CA1209503A - Immobilized microbial cells and processes for preparing and using same - Google Patents
Immobilized microbial cells and processes for preparing and using sameInfo
- Publication number
- CA1209503A CA1209503A CA000423176A CA423176A CA1209503A CA 1209503 A CA1209503 A CA 1209503A CA 000423176 A CA000423176 A CA 000423176A CA 423176 A CA423176 A CA 423176A CA 1209503 A CA1209503 A CA 1209503A
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- cells
- immobilized
- prepolymer
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
IMMOBILIZED MICROBIAL CELLS AND
PROCESSES FOR PREPARING AND USING THE SAME
ABSTRACT
A composition comprising immobilized cells having enzyme activity, the cells being immobilized by means of a fixed, insoluble, crosslinked polymer obtained by curing a curable prepolymer material selected from the group consisting of polyazetidine prepolymers, carboxymethyl cellulose, polyurethane hydrogel prepolymers and polymethylene isocyanates, said polymer being cured at a temperature below the temperature at which the enzyme activity of the microbial cells is significantly reduced, the cell/crosslinked polymer composition constituting a coating on a solid inert carrier. The composi-tion may be used in various ways, for example, to prepare L-aspartic acid.
PROCESSES FOR PREPARING AND USING THE SAME
ABSTRACT
A composition comprising immobilized cells having enzyme activity, the cells being immobilized by means of a fixed, insoluble, crosslinked polymer obtained by curing a curable prepolymer material selected from the group consisting of polyazetidine prepolymers, carboxymethyl cellulose, polyurethane hydrogel prepolymers and polymethylene isocyanates, said polymer being cured at a temperature below the temperature at which the enzyme activity of the microbial cells is significantly reduced, the cell/crosslinked polymer composition constituting a coating on a solid inert carrier. The composi-tion may be used in various ways, for example, to prepare L-aspartic acid.
Description
Lf~ r~ 3~
IMMOBILIZED MICROBIAL CELLS AND
PROCESSES FOR PREPARING AND US ING THE SAME
_ The present invention is concerned with the immobilization of microbial cells and processes 5 for preparing and using the same. The invention is particularly concerned with an improved process for manufacturing L-aspartic acid using immobilized microbial cells, notably Escherichia coli (~. coli) cells, which contain L-aspartase activity. However, lo the immobilization and use of other cells are also contemplated.
Backqround to The Invention There is a considerable amount of prior art regarding the immobilization of E. coli or other microbial cells for use in the preparation of L-aspartic acid. For example, U.S. patent 3,791,926 (Chibata et al) describes a process for the production of L-aspartic acid which involves polymerizing a monomer selected from acrylamide, 20 N,N'-lower alkylene-bis (ac--ylamide) and bis(acrylamidomethyl) ether in an aqueous suspension containing an aspartase-producing microorganism such as E. coli ATCC no. 11303. The resultant immobilized aspartase-producing micro-organism is treated with ammonium fumarate or amixture of fumaric acid or its salt and an inorganic ammonium salt whicil by enzymatic reaction gives L-aspartic acid.
The immobilization of E~ coli cells containing aspartase activity and use of the resulting immobilized cells for the production of L-aspartic acid are also described by Fusee et al, Applied and Environmental Microbioloqy, Vol. 42, ~.~g~$5~3 No. 4, pages 672-676 (October 1981). According to Fusee et al, the cells are immobilized by mixing a suspension of the cells with a liquid isocyanate-capped polyurethane prepolymer tHYPOL ~ ) so as to s form a "Eoam" containing the immob.ilized cells.
Sato et al (Biochimica et Biophysica Acta, 570tl979~ pages 179-186) have disclosed the immobilization of E. coli cells containing aspartase activity with K carrageenan, and use of the immobilized preparation for the production of L-aspartic acid.
Additional literature disclosures describing the immobilization of microbial cells in urethane prepolymers or polyurethanes or the like include the following:
(a) Immobilization of MicrobiaL Cells in Polyurethane Matrices, by Klein et al, Biotechnolo~ Lettes, Vol. 3, No. 2, pages 65-70 (1981);
(b) Hyrophilic Urethane Prepolymers:
Convenient Materials for Enzyme Entrapment, Biotechnoloqy_and Bioenqineering, Vol. XX, pages 1465-1469 (1978), (c) Transformation of Steroids by Gel-Entrapped Cells in Organic Solvent, by Omata et al, European J. Applied Microbioloqy and Blotechnoloqy, 8, pages 143-155 (1979); and (d) Entrapment of Microbial Cells and Organelles With Hydrophilic Urethane Prepolymers, by Tanaka et al, European J. Applied Microbioloqy and Biotech_ nology, 7, 351-354 (1979j~
`~ ~2~$~
The above-noted processes for preparing L~aspartic acid using immobilized microbial cells ~uffer from various dis advantages. For example, g-carrageenan gum and polyurethane "foam" as 5 disclosed by Fusee et al and Sato et al are relativel~ soft and compressible. Hence when these immobilized cell CGmpOSitiOnS are used, in a column through which ammonium fumarate is pa~sed for conversion to ammonium aspartase, they tend to be compressed and plug up, particularly where high flow rates and/or relatively tall column heights are involved.
Objects of the Invention The principal object of the present invention is to provide an improved process for preparing L aspartic acid using L-aspartase active microbial cells, preferably E. coli cells, which have been immobilized in a special way whereby the resulting composition is highly effective for the 20 preparation of L-aspartic acid batchwise or in continuous fashion.
A more specific object includes the provision of an improved process for preparing L-aspartic acid from ammonium fumarate using 25 immobilized E. coli cells which maintain optimum L-aspartase activity for relatively long periods of time. Another specific object of the invention is to provide novel immobilized microbial systems suitable for use in making L-aspartic acid which obviate problems encountered in prior procedures involving the use of immobilized cells. 0ther objects will also be hereinafter apparent.
5V;~
Summar~L_f ~ ~ veAtion According to one aspect of the invention, there is provided a process for preparing L-aspartic acid by contacting ammonium fumarate or its equivalent with an immobilized microbial cell composition comprising E. coli ATCC 11303 cells or equivalent cells containing h-aspartase activity, the cells being immobilized by means of a fixed~
insoluble, crosslinked polymer obtained by curing o a curable prepolymer material selected from the group consisting of polyazetidine prepolymers, carboxymethyl cellulose (CMC), polyurethane hydrogel prepolymers and polymethylene isocyanates cured at a temperature below the temperature at which the L-aspartase activity of the microbial cells is significantly reduced, the cell/cross-linked polymer composition constituting a coating on a solid inert carrier. The application of the immobilized cell/crosslinked polymer coating on the carrier provideq a highly advantageous form of the cell/polymer composition for use in preparing L-aspartic acid. It is a particularly advantageous feature that the cell/polymer systems of the invention can be provided as a dry coating on the carrier without significant loss of the L-aspartase activity.
An especially preferred a~pect of the invention contemplates the provision of immobilized E. coli cells having L-aspartase activity, the cells being immobilized by means of a crosslinked, water-insoluble polymer obta~ned by curing a polyazetidine prepolymer.
Broadly speaking, the invention i~
dependent on binding whole cells of E. coli, notably E. coli ATCC 11303, or the equivalent, s~
which are known to have L-aspartase activity and therefore are capable of producing L-aspartic acid (or ammonium salt thereof~ by conversion sf ammonium fumarate/ in a novel and useful configur-a~ion using a special crosslinked polymer systemas set forth above to bind the cells.
The indicated prepolymer systems can be crossLinked at temperature below 40C and in the presence of relatively large volumes of water lo containing E. coli cells without signiEicantly deteriorating the L-aspartase activity of the cells. It is a surprising aspect of the invention that such aqueous crosslinking conditions leave the E. coli cells with their L-aspartase activity even though the cells are immobilized in insoluble, crosslinked polymer networks.
Another unique aspect of the invention is that the wet dispersion of the E. coli cells iQ
the aqueous polymer solutions can be taken to dryness while the immobilized cells still surprisingly retain most of their original L-aspartase activity. The drying process has the advantage of providing strong, weLl-crosslinked, insoluble compositions in the form of coatings, 25 membranes, particles, etc. having high concentra-tions of cells retaining L-aspartase activity~
The novel immobilized L-asparta~e cell compositions disclosed herein have been found to outperform the immobilized L-aspartase active E. oli cell compositions previously reported by, fcr example, Fusee et al and Sato et al in the references noted above.
Prepolymer materials suitable for use to provide the crosslinked polymer net~ork for immobilizing th~ E. coli cells according to the invention include:
(1) polyazetidine prepolymers which may be crosslinked in aqueous solution by reaction with =NH, -SH, -UH, -COOH; or other polyaze-tidines which may be crosslinked by H20 removal, heat, or by changing to a more basic pH. The lo following is an idealiæed structure of a representative polyazetidine such as Polycup ~172 (Hercules, Inc.) which is useful for present purposes:
o O Cl-----C - R--CNEICH2 CH2~ N--CH2 - CH2NH--CH~ CH2 OH
where R is typically tCH2t4 (2~ carboxymethyl cellulose which can be crosslinked in aqueous solution by reaction with polyvalent ions such as Fe+~, Al++~, Ca++, Mg+~.
The following is an idealized structure of a carboxymethyl cellulose polymer such as CMC7HF
(Hercules, Inc.):
___ O
CH20CH2CONa OH
H / CH - O O CH CH H
\ / OH \ / \ / OH \ /
C / H H \ / C
/ CH- CH CH O
OH CH20CH2CONa (3) polyurethane hydrogel prepolymers which can be crosslinked in aqueous solution by reaction with -NH, -SH, -OH, -COOH or H20. The following is an idealized structure of a repre-sentative polyurethane hydrogel prepolymer which is made by taking a water soluble polyether polyol having greater than two hydroxyl groups per molecule and a moleculal weight greater than 3,000 lo and capping each hydroxyl group with a low molecular weight polyisocyanate:
R ~ (ocH2~H23 ~(~cEl2--cH~l ~cE~2cH ~ OCHNR2N(~;
where Rl typically ls a low molecular weight polyol fragment (from glyceroll pentaerithritol, sorbitolj etc.);
S~3 a and b are integers, eOg. a is typically>
10 and the ratio of a/b is generally from 2 to 5, the sequence of aerangement of the a and b segments being random or in blocks;
n is also an integer typically > 2; and R2 is typically a diisocyanate such as a toluene diisocyanate, methylene di(phenylisocyanate)) m-xylylene diisocyanate, isophorone-diisocyanate, hexamethylene diisocyanate, hexahydro-m-xylylene diisocyanate, dodecahydro-methylene di(phenylisocyanate) and the like; and (4) polymethylene isocyanates which can be cured by mixing with water such as the following:
[~L CE2 ~L C 2 [~
where n=0 to 4 Such polyisocyanates are commercially available and are commonly called "crude polymeric methylene dilphenylisocyanates)~. Other polyisocyanates which are convertible into crosslinked insoluble polymers with water are also thought to be useful, e.g. toluene dlisocyanates, methylene di(phenyl-isocyanate), m-xylylene diisocyanate, isophorone-diisocyanate, hexamethylene diisocyanate, hexahydro-m-xylylene diisocyanate, dodecahydro-methylene di(phenylisocyanate) and the like z~5~3 The isocyanates are usefully employed by coating an appropriate particulate substrate and then mixing with an aqueous slurry of E. coli cells~
Any of the above-noted polymer systems may be used to immobilize L-aspartase active E. coli cells to provide cell/polymer compositions having a variety of different forms and shapes~
For example, the immobilized cell/polymer composi-lo tion may be prepared in the form of membranes, filaments, fibers, tubes, beads or the like. A
particuldrly important embodiment oE the invention involves peoviding the immobilized cell/polymer composition as a coating on an appropriately shaped support which advantageously, although not necessarily, is in the form of solid or reticu-lated beads or particles composed of an inert solid organic or inorganic, porous or non~porous materialO As noted earlier, the composition of the invention can be cured and dried without substantively afecting the L-aspartase activity of the cells and, as a consequence, the composi tion can be made up in the form of dried coated beads or other particulate material and stored until needed fcr use. Typically, useful supports or carriers for the immobilized cell/crosslinked polymer compositions of the invention include the following in bead or particle form:
Molecular sieves Ion exchange resins Alumina Silica and silica ~el Foraminifera skeletons Polymer la~exes Metals 9S~3 It is a useful property of the polymer systems used in the present invention that they can be contacted or mixed in water wlth E. coli cells containing L~aspartase and then crosslinked (or cured) to form insoluble and relatively fixed polymer matrices which hold or otherwise immobilize the E. coli cells. It is particularly useful that the crosslinking conditions which need to be used are mild enough so that the L-aspartase lo activity oE the E. coli mutant cells is main~ained.
As indicated, a particularly desirable CompGsition according to the invention is obtained by applying the E. coli cells containing L-aspartase activity and the immobilizing polymer asa coating on hard inorganic or organic polymer beads~ Using these relatively noncompressible compositions an L-aspartase active catalyst bed capable of high throughput in a fixed bed or 20 fluidized bed is possible. As noted earlier, the L-aspartase active immobilized cell compositions previously described in the literature involving the use of relatively soft, compressible k-carrageenan gum or polyurathane foam are 2s compressible and therefore tend to plug under high flow rates, and/or tall column (bed) heights.
Various methods may be used to immobilize the E. coli ATCC 11303 cells~ while retaining their L-aspartase activity, by combination with cured or crosslinked forms of prepolymers according to the invention. The preferred method used in any specific situation will depend, at least to some extent, on the prepolymer involved.
Thus:
9Sj~3 a) In the case of the preferred poly-azetidine prepolymers, E. coli ATCC
11303 cells are advantageously mixed with an aqueous solution of the prepoly-mer so as to obtain a homogeneous mixture after which the polyazetidine may be cured or crosslinked to give an insoluble L-aspartase active composition by any of the following means:
lo Removal of part or nearly all of the water at temperatures below 60C
~usually between 40 and 0C) and pressures of 760 to 1~0 Torr;
raising the pH above 7.5; exposing the E. coli/polyazetidine mixture to polyamines (using, for example, 100 parts of the composition for each 1-2 parts/wt. of polyethylene imine, polyamino ion exchange re~ins, diethylene triamine, ethylene diamine, etc.) b) For carboxymethyl cellulose (CMC) gums, homogeneous mixtures with E. coli ATCC
11303 may be crosslinked (cured) to insoluble, L-aspartase active coatings, membranes, ibers, beads, etc.~ by contacting an aqueous mixture of, Eor example, 1.0 part/wt. of cell past/CMC
mixture with an aqueous solution of 0.001 to 1.0 parts/wtO of polyvalent cation salt or, ideally 0.01 to 0 4 parts/wt. of polyvalent cation salt per 1.0 part/wt. of the cell paste/CMC
mixture.
The resultant water insoluble immobilized cells/polymer network composition can be thus cured in the form of membranes, fibers or beads, but most efficiently as a coating around high sur~ace area particles. As an illustration lO to 1000 parts/wt. of the high surface area particles may be coated with 1.0 part/wt. of cell lo paste/CMC mixture and ideally 50 to 500 par~sJwt. of the particles per 1.0 partJw~. of cell paste/CMC mixture are used. Removal of 75% or more of the water contained in the cured coatings may be desirable to improve strength and bonding of the cell/polymer coating.
c) When polyurethane hydrogels are used~
homogeneous aqueous dispersions of E. coli ATCC 11303 (typically l.0 9 of cells per l.0 to 1000 ml H2O broadly and 10 to 190 ml H2O preferably) are mixed with the hydrogel prepolymer and the prepolymer is cured into a water-insoluble L-aspartase active composition by allowing the polyisocyanate to react with water or in a more rapid manner by either removing 50 to 100% of the available water 1000 to 0.1 Torr and below 50C (preferably 760 to 5.0 Torr and 0 to 30C) or by exposing the undried compositions to aqueous solu-tions o~ molecules having more than two primary or secondary amine groups.
Typically these polyamine molecules can range from hydrazine or ethylene diamine to polyethyleneimine. Generally for each l.0 g of hydrogel prepolymer used, from 0.01 to lO.0 g of the polyamine in from 1.0 to 500 ml of water could be used. Ideally 0 1 to l.0 9 of the polyamine and 10 to lO0 ml of water are employed per gram of hydrogel prepolymer used.
The above E. ~ hydrogel compositions can be fabricated into membranesp fibers, beads, etc., but a preferred configuration is to use the compositions as coatings onto high surface area lS particles, as discussed before.
Depending on the surface area of the particle employed from 0.1 to 100.0 g of the E. col~/hydrogel composition can be coated onto lO0 g of the particle.
d~ In the case where polyisocyanates are used, these are generally first applied to a surface and the cells of E. coli ATCC 11303 thereafter added thereto.
Usually, from 0.1 to 20.0 g of the polyisocyanate per lO.0 9 of the surface material can be employed, preferably 0.5 to 10.0 g of polyisocyanate per lO.0 9 of surface material. The polyisocyan-ates, if liquid, can ~e added undiluted to the substrate with good mixing (agitator or tumbling~. However, the preferable method is to use solutions of ~he polyisocyanates in inert, readily evaporated solvents such as toluene, ~ 2q:119S~3 acetone, chloroform, tetrahydrofuran~
dimethoxyethane, ethyl acetate, and the like. These solutions are added to the substrate, prefeeably dry particles of hi~h surface area, with good mixing and the solvents removed at their boiling points at 760 Torr or below while continuing good mixing to insure homogeneously coated, mobile substrate lo particles~ The amounts of solvents used will usually ran~e from 1.0 to 100 parts/wt. per 1.0 parts/wt. of polyisocyanate.
The immobilized cell/polymer compositions 1~ of the invention may be used to make aspartic acid by either a batch or continuous process. However, it is noted that these compositions, particularly when coated onto a bead or other particulate support, are especially effective for the continuous conversion of aqueous solutions of ammonium fumarate to ammonium L-aspartate according to the following equation:
O O
Il 11 --O--C ~ CO--\ / bed of immobilized C 3' CHNE~2 H~
NH4~ ll L-aspartase active /~ ~. coli compositions CH2 H 90 1 o NH4 O O
pH 8.5 pH 8.5 As illustrative of processes which may be used to prepare L-aspartic acid or ammonium L-aspartate according to the invention are the following:
(i) A batch type process wherein the catalyst compositions are stirred in from 0.1 to 5.0 molar (preferably 0.5 to
IMMOBILIZED MICROBIAL CELLS AND
PROCESSES FOR PREPARING AND US ING THE SAME
_ The present invention is concerned with the immobilization of microbial cells and processes 5 for preparing and using the same. The invention is particularly concerned with an improved process for manufacturing L-aspartic acid using immobilized microbial cells, notably Escherichia coli (~. coli) cells, which contain L-aspartase activity. However, lo the immobilization and use of other cells are also contemplated.
Backqround to The Invention There is a considerable amount of prior art regarding the immobilization of E. coli or other microbial cells for use in the preparation of L-aspartic acid. For example, U.S. patent 3,791,926 (Chibata et al) describes a process for the production of L-aspartic acid which involves polymerizing a monomer selected from acrylamide, 20 N,N'-lower alkylene-bis (ac--ylamide) and bis(acrylamidomethyl) ether in an aqueous suspension containing an aspartase-producing microorganism such as E. coli ATCC no. 11303. The resultant immobilized aspartase-producing micro-organism is treated with ammonium fumarate or amixture of fumaric acid or its salt and an inorganic ammonium salt whicil by enzymatic reaction gives L-aspartic acid.
The immobilization of E~ coli cells containing aspartase activity and use of the resulting immobilized cells for the production of L-aspartic acid are also described by Fusee et al, Applied and Environmental Microbioloqy, Vol. 42, ~.~g~$5~3 No. 4, pages 672-676 (October 1981). According to Fusee et al, the cells are immobilized by mixing a suspension of the cells with a liquid isocyanate-capped polyurethane prepolymer tHYPOL ~ ) so as to s form a "Eoam" containing the immob.ilized cells.
Sato et al (Biochimica et Biophysica Acta, 570tl979~ pages 179-186) have disclosed the immobilization of E. coli cells containing aspartase activity with K carrageenan, and use of the immobilized preparation for the production of L-aspartic acid.
Additional literature disclosures describing the immobilization of microbial cells in urethane prepolymers or polyurethanes or the like include the following:
(a) Immobilization of MicrobiaL Cells in Polyurethane Matrices, by Klein et al, Biotechnolo~ Lettes, Vol. 3, No. 2, pages 65-70 (1981);
(b) Hyrophilic Urethane Prepolymers:
Convenient Materials for Enzyme Entrapment, Biotechnoloqy_and Bioenqineering, Vol. XX, pages 1465-1469 (1978), (c) Transformation of Steroids by Gel-Entrapped Cells in Organic Solvent, by Omata et al, European J. Applied Microbioloqy and Blotechnoloqy, 8, pages 143-155 (1979); and (d) Entrapment of Microbial Cells and Organelles With Hydrophilic Urethane Prepolymers, by Tanaka et al, European J. Applied Microbioloqy and Biotech_ nology, 7, 351-354 (1979j~
`~ ~2~$~
The above-noted processes for preparing L~aspartic acid using immobilized microbial cells ~uffer from various dis advantages. For example, g-carrageenan gum and polyurethane "foam" as 5 disclosed by Fusee et al and Sato et al are relativel~ soft and compressible. Hence when these immobilized cell CGmpOSitiOnS are used, in a column through which ammonium fumarate is pa~sed for conversion to ammonium aspartase, they tend to be compressed and plug up, particularly where high flow rates and/or relatively tall column heights are involved.
Objects of the Invention The principal object of the present invention is to provide an improved process for preparing L aspartic acid using L-aspartase active microbial cells, preferably E. coli cells, which have been immobilized in a special way whereby the resulting composition is highly effective for the 20 preparation of L-aspartic acid batchwise or in continuous fashion.
A more specific object includes the provision of an improved process for preparing L-aspartic acid from ammonium fumarate using 25 immobilized E. coli cells which maintain optimum L-aspartase activity for relatively long periods of time. Another specific object of the invention is to provide novel immobilized microbial systems suitable for use in making L-aspartic acid which obviate problems encountered in prior procedures involving the use of immobilized cells. 0ther objects will also be hereinafter apparent.
5V;~
Summar~L_f ~ ~ veAtion According to one aspect of the invention, there is provided a process for preparing L-aspartic acid by contacting ammonium fumarate or its equivalent with an immobilized microbial cell composition comprising E. coli ATCC 11303 cells or equivalent cells containing h-aspartase activity, the cells being immobilized by means of a fixed~
insoluble, crosslinked polymer obtained by curing o a curable prepolymer material selected from the group consisting of polyazetidine prepolymers, carboxymethyl cellulose (CMC), polyurethane hydrogel prepolymers and polymethylene isocyanates cured at a temperature below the temperature at which the L-aspartase activity of the microbial cells is significantly reduced, the cell/cross-linked polymer composition constituting a coating on a solid inert carrier. The application of the immobilized cell/crosslinked polymer coating on the carrier provideq a highly advantageous form of the cell/polymer composition for use in preparing L-aspartic acid. It is a particularly advantageous feature that the cell/polymer systems of the invention can be provided as a dry coating on the carrier without significant loss of the L-aspartase activity.
An especially preferred a~pect of the invention contemplates the provision of immobilized E. coli cells having L-aspartase activity, the cells being immobilized by means of a crosslinked, water-insoluble polymer obta~ned by curing a polyazetidine prepolymer.
Broadly speaking, the invention i~
dependent on binding whole cells of E. coli, notably E. coli ATCC 11303, or the equivalent, s~
which are known to have L-aspartase activity and therefore are capable of producing L-aspartic acid (or ammonium salt thereof~ by conversion sf ammonium fumarate/ in a novel and useful configur-a~ion using a special crosslinked polymer systemas set forth above to bind the cells.
The indicated prepolymer systems can be crossLinked at temperature below 40C and in the presence of relatively large volumes of water lo containing E. coli cells without signiEicantly deteriorating the L-aspartase activity of the cells. It is a surprising aspect of the invention that such aqueous crosslinking conditions leave the E. coli cells with their L-aspartase activity even though the cells are immobilized in insoluble, crosslinked polymer networks.
Another unique aspect of the invention is that the wet dispersion of the E. coli cells iQ
the aqueous polymer solutions can be taken to dryness while the immobilized cells still surprisingly retain most of their original L-aspartase activity. The drying process has the advantage of providing strong, weLl-crosslinked, insoluble compositions in the form of coatings, 25 membranes, particles, etc. having high concentra-tions of cells retaining L-aspartase activity~
The novel immobilized L-asparta~e cell compositions disclosed herein have been found to outperform the immobilized L-aspartase active E. oli cell compositions previously reported by, fcr example, Fusee et al and Sato et al in the references noted above.
Prepolymer materials suitable for use to provide the crosslinked polymer net~ork for immobilizing th~ E. coli cells according to the invention include:
(1) polyazetidine prepolymers which may be crosslinked in aqueous solution by reaction with =NH, -SH, -UH, -COOH; or other polyaze-tidines which may be crosslinked by H20 removal, heat, or by changing to a more basic pH. The lo following is an idealiæed structure of a representative polyazetidine such as Polycup ~172 (Hercules, Inc.) which is useful for present purposes:
o O Cl-----C - R--CNEICH2 CH2~ N--CH2 - CH2NH--CH~ CH2 OH
where R is typically tCH2t4 (2~ carboxymethyl cellulose which can be crosslinked in aqueous solution by reaction with polyvalent ions such as Fe+~, Al++~, Ca++, Mg+~.
The following is an idealized structure of a carboxymethyl cellulose polymer such as CMC7HF
(Hercules, Inc.):
___ O
CH20CH2CONa OH
H / CH - O O CH CH H
\ / OH \ / \ / OH \ /
C / H H \ / C
/ CH- CH CH O
OH CH20CH2CONa (3) polyurethane hydrogel prepolymers which can be crosslinked in aqueous solution by reaction with -NH, -SH, -OH, -COOH or H20. The following is an idealized structure of a repre-sentative polyurethane hydrogel prepolymer which is made by taking a water soluble polyether polyol having greater than two hydroxyl groups per molecule and a moleculal weight greater than 3,000 lo and capping each hydroxyl group with a low molecular weight polyisocyanate:
R ~ (ocH2~H23 ~(~cEl2--cH~l ~cE~2cH ~ OCHNR2N(~;
where Rl typically ls a low molecular weight polyol fragment (from glyceroll pentaerithritol, sorbitolj etc.);
S~3 a and b are integers, eOg. a is typically>
10 and the ratio of a/b is generally from 2 to 5, the sequence of aerangement of the a and b segments being random or in blocks;
n is also an integer typically > 2; and R2 is typically a diisocyanate such as a toluene diisocyanate, methylene di(phenylisocyanate)) m-xylylene diisocyanate, isophorone-diisocyanate, hexamethylene diisocyanate, hexahydro-m-xylylene diisocyanate, dodecahydro-methylene di(phenylisocyanate) and the like; and (4) polymethylene isocyanates which can be cured by mixing with water such as the following:
[~L CE2 ~L C 2 [~
where n=0 to 4 Such polyisocyanates are commercially available and are commonly called "crude polymeric methylene dilphenylisocyanates)~. Other polyisocyanates which are convertible into crosslinked insoluble polymers with water are also thought to be useful, e.g. toluene dlisocyanates, methylene di(phenyl-isocyanate), m-xylylene diisocyanate, isophorone-diisocyanate, hexamethylene diisocyanate, hexahydro-m-xylylene diisocyanate, dodecahydro-methylene di(phenylisocyanate) and the like z~5~3 The isocyanates are usefully employed by coating an appropriate particulate substrate and then mixing with an aqueous slurry of E. coli cells~
Any of the above-noted polymer systems may be used to immobilize L-aspartase active E. coli cells to provide cell/polymer compositions having a variety of different forms and shapes~
For example, the immobilized cell/polymer composi-lo tion may be prepared in the form of membranes, filaments, fibers, tubes, beads or the like. A
particuldrly important embodiment oE the invention involves peoviding the immobilized cell/polymer composition as a coating on an appropriately shaped support which advantageously, although not necessarily, is in the form of solid or reticu-lated beads or particles composed of an inert solid organic or inorganic, porous or non~porous materialO As noted earlier, the composition of the invention can be cured and dried without substantively afecting the L-aspartase activity of the cells and, as a consequence, the composi tion can be made up in the form of dried coated beads or other particulate material and stored until needed fcr use. Typically, useful supports or carriers for the immobilized cell/crosslinked polymer compositions of the invention include the following in bead or particle form:
Molecular sieves Ion exchange resins Alumina Silica and silica ~el Foraminifera skeletons Polymer la~exes Metals 9S~3 It is a useful property of the polymer systems used in the present invention that they can be contacted or mixed in water wlth E. coli cells containing L~aspartase and then crosslinked (or cured) to form insoluble and relatively fixed polymer matrices which hold or otherwise immobilize the E. coli cells. It is particularly useful that the crosslinking conditions which need to be used are mild enough so that the L-aspartase lo activity oE the E. coli mutant cells is main~ained.
As indicated, a particularly desirable CompGsition according to the invention is obtained by applying the E. coli cells containing L-aspartase activity and the immobilizing polymer asa coating on hard inorganic or organic polymer beads~ Using these relatively noncompressible compositions an L-aspartase active catalyst bed capable of high throughput in a fixed bed or 20 fluidized bed is possible. As noted earlier, the L-aspartase active immobilized cell compositions previously described in the literature involving the use of relatively soft, compressible k-carrageenan gum or polyurathane foam are 2s compressible and therefore tend to plug under high flow rates, and/or tall column (bed) heights.
Various methods may be used to immobilize the E. coli ATCC 11303 cells~ while retaining their L-aspartase activity, by combination with cured or crosslinked forms of prepolymers according to the invention. The preferred method used in any specific situation will depend, at least to some extent, on the prepolymer involved.
Thus:
9Sj~3 a) In the case of the preferred poly-azetidine prepolymers, E. coli ATCC
11303 cells are advantageously mixed with an aqueous solution of the prepoly-mer so as to obtain a homogeneous mixture after which the polyazetidine may be cured or crosslinked to give an insoluble L-aspartase active composition by any of the following means:
lo Removal of part or nearly all of the water at temperatures below 60C
~usually between 40 and 0C) and pressures of 760 to 1~0 Torr;
raising the pH above 7.5; exposing the E. coli/polyazetidine mixture to polyamines (using, for example, 100 parts of the composition for each 1-2 parts/wt. of polyethylene imine, polyamino ion exchange re~ins, diethylene triamine, ethylene diamine, etc.) b) For carboxymethyl cellulose (CMC) gums, homogeneous mixtures with E. coli ATCC
11303 may be crosslinked (cured) to insoluble, L-aspartase active coatings, membranes, ibers, beads, etc.~ by contacting an aqueous mixture of, Eor example, 1.0 part/wt. of cell past/CMC
mixture with an aqueous solution of 0.001 to 1.0 parts/wtO of polyvalent cation salt or, ideally 0.01 to 0 4 parts/wt. of polyvalent cation salt per 1.0 part/wt. of the cell paste/CMC
mixture.
The resultant water insoluble immobilized cells/polymer network composition can be thus cured in the form of membranes, fibers or beads, but most efficiently as a coating around high sur~ace area particles. As an illustration lO to 1000 parts/wt. of the high surface area particles may be coated with 1.0 part/wt. of cell lo paste/CMC mixture and ideally 50 to 500 par~sJwt. of the particles per 1.0 partJw~. of cell paste/CMC mixture are used. Removal of 75% or more of the water contained in the cured coatings may be desirable to improve strength and bonding of the cell/polymer coating.
c) When polyurethane hydrogels are used~
homogeneous aqueous dispersions of E. coli ATCC 11303 (typically l.0 9 of cells per l.0 to 1000 ml H2O broadly and 10 to 190 ml H2O preferably) are mixed with the hydrogel prepolymer and the prepolymer is cured into a water-insoluble L-aspartase active composition by allowing the polyisocyanate to react with water or in a more rapid manner by either removing 50 to 100% of the available water 1000 to 0.1 Torr and below 50C (preferably 760 to 5.0 Torr and 0 to 30C) or by exposing the undried compositions to aqueous solu-tions o~ molecules having more than two primary or secondary amine groups.
Typically these polyamine molecules can range from hydrazine or ethylene diamine to polyethyleneimine. Generally for each l.0 g of hydrogel prepolymer used, from 0.01 to lO.0 g of the polyamine in from 1.0 to 500 ml of water could be used. Ideally 0 1 to l.0 9 of the polyamine and 10 to lO0 ml of water are employed per gram of hydrogel prepolymer used.
The above E. ~ hydrogel compositions can be fabricated into membranesp fibers, beads, etc., but a preferred configuration is to use the compositions as coatings onto high surface area lS particles, as discussed before.
Depending on the surface area of the particle employed from 0.1 to 100.0 g of the E. col~/hydrogel composition can be coated onto lO0 g of the particle.
d~ In the case where polyisocyanates are used, these are generally first applied to a surface and the cells of E. coli ATCC 11303 thereafter added thereto.
Usually, from 0.1 to 20.0 g of the polyisocyanate per lO.0 9 of the surface material can be employed, preferably 0.5 to 10.0 g of polyisocyanate per lO.0 9 of surface material. The polyisocyan-ates, if liquid, can ~e added undiluted to the substrate with good mixing (agitator or tumbling~. However, the preferable method is to use solutions of ~he polyisocyanates in inert, readily evaporated solvents such as toluene, ~ 2q:119S~3 acetone, chloroform, tetrahydrofuran~
dimethoxyethane, ethyl acetate, and the like. These solutions are added to the substrate, prefeeably dry particles of hi~h surface area, with good mixing and the solvents removed at their boiling points at 760 Torr or below while continuing good mixing to insure homogeneously coated, mobile substrate lo particles~ The amounts of solvents used will usually ran~e from 1.0 to 100 parts/wt. per 1.0 parts/wt. of polyisocyanate.
The immobilized cell/polymer compositions 1~ of the invention may be used to make aspartic acid by either a batch or continuous process. However, it is noted that these compositions, particularly when coated onto a bead or other particulate support, are especially effective for the continuous conversion of aqueous solutions of ammonium fumarate to ammonium L-aspartate according to the following equation:
O O
Il 11 --O--C ~ CO--\ / bed of immobilized C 3' CHNE~2 H~
NH4~ ll L-aspartase active /~ ~. coli compositions CH2 H 90 1 o NH4 O O
pH 8.5 pH 8.5 As illustrative of processes which may be used to prepare L-aspartic acid or ammonium L-aspartate according to the invention are the following:
(i) A batch type process wherein the catalyst compositions are stirred in from 0.1 to 5.0 molar (preferably 0.5 to
2.0 molar3 solutions of ammonium fumarate in water at 5.0 to 10.0 pH
lo (preferably 7.5 to 9.5 pH) for periods of 1.0 to 100 hours (preEerably B to 48 hours) at temperatures below 50C
(preferably 20 to 40C). Broadly from 0.05 to 50 g of immobilized cells, preferably from 1.0 to 15 9, are used per 1.0 mole of starting ammonium fumarate. After the conversiont the catalyst compositions may be removed by filtration or the equivalent for reuse in converting fresh batches o fumarate solutions. The produ~t solutions are obtained in a form suitable for conven-tional processing to isolate the L-aspartic acid (acidification, precipitation, filtration, washing, recrystallization, drying).
(ii) A continuous process wherein the catalyst compositions, e.g. coated beads, are placed in columns and the solutions of ammonium umarate (concen-trations, pH's, and temperatures are the same as described above for the batch processes) are passed through the catalyst beds either from above or from 5~3 below (fluidized bPd mode). The rates of passage of these fumarate solutions may range from 0.1 to 1000 space velocities/hour. For example~ 5.0 liters of solution per hour may be passed through 1.0 liter of catalyst bed repre-senting 5.0 space velocities (S.V.) per hour. Preferably the fumarate solution flow rates which yield essentially 100%
lo conversion of the fumarate to the L-aspartate fall in the range of 0.5 to 20.0 S.V./hour. The effluent from these columns of catalyst beds is suitable for conventional processing to isolate L-aspartic acid (as outlined above in the batch processes).
The invention is illustrated by the following Examples:
Example 1.
Preparation of E. coli Containing L-Aspartase Immobilized in CMC Gum.
To 10 9 of a 1% aqueous solution of high molecular weight carboxymethyl cellulose (Hercules 7 HF CMC gum, approximate molecular weight of 2s 70D,000 and 0.65 to 0.85 carboxymethyl yroups per anhydroglucose units~ was added 0.5 g of a paste of the E. coli cells, ATCC 11303. A thoroughly homogeneous dispersion was made by hand mixing with a wooden stick . A 2.0 9 aliquot of this dispersion was placed onto 10 9 of hydrated 5 molecular sieve beads (8-12 mesh, previously hydrated by mixing 100 g of the beads with.200 9 of water following by decanting the excess water and allowing the beads to dry at 25 C in air).
After a thorough mixing by hand for five to ten minutes, uniformly coated, damp, but free flowing, beads were obtained. These coated beads were then poured in~o 40 ml of a 0.1% solution of FeSO~.nH20 in water. After 4 hours of periodic gentle stirring at 25C the liquid was decanted (considerable gel microparticles were noted in the liquid~ and the beads washed four times with 0.1 M
lo sodium phosphate buffer solution. A total of 22.2 of wet coated beads were obtained. The beads were stored at 5C in damp air. For L-aspartase activity (see Table I at 5.0 ml aliquot of these wet beads was taken. This aliquot weighed 6.3 and was estimated to contain d.027 9 of the E. coli cells (0 5 g X 2 . 0 9 X 26 2 g a 0 . 027 g) Example 2 Preparation of E. coli Cells Containing L-Aspartase Immobiliæed In a Polyurethane Hydrogel.
a) A polyurethane hydrogel prepolymer was prepared by capping a 7,000 molecular weight water soluble polyether (high level of polyoxy-ethylene segments) triol, Plueacol ~V 7 from BASF
Wyandotte, with three moles of dodecahydro-methylene di(phenylisocyanate), Desmodur ~ W from Mobay Chemical Company. A solution of 1.0 9 of this hydrogel prepolymer in 9.0 g of water was thoroughly mixed with 1.0 9 of E. coli ATCC 11303 cell paste at 25C. To 20 g oE hydrated 5A, 8-12 mesh, molecular sieve beads was added a 2.0 g aliquot of the E. coli-hydrogel prepolymer aqueous - `~2~5C~3 dispersion. The resultant mixture ~as stirred ovet a period of 10 minutes by hand with a wooden stick at 25C to give 22.0 9 of wet but free flowing coated beads~ Preliminary experiments showed that 10% solutions of this hydrogel prepolymer in water formed water soluble gels upon drying at 25C in air for 8 to 24 hours.
Thus, the wet coated beads were allowed to dry in the open air at 25C for 16 hours to give 20.1 9 lo of free flowing cured coated beads. A 4.3 9 aliquot of these coated beads had a volume of 5O0 ml and an estimated E~ coli cell content of 0.039 9 (1 0 ~ X 2.0 9 X 4 3 q = 0.039 g) -This 5.0 ml sample was used for L-aspartase activity measurements (see Table 1).
b) A 1.0 9 aliquot of the aqueous E~ coll-hydrogel prepolymer dispersion described above was also coated onto 10.0 g of 5A, 8-12 mesh, hydrated molecular sieve beads at 25C~
After stirring for S to 10 minutes the resultant free flowing wet beads were poured with good mixing into 40 ml of a 0.1% solution o poly-ethylene imine (Aldrich Chemical Co. ~181g7-8) in water. Preliminary experiments showed that a fresh, less than 15 minute old, solution of 1.0 9 of hydrogel prepolymer in 10 ml H2O formed a water insoluble gel immediately upon contacting a 0.1%
aqueous solution oE the polyethylene imine. The slurry of coated beads in the polyethylene imine solution were gently stirred for 30 minutes at 25C
and then washed four times with 40 ml portions of 0.1 M sodium phosphate buffer solution (pH 7.5) to give 1105 g damp, free Elowing beads. A 5.0 ml ali~uot of those beads weighed 5.1 g and was g503 estimated to contain 0~040 g of the E. coli cells 02 9 X 1.0 g X 15 l q = 0.041 ~ -This 5~0 ml aliquot was used for L-aspartase measurements (see Table I).
c) A polyurethane hydrogel membrane having E. coli ATCC 11303 containing L-aspartase was prepared in the Eollowing manner:
A sLurry of 10.0 9 of E. coli ATCC 11303 cell paste ~n 10.0 9 of phosphate buffered saline lo water (pH 7.4) was mixed for 30 minutes at 25C to insure a homo~eneous dispersion. A solution of 5.0 9 of the above-mentioned hydrogel prepolymer (7,000 molecular weight triol capped with three moles of Desmodur ~W) in 10.0 g of deionized 1S water was formed by vigorous stirring Eor one minute at 25C. This solution was mixed with the homogeneous cell paste slurry. Within 20 seconds of vigorous stirring the resultant mixture became quite thick and was immediately poured onto a Teflon ~sheet. Within a few seconds at 25C the mixture became an elastomeric gel. After 45 minutes in air at 25C the resultant elastomeric membrane composite (1/16" to 1/4" thick by 4"
diameter) was stripped off the Teflon ~surface.
2s The membrane was placed in 500 ml of 105 M ammonium fumarate solution and shaken for 30 minutes. Considerable turbidity developed in the aqueous solution indicating cells and/or gel were washed out of the membrane. This washing process was repeated with one liter of the 1.5 M ammonium fumarate solution at 25C for 64 hour~. The solu-tion was only very slightly hazy and its optical density at 280 nm (1/1000 dilution3 was 0.005 "` ~Z~ 3 2~
compared to 0.43 (1/1000 dilution) at the start.
This indicates that essentially all the ammonium fumarate had been consumed.
The membrane was removed intact and s placed in 750 ml fresh 1.5 M ammonium fumarate.
After 16 hours of gentle stirring at 25C the clear product liquid over the membrane had an optical density at 280 nm (1/1000 dilution) of 0.005. This process was repeated with 750 ml more lo of the ammonium Eumarate for 20 hours at 25C.
The combined 15,000 ml oE product solution was clear and had an optical density at 280 nm of 0.~05.
A 1000 ml aliquot of the combined product solution was acidified to a pH of 2.3 using 145 9 of concentrated hydrochloric acid. The resultant slurry of white crystals was kept at 5C for 16 hours and then filtered. The damp filter cake was then stirred in 1000 ml oE deion;zed water at 80 to 90C Eor 2 hours and then allowed to cool to 30C.
The white crystalline solids were collected by filtration and washed with 250 ml deionized water.
The solids were dried at 25C to 100C and 5 to 20 Torr for 16 hours to a constant weight of 178.0 g which represents an 89.4% yield of L-aspartic acid (78.0 X 133 = 0.894) The optical rotation of a solution of 2.04 9 of the white solid in 100 ml of 6.0 N HCl was measured on a Perkin-Elmer polari-meter at 25C and found to be +0.497. On the same instrument a solution of 2.04 9 of an authentic sample of L-aspartic acid (Sigma Chemical Co~ ~A-9256) had a rotation of ~0.495.
5~3 Preparation of E. coli Containing L-Aspartase Immobilized On Surfaces Pretreated with Polyisocyanates.
a) A 10.0 9 sample of activated anhydrous 5A, 8-12 mesh molecular sieve beads was mixed in a rotary evaporation apparatus with a solution of 2.0 9 oE a crude polymerîc aromatic polyisocyanate (Upjohn Chemical Company PAPI ~320, 7.1 meg NCO/g) in 50.0 9 of dry toluene. The toluene was removed at 85C and 10 to 20 Torr over a period of an hour of continued tumbling~ ~he free flowing impregnated beads were cooled to 25C
and a slurry of 1.0 g E. coli ATCC 11303 paste in 1.0 9 of 0.1 M sodium phosphate bufEer (pH 7.5) was added. The resultant bead mixture was tumbled slowly Eor four hours at 25~C and one atmosphere to give 12.9 9 dry free flowing beads. A 5.0 ml aliquot of the dry beads weighed 4.9 g. This volume did not change after soaking in 1.5 M
ammonium fumarate solution. An estimated 0~38 g of ~. coli cells were on this 5.0 ml aliquot (4 9 X 1.0 9 = 0.38 ~ which was used to measure 5 L-aspartase activity (see Table I).
b) In a similar manner 10.0 g of anhydrous, weakly basic, =N-H form, ion exchange beads (Rohm & Haas, Amberlite ~ IRA 45, 0.42 mm diameter dried at 85 to 90C and 5 to 20 Torr for several hours~ were treated with a solution of 2.0 g P~PI ~ 20 in 50 ml dry toluene. Removal of the toluene at 85 to 90C and 5 to 20 ~orr over a period of an hour gave a somewhat sticky but still mobile mass of coated beads. To this tumbled mixture was added a slurry of 1.0 9 E. coli ATCC
lo (preferably 7.5 to 9.5 pH) for periods of 1.0 to 100 hours (preEerably B to 48 hours) at temperatures below 50C
(preferably 20 to 40C). Broadly from 0.05 to 50 g of immobilized cells, preferably from 1.0 to 15 9, are used per 1.0 mole of starting ammonium fumarate. After the conversiont the catalyst compositions may be removed by filtration or the equivalent for reuse in converting fresh batches o fumarate solutions. The produ~t solutions are obtained in a form suitable for conven-tional processing to isolate the L-aspartic acid (acidification, precipitation, filtration, washing, recrystallization, drying).
(ii) A continuous process wherein the catalyst compositions, e.g. coated beads, are placed in columns and the solutions of ammonium umarate (concen-trations, pH's, and temperatures are the same as described above for the batch processes) are passed through the catalyst beds either from above or from 5~3 below (fluidized bPd mode). The rates of passage of these fumarate solutions may range from 0.1 to 1000 space velocities/hour. For example~ 5.0 liters of solution per hour may be passed through 1.0 liter of catalyst bed repre-senting 5.0 space velocities (S.V.) per hour. Preferably the fumarate solution flow rates which yield essentially 100%
lo conversion of the fumarate to the L-aspartate fall in the range of 0.5 to 20.0 S.V./hour. The effluent from these columns of catalyst beds is suitable for conventional processing to isolate L-aspartic acid (as outlined above in the batch processes).
The invention is illustrated by the following Examples:
Example 1.
Preparation of E. coli Containing L-Aspartase Immobilized in CMC Gum.
To 10 9 of a 1% aqueous solution of high molecular weight carboxymethyl cellulose (Hercules 7 HF CMC gum, approximate molecular weight of 2s 70D,000 and 0.65 to 0.85 carboxymethyl yroups per anhydroglucose units~ was added 0.5 g of a paste of the E. coli cells, ATCC 11303. A thoroughly homogeneous dispersion was made by hand mixing with a wooden stick . A 2.0 9 aliquot of this dispersion was placed onto 10 9 of hydrated 5 molecular sieve beads (8-12 mesh, previously hydrated by mixing 100 g of the beads with.200 9 of water following by decanting the excess water and allowing the beads to dry at 25 C in air).
After a thorough mixing by hand for five to ten minutes, uniformly coated, damp, but free flowing, beads were obtained. These coated beads were then poured in~o 40 ml of a 0.1% solution of FeSO~.nH20 in water. After 4 hours of periodic gentle stirring at 25C the liquid was decanted (considerable gel microparticles were noted in the liquid~ and the beads washed four times with 0.1 M
lo sodium phosphate buffer solution. A total of 22.2 of wet coated beads were obtained. The beads were stored at 5C in damp air. For L-aspartase activity (see Table I at 5.0 ml aliquot of these wet beads was taken. This aliquot weighed 6.3 and was estimated to contain d.027 9 of the E. coli cells (0 5 g X 2 . 0 9 X 26 2 g a 0 . 027 g) Example 2 Preparation of E. coli Cells Containing L-Aspartase Immobiliæed In a Polyurethane Hydrogel.
a) A polyurethane hydrogel prepolymer was prepared by capping a 7,000 molecular weight water soluble polyether (high level of polyoxy-ethylene segments) triol, Plueacol ~V 7 from BASF
Wyandotte, with three moles of dodecahydro-methylene di(phenylisocyanate), Desmodur ~ W from Mobay Chemical Company. A solution of 1.0 9 of this hydrogel prepolymer in 9.0 g of water was thoroughly mixed with 1.0 9 of E. coli ATCC 11303 cell paste at 25C. To 20 g oE hydrated 5A, 8-12 mesh, molecular sieve beads was added a 2.0 g aliquot of the E. coli-hydrogel prepolymer aqueous - `~2~5C~3 dispersion. The resultant mixture ~as stirred ovet a period of 10 minutes by hand with a wooden stick at 25C to give 22.0 9 of wet but free flowing coated beads~ Preliminary experiments showed that 10% solutions of this hydrogel prepolymer in water formed water soluble gels upon drying at 25C in air for 8 to 24 hours.
Thus, the wet coated beads were allowed to dry in the open air at 25C for 16 hours to give 20.1 9 lo of free flowing cured coated beads. A 4.3 9 aliquot of these coated beads had a volume of 5O0 ml and an estimated E~ coli cell content of 0.039 9 (1 0 ~ X 2.0 9 X 4 3 q = 0.039 g) -This 5.0 ml sample was used for L-aspartase activity measurements (see Table 1).
b) A 1.0 9 aliquot of the aqueous E~ coll-hydrogel prepolymer dispersion described above was also coated onto 10.0 g of 5A, 8-12 mesh, hydrated molecular sieve beads at 25C~
After stirring for S to 10 minutes the resultant free flowing wet beads were poured with good mixing into 40 ml of a 0.1% solution o poly-ethylene imine (Aldrich Chemical Co. ~181g7-8) in water. Preliminary experiments showed that a fresh, less than 15 minute old, solution of 1.0 9 of hydrogel prepolymer in 10 ml H2O formed a water insoluble gel immediately upon contacting a 0.1%
aqueous solution oE the polyethylene imine. The slurry of coated beads in the polyethylene imine solution were gently stirred for 30 minutes at 25C
and then washed four times with 40 ml portions of 0.1 M sodium phosphate buffer solution (pH 7.5) to give 1105 g damp, free Elowing beads. A 5.0 ml ali~uot of those beads weighed 5.1 g and was g503 estimated to contain 0~040 g of the E. coli cells 02 9 X 1.0 g X 15 l q = 0.041 ~ -This 5~0 ml aliquot was used for L-aspartase measurements (see Table I).
c) A polyurethane hydrogel membrane having E. coli ATCC 11303 containing L-aspartase was prepared in the Eollowing manner:
A sLurry of 10.0 9 of E. coli ATCC 11303 cell paste ~n 10.0 9 of phosphate buffered saline lo water (pH 7.4) was mixed for 30 minutes at 25C to insure a homo~eneous dispersion. A solution of 5.0 9 of the above-mentioned hydrogel prepolymer (7,000 molecular weight triol capped with three moles of Desmodur ~W) in 10.0 g of deionized 1S water was formed by vigorous stirring Eor one minute at 25C. This solution was mixed with the homogeneous cell paste slurry. Within 20 seconds of vigorous stirring the resultant mixture became quite thick and was immediately poured onto a Teflon ~sheet. Within a few seconds at 25C the mixture became an elastomeric gel. After 45 minutes in air at 25C the resultant elastomeric membrane composite (1/16" to 1/4" thick by 4"
diameter) was stripped off the Teflon ~surface.
2s The membrane was placed in 500 ml of 105 M ammonium fumarate solution and shaken for 30 minutes. Considerable turbidity developed in the aqueous solution indicating cells and/or gel were washed out of the membrane. This washing process was repeated with one liter of the 1.5 M ammonium fumarate solution at 25C for 64 hour~. The solu-tion was only very slightly hazy and its optical density at 280 nm (1/1000 dilution3 was 0.005 "` ~Z~ 3 2~
compared to 0.43 (1/1000 dilution) at the start.
This indicates that essentially all the ammonium fumarate had been consumed.
The membrane was removed intact and s placed in 750 ml fresh 1.5 M ammonium fumarate.
After 16 hours of gentle stirring at 25C the clear product liquid over the membrane had an optical density at 280 nm (1/1000 dilution) of 0.005. This process was repeated with 750 ml more lo of the ammonium Eumarate for 20 hours at 25C.
The combined 15,000 ml oE product solution was clear and had an optical density at 280 nm of 0.~05.
A 1000 ml aliquot of the combined product solution was acidified to a pH of 2.3 using 145 9 of concentrated hydrochloric acid. The resultant slurry of white crystals was kept at 5C for 16 hours and then filtered. The damp filter cake was then stirred in 1000 ml oE deion;zed water at 80 to 90C Eor 2 hours and then allowed to cool to 30C.
The white crystalline solids were collected by filtration and washed with 250 ml deionized water.
The solids were dried at 25C to 100C and 5 to 20 Torr for 16 hours to a constant weight of 178.0 g which represents an 89.4% yield of L-aspartic acid (78.0 X 133 = 0.894) The optical rotation of a solution of 2.04 9 of the white solid in 100 ml of 6.0 N HCl was measured on a Perkin-Elmer polari-meter at 25C and found to be +0.497. On the same instrument a solution of 2.04 9 of an authentic sample of L-aspartic acid (Sigma Chemical Co~ ~A-9256) had a rotation of ~0.495.
5~3 Preparation of E. coli Containing L-Aspartase Immobilized On Surfaces Pretreated with Polyisocyanates.
a) A 10.0 9 sample of activated anhydrous 5A, 8-12 mesh molecular sieve beads was mixed in a rotary evaporation apparatus with a solution of 2.0 9 oE a crude polymerîc aromatic polyisocyanate (Upjohn Chemical Company PAPI ~320, 7.1 meg NCO/g) in 50.0 9 of dry toluene. The toluene was removed at 85C and 10 to 20 Torr over a period of an hour of continued tumbling~ ~he free flowing impregnated beads were cooled to 25C
and a slurry of 1.0 g E. coli ATCC 11303 paste in 1.0 9 of 0.1 M sodium phosphate bufEer (pH 7.5) was added. The resultant bead mixture was tumbled slowly Eor four hours at 25~C and one atmosphere to give 12.9 9 dry free flowing beads. A 5.0 ml aliquot of the dry beads weighed 4.9 g. This volume did not change after soaking in 1.5 M
ammonium fumarate solution. An estimated 0~38 g of ~. coli cells were on this 5.0 ml aliquot (4 9 X 1.0 9 = 0.38 ~ which was used to measure 5 L-aspartase activity (see Table I).
b) In a similar manner 10.0 g of anhydrous, weakly basic, =N-H form, ion exchange beads (Rohm & Haas, Amberlite ~ IRA 45, 0.42 mm diameter dried at 85 to 90C and 5 to 20 Torr for several hours~ were treated with a solution of 2.0 g P~PI ~ 20 in 50 ml dry toluene. Removal of the toluene at 85 to 90C and 5 to 20 ~orr over a period of an hour gave a somewhat sticky but still mobile mass of coated beads. To this tumbled mixture was added a slurry of 1.0 9 E. coli ATCC
3;~ 3 11303 paste in 1.0 ml of 0.1 M sodium phosphate (pH 7.5) buffer solution. To aid in achieving a homogeneous coating of this rather gummy mass, sixty 0025" diameter Teflon ~balls were added.
s The resultant mixture was tumbled at 25C and 5 to 20 Torr for two hours to yive, after removal of the Teflon ~ balls, 15.6 9 of dry free flowing coated beads. A 5.0 ml aliquot of these coated beads weighed 2.B g and did not change volume lo after soaking in 1.5 M ammonium fumarate solution. This 5.0 ml aliquot was used for L-aspartase measurements (see Table I) and was estimated to contain 0.18 9 of E. Coli (15 0 q X 2.8 g = ~.18 ~ -Example 4 Preparation of E coli Containing L-Aspartase Immobilized With Polyazetidine Gels.
a) A mixture of 2~0 g of an aqueous polyazetidine solution (Hercules Polycup ~ 172, as received 12~ solids in H20) and 2.0 g of E. coli ATCC 11303 paste was stirred rapidly with a magnetic stirring bar for five minutes at 25C, to insure a homogeneous dispersion. This mixture was then poured out onto a polystyrene surface to dry at 16 hours at 25C. The resultant 2" X 2" X
1/16" thick flexible membrane was stripped from the surface and found to weight 0.7 9. ~his film was used in L-aspartase measurements (see Table I3 and was estimated to contain 2.0 g of ~. coli 0 cells (hydrated state).
b) A homogeneous mixture of 2.0 g Polycup ~ 172 and 2.0 9 of E. coli ATCC 11303 was disteib~ted onto 30 g of hydrated 5A, 8-12 mesh ~2a~95(?3 molecular sieve beads by means of tumbling at 25C
and 5 to 20 Torr for 45 minutes. After 16 more hours drying at 25~C in open air the resultant coated beads weighed 30.4 9. A 5.0 ml aliquot of these coated beads weighed 3.9 9. This sample was usPd for L-aspartase ~ctivity (see Table I) and was estimated to have 0.26 g of E. coli cells (3 9 X 2.0 g = 0.2~ g) -lo c) A homogeneous dispersion of 2.0 9 E. coli ATCC 11303 in 2.0 g of Polycup ~ 172 at 25C was dispersed onto 30.0 9 of Amberlite R IRA
45 ion exchange beads (as received, 42% H20, =N-H
form~ by hand mixing with a wooden stisk for 30 minutes at 25C. A thin layer of the somewhat sticky mixture was allowed to stand in air at 25C
for 16 hours. The resultant free flowing beads weighed 25.8 9. ~ 3.0 9 sample of these dry coated beads had a volume of 5.0 ml. Upon soaking in 1.5 M ammoniwm fumarate solution the volume expanded to 5.5 ml. A 5.0 ml aliquot of these wet coated beads was taken for L-aspartase measurements ~see Table I) and the estimated E. coli content was 0.21 9 (3 0 9 X 200 9 X 55-~ = 0.21 9) -d) A homogeneous dispersion of 2.0 g E. coli ATCC 11303 in 4.0 g deionized water and 2.0 g Polycup @9 172 was added over a period of 3.5 hours in three equal portions to 10.0 g of Amberlite ~IRA 45 (as received, 42~ H20, free =N-H
form) containing sixty 0.25 diameter ~eflon ~
balls. This mixture was tumbled at 25C at 5 to 20 Torr. After a total of five hours of drying at reduced pressure the resultant dry~ coated beads (Teflon ~ balls removed) weighed 7.8 g, A 5.0 ~20956~3 aliquot of these dry, free ~lowing coated beads weighed 2.9 g. After saturating with 1.5 M ammonium fumarate solution the volume swelled to 6.25 ml7 A
5.0 ml aliquot of these wet, coated beads was taken for L-aspartase measurements (see Table I)o The estimated E. coli content o this aliquot was ~( C_~ X 2.9 9 X 56 5 = 0-59 9) -e) A homogeneous dispersion of 3.0 9 lo E. coli ATCC 11303 in 6.0 ml deionized water plus 3.0 g of Polycup ~ 172 was spread onto 10.0 g of Amberlite ~IRA 45 (as received, 42% H20; free =N-~
form) in the same manner of drying at reduced pressure described above. A total of 10.79 dry, coated, free flowng beads were obtained. A 5.0 ml aliquot oE dry coated beads weighed 3.0 g. After soaking in 1.5 M ammonium fumarate solution this volume swelled to 5~75 ml~ A 5.0 ml aliquot of the wet, coated beads was taken for L-aspartAse measurement (see Table 1)~ The estima~ed E. coli content of this 5.0 ml aliquot was 0.73 g 3O~ g X 5 -5- 0-73 9) -f) A homogeneous dlspersion of 4.0 9 E. coli ATCC 11303 in 4.0 9 of Polycup ~ 172 and 8.0 g deionized water was added in approximatelysix equal portions over a 5.5 hour pericd at 25C
and 5 to 20 Torr to 10.0 9 of Amberlite ~ IR~ 938 beads (Rohm and Haas, 73% H20, 0.38 mm diameter, tert. amine chloride salt form) containing eight 0.5 inch diameter Teflon ~ balls. A~ter 6 hours total of tumbling at 25C and 5 to 20 Torr the resultant dry, coated free flowing beads weighed 10.3 g and had a volume of 16~6 ml, Soaking ~hese beads in excess 1.5 M ammonium fumarate solution ~L2'~ 3 (pH B.5) for 16 hours at 25C gave 17.5 ml wet beads. A 5.0 ml aliquot of the wet beads, estimated to contain 1.14 9 E. coli ATCC 11303 (7 gmlX S.0 ml = 1.14 9) ~ was taken for L-aspartase activity measurements (see Table I).
Example 5 Coating Molecular Sieve Bead~ and Ion Exchange Resin Beads with E. coli Cells Containing L-Aspartase Using No Polymer Binders.
lo To demonstrate the utility of the above described polymer systems used to immobilize the Eo coli cells containing L-aspartase, the _ _ following experiments were conducted to coat materials with the cells containing L-aspartase alone, i.e., with no polymer bindees.
a) A homogeneous slurry of 1.0 g E. coli cells, ATCC 11303, containing L-aspartase in 5.0 ml of Na phosphate buffer (0.1 M, pH 7.5) was added in four equal portions over a period of 3.5 hours to 10.0 9 of hydrated 5A molecular sieve beads (8-12 mes~) also containing ten O.S inch diameter TeElon @~ balls. This mixture was tumbled at 25~C and 5 to 10 Torr. After a total of about
s The resultant mixture was tumbled at 25C and 5 to 20 Torr for two hours to yive, after removal of the Teflon ~ balls, 15.6 9 of dry free flowing coated beads. A 5.0 ml aliquot of these coated beads weighed 2.B g and did not change volume lo after soaking in 1.5 M ammonium fumarate solution. This 5.0 ml aliquot was used for L-aspartase measurements (see Table I) and was estimated to contain 0.18 9 of E. Coli (15 0 q X 2.8 g = ~.18 ~ -Example 4 Preparation of E coli Containing L-Aspartase Immobilized With Polyazetidine Gels.
a) A mixture of 2~0 g of an aqueous polyazetidine solution (Hercules Polycup ~ 172, as received 12~ solids in H20) and 2.0 g of E. coli ATCC 11303 paste was stirred rapidly with a magnetic stirring bar for five minutes at 25C, to insure a homogeneous dispersion. This mixture was then poured out onto a polystyrene surface to dry at 16 hours at 25C. The resultant 2" X 2" X
1/16" thick flexible membrane was stripped from the surface and found to weight 0.7 9. ~his film was used in L-aspartase measurements (see Table I3 and was estimated to contain 2.0 g of ~. coli 0 cells (hydrated state).
b) A homogeneous mixture of 2.0 g Polycup ~ 172 and 2.0 9 of E. coli ATCC 11303 was disteib~ted onto 30 g of hydrated 5A, 8-12 mesh ~2a~95(?3 molecular sieve beads by means of tumbling at 25C
and 5 to 20 Torr for 45 minutes. After 16 more hours drying at 25~C in open air the resultant coated beads weighed 30.4 9. A 5.0 ml aliquot of these coated beads weighed 3.9 9. This sample was usPd for L-aspartase ~ctivity (see Table I) and was estimated to have 0.26 g of E. coli cells (3 9 X 2.0 g = 0.2~ g) -lo c) A homogeneous dispersion of 2.0 9 E. coli ATCC 11303 in 2.0 g of Polycup ~ 172 at 25C was dispersed onto 30.0 9 of Amberlite R IRA
45 ion exchange beads (as received, 42% H20, =N-H
form~ by hand mixing with a wooden stisk for 30 minutes at 25C. A thin layer of the somewhat sticky mixture was allowed to stand in air at 25C
for 16 hours. The resultant free flowing beads weighed 25.8 9. ~ 3.0 9 sample of these dry coated beads had a volume of 5.0 ml. Upon soaking in 1.5 M ammoniwm fumarate solution the volume expanded to 5.5 ml. A 5.0 ml aliquot of these wet coated beads was taken for L-aspartase measurements ~see Table I) and the estimated E. coli content was 0.21 9 (3 0 9 X 200 9 X 55-~ = 0.21 9) -d) A homogeneous dispersion of 2.0 g E. coli ATCC 11303 in 4.0 g deionized water and 2.0 g Polycup @9 172 was added over a period of 3.5 hours in three equal portions to 10.0 g of Amberlite ~IRA 45 (as received, 42~ H20, free =N-H
form) containing sixty 0.25 diameter ~eflon ~
balls. This mixture was tumbled at 25C at 5 to 20 Torr. After a total of five hours of drying at reduced pressure the resultant dry~ coated beads (Teflon ~ balls removed) weighed 7.8 g, A 5.0 ~20956~3 aliquot of these dry, free ~lowing coated beads weighed 2.9 g. After saturating with 1.5 M ammonium fumarate solution the volume swelled to 6.25 ml7 A
5.0 ml aliquot of these wet, coated beads was taken for L-aspartase measurements (see Table I)o The estimated E. coli content o this aliquot was ~( C_~ X 2.9 9 X 56 5 = 0-59 9) -e) A homogeneous dispersion of 3.0 9 lo E. coli ATCC 11303 in 6.0 ml deionized water plus 3.0 g of Polycup ~ 172 was spread onto 10.0 g of Amberlite ~IRA 45 (as received, 42% H20; free =N-~
form) in the same manner of drying at reduced pressure described above. A total of 10.79 dry, coated, free flowng beads were obtained. A 5.0 ml aliquot oE dry coated beads weighed 3.0 g. After soaking in 1.5 M ammonium fumarate solution this volume swelled to 5~75 ml~ A 5.0 ml aliquot of the wet, coated beads was taken for L-aspartAse measurement (see Table 1)~ The estima~ed E. coli content of this 5.0 ml aliquot was 0.73 g 3O~ g X 5 -5- 0-73 9) -f) A homogeneous dlspersion of 4.0 9 E. coli ATCC 11303 in 4.0 9 of Polycup ~ 172 and 8.0 g deionized water was added in approximatelysix equal portions over a 5.5 hour pericd at 25C
and 5 to 20 Torr to 10.0 9 of Amberlite ~ IR~ 938 beads (Rohm and Haas, 73% H20, 0.38 mm diameter, tert. amine chloride salt form) containing eight 0.5 inch diameter Teflon ~ balls. A~ter 6 hours total of tumbling at 25C and 5 to 20 Torr the resultant dry, coated free flowing beads weighed 10.3 g and had a volume of 16~6 ml, Soaking ~hese beads in excess 1.5 M ammonium fumarate solution ~L2'~ 3 (pH B.5) for 16 hours at 25C gave 17.5 ml wet beads. A 5.0 ml aliquot of the wet beads, estimated to contain 1.14 9 E. coli ATCC 11303 (7 gmlX S.0 ml = 1.14 9) ~ was taken for L-aspartase activity measurements (see Table I).
Example 5 Coating Molecular Sieve Bead~ and Ion Exchange Resin Beads with E. coli Cells Containing L-Aspartase Using No Polymer Binders.
lo To demonstrate the utility of the above described polymer systems used to immobilize the Eo coli cells containing L-aspartase, the _ _ following experiments were conducted to coat materials with the cells containing L-aspartase alone, i.e., with no polymer bindees.
a) A homogeneous slurry of 1.0 g E. coli cells, ATCC 11303, containing L-aspartase in 5.0 ml of Na phosphate buffer (0.1 M, pH 7.5) was added in four equal portions over a period of 3.5 hours to 10.0 9 of hydrated 5A molecular sieve beads (8-12 mes~) also containing ten O.S inch diameter TeElon @~ balls. This mixture was tumbled at 25~C and 5 to 10 Torr. After a total of about
4.0 hours the resultant dry, coated, free flowing beads weighed 9.9 9. A 5.0 ml aliquot of these dry beads weighed 4.2 g. The volume did not change upon soaking in 1.5 M ammonium fumarate solution. The estimated E. coli cell content o this aliquot of coated beads was 0~42 9 (1 902 9 X 4.2 = 0.42 ~ ~ A 5.0 ml aliquot of the coated beads was gently stirred in 50 ml 1.5 M
ammonium fumarate solution at 25C for 24 hours~
~he solution became very turbid indicating con-siderable amounts of material were sloughing off the beads. The beads were isolated by decantation and again gently stirred in 50.0 ml fresh 1.5 M
ammonium fumarate at 25C for L-aspartase activity measurement (see Table I). However, even after one hour the solution became very turbid again.
On several more occasions the beads were isola~ed and gently stirred in feesh l.S M ammonium lo fumarate solution. The beads continued to give ofE considerable amounts of material to make the solutions very turbid. This behavior was not observed in any of the preparations of L-aspartase active cell coatings of beads where the polymer binders were also added to the formula~ion. In all the polymer examples the ammonium fumara~e solutions over the coated beads remained clear.
b) In a similar wayy a slurry of 10.0 9 E. coli ATCC 11303 in 30 9 of aqueous phosphate bufEer solution (O.i M, p~ 7.5)~ but no polymer binders, was coated onto 50.0 g of Amberlite ~IRA
4~ (as received from Rohm and Haas, 42% H20, =N-H
form) over a period of 4.5 hours at 25C and 5 to 20 Torr. The resultant 47.6 g of free Elowing dry, coated beads had a volume of 83.5 ml. Upon soaking these beads in excess 1.5 M aqueous ammonium fumarate the solution became thick and opaque from the massive amount of material detached from the beads. All attempts to Eilter (cotton wadding, fiberglass mat, 43 mesh nylon screen, medium porosity sintered glass3 off the beads from this opaque mother liquid failed because the thick gelatinous particles quickly plugged the filters. This behavior was not observed in any of the examples where the polymer ~2~5~
binders were incorporated into the formulation.
In these cases the aqueous solutions over the beads remained clear at all times, and flowed quickly through filters.
ExamPle 6 L~Aspartase Activity Measurements Shown in Table I are the L-aspartase activities of the immobilized Eo coli ATCC 11303 compositions described in the preceding examples.
In each case the samples were preconditioned by gently shaking in 50 ml of a 1.5 M ammonium fumarate solution in deionized water (pH 8.5) containing 0.002 M MgSO4 for a period of 8 to 16 hours at 25C. The solution was then drained from each sample and 50 ml fresh fumarate solution added p~ior to gentle shaking again for 8 to 16 hours at 25C. At this point all the samples prepared with the polymer binders discussed in the previous examples showed no evidence of material sloughiny off the solid supports; i.e., their supernatant solutions were all clear. 5amples prepared using no polymer binders (Example S) continued to give very turbid supernatant solutions even after several washings. This indicate~ the cell material was not securely bound to the solid supports.
The next step in each example was to take a 5.0 ml aliquot ~or in the case of the membrane, a 2 inch X 2 inch X 0.125 inch section3 of the washed and well drained composite~ and gently shake in a fresh 50.0 ml of the above mentioned ammonium fumarate solutin at 25C. Samples were removed after 60 minutes and diluted 1/1000 with deionized water. The decrease in optical density
ammonium fumarate solution at 25C for 24 hours~
~he solution became very turbid indicating con-siderable amounts of material were sloughing off the beads. The beads were isolated by decantation and again gently stirred in 50.0 ml fresh 1.5 M
ammonium fumarate at 25C for L-aspartase activity measurement (see Table I). However, even after one hour the solution became very turbid again.
On several more occasions the beads were isola~ed and gently stirred in feesh l.S M ammonium lo fumarate solution. The beads continued to give ofE considerable amounts of material to make the solutions very turbid. This behavior was not observed in any of the preparations of L-aspartase active cell coatings of beads where the polymer binders were also added to the formula~ion. In all the polymer examples the ammonium fumara~e solutions over the coated beads remained clear.
b) In a similar wayy a slurry of 10.0 9 E. coli ATCC 11303 in 30 9 of aqueous phosphate bufEer solution (O.i M, p~ 7.5)~ but no polymer binders, was coated onto 50.0 g of Amberlite ~IRA
4~ (as received from Rohm and Haas, 42% H20, =N-H
form) over a period of 4.5 hours at 25C and 5 to 20 Torr. The resultant 47.6 g of free Elowing dry, coated beads had a volume of 83.5 ml. Upon soaking these beads in excess 1.5 M aqueous ammonium fumarate the solution became thick and opaque from the massive amount of material detached from the beads. All attempts to Eilter (cotton wadding, fiberglass mat, 43 mesh nylon screen, medium porosity sintered glass3 off the beads from this opaque mother liquid failed because the thick gelatinous particles quickly plugged the filters. This behavior was not observed in any of the examples where the polymer ~2~5~
binders were incorporated into the formulation.
In these cases the aqueous solutions over the beads remained clear at all times, and flowed quickly through filters.
ExamPle 6 L~Aspartase Activity Measurements Shown in Table I are the L-aspartase activities of the immobilized Eo coli ATCC 11303 compositions described in the preceding examples.
In each case the samples were preconditioned by gently shaking in 50 ml of a 1.5 M ammonium fumarate solution in deionized water (pH 8.5) containing 0.002 M MgSO4 for a period of 8 to 16 hours at 25C. The solution was then drained from each sample and 50 ml fresh fumarate solution added p~ior to gentle shaking again for 8 to 16 hours at 25C. At this point all the samples prepared with the polymer binders discussed in the previous examples showed no evidence of material sloughiny off the solid supports; i.e., their supernatant solutions were all clear. 5amples prepared using no polymer binders (Example S) continued to give very turbid supernatant solutions even after several washings. This indicate~ the cell material was not securely bound to the solid supports.
The next step in each example was to take a 5.0 ml aliquot ~or in the case of the membrane, a 2 inch X 2 inch X 0.125 inch section3 of the washed and well drained composite~ and gently shake in a fresh 50.0 ml of the above mentioned ammonium fumarate solutin at 25C. Samples were removed after 60 minutes and diluted 1/1000 with deionized water. The decrease in optical density
5~;~
at 280 nm from the starting 1.5 M ammonium fumarate solution (observed to be 0.43 at 1/1030 dilution~
indicated the L-aspartase activity. The optical density o a 1 5 M L-aspartic acid ammonium salt ~pH 8.5) at 280 nm is essentially zero O0.005).
The observed conversion of the fumarate to the L-aspartate is shown in Table I as moles of aspartic acid/hr/liter of catalyst bed and as moles oE aspartic acid/hr/kg we~ E. coli. For lo example in experiment ~4d using polyazetidines, cell paste and ion exchange beads, the optical density at 280 nm dropped from 0.43 to 0.32 after 60 minutes at 25C. The ¢alculations are:
Q-43-0-32 X 1.5 mol~s X 50 ml X 1000 ml 0.43 1000 ml 5.0 ml = 3.8 moles ASP/hr/liter catalyst bed 0.43-0~32 X 1.5 moles X 1000 g/kq 0.43 0.59 g wet cells 1000 ml 32t5 mlles ASP/hr~kg Also shown in Table I are values calculated erom data in the literature showing the L-aspartase 20 performance at 37C of a polyurethane foam (HYPOL ~ containing E~ coli ATCC 11303. Here a 0.777 liter bed having 126 9 o cells required 32 minutes to convert 2.0 liters of 1.0 M fumarate 100% to L-aspartate at 37C. These workers also report the average rate of conversion of 1.5 M
fumarate to be 8% lower than 1.0 M fumarate solution. They also observed rates at 37C to be 1.67 times rates at 25C. Thus a fair estimate of their L-aspartic acid production rate for comparison to our data (25Ct 1.5 M fumarate) is:
5~3 _ 2.0 1 - X 1.0 moles/l X 0.92 X
32 hrs X 0.77 1 bed 1.67 = 2.69 moles L-ASP
hr X 1 cat. bed _ 2.0 1 X 1.0 moles/l X 0.92 X
0.126 k~ cell X 32 hrs 1.67 = 16 4 moles L-ASP
hr X kg ~ells Comparing these values to the data obtained repre-sentative of the present invent;on shows the superiority of the present process over the polyurethane foam system.
~IL;2~5~51~3 ASPARTASE ACTIVITY OF BOU~D
Moles of _SP
5 EX~MPLE ~ B INDING MF.THt)D lhr X li ter cat . bed ) CMC + Fe~++ on M.S. beads0.87 2a P.U. Hydrogel via drying on 0.45 M . S O beads 2b POU. Hydrogel via poly EI0 . 27 lo on M.S. bead~
3a PAPI 20 on M.5. beads 2.5 3b PAPI 20 e)n I .E . beads1. 2 4a Polycup 6~) 172 film 4b Polycup (~) 172 on M.S. beads 0.6 4c Polycup ~) 172 on I.E. beads 1.0 4d Polycup ~)172 on I ~E. beads 3.8 4e Polycup g) 172 on I.E. beads 3.9 4f Polycup ~)172 on I.G. beads 12.75 5a E. coli alone (No binder)2.6*
on M.S. beads -~ HYPOL ~ foam ~estimate from 2.69 data of Fusee et al) 2 * Massive amounts of cells sloughed of f the beads, thus much of the activity shown i5 2 s caused by unbound cells .
-5(~3 TABLE 1 (Continued) ASPARTASE ACTIVITY ~F BOUND
E COLI (ATCC 11303) AT 25C
Est. g of E. Coli Moles sf ASPused (per 5.0 ml EXAMPLE # (hr X kg wet E. coli) bed sample) 1 185 ~.027 2a 57.7 0.039 2b 33.8 00040 3a 32.9 0038 3b 33.3 0.18 4~ 65.0 0.30 4b 11.5 0.26 4c 23~8 0.21 4d 32.5 0.59 4~ 2Ç~7 0.73 4f 55.9 lo 14 5a 30.9* 0~42*
~~ 16.~
20 * Ma~sive amounts of cells sloughed off the beads, thus much of the activity shown is caused by unbound cells.
~2~9~3 Example 7 Continuous Produc~ion of L-Aspart.i~ Acid ~as ammonium L~aspartate).
A 5.0 ml aliquot of the wet beads from Example ~4d were placed in a 0.7 cm diameter X 15 cm jacketed glass column. The column temperatu~e was maintained at 37C. A fresh 1.5 M ammonium fumarate solution in deionized water containin~
0.002 m MgSO4 (all at pH B.5) was continuously lo passed up thorugh the bed of beads at a rate of 0.5 ml~min. Samples of the effluent product solution were taken periodically and analyzed for L-aspartate contenk as 1/1000 dilutions in deionized water as described in Example 6. The bulk of the efEluent was collected but not returned to the column. The Eollowing data shows that over a period of 50 days of continuous running the L-aspartase activity of the coated beads remained essentially unchanged.
L-aspartate content Time~ day~L f effluent (Percent 5g .6-) 61~5 ~ 62~5 average
at 280 nm from the starting 1.5 M ammonium fumarate solution (observed to be 0.43 at 1/1030 dilution~
indicated the L-aspartase activity. The optical density o a 1 5 M L-aspartic acid ammonium salt ~pH 8.5) at 280 nm is essentially zero O0.005).
The observed conversion of the fumarate to the L-aspartate is shown in Table I as moles of aspartic acid/hr/liter of catalyst bed and as moles oE aspartic acid/hr/kg we~ E. coli. For lo example in experiment ~4d using polyazetidines, cell paste and ion exchange beads, the optical density at 280 nm dropped from 0.43 to 0.32 after 60 minutes at 25C. The ¢alculations are:
Q-43-0-32 X 1.5 mol~s X 50 ml X 1000 ml 0.43 1000 ml 5.0 ml = 3.8 moles ASP/hr/liter catalyst bed 0.43-0~32 X 1.5 moles X 1000 g/kq 0.43 0.59 g wet cells 1000 ml 32t5 mlles ASP/hr~kg Also shown in Table I are values calculated erom data in the literature showing the L-aspartase 20 performance at 37C of a polyurethane foam (HYPOL ~ containing E~ coli ATCC 11303. Here a 0.777 liter bed having 126 9 o cells required 32 minutes to convert 2.0 liters of 1.0 M fumarate 100% to L-aspartate at 37C. These workers also report the average rate of conversion of 1.5 M
fumarate to be 8% lower than 1.0 M fumarate solution. They also observed rates at 37C to be 1.67 times rates at 25C. Thus a fair estimate of their L-aspartic acid production rate for comparison to our data (25Ct 1.5 M fumarate) is:
5~3 _ 2.0 1 - X 1.0 moles/l X 0.92 X
32 hrs X 0.77 1 bed 1.67 = 2.69 moles L-ASP
hr X 1 cat. bed _ 2.0 1 X 1.0 moles/l X 0.92 X
0.126 k~ cell X 32 hrs 1.67 = 16 4 moles L-ASP
hr X kg ~ells Comparing these values to the data obtained repre-sentative of the present invent;on shows the superiority of the present process over the polyurethane foam system.
~IL;2~5~51~3 ASPARTASE ACTIVITY OF BOU~D
Moles of _SP
5 EX~MPLE ~ B INDING MF.THt)D lhr X li ter cat . bed ) CMC + Fe~++ on M.S. beads0.87 2a P.U. Hydrogel via drying on 0.45 M . S O beads 2b POU. Hydrogel via poly EI0 . 27 lo on M.S. bead~
3a PAPI 20 on M.5. beads 2.5 3b PAPI 20 e)n I .E . beads1. 2 4a Polycup 6~) 172 film 4b Polycup (~) 172 on M.S. beads 0.6 4c Polycup ~) 172 on I.E. beads 1.0 4d Polycup ~)172 on I ~E. beads 3.8 4e Polycup g) 172 on I.E. beads 3.9 4f Polycup ~)172 on I.G. beads 12.75 5a E. coli alone (No binder)2.6*
on M.S. beads -~ HYPOL ~ foam ~estimate from 2.69 data of Fusee et al) 2 * Massive amounts of cells sloughed of f the beads, thus much of the activity shown i5 2 s caused by unbound cells .
-5(~3 TABLE 1 (Continued) ASPARTASE ACTIVITY ~F BOUND
E COLI (ATCC 11303) AT 25C
Est. g of E. Coli Moles sf ASPused (per 5.0 ml EXAMPLE # (hr X kg wet E. coli) bed sample) 1 185 ~.027 2a 57.7 0.039 2b 33.8 00040 3a 32.9 0038 3b 33.3 0.18 4~ 65.0 0.30 4b 11.5 0.26 4c 23~8 0.21 4d 32.5 0.59 4~ 2Ç~7 0.73 4f 55.9 lo 14 5a 30.9* 0~42*
~~ 16.~
20 * Ma~sive amounts of cells sloughed off the beads, thus much of the activity shown is caused by unbound cells.
~2~9~3 Example 7 Continuous Produc~ion of L-Aspart.i~ Acid ~as ammonium L~aspartate).
A 5.0 ml aliquot of the wet beads from Example ~4d were placed in a 0.7 cm diameter X 15 cm jacketed glass column. The column temperatu~e was maintained at 37C. A fresh 1.5 M ammonium fumarate solution in deionized water containin~
0.002 m MgSO4 (all at pH B.5) was continuously lo passed up thorugh the bed of beads at a rate of 0.5 ml~min. Samples of the effluent product solution were taken periodically and analyzed for L-aspartate contenk as 1/1000 dilutions in deionized water as described in Example 6. The bulk of the efEluent was collected but not returned to the column. The Eollowing data shows that over a period of 50 days of continuous running the L-aspartase activity of the coated beads remained essentially unchanged.
L-aspartate content Time~ day~L f effluent (Percent 5g .6-) 61~5 ~ 62~5 average
6~.5~
At the 62.5% conversion rate or a flow of 6.0 bed volumes/hr ~0.5 ml/min through 5.0 ml of catalyst bed) the production rate o L-aspartic acid iso 6.n 1 X 0.625 X 1 5 mol~s _ 5.6 moles L-ASP
hr X 1 cat bed 1 hr X 1 cat bed ';~
For a period of one hour at 37C the same column was run at the rate of 4.0 bed volumes/hr (0O33 ml/min through 5.0 ml of catalyst bed) and the average rate of Eumarate (1.5 M) conversion was 80.0~. This corresponds to:
~ X 5.6 - 7-2 moles L-ASP
62.5 hr X 1 cat. bed Based on these conversion rates of 6205% and 80.0%
at 6.0 and 4.0 ~ed vol/hr respectively, a conservative estimate by graphical methods, of the flow rate required to achieve 100% conversion is 1.5 bed volume~. At 1.5 bed volumeS/hr the L-~SP
hr production rate is:
_ 1.5 1 X l.5 moles L-~SP = 2.25 moles L~ASP
hr X l bed 1 hr X l cat bed These values (100% conversion at 1.5 bed vol/hr, and 2.25 moles h-ASP are significantly better than hr X 1 cat bed values of 1.1 bed vol/hr for 100% conversion and 1.65 moles L-ASP repor~ed for the continuous hr % l cat bed operation of a column of E. coli ATCC 11303 cells immobilized on k -carrageenan gel.
In a fuether variation of the invention, it is contemplated that the aqueous ammonium L-aspartate solution obtained in Example 7 may be used directly, i.e. without recovery or separation of dry, solid aspartic acid, for reaction with benzyl chloroformate to form the benzyloxy carbamate of the amine oE aspartic acid, this carbamate being an important intermediate in the preparation of the commercially important sweetener, L-aspartyl-L-phenylalanine methyl ester. Presently this benzyloxy carbamate i~ made by starting with dry, crystalline solid L-aspartic acid. The acid is dissolved in aqueous sodium hydroxide prior to reaction with benzyl chloroformate according to the following reactions:
Q O
Il ll HOCCH2-CH-COH + 2 NaOH
~H2 dry solid aqueous ~' O O
~1 11 NaOCCH2-CH~CONa ~ 2H20 0cH
O O
Il 11 NaOCCH2fHCOH + NaCl (aq solu) The immobilized whole cell enzymatic process for converting ammonium Eumarate to ammonium L-lo aspartate according to the invention provides ~3 directly ~ basic aqueous produc~ stream of, for example, 1.5 molar 98.5% ammonium L aspar~ate solution which, after removal of ammonia, is suitable for direct use in the reaction of enzyl chlo~oformate (or other chloroformate) to make the desired aqueous solution of benzyl (or other) oxycarbamates. This shsuld make it possible to avoid the need to isolate dry aspartic acid crystals and the time and expense incident lo thereto. For example, the isolation of dry aspartic acid crystals from the ammonium L-aspar~ate solution of Example 7 would generally r~quire the following:
(1) acidification of the 1.5 molar ammonium L aspartate product (L-ASP) stream using approximately 0.7 to 0.8 moles of ~2S04 to precipitate th L~ASP at pH of 2.8.
This offers a prospective corrosion problem and the expense involved in disposing of the ammonium sulfate by-product;
(2) although the L-ASP product stream may contain 98.5~ L-ASP (the rest is ammonium fumarate), the precipitate of L-ASP and washing with water gives only an 89-93% yield of solid L-ASP. The retention of 5-9% of the 1-ASP in the mother liquor is an appreciable loss, considering the value of the L-ASP.
(3) subsequent drying of the precipitated L-A5P crystals is also costly (estimated at about 5-10 cents per pound3.
Use of the aqueous ammonium L-ASP product directly, according to the invention, eliminatQs the disadvantages inherent in steps ~1) (3~.
3~
Ad~ustment of the ammonium L-ASP product stream is appropriate to obtain optimum results. For example, the addition of sodium hydroxide (in the order of 0.5 to 2.0 moles/mole ammonium L-ASP) results in displacement o~ the NH4~ as NH3 which can be boiled off and used to make additonal ammonium fumarate. Most of the NH4~ ion should be removed to facilitate the chloroformate reactionO
The invention is not limited to the lo immobil~zation oE _ coli cells or the use of such immobilized cells for the production of L-aspartic acid. Thus, other cells may be immobilized in generally similar fashion using the indicated curable polymeric mater;als. Such further immo-bilized cell variations, and uses thereof, areexemplified in ~i~e following examples:
Example 8 Production of L-Alanine Via Immobiliza-tion of a Pseudomonas dacunhae Immobilized with Polyazetidine Gels.
Pseudomonas dacunh _ ATCC 21192 was cultured on a medium of peptone (Bacto--Difco), 9.0 g/liter~ casein hydrolysate (Casamino acids;
Difco), 2.0 g/liter; KH2P04, 0.5 g/liter;
MgS0~-7H~0, 0.1 g/liter; and L-glutamic acid sodium salt, 15.0 g/liter, pH adjusted to 7.2 with NH40H. The Eermentation was carried out under aerobic conditions for 23 hours at 30C, 300 rotations/min. A mixture of 3.62 grams ~wet weight) of cell paste and 3.6 grams uf aqueous polyazetidine solution (Hercules Polycup ~172) was stirred to homogenity at 25C by hand mixing with a wooden stick. This mixture was dispersed on 3.6 9 of Amberlite ~IRA 938 ion exchange bead 5a!3 (Rohm & 8aas) which had ~een previously air dried at 25-30C for 60 hours to remove greater than 85% of the moisture associated with the beads.
The thin film of the mixture on the beads wa~
allowed to air dry at 25C for 24 hour~. The resulting free flowing beads occupied a volume of 20 ml. These beads were washed with 5 volumes of normal saline and an 18 ml. ali~uot was placed in a flask containing .5 liters oE 1.5 M ammonium aspartate solution, pH 5.5 (adjusted with ~2SO4) which was held at 37C. The entiee mixture was stirred for a period of 48 hours while the pH was maintained by the addition of sulfuric acid as needed.
Analysis of the liquid at the end of this period on HP~C (uNH2 Bondapak column, Waters Assoc.; eluant CH3CN, 25~, 5 mM KHPO4 pH 4.4, 75%3 showed that L-aspartic a~id had been decarboxy-lated to form L-alanine in 98% yield.
Example 9 Preparation of Penicillin-G Acylase Containing Bacillus meqaterium Immobilized With Polyazetidine Gels.
Bacillus meqaterium ATCC 14945 was cultured under aerobic conditions as previously described (U.S. Patent 3,446,705). A mixture of 10.5 9 (wet weight) of the cell material and 10.5 g of an aqueous polyazetidine solution (Hercules Polycup ~172) was stirred to homogeneity at 25C
by hand mixing with a wooden stick. This mixture was dispersed onto 9.8 9 of Amberlite ~IRA 938 ion exchange beads (Rohm and Haas) which had been previously air-drled at 25-30C for 60 hou,rs which removed> 85% of the moisture from the beads.
~3 A thin layer of the mixture was allowed to air dry at 25C for 24 hours. The resultant free flowing beads weighed 13.1 g and occupied a volume of 53 ml. A 1247 mg aliquot of these beads corres-ponding to 1 gram of ree cells was assayed forpenicillin acylase activity. 6-Aminopenicillanic acid production was quantitated by high pressure liquid chromatography (HPLC). The activity of the immobilized preparation was 19.7 ~ moles hr 1 9 lo cell paste 1. This value was 67% of the free cell activity.
Ex amPl e 10 Preparation of Penicillin-G Acylase Containing Escherichia coli Immobilized With Polyazetidine Gels.
Escherichia coli ATCC 9637 was cultured aerobically as described by 5ato et al, Eur. J.
A~pl. Microbiol. 2:153-160 (1976). A mixture o 20.7 9 (wet weight) of cell paste and 20.7 g of ~o aqueous polyazetidine solution ~Hercules Polycup 172) was stirred to homogeneity at 25C by hand mixing with a wooden stick. This mixture was dispersed onto 19.3 g of Amberlite ~IRA 938 ion exchange beads (Rohm and Haas) which had been previously air-dried at 25-30C for 60 hours to remove >85% of the moisture associated with the beads. A thin film o~ the mixture was allowed to air dry at 25C for 24 hours. The resultant free flowing beads weighed 27.4 9 and occupied a volume of 95 ml. A 1325 mg aliquot of these ~eads corresponding to 1 gram of free cells was assayed for penicillin acylase activity. 6-Amino penicillanic acid production was quantitate~ by high-pressure li~uid chromatography (HPLC). The activity of the immobilized preparation was 53.6 ~ moles hr~l gram cell paste~l which was 99%
of the free cell activity.
ExamPle 11 Immobilization of Arthrobacter species Containing Glucose Isomerase Activity for Produc-tion of ~igh Fructose Corn Syrup.
Arthrobacter species ATCC 21748 was grown under submerged aerobic conditions and the cells lo were harvested. The cells had a glucose isomerase activity of 420~ moles hr~lgm~l cell paste (wet weight) at 60~C.
To 47 gm of Polycup ~ 172 was added 47 gm of Arthrobacter species ATCC 21748 (wet weight~
and the mixture was stirred vigorously for 5-10 minutes. The Polycup ~/cell mixture was then added slowly to 46.4 gm of air dried IRA @9938 beads with mixing and the resultant beads were dried at rsom temperature for 24 hours.
To a flask containing 5 ml oE the Arthrobacter species ATCC 21748 immobilized beads was added 50 ml of 0.2 M phosphate buffer, pH 7.5, 10 mM MgS04, 1 mM CoC12 and the solution was heated to 60C with stir~ing. 450 mg ofO~-D-glucose was added to the flask and the flask was placed in an incubated shaker at 60C for 1 hour at 150 rpm. The glucose isomerase activity of the immobilized Arthrobacter species ATCC 21748 was 530~ moles-hr l gm 1 cell paste (wet weight) at 60C as determined by the cysteine/H2SO4 reaction.
[(Dische, Biochim. Biophys. Acta, 39, 140 (1960)].
10 Ml of the Arthrobacter species ATCC
21748 immobilized beads were added to a 0.9 x 30 cm jacketed glass column which was maintained at ~g~ 3 60C by a circulating waterba~h. Prewarmed substrate solution containing 250 mM glucose, 5 mM
MgS04 and 0.5 mM CoC12 in 50 mM Tris-HCl buffer, p~ 7.5, was passed through the column. After 6 days of continuous operation the glucose isomerase activity of the immobilized Arthrobacter species ATCC 21748 was 280 ~ moles-hr l gm 1 cell paste (wet weight) at 60C and after 37 days the glucose isomerase activi~y was 18 ~ moles hr~l-gm~~ cell lo paste (wet weight) at 60C.
Example 12 Immobilization of Arthrobacter simplex Having Steroid Dehydrogenase Activity for The Production of Prednisolone or Related Steroids.
Arthrobacter simplex ATCC 6946 was grown under submerged aerobic conditions and the cells were harvested. The cells have a ~ -l-dehydro-genase activity of 980 ~ moles-hr l-gm~~ cell paste ~wet weight) at 34C.
To 20 gm of Polycup ~172 was added 20 gm of A. ~ ATCC 6946 and the mixture was stirred vigorously for 5-10 minutes. The Polycup ~/cell mixture was then added slowly ~o 18.6 gm of air dried IRA ~938 beads with mixing and the resultant beads were dried at room temperature for 24 hours.
To a Elask containing 5 Ml of the A ~
simplex ATCC 6946 immobilized heads was added 36 Ml of 20 mM phosphate buffer (pH 7.0) followed by 4 Ml of ethanol containing 60 mg of hydrocortisone.
The flask was placed in an incubated shaker at 34C for 1 hr at 150 rpm~ The~ dehydrogenase activity of the immobilied A. simplex ATCC 6946 ~Z(~95C~3 was 440 ~ moles hr~l gm ~ cell paste (wet weight) at 34C as determined by absorbance at 285 nm.
Example 13 Immobilization of a StrePtomyces species Having Glucose Isomerase Activity for the Produc~
tion of High Fructose Corn Syrup.
Streptomyces phaeochromogenes NRRL B3559 was grown under submerged ae~obic conditions and the cells were harvested. The cells had a glucose lo isomerase activity of 7.7 ~ moles min~l-gm~l cell paste (wet weight) at 60C.
To 64.5 gm of Polycup ~ 172 was added 64.5 gm of S. ~haeochromogenes NRR~ B35S9 and the mixture was vigorously stirred 5-10 minutes. The Polycup ~/cell mixture was then slowly added to 60.0 gm of air dried IRA ~938 beads with mixing and the resultant beads were dried at room temperature or 24 hours.
10 Ml of the S. phaeochromo~enes NRRL
B3559 immobilized beads were added to a 0.9 x 30 cm jacketed glass column which was maintained at 60C by a circulating waterbath. Prewarmed substrate solution containing 250 mM glucose, 100 mM Na2S03, 10 mM Mg504 and 1 mM CoC12 adjusted to pH 7.0 with HCl was passed through the column at a flowrate of 28 ml/hr~ The glucose isomerase activity of the immobilized S. phaeochromo~enes NRRL B3559 was 7.7~ moles-min l~gm 1 cell paste (wet weight) at 60 C as determined by the cysteine/H2SO4 reaction (Dische et al, supra~.
~.~09~03 Example 14 Immobiliztion of a Rhodosporidium Species High In Phenylalanine ~mmonia Lyase for the Production of Phenylalanine~
Rhodos~or dium toruloides ATCC 10788 was grown under submerged aerobic conditions and the cells were harvested. The cells had an L-phenylalanine ammonia-lyase activity of 112~x moles~hr~l~gm~l cell paste (wet weight) at 30C.
To 14.9 gm of Polycup ~ 172 was added 14.9 gm of R7 toruloides ATCC 10788 and the cells mixture was vigorously stirred for 5-10 minutes.
The Polycup ~/cell mixture was then added slowly to 13.8 gm of air dried IRA ~938 beads with lS mixing and the resultant beads were dried at room temperature for 24 hours7 To a flask containing 1 Ml of the R_ toruloides ATCC 10788 immobilized beads was added 5 Ml of 25 mM Tris-HCl bufEer (pH 808), 25 mM ~-phenylalanine, 0.005% cetyl pysîdinium chloride.
The flask was placed in a Dubnoff shaking incubator at 30C for 1 hour at 100 rpm. The phenylalanine ammonia-lyase activity of the immobilized R. toruloides ATCC 10788 was 10~
moles~hr l gm 1 cell paste ~wet weight) at 30~C as determined by absorbance at 278 nm, Those in the art will appreciate from the prior literature how the immobilized cell/polymer compositions disclosed in Examples 8-14 can be used to make the products indicated. Thus, with respect to Example 8, it is known that the production of L-alanine via decarboxylation of aspartic acid is mediated by the enzymP, L-aspartic-~ -decarboxylase. The presence of this enzyme i5 well known in many genera of micro~
~L2~5Q3 ~3 organisms, as reported by Chibata et al, Applied Microbioloq~, Vol. 13V No. 5, pages 638-645 (1965). It is produced by Clostridium perfri~ens, Desulfovibrio desulfuricans, Nocardia , globerula, P _ domonas eeptilivora, Acetobacter species, Achromobacter species, and Alcaliqenes faecalisc =
Chibata et al determined that alanine forming strains were fairly common in the genera lo Acetobacter, Achromobacter, Pseudomonas, Torula, Torulopsis, Absidia, ~ Mucor, and Oospora. Chibata et al increased the knowledge p~eviously ob~ained by Meister and coworkers and determined tha~ L-aspartic acid was converted stoichiometrically to alanine. Isolated yields of L-alanine over 90% from L-aspartic acid were easily obtained. This process is described with respe~t to the organisms, Pseudomonas dacunhae and Achromobacter Pestifer in U.S. Patent No.
3,458,4~0, which claims a process for production oE L-alanine in an aqueous nutrient medium, by dried living cells, or by cell free extracts. A
modification of this process was patented by Chlbata et al, 1975, U.S. Patent No. 3,898,1285 by im~obilization of the microorganism in acrylamide polymers. A further patent on the use oE the enzyme from Pseudomonas dacunhae was obtained in 1969, U.9. Patent No. 3,463,704. Continued research by Shibatani et al, Applied ~ Environ-mental Microbiology, Vol 38, No~ 3, pages 359 364 (1~79) has shown that additional species have been found by other workers to possess L-aspartic-~ -decarboxylase and shows that production of this enzyme can be stimulated by the addition of certain amino acids, such as glutamate. An improved continuous method of production was published by ~amamoto et al, Biotechnoloqy ~
Bioenqineerin~l, Yol. XXII, pages 2045-2054 ~1980), when the whole OrganiSM was immobilized in s carrageenan gel. Additionally, others have worked on the production of alanine from Pseudomonas .
dacunhae and Alcaligenes faecalis immobilized in HYPOL ~foam lFusee et al, American SocietY for Microbiology Abstracts, Dallas (March 1980~].
The present invention contemplates using the exemplified cell/polymer composition to produce L-alanine from aspartic acid in lieu of the previously used cell compositions.
A great deal of work has also previously been done with respect to the immobilization of the enzyme penicillin acylase~ Seet for example, Biochimie, 62: 317-321 (1980). Marconi et al have immobiliæed penicillin acylase in cellulose triacetate fibers [Biotechnolo~ and Bioen~ineerinq 22:735~756 ~1980); Biotechnoloqy and Bioen~ineerin~ _:1057-1073 (1979)], and a numbee of U.S. patents have issued on the enzyme immobilization: U.S. patents 3,278,391; 3,116,218;
3,190,~86; 3,446,705; 3,622,46~; 3,736,230;
2s 3,766,00~; 3,801,962; 3,~83,394; 3,900,488;
3,499,909; 3,736,230; 4,001,264; 4,113,566; and 4,230,804.
It appears that relatively few papers on whole cell immobilization are available. Tanabe 30 Seiyaku has published a report of cells entrapped in polyacrylimide gel. Chîbata et al (1974), German Patent 2 9 414,128, and Mandel et al, published on entrapment in polyacrylamide gel [Prikl. Blkhim. Mikrobiol. 11-219-225 (1975)3.
Immobilized whole cells of Bacillu~ meqaterium or 9L;~ Q~
Achromobacter adsorbed on DE~E cellulose have been used by Toyo Jozo, Fujii et al, Japanese patent 73 99393 and Sato et al, 1976, Eur. J. A~plied Microbiolo~y 2:153-160 ~1976), have repo~ted on the production oE 6-APA from pencillin-G by using immobilized E. coli having high penicillin acylase activity.
It will be appreciated that various other modifications may also be made without deviating from the invention, the scope of which is defined in the following claims wherein:
At the 62.5% conversion rate or a flow of 6.0 bed volumes/hr ~0.5 ml/min through 5.0 ml of catalyst bed) the production rate o L-aspartic acid iso 6.n 1 X 0.625 X 1 5 mol~s _ 5.6 moles L-ASP
hr X 1 cat bed 1 hr X 1 cat bed ';~
For a period of one hour at 37C the same column was run at the rate of 4.0 bed volumes/hr (0O33 ml/min through 5.0 ml of catalyst bed) and the average rate of Eumarate (1.5 M) conversion was 80.0~. This corresponds to:
~ X 5.6 - 7-2 moles L-ASP
62.5 hr X 1 cat. bed Based on these conversion rates of 6205% and 80.0%
at 6.0 and 4.0 ~ed vol/hr respectively, a conservative estimate by graphical methods, of the flow rate required to achieve 100% conversion is 1.5 bed volume~. At 1.5 bed volumeS/hr the L-~SP
hr production rate is:
_ 1.5 1 X l.5 moles L-~SP = 2.25 moles L~ASP
hr X l bed 1 hr X l cat bed These values (100% conversion at 1.5 bed vol/hr, and 2.25 moles h-ASP are significantly better than hr X 1 cat bed values of 1.1 bed vol/hr for 100% conversion and 1.65 moles L-ASP repor~ed for the continuous hr % l cat bed operation of a column of E. coli ATCC 11303 cells immobilized on k -carrageenan gel.
In a fuether variation of the invention, it is contemplated that the aqueous ammonium L-aspartate solution obtained in Example 7 may be used directly, i.e. without recovery or separation of dry, solid aspartic acid, for reaction with benzyl chloroformate to form the benzyloxy carbamate of the amine oE aspartic acid, this carbamate being an important intermediate in the preparation of the commercially important sweetener, L-aspartyl-L-phenylalanine methyl ester. Presently this benzyloxy carbamate i~ made by starting with dry, crystalline solid L-aspartic acid. The acid is dissolved in aqueous sodium hydroxide prior to reaction with benzyl chloroformate according to the following reactions:
Q O
Il ll HOCCH2-CH-COH + 2 NaOH
~H2 dry solid aqueous ~' O O
~1 11 NaOCCH2-CH~CONa ~ 2H20 0cH
O O
Il 11 NaOCCH2fHCOH + NaCl (aq solu) The immobilized whole cell enzymatic process for converting ammonium Eumarate to ammonium L-lo aspartate according to the invention provides ~3 directly ~ basic aqueous produc~ stream of, for example, 1.5 molar 98.5% ammonium L aspar~ate solution which, after removal of ammonia, is suitable for direct use in the reaction of enzyl chlo~oformate (or other chloroformate) to make the desired aqueous solution of benzyl (or other) oxycarbamates. This shsuld make it possible to avoid the need to isolate dry aspartic acid crystals and the time and expense incident lo thereto. For example, the isolation of dry aspartic acid crystals from the ammonium L-aspar~ate solution of Example 7 would generally r~quire the following:
(1) acidification of the 1.5 molar ammonium L aspartate product (L-ASP) stream using approximately 0.7 to 0.8 moles of ~2S04 to precipitate th L~ASP at pH of 2.8.
This offers a prospective corrosion problem and the expense involved in disposing of the ammonium sulfate by-product;
(2) although the L-ASP product stream may contain 98.5~ L-ASP (the rest is ammonium fumarate), the precipitate of L-ASP and washing with water gives only an 89-93% yield of solid L-ASP. The retention of 5-9% of the 1-ASP in the mother liquor is an appreciable loss, considering the value of the L-ASP.
(3) subsequent drying of the precipitated L-A5P crystals is also costly (estimated at about 5-10 cents per pound3.
Use of the aqueous ammonium L-ASP product directly, according to the invention, eliminatQs the disadvantages inherent in steps ~1) (3~.
3~
Ad~ustment of the ammonium L-ASP product stream is appropriate to obtain optimum results. For example, the addition of sodium hydroxide (in the order of 0.5 to 2.0 moles/mole ammonium L-ASP) results in displacement o~ the NH4~ as NH3 which can be boiled off and used to make additonal ammonium fumarate. Most of the NH4~ ion should be removed to facilitate the chloroformate reactionO
The invention is not limited to the lo immobil~zation oE _ coli cells or the use of such immobilized cells for the production of L-aspartic acid. Thus, other cells may be immobilized in generally similar fashion using the indicated curable polymeric mater;als. Such further immo-bilized cell variations, and uses thereof, areexemplified in ~i~e following examples:
Example 8 Production of L-Alanine Via Immobiliza-tion of a Pseudomonas dacunhae Immobilized with Polyazetidine Gels.
Pseudomonas dacunh _ ATCC 21192 was cultured on a medium of peptone (Bacto--Difco), 9.0 g/liter~ casein hydrolysate (Casamino acids;
Difco), 2.0 g/liter; KH2P04, 0.5 g/liter;
MgS0~-7H~0, 0.1 g/liter; and L-glutamic acid sodium salt, 15.0 g/liter, pH adjusted to 7.2 with NH40H. The Eermentation was carried out under aerobic conditions for 23 hours at 30C, 300 rotations/min. A mixture of 3.62 grams ~wet weight) of cell paste and 3.6 grams uf aqueous polyazetidine solution (Hercules Polycup ~172) was stirred to homogenity at 25C by hand mixing with a wooden stick. This mixture was dispersed on 3.6 9 of Amberlite ~IRA 938 ion exchange bead 5a!3 (Rohm & 8aas) which had ~een previously air dried at 25-30C for 60 hours to remove greater than 85% of the moisture associated with the beads.
The thin film of the mixture on the beads wa~
allowed to air dry at 25C for 24 hour~. The resulting free flowing beads occupied a volume of 20 ml. These beads were washed with 5 volumes of normal saline and an 18 ml. ali~uot was placed in a flask containing .5 liters oE 1.5 M ammonium aspartate solution, pH 5.5 (adjusted with ~2SO4) which was held at 37C. The entiee mixture was stirred for a period of 48 hours while the pH was maintained by the addition of sulfuric acid as needed.
Analysis of the liquid at the end of this period on HP~C (uNH2 Bondapak column, Waters Assoc.; eluant CH3CN, 25~, 5 mM KHPO4 pH 4.4, 75%3 showed that L-aspartic a~id had been decarboxy-lated to form L-alanine in 98% yield.
Example 9 Preparation of Penicillin-G Acylase Containing Bacillus meqaterium Immobilized With Polyazetidine Gels.
Bacillus meqaterium ATCC 14945 was cultured under aerobic conditions as previously described (U.S. Patent 3,446,705). A mixture of 10.5 9 (wet weight) of the cell material and 10.5 g of an aqueous polyazetidine solution (Hercules Polycup ~172) was stirred to homogeneity at 25C
by hand mixing with a wooden stick. This mixture was dispersed onto 9.8 9 of Amberlite ~IRA 938 ion exchange beads (Rohm and Haas) which had been previously air-drled at 25-30C for 60 hou,rs which removed> 85% of the moisture from the beads.
~3 A thin layer of the mixture was allowed to air dry at 25C for 24 hours. The resultant free flowing beads weighed 13.1 g and occupied a volume of 53 ml. A 1247 mg aliquot of these beads corres-ponding to 1 gram of ree cells was assayed forpenicillin acylase activity. 6-Aminopenicillanic acid production was quantitated by high pressure liquid chromatography (HPLC). The activity of the immobilized preparation was 19.7 ~ moles hr 1 9 lo cell paste 1. This value was 67% of the free cell activity.
Ex amPl e 10 Preparation of Penicillin-G Acylase Containing Escherichia coli Immobilized With Polyazetidine Gels.
Escherichia coli ATCC 9637 was cultured aerobically as described by 5ato et al, Eur. J.
A~pl. Microbiol. 2:153-160 (1976). A mixture o 20.7 9 (wet weight) of cell paste and 20.7 g of ~o aqueous polyazetidine solution ~Hercules Polycup 172) was stirred to homogeneity at 25C by hand mixing with a wooden stick. This mixture was dispersed onto 19.3 g of Amberlite ~IRA 938 ion exchange beads (Rohm and Haas) which had been previously air-dried at 25-30C for 60 hours to remove >85% of the moisture associated with the beads. A thin film o~ the mixture was allowed to air dry at 25C for 24 hours. The resultant free flowing beads weighed 27.4 9 and occupied a volume of 95 ml. A 1325 mg aliquot of these ~eads corresponding to 1 gram of free cells was assayed for penicillin acylase activity. 6-Amino penicillanic acid production was quantitate~ by high-pressure li~uid chromatography (HPLC). The activity of the immobilized preparation was 53.6 ~ moles hr~l gram cell paste~l which was 99%
of the free cell activity.
ExamPle 11 Immobilization of Arthrobacter species Containing Glucose Isomerase Activity for Produc-tion of ~igh Fructose Corn Syrup.
Arthrobacter species ATCC 21748 was grown under submerged aerobic conditions and the cells lo were harvested. The cells had a glucose isomerase activity of 420~ moles hr~lgm~l cell paste (wet weight) at 60~C.
To 47 gm of Polycup ~ 172 was added 47 gm of Arthrobacter species ATCC 21748 (wet weight~
and the mixture was stirred vigorously for 5-10 minutes. The Polycup ~/cell mixture was then added slowly to 46.4 gm of air dried IRA @9938 beads with mixing and the resultant beads were dried at rsom temperature for 24 hours.
To a flask containing 5 ml oE the Arthrobacter species ATCC 21748 immobilized beads was added 50 ml of 0.2 M phosphate buffer, pH 7.5, 10 mM MgS04, 1 mM CoC12 and the solution was heated to 60C with stir~ing. 450 mg ofO~-D-glucose was added to the flask and the flask was placed in an incubated shaker at 60C for 1 hour at 150 rpm. The glucose isomerase activity of the immobilized Arthrobacter species ATCC 21748 was 530~ moles-hr l gm 1 cell paste (wet weight) at 60C as determined by the cysteine/H2SO4 reaction.
[(Dische, Biochim. Biophys. Acta, 39, 140 (1960)].
10 Ml of the Arthrobacter species ATCC
21748 immobilized beads were added to a 0.9 x 30 cm jacketed glass column which was maintained at ~g~ 3 60C by a circulating waterba~h. Prewarmed substrate solution containing 250 mM glucose, 5 mM
MgS04 and 0.5 mM CoC12 in 50 mM Tris-HCl buffer, p~ 7.5, was passed through the column. After 6 days of continuous operation the glucose isomerase activity of the immobilized Arthrobacter species ATCC 21748 was 280 ~ moles-hr l gm 1 cell paste (wet weight) at 60C and after 37 days the glucose isomerase activi~y was 18 ~ moles hr~l-gm~~ cell lo paste (wet weight) at 60C.
Example 12 Immobilization of Arthrobacter simplex Having Steroid Dehydrogenase Activity for The Production of Prednisolone or Related Steroids.
Arthrobacter simplex ATCC 6946 was grown under submerged aerobic conditions and the cells were harvested. The cells have a ~ -l-dehydro-genase activity of 980 ~ moles-hr l-gm~~ cell paste ~wet weight) at 34C.
To 20 gm of Polycup ~172 was added 20 gm of A. ~ ATCC 6946 and the mixture was stirred vigorously for 5-10 minutes. The Polycup ~/cell mixture was then added slowly ~o 18.6 gm of air dried IRA ~938 beads with mixing and the resultant beads were dried at room temperature for 24 hours.
To a Elask containing 5 Ml of the A ~
simplex ATCC 6946 immobilized heads was added 36 Ml of 20 mM phosphate buffer (pH 7.0) followed by 4 Ml of ethanol containing 60 mg of hydrocortisone.
The flask was placed in an incubated shaker at 34C for 1 hr at 150 rpm~ The~ dehydrogenase activity of the immobilied A. simplex ATCC 6946 ~Z(~95C~3 was 440 ~ moles hr~l gm ~ cell paste (wet weight) at 34C as determined by absorbance at 285 nm.
Example 13 Immobilization of a StrePtomyces species Having Glucose Isomerase Activity for the Produc~
tion of High Fructose Corn Syrup.
Streptomyces phaeochromogenes NRRL B3559 was grown under submerged ae~obic conditions and the cells were harvested. The cells had a glucose lo isomerase activity of 7.7 ~ moles min~l-gm~l cell paste (wet weight) at 60C.
To 64.5 gm of Polycup ~ 172 was added 64.5 gm of S. ~haeochromogenes NRR~ B35S9 and the mixture was vigorously stirred 5-10 minutes. The Polycup ~/cell mixture was then slowly added to 60.0 gm of air dried IRA ~938 beads with mixing and the resultant beads were dried at room temperature or 24 hours.
10 Ml of the S. phaeochromo~enes NRRL
B3559 immobilized beads were added to a 0.9 x 30 cm jacketed glass column which was maintained at 60C by a circulating waterbath. Prewarmed substrate solution containing 250 mM glucose, 100 mM Na2S03, 10 mM Mg504 and 1 mM CoC12 adjusted to pH 7.0 with HCl was passed through the column at a flowrate of 28 ml/hr~ The glucose isomerase activity of the immobilized S. phaeochromo~enes NRRL B3559 was 7.7~ moles-min l~gm 1 cell paste (wet weight) at 60 C as determined by the cysteine/H2SO4 reaction (Dische et al, supra~.
~.~09~03 Example 14 Immobiliztion of a Rhodosporidium Species High In Phenylalanine ~mmonia Lyase for the Production of Phenylalanine~
Rhodos~or dium toruloides ATCC 10788 was grown under submerged aerobic conditions and the cells were harvested. The cells had an L-phenylalanine ammonia-lyase activity of 112~x moles~hr~l~gm~l cell paste (wet weight) at 30C.
To 14.9 gm of Polycup ~ 172 was added 14.9 gm of R7 toruloides ATCC 10788 and the cells mixture was vigorously stirred for 5-10 minutes.
The Polycup ~/cell mixture was then added slowly to 13.8 gm of air dried IRA ~938 beads with lS mixing and the resultant beads were dried at room temperature for 24 hours7 To a flask containing 1 Ml of the R_ toruloides ATCC 10788 immobilized beads was added 5 Ml of 25 mM Tris-HCl bufEer (pH 808), 25 mM ~-phenylalanine, 0.005% cetyl pysîdinium chloride.
The flask was placed in a Dubnoff shaking incubator at 30C for 1 hour at 100 rpm. The phenylalanine ammonia-lyase activity of the immobilized R. toruloides ATCC 10788 was 10~
moles~hr l gm 1 cell paste ~wet weight) at 30~C as determined by absorbance at 278 nm, Those in the art will appreciate from the prior literature how the immobilized cell/polymer compositions disclosed in Examples 8-14 can be used to make the products indicated. Thus, with respect to Example 8, it is known that the production of L-alanine via decarboxylation of aspartic acid is mediated by the enzymP, L-aspartic-~ -decarboxylase. The presence of this enzyme i5 well known in many genera of micro~
~L2~5Q3 ~3 organisms, as reported by Chibata et al, Applied Microbioloq~, Vol. 13V No. 5, pages 638-645 (1965). It is produced by Clostridium perfri~ens, Desulfovibrio desulfuricans, Nocardia , globerula, P _ domonas eeptilivora, Acetobacter species, Achromobacter species, and Alcaliqenes faecalisc =
Chibata et al determined that alanine forming strains were fairly common in the genera lo Acetobacter, Achromobacter, Pseudomonas, Torula, Torulopsis, Absidia, ~ Mucor, and Oospora. Chibata et al increased the knowledge p~eviously ob~ained by Meister and coworkers and determined tha~ L-aspartic acid was converted stoichiometrically to alanine. Isolated yields of L-alanine over 90% from L-aspartic acid were easily obtained. This process is described with respe~t to the organisms, Pseudomonas dacunhae and Achromobacter Pestifer in U.S. Patent No.
3,458,4~0, which claims a process for production oE L-alanine in an aqueous nutrient medium, by dried living cells, or by cell free extracts. A
modification of this process was patented by Chlbata et al, 1975, U.S. Patent No. 3,898,1285 by im~obilization of the microorganism in acrylamide polymers. A further patent on the use oE the enzyme from Pseudomonas dacunhae was obtained in 1969, U.9. Patent No. 3,463,704. Continued research by Shibatani et al, Applied ~ Environ-mental Microbiology, Vol 38, No~ 3, pages 359 364 (1~79) has shown that additional species have been found by other workers to possess L-aspartic-~ -decarboxylase and shows that production of this enzyme can be stimulated by the addition of certain amino acids, such as glutamate. An improved continuous method of production was published by ~amamoto et al, Biotechnoloqy ~
Bioenqineerin~l, Yol. XXII, pages 2045-2054 ~1980), when the whole OrganiSM was immobilized in s carrageenan gel. Additionally, others have worked on the production of alanine from Pseudomonas .
dacunhae and Alcaligenes faecalis immobilized in HYPOL ~foam lFusee et al, American SocietY for Microbiology Abstracts, Dallas (March 1980~].
The present invention contemplates using the exemplified cell/polymer composition to produce L-alanine from aspartic acid in lieu of the previously used cell compositions.
A great deal of work has also previously been done with respect to the immobilization of the enzyme penicillin acylase~ Seet for example, Biochimie, 62: 317-321 (1980). Marconi et al have immobiliæed penicillin acylase in cellulose triacetate fibers [Biotechnolo~ and Bioen~ineerinq 22:735~756 ~1980); Biotechnoloqy and Bioen~ineerin~ _:1057-1073 (1979)], and a numbee of U.S. patents have issued on the enzyme immobilization: U.S. patents 3,278,391; 3,116,218;
3,190,~86; 3,446,705; 3,622,46~; 3,736,230;
2s 3,766,00~; 3,801,962; 3,~83,394; 3,900,488;
3,499,909; 3,736,230; 4,001,264; 4,113,566; and 4,230,804.
It appears that relatively few papers on whole cell immobilization are available. Tanabe 30 Seiyaku has published a report of cells entrapped in polyacrylimide gel. Chîbata et al (1974), German Patent 2 9 414,128, and Mandel et al, published on entrapment in polyacrylamide gel [Prikl. Blkhim. Mikrobiol. 11-219-225 (1975)3.
Immobilized whole cells of Bacillu~ meqaterium or 9L;~ Q~
Achromobacter adsorbed on DE~E cellulose have been used by Toyo Jozo, Fujii et al, Japanese patent 73 99393 and Sato et al, 1976, Eur. J. A~plied Microbiolo~y 2:153-160 ~1976), have repo~ted on the production oE 6-APA from pencillin-G by using immobilized E. coli having high penicillin acylase activity.
It will be appreciated that various other modifications may also be made without deviating from the invention, the scope of which is defined in the following claims wherein:
Claims (32)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A composition comprising immobilized cells having enzyme activity, the cells being immobilized by means of a fixed, insoluble, crosslinked polymer obtained by curing a curable prepolymer material selected from the group consisting of polyazetidine prepolymers, carboxymethyl cellulose, polyurethane hydrogel prepolymers and polymethylene isocyanates, said polymer being cured at a temperature below the temperature at which the enzyme activty of the microbial cells is significantly reduced, the cell/crosslinked polymer composition constituting a coating on a solid inert carrier.
2. A composition according to claim 1 wherein the cells are whole cells of E. coli which have L-aspartase activity.
3. A composition according to claim 1 comprising immobilized E. coli cells having L-aspartase activity, the cells being immobilized by means of a fixed, insoluble, crosslinked polymer obtained by curing a curable prepolymer material selected from the group consisting of poly-azetidine prepolymers, carboxymethyl cellulose, polyurethane hydrogel prepolymers and polymethy-lene isocyanates, said polymer being cured at a temperature below the temperature at which the L-aspartase activity of the microbial cells is significantly reduced, the cell/crosslinked polymer composition constituting a coating on a solid inert carrier.
4. A composition according to claim 1 wherein the cells are whole cells of Pseudomonas dacunhae, Bacillus megaterium, Arthrobacter species, Arthrobacter simplex, Streptomyces phaeochromogenes or Rhodosporidium toruloides.
5. A composition according to claim 1 wherein the prepolymer material is a polyazetidine prepolymer.
6. A composition according to claim 1 wherein the prepolymer material is carboxymethyl cellulose.
7. A composition according to claim 1 wherein the prepolymer material is a polyurethane hydrogel.
8. A composition according claim 1 wherein the prepolymer material is a polymethylene isocyanate.
9. A composition according to claim 1 wherein the carrier is a particulate support.
10. A composition comprising immobilized E. coli cells having L-aspartase activity, the cells being immobilized by means of a crosslinked, water-insoluble polymer obtained by curing a polyazetidine prepolymer in admixture with the cells.
11. A composition according to claim 9 and comprising a coating of the immobilized cells on a particulate support.
12. A process for preparing an immobilized microbial cell composition according to claim 1 which comprises mixing together an aqueous dispersion of the cells and a prepolymer which is capable of being cured into a crosslinked polymer and curing the prepolymer in said mixture.
13. The process of claim 12 wherein the mixture is cured after application as a coating to a carrier.
14. The process of claim 12 wherein the coating is dried.
15. A process for preparing L-aspartic acid which comprises contacting ammonium fumarate with an immobilized microbial cell composition comprising cells containing L-aspartase activity immobilized by means of a fixed, insoluble, crosslinked polymer obtained by curing a curable prepolymer material selected from the group consisting of polyazetidine prepolymers, carboxymethyl cellulose, polyurethane hydrogel prepolymers and polymethylene isocyanates, said polymer being cured at a temperature below the temperature at which the L-aspartase activity of the microbial cells is significantly reduced, the cell/crosslinked polymer composition constituting a coating on a solid inert carrier.
16. A process according to claim 15 wherein the cells are whole cells of E. coli 11303.
17. A process according to claim 15 wherein the prepolymer is a polyazetidine prepolymer.
18. A process according to claim 15 wherein the prepolymer material is carboxymethyl cellulose.
19. A process according to claim 15 wherein the prepolymer material is a polyurethane hydrogel.
20. A process according to claim 15 wherein the prepolymer material is a polymethylene isocyanate.
21. A process according to claim 15 wherein the carrier comprises beads or other particulate material.
22. A process for preparing L-alanine which comprises contacting L-aspartic acid solution with an immobilized cell/polymer composition comprising cells containing L-aspartate decarboxylase activity immobilized by means of a fixed, insoluble, crosslinked polymer obtained by curing a curable prepolymer of polyazetidine, said composition comprising a coating on a solid inert carrier.
23. A process according to claim 22 wherein the carrier comprises beads or other particulate material.
24. A process according to claim 22 wherein the cell/polymer composition comprises Pseudomonas dacunhae immobilized in polyazetidine prepolymer.
25. A process for preparing 6-Amino-penicillanic acid which comprises using an immobilized cell/polymer composition comprising cells containing penicillin-G acylase activity immobilized by means of a fixed, insoluble, crosslinked polymer obtained by curing a curable prepolymer of polyazetidine, said composition comprising a coating on a solid inert carrier.
26. A process according to claim 25 wherein the carrier comprises beads or other particulate material.
27. A process for producing high fructose corn syrup which comprises using an immobilized cell/polymer composition comprising cells containing glucose isomerase activity immobilized by means of a fixed, insoluble, crosslinked polymer obtained by curing a curable prepolymer of polyazetidine, said composition comprising a coating on a solid inert carrier.
28. A process according to claim 27 wherein the carrier comprises beads or other particulate material
29. A process for producing prednisolone or related steroids which comprises using an immobilized cell/polymer composition comprising cells having steroid dehydrogenase activity immobilized by means of a fixed, insoluble, crosslinked polymer obtained by curing a curable prepolymer of polyazetidine, said composition comprising a coating on a solid inert carrier.
30. A process according to claim 29 wherein the carrier comprises beads or other particulate material.
31. A process for producing phenyl-alanine which comprises using an immobilized cell/polymer composition comprising cells containing phenylalanine ammonia-lyase activity immobilized by means of a fixed, insoluble, crosslinked polymer obtained by curing a curable prepolymer of polyazetidine, said composition comprising a coating on a solid inert carrier.
32. A process according to claim 31 wherein the carrier comprises beads or other particulate material.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US358,874 | 1982-03-16 | ||
US06/358,874 US4466961A (en) | 1981-03-17 | 1982-03-16 | Composition for the treatment of epithelial injuries and process for the preparation thereof |
US465,551 | 1983-02-10 | ||
US06/465,551 US4732851A (en) | 1982-03-16 | 1983-02-10 | Immobilization of cells with a polyazetidine prepolymer |
Publications (1)
Publication Number | Publication Date |
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CA1209503A true CA1209503A (en) | 1986-08-12 |
Family
ID=27000233
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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CA000423176A Expired CA1209503A (en) | 1982-03-16 | 1983-03-09 | Immobilized microbial cells and processes for preparing and using same |
Country Status (1)
Country | Link |
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CA (1) | CA1209503A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114921401A (en) * | 2022-05-24 | 2022-08-19 | 灵知蓝诺(北京)生物技术有限公司 | Method for extracting cells from mucus based on liquid-phase molecular sieve |
-
1983
- 1983-03-09 CA CA000423176A patent/CA1209503A/en not_active Expired
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114921401A (en) * | 2022-05-24 | 2022-08-19 | 灵知蓝诺(北京)生物技术有限公司 | Method for extracting cells from mucus based on liquid-phase molecular sieve |
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