycidyl, lower alkylethyl, N, N- (disubstituted) aminoalkylesters, amides, lower alkylsubstituted amides, methylol Substituted amides, N-monosubstituted aminoalkylamides and N, N-disubstituted aminoalkylamides, styrene, butadiene and isoprene. Preferred comonomers are styrene, acrylic acid, [2-hydroxy-3- (1- [4-methylpiperazinyl) -propyl] methacrylate, and especially methylmethacrylate.

Various crosslinking monomers may be used that impart steric properties and water solubility to the final polymer, such as acrylic monomers or olefin compounds and other known ones. Representative examples of such monomers include 1,3-butylene diacrylate, ethylene glycol dimethacrylate, 1,3-butylene dimethacrylate, 1,6-hexamethylene diacrylate, ethylenediacrylate, diethylene Glycol dimethacrylate, N, N'-methylenebis acrylamide, neopentyl glycol dimethacrylate, 1,1,1-trimethylol ethane trimethacrylate and divinylbenzene. Preferred crosslinking monomers are N, N'-methylenebisacrylamide.

Polymerization and copolymerization reactions are initiated by glass reactors. Suitable free reactor initiator systems are those which generate the free reactor at a rate suitable for polymerization or copolymerization under the mild conditions required by the heat sensitivity of the cellular enzyme moisture. For this reason, redox initiator systems are preferred. Representative examples of such systems are sodium thiosulfate, sodium metabisulfite, perus ammonium sulfate hexahydrate, dimethyl amino in combination with ammonium, sodium or potassium percellate, benzoylperoxide and di (secondary butyl) -peroxy dicarbonate. A reducing agent such as propionitrile or riboflavin is combined. Preferred initiator systems are dimethylamino propionitrile-ammonium persulfate.

The polymer composition can vary widely, with preferred compositions being 15-90% cytoreactive monomer, 0-60% comonomer and 10-40% crosslinking monomer relative to the mole percent of total monomers. This ratio is desirable whether the cells bind to monomers or to preformed polymers.

In some cases, the cells are treated with a multifunctional crosslinking reagent to detect enzyme loss from the cells. The exact mechanism for this is not fully understood, but one group of multifunctional reagents reacts with and cross-links the amino groups of the cell membrane to effectively reduce the pores of the membrane, while the other group activates the carboxyl groups present on the membrane to form amide bonds. It is believed that the above reaction is achieved by reacting with an amino group. Typical examples of the first group include pyruvicaldehyde, glyoxal, hydroxy adialdehyde, cyanuric chloride, tetraazatiated 0-diazinidine, bis-diazatized benzidine, 1,3-difluoro- Reagents such as 4,6-dinitrobenzene, toluene-2,4-diisocyanate, and in particular glutaraldehyde, the second group being ethylchloro formate and water-soluble carbodiides such as 1-ethyl- Reagents such as 3- (3′-dimethylaminopropyl) carbodiimide hydrochloride.

The cells can be treated before, during or after attachment to the reactive monomer or polymer. Like cell binding, this treatment is carried out in an aqueous medium. Cells can be effectively treated over a wide range of temperatures, with a preferred range of 5-50 ° C., particularly 10-30 ° C. Depending on the temperature and the reagents used, the process usually requires 0.5-10 hours, preferably 2-4 hours. The amount of reagent required can vary widely and is usually 1-50% by weight, preferably 5-25% by weight of the cells (dry).

As described above, if the cells are immobilized to a non-aqueous polymer through chemical covalent bonds and optionally treated to minimize the leakage of enzymes from the cells, a stable, unmigrated enzyme system is obtained without the need to separate the required enzymes.

Due to their stability and easily particulate form, the product can be effectively used in aqueous contact chemistry methods that require recycle dilution or continuous processing.

Example 1

30 g of N, N'-methylenebis acrylamide was added to 80 g of glycidyl methacrylate and 1.0 L of water while stirring. After the mixture was stirred at room temperature for 15 minutes, 10 g of methyl methacrylate was added, the mixture was cooled to about 5 ° C., and nitrogen gas was passed through a cold mixture for 15 minutes. Next, 20 ml of dimethylamino propionitrile and 2 g of ammonium persulfate were added. The mixture was stirred at 10 ° C. under nitrogen until polymerization started and then further stirred at room temperature for 1 hour and finally left for another 2 hours. About 750 ml of water was added to the resulting solid and the mixture was vigorously stirred to destroy the polymer. The solid was filtered off, washed with water and air dried to yield 120 g of a white particulate polymer.

The Aspergillus Niger (NRRL-3; Pfizer Culture FD2454) fermentation broth cultured on air glucose conditions at 33 ° C. and pH 5.8-7.0 under air soaking conditions was filtered and the cells washed with water to give a semi-dry paste. Cells (250 g, 55 g of dry matter) were placed in a solution containing 22 g of 25% by weight aqueous glutaraldehyde in 1,000 ml of water. The suspension was adjusted to pH 6.2 and stirred at room temperature for 1-3 / 4 hours. The solid was filtered off and washed with water to give 104 g of wet treated cells with 76% of the original glucose oxidase activity.

A suspension of 32 g of wet cells and 16 g of glycidyl methacrylate polymer in 240 ml of water was stirred at room temperature for 30 minutes and then filtered. Washing the solids with water yielded 126 g of wet non-migrated cells with 58% of the glucose oxidase activity of the original untreated cells.

Example 2

600 mg of methacryloyl chloride and 400 mg of triethylamine were simultaneously added to a slurry of 600 mg of the wet-treated Aspergillus Niger cells prepared in Example 1 in 20 ml of acetonitrile. The mixture was stirred at room temperature for 2 hours and then placed in 200 ml of water (final pH: 6.03), 1.3 g of N, N'-methylenebisacrylamide, 750 mg of methyl methacrylate and 300 mg of acrylic acid were added to the aqueous mixture under nitrogen. The mixture was adjusted to pH 6.7 and then treated with 0.5 ml of dimethylaminopropionnitrile and 250 mg of ammonium persulfate. After 2 hours the reaction was further treated with 150 mg ammonium persulfate and then further stirred for 1 hour. An equal volume of water was added to the reaction and the mixture was centrifuged. The residue was filtered and washed with water to give 35.25 g of wet non-migrated cells containing 78% of the glucose oxidase activity of the original untreated cells.

Example 3

The wet non-migrated cells of Example 1 (47.3 g) were placed in 100 ml of an aqueous solution containing 10 g of glucose, and the mixture was stirred with aeration at room temperature for 21 hours. The conversion was 99% with a mixture of gluconic acid and deltagluconol acetone, and 75% of the original enzyme activity was recovered.

This oxide can be repeated using a dispersion of 30 g of the wet non-migrated cells of Example 2 in 20 ml of an aqueous solution containing 2 g of glucose, resulting in comparative conversion and recovery of enzyme activity.

Example 4

After centrifugation of the proteus retogeri (ATCC 9250; Pfizer Culture FD 18470-76-60) fermentation broth cultured under air-sedimentation conditions at 28 ° C. and pH 6.8-7.0 on a lactic casein substrate, the cells are repulped in water and half Marked as dry paste. 4 g of 25% by weight aqueous glutaraldehyde was added to a slurry of wettable cells (dry weight 7.5 g) in 100 ml of water. The mixture was stirred at pH 7.0 for 3 hours and then centrifuged. The treated cells were resuspended in 100 ml of water, and the glycidyl methacrylate polymer 7.5 of Example 1 was added with stirring. The mixture was stirred for 30 minutes at pH 7.0 and room temperature. The solid was filtered off, washed with water and stored as a wet cake until use. Unmigrated cells contained 65% of the activity of the original untreated cells as measured by the conversion rate of penicillin G to 6-amino peniclanic acid.

Example 5

5 g of potassium penicillin G was added to a slurry of 20 g (dry weight 5.0 g) of the non-migrated Proteus letgeri cells of Example 4 in 500 ml of water at pH 8.0 and 37 ° C. 1N sodium hydroxide was added to maintain the pH of the slurry at 8.0, and hydrolysis was complete after 3 hours. Unmigrated cells were filtered, washed with water and stored as a wet cake until use. 2N hydrochloric acid was added to the filtrate to adjust the pH to 2, and then extracted with ethyl acetate. 2N sodium hydroxide was added to the resulting aqueous layer, adjusted to pH 4.3, concentrated and cooled to give a white crystalline solid. The crystals were filtered off, washed with water and air dried to yield 2.17 g (75% yield) of pure 6-amino penicsilane acid. The unmigrated cells retained about 50% of the penicillin acylase activity of the original non-migrated cells of Example 4 after the 20th position hydrolysis cycle.

Example 6

375 mg of N, N'-methylenebisacrylamide was added to the mixture which stirred 3.6 g of bromoacetyl hydroxyethyl methacrylate in 15 ml of water under nitrogen. The mixture was cooled to 5 ° C. and then treated with 0.25 ml of dimethylaminopropionitrile and 38 mg of ammonium persulfate. The mixture was stirred slowly at room temperature until polymerization started. After stirring was stopped, the reaction mixture was left at room temperature for 2 hours. The resulting slurry was diluted with 25 ml of water and then filtered. The cake was washed with water and air dried to yield 3.7 g of particulate polymer.

Wetted proteus Letgeri cells cultured and isolated as in Example 4 were placed in a solution containing 0.8 g of 25% by weight aqueous glutaraldehyde in 50 mlso of water. The suspension was adjusted to pH 7.0, stirred at room temperature for 3 hours and then centrifuged. Treated cells were repulped in 50 ml of water and marked as semi-dry paste.

A suspension of 10 g of treated cells and 120 g of bromoacetylhydroxyethyl methacrylate polymer in 120 ml of water was stirred at room temperature and pH 7.0 for 30 hours. The mixture was filtered and the cake washed with water to give 36.8 g of wet non-migrating cells with 75% of the penicillin acylase activity of the original untreated cells.

Example 7

To a slurry of 46 g of wet non-migrating cells of Example 6 in 500 ml of water was added 5 g of potassium penicillin G at pH 8.0 and 37 ° C. 1N sodium hydroxide was added to maintain the pH of the slurry at 8.0, and hydrolysis was complete after 3 hours. Non-migrating cells were filtered off, washed with water and then stored as a wet cake until use. 2N hydrochloric acid was added, the filtrate was adjusted to pH 2 and extracted with ethyl acetate. 2N sodium hydroxide was added to the resulting aqueous layer, adjusted to pH 4.3, concentrated, and cooled to obtain crystalline 6-aminophenicylanic acid.

Example 8

A suspension of 25 g of the wet treated Aspergillus Niger cells of Example 1 and 2 g of glycidyl methacrylate in 60 ml of water was stirred at 7 ° C. for 17 hours. The suspension was then stirred at pH 6.8 at 2.2 g of N, N'-methylenebisacrylamide, 40 ml of styrene, 2 ml of styrene, 450 ml of methyl methacrylate, 0.5 ml of dimethylaminopropionitrile and [2-hydroxy-3- (1- [4-methylpiperazinyl]) propyl] methacrylate was added to a mixture of 1.5. The mixture was treated with 200 mg of ammonium persulfate under nitrogen and then stirred at room temperature for 3 hours. The reaction solid was filtered and washed with water to give 43.6 g of non-migrating cells with 75% of the glucose oxidase activity of the original untreated cells.

Example 9

The wet non-migrating cells (28.3 g) of Example 8 were placed in 200 ml of an aqueous solution containing 20 g of glucose, and the mixture was stirred for 21 hours while being aerated at room temperature. A mixture of gluconic acid and delacloconol acetone was obtained in 80% yield. The reaction slurry was filtered and the solid was washed with water to give 28 g of wet non-migrating cells having 82% of the original enzyme activity.

Example 10

A suspension of 5 g of wetted Proteus letgeri cells and 10 g of glycidyl methacrylate polymer of Example 1 was cultured and separated as in Example 4 in 65 ml of water at room temperature for 24 hours. The mixture was filtered to wash the filter cake with water to give 26.9 g of wet non-migrating cells with 62% of the original penicillin acylase activity.

Non-migrating cells (25 g, dry cell weight 1 g) were placed in a solution containing 0.4 g of 25% by weight aqueous glutaraldehyde in 50 ml of water. The suspension was adjusted to pH 7.0 and stirred at rt for 3 h. The slurry was filtered to wash the filter cake with water to give 23.2 g of wettable non-immobilized cells with 79% of the penicillin acylase activity of untreated non-immobilized cells.

Example 11

1 g of potassium penicillin G was added to a slurry of 12 g of the wettable non-immobilized cells of Example 10 in 100 ml of water at pH 8.0 and 37 ° C. 1N sodium hydroxide was added to maintain the pH of the reaction at 8.0 and the reaction was complete after 3 hours. Non-migrating cells were filtered off, washed with water and stored as a wet cake until use. 2N hydrochloric acid was added, the filtrate was adjusted to pH 2 and extracted with ethyl acetate. 2N sodium hydroxide was added to the resulting aqueous layer, adjusted to pH 4.3, concentrated and cooled to obtain crystalline 6-APA.

Example 12

A suspension of aspergillus niger cells (20 g, dry weight 4 g) and 8 g glycidyl methacrylate as in Example 1 in 100 ml of water was adjusted to pH 7.5 and shaken for 18 hours at room temperature. . N, N-methylenebis acrylamide (1.5 g) was added and the resulting slurry was stirred under nitrogen for 15 hours, cooled to 5 ° C, treated with 150 mg of ammonium persulfate and 1 ml of dimethylaminopropionitrile, Stirred for hours. The reaction solid was filtered and washed with water to give 71 g of wet non-migrating cells with 62% of the original glucose oxidase activity.

Non-migrating cells (18 g, dry cell weight 1 g) were placed in a solution containing 0.4 g of 25% by weight aqueous glutaraldehyde in 100 ml of water. The suspension was adjusted to pH 7.0 and stirred at room temperature for 3 hours. The solids were filtered and washed with water to give 17.3 wet treated non-migrating cells with 75% of the glucose oxidase activity present in the non-migrating cells prior to glutaraldehyde treatment.

Example 13

The wet non-immobilized cells of Example 12 (14 g) were placed in 15 ml of an aqueous solution containing 1.5 g of glucose, and the mixture was stirred at room temperature for 21 hours while the mixture was aerated. The conversion of gluconic acid and the recovery of enzymatic activity are similar to those in Example 3.

Example 14

Rhotorola lugralis cells (NRRL Y 1091; Pfizer culture FD 6521) were isolated by centrifugation from fermentation broth cultured at 28 ° C. and pH 6.8 to 7.0 under air soaking conditions on glucose substrate. 50 g of washed cells in 250 ml of water were treated with 5 g of the glycidyl methacrylate polymer of Example 1. The mixture was stirred at rt for 20 h. The suspension was filtered to wash the filter cake with water, resulting in 63 g of wet non-migrating cells with 49% of the original phenylalanine ammonia-lyase activity.

Example 15

A suspension of 14.7 g of wet non-migrating cells of Example 14 in 30 ml of water containing 2.25 g of trans-cinnamic acid, 652 mg of ammonium chloride and 3,3 ml of concentrated ammonium hydroxide was adjusted to pH 9.5. The mixture was shaken at 37 ° C. for 18 hours, yielding about 90% yield of L-phenylalanine based on the recovered cinnamic acid. The reaction solid was filtered and washed with water to give 14 g of wet non-migrating cells that contained 77% of the original phenylalanine ammonia-lyase activity. L-renylalanine was isolated from the filtrate using standard methods.

Example 16

A suspension of 40 g of Rodotorulagracillis and 8 g of glycidyl methacrylate incubated and isolated as in Example 14 in 80 ml of water was adjusted to pH 7.2 and stirred at room temperature for 1.5 hours. N, N'-methylene acrylamide (1.5 g) was added, the resulting slurry was stirred for 10 minutes under nitrogen, cooled to 5 ° C, treated with 150 mg of ammonium persulfate and 1 ml of dimethylaminopropionitrile, and then 3 hours at room temperature. Was stirred.

The final reaction slurry was diluted with water and filtered. Washing the polymer cake with water yielded 62.3 g of wet non-migrating cells with 44% of the original phenylalanine ammonia lyase activity.

Example 17

The suspension of 15 g of wet non-migrating cells of Example 16 in 30 ml of water containing 1.12 g of trans-cinnamic acid, 320 mg of ammonium chloride and 1.6 ml of concentrated ammonium hydroxide was adjusted to pH 9.5. The mixture was shaken at 37 ° C. for 18 hours, resulting in L-phenyl alanine as in Example 15.

Example 18

Suspension of 15 g of the wetted Proteus Letgeri and 30 g of the glycidyl methacrylate polymer of Example 1 in culture and separated as in Example 4 in 100 ml of water was stirred at room temperature and pH 7 for 18 hours. Centrifugation of the suspension followed by washing of the solid with water yielded 100 g of wet-cell immobilized cells containing 100% of the original penicillin acylase activity.

To a slurry of 25 g of wet non-migrating cells in 70 ml of water was added 1 g of potassium penicillin G at pH 8.0 and 37 ° C. The hydrolysis carried out with the addition of 1N sodium hydroxide and maintained at pH 8.0 was completed in 3 hours. The slurry was filtered to wash non-immobilized cells with water and then stored as a wet cake until use. The effect of this cell to prepare 6-amino penicilanic acid is not as great as the non-migrating cells of Example 4, because it retains only 15% of the penicillin acylase activity of the original cell after only two hydrolysis cycles. Because.

Example 19

To 25 g of 2-hydroxyethyl methacrylate in 100 ml of water, 12.5 g of cyanogen bromide dissolved in 100 ml of water was added. 5N sodium hydroxide was added to the mixture immediately to pH 11, and the reaction temperature rose to 46 ° C, after which the pH was maintained until no more base was required. The mixture was then cooled to room temperature, adjusted to pH 6.5, and then treated with slurry of 50 g of lyophilized proteus letgeri isolated and cultured as in Example 4 before drying in 500 ml of water.

The resulting suspension was stirred for 45 minutes. Next, 25 g of acrylamide and 2.5 g of N, N'-methylenebisacrylamide were added, the suspension was stirred for 15 minutes, cooled to 4 ° C, and treated with 4 ml of dimethylamino propionitrile and 425 mg of ammonium persulfate. The polymerization mixture was allowed to warm to room temperature and then left for 1 hour. The mixture was then mixed using a Waring mixer and centrifuged. The gelled centrifuged solid was resuspended in water, centrifuged again, washed with water and lyophilized to give 112 g of dry, non-immobilized cells containing 19% of the original penicillin acylase activity.

Example 20

To 80 g of water, a suspension of 20 g of wetted Proteus Letgeri cultured and isolated as in Example 4 was added 1 g of 25% by weight aqueous glutaraldehyde and 10 g of glycyryl methacrylate. The suspension was adjusted to pH 6.8 and then stirred at room temperature for 2 hours. The mixture was treated with 1.9 g of N, N'-methylenebisacrylamide and 2 ml of dimethylaminopropionitrile under nitrogen, then adjusted to pH 7 and treated with 200 mg of ammonium persulfate. The resulting suspension was stirred for 1/2 hour and then left at room temperature for 1½ hour. The solid was filtered and washed with water to give 81.3 g of wet non-migrating cells containing 54% of the original penicillin acylase activity of untreated cells.

Wet non-migrating cells are useful for converting potassium penicillin G to 6-aminophenicylanic acid as in Example 7, with similar results as in Example 7.

Example 21

After centrifugation of Bacterium Cadabers (ATCC 9760; Pfizer Culture FD 24016) fermentation broth incubated at 28 ° C. and pH 6-8 on protein lysate substrates, the cells were repulped in water and semi-dried. Marked with paste. 4 g of 25% by weight aqueous glutaraldehyde was added to a slurry of wettable cells (dry weight 7.5 g) in 100 ml of water. The mixture was stirred at pH 7 for about 2 hours and then centrifuged. After the treated cells were resuspended in 100 ml of water, 7.5 g of the glycidyl methacrylate polymer of Example 1 was added with stirring. The mixture was stirred at about pH 7.0 and at room temperature for 30 hours. The solid was filtered off, washed with water and stored as a wet cake until use.

Example 22

Fumaric acid (120 g) was added to 140 ml of concentrated sodium hydroxide. Ammonium hydroxide was added to adjust the mixture to pH 8.5, followed by water to adjust the volume of the reaction to 600 ml. The non-migrated cells of Example 21, containing 7,000μ asphaltate ammonia-lyase activity, were added and then the mixture was stirred at 37 ° C. for 4 hours. After the reaction slurry was filtered, sulfuric acid was added to adjust the filtrate to pH 2.8 to precipitate aspartic acid.

Claims (1)

Bacterial, fungal and yeast cells are reacted with methacrylate monomers or polymers to form covalent bonds between the microbial cells and methacrylate material, and if monomers are used, the monomers are then polymerized to make the microbial cells insoluble A method for non-migrating microbial cells, characterized in that the product is covalently bound to the particulate polymer matrix.