AU613387B2 - An inorganic carrier element comprising an amine-containing surface layer for the immobilization of microorganisms or cells, a process for the preparation thereof - Google Patents

Description

lur: COHNNON EALTH OF AUSTRALIA PATENT ACT 1952 COMPLETE SPECIFICATION 6 1 W

(ORIGINAL)

FOR OFFICE USE CLASS INT. CLASS Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority: Related Art: 04 00 00r 00O 0 04 0 bI 0 *0 08 00 0n 4 00 "FORSCHUNGSZENTRUM JULICH GmbH and CARL-ZEISS- STIFTUNG trading as SCHOTT GLASWERKE" NAME OF APPLICANT: ADDRESS OF APPLICANT: Postfach 1913, 5170 Julich, Federal Republic of Germany Postfach 2480, 6500 Mainz 1, Federal Republic of Germany, and respectively.

NAME(S) OF INVENWOR(S) 00 4 0 .0 Jochen BUCHS Christian WANDREY Maria-Regina KULA Manfred RADKE DAVIES COLLISON, Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

ADDRESS FOR SERVICE: COMPLETE SPECIFIPXCATION FOR TBE INVENTION NTITLED: "AN INORGANIC CARRIER ELEMENT COMPRISING AN AMINE-CONTAINING SURFACE LAYER FOR THE IMMOBILIZATION OF MICROORGANISMS OR CELLS, A PROCESS FOR THE PREPARATION THEREOF" The following statement is a ful. description of this invention, including the best method of perfoxragit knaovn to us

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Background of the Invention This invention relates to a carrier, in particular, a carrier comprising glass or ceramic, having a basic (alkaline), amine-containing surface layer for the immobilization of microorganisms or of human, animal or plant cells, as well as to the preparation and use thereof.

o o 4 0 0 The immobilization of microorganisms has been So° o for some time of considerable importance in the implementation of bittechnologial processes because 0 15 it is possible in this way to provide considerable 9 localized concentrations of active biomass, from which it is relatively easy to separate the reaction mixture, which is generally liquid.

o 0o 0 0 0 0 .0 -1A- Thus, organic carrier materials such as beechwood turnings, straw, sawdust, chopped alfalfa, etc., have long been part of the state of the art, as have inorganic materials in the form of glass beads, chippings, earthenware, pumice, lava, sand and the like. Also already known are synthetic carrier materials composed of carboxymethylcellulose, polyurethane foam, ethylene/maleic anhydride copolymers, cellulose triacetate and the like, which have a relatively large surface area.

It has also been known for a long time to modify glass surfaces (especially of porous membranes) by polymer films or by reacting the silanol groups with organic molecules having finctional groups (German Patent 2,454,111).

B oKnown for chromatographic purposes are glass o' supports coated with optionally crosslinked 0 00 0 t *polysaccharide which is modified by grafted-on 00 0 molecules or macromolecules having amino groups (FR- 0000 00 B 20 Al 2,422,699; a specific example mentioned here is microporous glass having a layer of cellulose or crosslinked DEAE-dextran, which are modified by agrafted-on biospecific compounds) or else glass o supports having chemically bonded polye'byleneimine Chromat. 120: 321 333 (1976)].

Such techniques for modifying porous glass surfaces have also already been provided for carrier elements for microorganisms: thus, the PCT application WO 87/02703 describes so-called microcarriers of porous glass which are coated with polymers having amino or hydroxyl groups. From the large number of stated options, emphasized therein are chitosan, cellulose, alginic acid and -2r subsequently crosslinked coatings composed of agar, gelatin or K-carrageenan.

Acccrding to U.S. Patent 4,153,510, also provided for the immobilization of cells and microorganisms is a chemical coupling of the cells themselves.

Furthermore, U.S. Patent 4,189,534 discloses microcarriers for cell cultures, which are composed of crosslinked DEAE-dextran. The attachment of cells to DEAE-Sephadex has also already been reported by van Welzel in Nature 216: 64 65 (1967).

However, a study by C.D. Buck et al., [S.

Virol. Methods 10: 171 184 (1985)] reports on a comparison of microcarriers composed of o 13 polyacrylamide, polystyrene and crosslinked DBAE- 0 0 dextran, in which the behavior of the microcarriers 0 00 o oo composed of crosslinked DEAE-dextran was found to be 0 0 inadequate.

0 S0 a0 o Summary of the Invention 0 00 It is therefore an object of the present 00 0 invention to provide a carrier for immobilization of 0o°.0 microorganisms and of human, animal and plant cells 0 (hereafter such microorganisms and cells are 0 a 0 collectively referred to as "cells"), wherein the carrier is capable of supporting the attachment of a higher number of cells than that supported by the currently available carriers.

It is also an object of the present invention to provide a carrier as above that is capable of supporting the production of a larger amount of -3- 'S.t cellular metabolites than that supported by the currently available carriers.

It is another object. of the present invention to provide a carrier as above that is capable of supporting a higher and/or faster rate of growth of cells than that supported by the currently available carriers.

It is a further object of the present invention to provide a carrier as above that is capable of providing a better growing environment for cells than that provid-d by the currently available carriers.

In aog these a nthr objects, accordance with one aspect of the present invention//a carrier for immobilization of microorganisms or of human, animal or plant cells comprising a basic, amine-containing surface 0 C'4 0 layer and an inorganic element disposed under the surface layer, wherein the surface layer is comprised 0.00 20 of uncrosslinked dialkylaminoalkyl-dextran that is 0 00 bound by adsorption or by covalent bonding to the inorganic element.

In accordance with another aspect of the 0 0 0o 0 present invention, there has been provided a carrier as above wherein the inorganic element comprises a 00000 plurality of pores.

o o0 In accordance with a further aspect of the present invention, there has been provided a process for the preparation of a carrier as above comprising the steps of boiling the inorganic element for at least one hour in a strong acid of not less than about -4washing and drying the boiled inorganic element; contacting the dried inorganic element with a DAAA-dextran solution of not less than about 1% strength, under reduced pressure, to allow the solution to penetrate into the pores to form the carrier; freeing the carrier from the DAAAdextrs 1 solution; and drying or allowing the carrier to dry.

In accordance with yet another aspect of the present invention, there has been provided a method 15 of using the carrier as abovc in a biotechnological o0 o o o process comprising the step of contacting the carrier o with microorganisms or with human, animal or plant So"n cells.

o Further objects, features and advantages of a 20 the present invention will become apparent from the S" following detailed description. It should be understood, however, that the detailed description o.q and specific examples, while indicating the preferred 0. embodiments of the invention, are given -y way of illustration only, since various changes and o modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

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Brief Description of the Drawinas Figs. 1 to 3 show the yield of leucine as a function of time when various carriers within the present invention are used.

Figs. 4 and E-8 show the number of microorganisms immobilized per gram of carrier.

Fig. 5 shows the number of microorganisms immobilized per gram of carrier after different residence time on DEAE-dextran coatings .btained from solutions of different DEAE-dextran concentrations.

Detailed Description of the Preferred Embodiments o o 00o o0 a o o o0 0 00 o a0 0 00 0 0 0 0 00 o oo It has now been established, surprisingly, that it is possible, by coating glass with uncrosslinked dialkylaminoalkyl-dextran (hereafter "DAAA-dextran"), especially diethylaminoethyl-dextran (hereinafter "DEAE-dextrau"), to achieve particularly good immobilization of microorganisms on inorganic carrier elements, especially glass. Accordingly, the carrier according to the present invention comprises a surface layer which is formed of uncrosslinked dialkylaminoalkyl-dextran, in particular DEAEdextran, and is bound by adsorption or, in particular, is covalently bonded to the inorganic element.

Such elements provided with surface layers composed of uncrosslinkedL dialkylaminoalkyl-dextran, which are bound by adsorption or, in particular, are covalently bonded, exhibit a distinctly improved attachment capacity for microorganisms and cells and 0000 a PL 0 40 00, 0 thus can be used particularly beneficially in biotechnologicPl processes.

The inorganic elements to be coated are, in particular, elements in the form of oxides or silicates, for example composed of glass, silica, alumina, zirconia, etc., with glass being preferred because it is relatively simple to obtain defined elements.

The elements to be coated can be in the form of membranes, fibers or filaments, tubes, capillaries, microcarriers or, in particular, porous carrier elements of any desired shape.

Particularly preferred are porous carrier elements which have the specific pore structure described in German Offenlegungsschrift 3,410650 and a 0° 0embrace the porosity-determining continuous macropores (of, in particular, about 20 um to about o 500 am) for unimpeded exchange of fluid and gas from g the interior of the carrier to the surroundings, as o well as mic 'opores (of, in particular, about 1 pm to about 10 pm) which pass through and are disposed in the walls o(f tie macropores and whose size ought to °00g do be in the region of the size of the microorganisms or cells.

The carrier coated according to the present invention can be autoclaved without restriction.

004 They are suitable, in particular, for the sterile cultivation of microorganisms or cells which adhere or grow only poorly on customarily untreated glass or ceramic surfaces. Particularly suitable are coated porous carriers, because of their stability of shape and high density, for fixed bed and fluidized bed reactors in which are carried out aerobic or f 0 0 00 0 0 0 0 0 0 0 0 00 anaerobic processes with extensive gas evolution or processes having the risk of transport limitation, in which case carriers having a high proportion of open pores are preferably used.

Carrif.rs composed of glass with a DEAE-doxtran coating have been particularly investigated. After this, those particularly preferably used are DEAEdextrans with a high nitrogen content but relatively small proportion of dimeric DEAE groups and a specific size range of molecular weights.

Since single DEAE grcups have a pK of about 9.2 but dimeric groups have a pK of about 6.3, under the operating conditions substantially customary in the biotechnological sector (around pH nly a 15 small fraction of the dimeric groups are dissociated and are, hence, less useful. The proportion of dimeric groups should, preferably, be below about in particular, not above about The molecular weight of the DEAE-dextran used 20 for the coating ought to be between about 100,000 and 2,000,000, preferably 10 6 there being, however, in the upper ranges the onset of folding of the otherwise elongated DEAE-dextran molecules, which is undesirable. Hence molecular weights around 600,000 are preferred.

The nitrogen content should, preferably, be between about 0.5% and about in particular about 3% to about good results having been obtained with a material with an N content of about 3.3%.

It is preferable for these adhesion-promoting layers to be covalently coupled to the inorganic element, and they are then stably absorbed on the carrier, which can be exposed to a variety of -8-

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Ii~, conditions without the need to worry about detachment of the adhesion-promoting layer. In particular, this means that there are no problems with purif:' ation and regeneration of the carrier material between two biotechnological uses.

Suitable for such binding of the dialkylaminoalkyl-dextran to the glass element are those having a glycidyloxyalkyl-glass (epoxyalkylglass) or chloroalkyl-glass surface or else having an aminoalkyl-glass surface, coupling to the latter taking place with the aid of diisocyanates.

Binding of dialkylaminoalkyl-dextrans to the inorganic element containing hydroxyl groups can take place analogously.

The alkyl groups of the dialkylaminoalkyldextran can be identical or different and are, in particular, lower alkyl groups having 1 to 4 carbon atoms, specifically 1 or 2 carbon atoms. The present invention is exemplified by the use of diethylaminoethyl-dextran ("DEAE-dextran").

The procedure for the coupling rerations is in accordance with generally established techniques. To generate simple adsorption layers on porous inorganic elements, the latter are preferably added to an aqueous DEAE-dextran solution, in particular an approximately 3% strength DEAE-dextran solution, and the pressure is reduced to ensure satisfactory penetration of the solution to the inner surfaces of the inorganic elements.

The latter are preferably treated beforehand with strong acid, with prolonged boiling in nitric acid having been proven to be appropriate.

Specifically, the dried inorganic elements are boiled 0a 0 o oO 0 0- 84 0 Oo 0s @4 4 S 4 Il -9- 1

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.9 0 0 0 o a O 0 0 0 00 0*no 0 o 00 0 0 0 0 i; U in approximately 5% strength nitric acid for some hours (for example for about 4 hours), removed from the acid, washed, dried and then left in a reduced pressure chamber with about 3% strength DAE-dextran solution at room temperature for about 1 day, or autoclaved after reducing the pressure. Finally, the coated inorganic elements that have been removed from the dextran solution are freed of excess solution and dried, and can then be used immediately, or after storage, for the immobilization of microorganisms.

The present invention is further described below by reference to the following examples. Some of these examples demonstrate the advantageous effect of the coating, according to the present invention, of the inorganic elements by showing the production of L-leucine from a-ketoisocaproate using Corynebacterium glutamicum ATCC 13032. The utility of the invention has been explained hereinbefore on the basis of investigations using corynebacterium crlutamicum. Of course, the invention can be used with any microorganisms, human, animal or plant cells and is not confined to the use of the exemplified bacteria.

Example t, The binding of uncrossed-linked DEAEdextran to glycidyl-glass in dimethyl sulfoxide.

110 g of sintered glass material according to German Offenlegungsschrift 3,410,650 which have been boiled in 5% strength INO 3 for 4 hours, rinsed and dried were added to 10 ml of gamma-glycidyloxypropyltrimethoxysilane and 490 ml of anhydrous acetone, and the mixture was degassed by applying a vacuum and was left at room temperature for one day, i- The material was subsequently wa sed with anhydrous acetone three times and then dried at 60*C for two hours and at room temperature for one day. Thereafter the sintered glass which had been treated in this way was added to a 3% strength solution co DEAEdextran in anhydrous dimethyl sulfoxide, and the mixture was degassed by vacuum and shaken at room temperature for three days and then at 60°C for one day, and the material was then washed and dried.

o 10 It is also possible to use water in place of dimethyl sulfoxide as solvent, Example 2. Production of L-Loucine by c, glutamicum immobilized on carrier, Figs. 1 3 compare the production of Lleucine from a-ketoisocaproate using C. glutamicum ATCC 13032 under comparable conditions using glass carriers which are composed of open-pore sintered glass according to German offenlegungsschrift 3,410,650 and have been treated in different ways.

As is evident from Fig. 1, the productivity obtained by the use of DEAE-doxtran coating of the qlass is o o distinctly higher than with uncoated glass or ,Tass coated with other materials, including the priordisclosed chitosan, and the productivity derives from improved immobilization and activity of the immobilized organisms.

Before the coating, all the glasses had been boiled with 5% strength nitric acid solution for hours, then thoroughly washed and dried.

Fig. 2 shows the results of comparison experiments which reveal the advantageous effect of -i1o the pretreatment of the glass with strong acid before the immobilization-promoting coating.

Finally, Fig. 3 is a comparison plot of data obtained with carrier elements coated with DEAEdextran after pretreatment with nitric acid: it is seen that a relatively low proportion of dimers, a high nitrogen content and a limited chain length promote the immobilization of productive microorganisms.

o a Qo Example 3. The number of microorganisms immobilized 0o1 on carrier as a function of time.

4440 0 a Fig. 4 is a plot o' the number of microorganisms immobilized on glass coated with DEAEdextran (109 microorganisms per gram of glass) against the residence time in a suspension of C.

glutamicum ATCC 13032 cells in 2% strength NaCI S" solution (corresponding to the ionic strength customary under fermentation conditions). This entailed 20 g of borosilicate glass carri'er being shaken in 40 ml of uell suspension containing about 2 x 101 0 cells/ml. Untreated glass has been compared 0 o with glass treated with DEAE-dextran. In addition, glass treated with DEAE-deXtran was previously rinsed with 2% strength NaC1 solution for two days in order to test the adhesion of the DEAE-dextran adsorption layer on the glass. It is seen that the immobilization of microorganisms on the previously rinsed glass is reduced to only an inconsiderable extent. The absolute values of the biomass are very high and almost reach the maximum possible occupation of the carrier corresponding to the geometric dimensions, which has also been verified by microscopic examinations.

In practical operation, continuous fermentations have been carried out successfully for almost three months with the immobilization of biomass on the carriers coated with DEAE-dextran continuing to be satisfactory.

Example 4. The effect of DEAE-dextran concentration on the number of microorganisms immobilization on the carrier.

Finally, Fig. 5 illustrates the effect of different concentrations of DEAE-dextran solution, used for coating the glass element, on the number of microorganisms attached after various residence time Residence timq4 is defined as the amount of time the coated glass carriers are kept in a suspension of microorganisms (each containing about 1.6 x I01 0 cells per ml of 2% strength NaCI solution). The glass elements were autoclaved and pretreated with acid before the application of the DEAE-dextran coating.

It is seen that the DEAE-dextran concentration of the coating solution ought to be at least about 0,3% strength, but preferably about 3% strength.

Obviously, the residence time in the suspension of microorganisms has a considerable effect on the number of them adsorbed.

Example 5. Number of E. coli and yeast cells immobilized on the carrier as a function of time.

-13- Fig. 6 shows the results of experiments with Escherichia coli K2, mutant DH5 [see, DNA Cloning vol. 1, Glover D.M. ed., (IRL Press Oxford, 1985)] and Bakers' Yeast (Uniferm GmbH Co., 4712 Werne, FRG, batch 631228).

The number of microorganisms immobilized on glass coated with D)EAE-dextran is plotted against the residence time in the particular cell suspension in 2% strength NaCl solution having an initial concentration of 1.1 x 1010/ml coli) or 4.3 x 10 8 /ml (bakers' yeast).

For the investigations, 20 g portions of the coated carrier elements comosed of open-pore sintered glass according to German Offenlegungsschbift 3,410,650 were shaken in 40 ml of cell suspension. It is seen that the two microorganisms were likewise taken up well by the carrier element coated with DEAE-dextran. The lower occupancy of the glass with bakers' yeast organisms compared with E.

ci is essentially consistent with the relative sizes of the cells, so that it can be assumed that the cells of the two microorganisms are adsorbed onto the glass carrier with approximately comprable surface densities.

Example 6. Immobilization of C. glutamicum on different carriers.

Figs. 7 and 8 compare the figures for the immobilization of Corynebacterium glutamicum ATCC 13032, under comparable conditions in each casea, vth variously treated carrier elements composed of openpore sintered glasc according to German Offenlegungsschrift 3,410,650.

Washed microorganisms were suspended in 2% strength NaCI solution (about 2 x 101 0 cells/ml in each case), and, in each case, 40 ml of this suspension were added to 20 g of carrier elements of the specification mentioned in each case, and shaken at room temperature.

Fig. 7 shows the attachment capacity of carrier elements modified with DEAE-dextran by adsorption or by covalent bonding. DEAE-dextran was coupled covalently from a 3% strength solution, once in dqueous phase and once in anhydrous dimethyl sulfoxide, to glass treated with gLycidylsilane. It 15 is seen that the immobilizing capacities of the three types of glass treated with DEAE-dextran are approximately the same and distinctly better than o %those of untreated glass.

Fig. 8 illustrates the superior properties of S" 20 the covalently modified carrier elements compared %o ,with carrier elements modified only by adsorption in the case of the material being rinsed for 1 week with 1M Na 2

CO

3 of pH 11.5 before the immobilization test.

All percentages are by weight with the exception of the percentages of the dimeric DEAE 9,jups: these refer to the total of the DEAE groups within the DEAE dextran.

Claims (19)

1. A carrier for immobilization of microorganisms or of human, animal, or plant cells comprising a basic, amino-containing surface layer and an inorganic element disposed under the surface layer, wherein the surface layer is comprised of uncrosslinked dialkylaminoalkyl-dextran which is bound by adsorption or by covalent bonding to the inorganic element.

2. A carrier as claimed in claim 1, wherein the alkyl groups of the dialkylaminoalkyl-dextran are the o same or different and each is a lower alkyl comprising 1 o f to 4 carbon atom.

3. A carrier as claimed in claim 2, wherein the 'dialkylaminoalkyl-dextran comprises a DEAE-dextran.

4. A carrier as claimed in claim 3, wherein said DEAE-dextran comprises not more than about 50% of dimeric DEAE groups, a molecular weight in the range of 100,000 to 2 x 106 and a nitrogen content of 0.5% to o

5. A carrier as cl., imed in claim 4, wherein said DEAE-dextran comprises about 25% of dimeric DEAE groups, a molecular weight of about 600,000 and a nitrogen content of about 3.3%.

6. A carrier as claimed in claim 3, wherein the DEAE-dextran is derived from a solution comprising DEAE- dextran at a concentration of at least about 0.3%.

7. A carrier as claimed in claim 1, wherein the inorganic element comprises hydroxyl groups. L_ y t I- lk^ iB Q '0 0C Qo 0 V 0 0 ,J

8. A carrier as claimed in claim 1, wherein the inorganic element is a glass, with an epoxyalkyl-glass, a chloroalkyl-glass or an aminoalkyl-glass surface entering into covalent bonding to the said surface layer.

9. A carrier as claimed in claim 1, wherein the inorganic element is a sintered glass.

A carrier as claimed in claim 1, wherein said inorganic element comprises a plurality of pores.

11. A carrier as claimed in claim 10, wherein the inorganic element comprises a dual pore structure comprising a plurality of porosity-determining continuous macropores and a plurality of micropores, the micropores being disposed in the walls of the macropores.

12. A carrier as claimed in claim 11, whereij the diameter of each of said macropores is 20pm to 500pm.

13. A carrie- as claimed in claim 11, wherein the diameter of each of said micropores is 1pm to

14. A process for the preparation of a carrier as claimed in claim 10 comprising the steps of boiling the inorganic element for at least one hour in a strong acid of not less than about washing and drying the boiled inorganic element; contacting the dried inorganic element with a DAAA-dextran solution of not less than about 1% strength, under reduced pressure, to allow the solution to penetrate into the pores to form the carrier; 0 r~ 3 b17 :Etij L 11 I freeing the carrier from the DAAA-dextran solution; and drying or allowing the carrier to dry.

The process as claimed in claim 14, wherein the inorganic element is boiled in an approximately strength nitric acid.

16. The process as claimed in claim 14, wherein step is performed in a DAAA-dextran solution of about either at room temperature for about one day or is autoolaved in the DAAA-dextran solution after degassing under reduced pressure.

17. The process as claimed in claim 16, wherein said autoclaving is performed at 120°C for 20 minutes 0 under 1 atmosphere gauge pressure.

18. The process as claimed in claim 14, wherein after step and before step the process further comprises the step of a contacting the dried inorganic element with Sgamma-glycidyloxypropyltrimethoxysilane and anhydrous acetone; o (ii) degassing the mixture; and (iii) washing and drying the above-treated inorganic element.

;19. A method of using the carrier as claimed in cliim 1 in a biotechnological process comprising the step of contacting the carrier with microorganisms or with human, animal or plant cells. c;rc-~, A carrier as claimed in claim 1, or a method of preparation or use thereof substantially as hereinbefore described with reference to the drawings and/or Examples. Dated this 16th day of May, 1991, FORSCHUNGSZENTRUM JULICH GmbH and CARL-ZEISS-STIFTUNG trading es SCHOTT GLASWERKE, By their Patent Attorneys, DAVIES COLLISON i