CA1076502A

CA1076502A - Mineral carriers grafted by silicon derivatives

Info

Publication number: CA1076502A
Application number: CA259,941A
Authority: CA
Inventors: Francois Meiller; Pierre Monsan
Original assignee: Rhone Poulenc Industries SA
Current assignee: Rhone Poulenc Industries SA
Priority date: 1975-08-28
Filing date: 1976-08-26
Publication date: 1980-04-29
Also published as: FR2322155B1; GB1530369A; DE2638467A1; FR2322155A1; NL7609495A; JPS5256092A; SE7609415L; BE845561A; DK385876A

Abstract

Abstract of the Disclosure Mineral carriers grafted with silicon derivatives comprixing oxides, hydroxides and other insoluble, porous, mineral compounds grafted with silicon radicals having acetal or aldehyde groups, said grafted mineral carriers being suit-able for use in chromatography, affinity chromatography, and fixing enzymes.

Description

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The invention relates to mineral carriers grafted with silicon derivatives. It also relates to a method of preparing such carriers and their application particularly to fixing enzymes.
~ nzymes are generally fixed on carriers based on modi-fied cellulose or dextran. However, such carriers have no mechan-ical properties, they swell in water and organic solvents and have no resistance to temperature and pressure.
Mineral carriers are known products but, for certain applications such as fixing enzymes, it is necessary to modify the chemical nature of the mineral carrier by grafting on to it a group which will improve the bond between the carrier and the enzyme. Thus it has been proposed to graft silanes with various substi~uents onto mineral carriers, to permit a certain number of direc~ fixing reactions between the carriers and certain enzymes.
-~ Ln other cases of coupling between the grafted carrier ~ and the enzyme, it is often necessary to use intermediate com-.( pounds such as dicyclohexylcarbodi-im:ide, thiophosgene or glutar-aldehyde, if the bond is required to be stable in ti~e.
The carriers of the invention described and claimed herein, may be used in many fields and are insensitive to solvents, temperature and pressure. When applied to fixing enzymes, they make it possible not only to carry out new reactions and avoid the use of intermediate compounds but also to obtain enzyme bonds .:, ~; which are stable and resistant to denaturing factors, and it is an object of this invention to produce such carriers in a simple manner and with excellent yie]ds.
The present invention provides grafted mineral carriers suitable as supports for fixing enzymes, comprising an insoluble porous material having a particle size of from 40 ~m to 5 mm, a specific surface area of 2 to 600 m /g, a pore diameter of 600 to . ~I

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10,000 A and a pore volume of 0.5 to 1.8 ml/g, said insoluble porous material having hydroxy groups to which have been grafted silicon containing radicals bearing an acetal or aldehyde sub-stituent from a silane having the general formula:

A\
/ Si ~ (CH2)n Y
C

. where each of A, B and C, which are similar or different, is a methoxyg ethoxy, methyl or ethyl group, provided that at least one of A, B and C is capable of reacting with a hydroxyl group in the carrier; n is 2 or 3, and Y is an acetal or aldehyde group.
. The present invention also provides a method of pre-paring carriers ~' .

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5~2 with an acetal group as claimed above, comprising reacting a mineral carrier having hydroxyl groups, a particle size of 40 ~m to 5 mm, a specific surface area of 2 to 600 m2/g, a pore diameter of 600 to 10,000 A and a pore vol~e of 0.5 to 1.8 ml/g with a silane having the general fo~mula:

;~ A
~ B ~ Si-(CH2) -Y
C
. ~ ' where each of A, B and C, whlch are similar or dlfferent, is a methoxyJ
ethyoxy, methyl or ethyl group, provided that at least one of A, B and C is capable of reacting with a hydroxyl group in the carrier; _ is 2 or 3, and Y
represents a -CH-(OR)2 or -CH2-0-C6H4-CH-(OR)2 group in which R represents a methyl or ethyl group.
Carriers grafted by silicon derivatives ha~ing an acetal sub-stituent are obtained by reacting a mineral carrier having hydroxyl groups with a silane having an acetal substituent and 1 to 3 groups which will react ~i~ with the hydroxyl groups of the carrier.
The mineral carrier must include hydroxyl groups and must have a particle size from 40~um to 5 mm, a specific surface area of approximately 2 ~: 2 to 600 m ~g, and preferably 2 to 100 m2/g, when used for fixlng enzymes, a pore diameter of approxlmately 600 ~o 10000 A and a pore volume of 0.5 to 1,8 - ml/g.
Carriers which have these features include brick, glass~ mineral silicates, metallic oxides such as aluminas and more particularly silicas.
- The compound to be grafted is a silane which has an acetal sub-stituent and is of the general formula A

B ~ ( 2)n C

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765~2 - A3 B and C, which may be similar or different, represent a methoxy, ethoxy, methyl or ethyl group, provided that at leas~ one of substituents A, B or C
is capable of reacting with a hydroxyl group of the mineral carrier, - n is 2 or 3.
- Y is a -CH-(OR)2 or CH2-O- ~ C~-(OR) group, wherein R represents a methyl or ethyl radical.
Some examples of compounds more partlcularly suitable :; :
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765~2 : for grafting include the following Eormulae: -~
` ~ (CH30)~ - SI - CH2 - CH2 - CH - (OCH3)2 ; (C2H50)3 - Si - CH2 - CH2 - CH - (OC2H5)2 (C2X5~)3 - Si - CH2 - CH2 - CH2 - CH~(OC2H5)2 (CH30)2 - li - CH2 - CH2 - CH - (OC2H5)2 9 ;' ~H - (OC2H5)2 (C2H50)3 - SI - CH2 - CH2 - CH2 - O ~ t (CH30)3 - Si - ~H2 ~ CH2 ~ CH2 ~ ~ ~ -CH - (OCH3)2 .'~
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,~ The grafting reaction may be carried out by any known methods in a solvent or aqueous medium, at atmospheric pressure or under pressure and generally at elevated temperature.
If carriers with the silicon graft carrying an acetal ; substituent are hydrolyzed in a hydrochloric medium, the silicon graft of the carriers obtained will carry a corresponding alde-hyde substituent.
10Hydrolysis may be brought about e.g. by dispersing the ; carrier, with the silicon graft carrying an acetal substituent 3 in a concentrated aqueous solut~on of hydrochloric acid then by heating the dispersion at boiling point for 15 to 20 hours. The carrier is then saparated, then washed with water.
Grafted carriers, according to the invention, may be used in chromatography, affinity chromatography and more partic-ularly for fixing enzymes.
In this case a great many enzymes may be fixed, such as trypsin, glucoamylase, invertase, papain, urease and ~-galactos-idase, although the invention is not restricted to this list.
The enzyme may be fixed by any known methods, either . . , ~7~i5~;~

~old in aqueous solution, buffered according to the pH most compatible with the enzyme, or hot in hydrocar~on or chlorin-ated solventsO by the method disclosed in application Serial ` ~o~ 2079447 , filed August20, 19740 and entitled "Process for Fixing En~ymes on Supports'~
Choice of one of the methods depends on the nature of the enzyme. Similarly the choice of carrier and silicon graft dep~nds on the specificness and the peculiar characteristics of the enzyme reaction.
Thus~ for enzymes which are difficult to react, it is preferable to use a carrier where the graft carries an al~
;~ dehyde substituent, which is more reactive than the ac~tal substituent. This is the case of glucoamylase among other ' materials; glucoamylase can be fixed more ea~ily on the alde-hyde substituent than on the acetal substituent.
Enzynes thus fixed are stable and resistant to fact- -ors of denaturing, pH and temperature.
However, it is possible to increase the stabili~y o~
carrier-enzyme complexes by hydrogenating the complex, more particularly w~en the graft is aliphatic.
- This may be done by any of the known methods of hy-droge~ation in aqueo~s solution, using compounds which liberate nascent hydrogen, such as sodium borohydride, with an alkaline pH or aluminum lithium hydride, in a quantity sufficient to ob-tain complete hydrogenation.
Some non restrictive examples will now be given to illustrate the invention~

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100 g of silica micro s~heres of a particle size rang-ing from 100 to 200 ~m, a specific s~irface araa o 44 m2/g~ a ~7~5~2 pore diameter of ~00 A and a pore volume of 0.97 ml/g is dried at 150C under vacuum for 4 hours.
The dried silica is admixed with 200 ml of ~ylene containing, in solution, 20 g of silane of the formula (C2H50)3-Si-(CH2)2-CH-(oC2H5)2, then the dispersion obtained is heated at boiling point for 4 hours and stirred.
~hen it has cooled, the silica is filtered, washed with acetone and finally dried.
The product contains 1% by weight o ca~o~, deter-mined by micro analysis.
Exam~le 2 50 g of the grafted silica from Example 1 is treated with 100 ml of an aqueous solution of 6 ~ hydrochloric acid at boiling point for 20 hours. When it has cooled, the carrier i~ sep~ated, then washed with water until it is neutral.
The grafted silica obtained is the same as in Example 1 but with an aldehyde group. The carbon content is 0.8% by weight.
Example 3 Example 1 is repeated, but with the 20 g of silane replaced by 15 g of silane of the formula (C2~so)3-si-~cH2~3 o ~
-(OC2M5~2-The product contains 1% by weight of carbon.
Example 4 20 g of the grafted silica from Example 3 is treated with 40 ml of an aqueous solution of 6 N hydrochloric acid ~or 18 hours at ambient temperature. The carrier is separated and then washed with distilled water.
q~he silica obtained i grafted by a silane carrying t ~7~5~;~

an aldehyde group. The carbon content is 0.~/0 by weight.
Example 5 - Fixing of trypsin.
100 mg of the silica ~rom Example 1 is added to 5 ml of a 2 g/l solution of trypsin in 0~05 M Phosphate buffer, 5 pH 7, at 4C~ The dispersion obtained is agitated continuously ~t 4C for 15 hours.
When it has been decanted, the carrier is washed 6 -; times by suspending it in distilled water and its activity is measured: a mlxture of 100 mg of the carrier-enzyme complex and 10 ml of 0.6 M methyl DL-lysinate solution in distilled ; water is kept at 30C and pH 5.5.
The speed at which L-lysine is liberated is followed by means of a Phstat, by measuring the volume of 0.1 ~ caustic soda required to keep the pH at 5.5, as a function of time.
244 micro-moles of L-lysine is found to be liberatedper minute per gram of complex.
Example 6 - Fixing of trypsin.
1000 mg of the grafted silica ~rom Example 1 and 40 ml of a o.l M pho~p~ate buf~er solution at pEI 7 of trypsin containing 2 g/l are put into contact fox 18 hours at 4C.
When the carrier-enzyme complex has been decanted, it is washed 3 times by suspending it in 20 ml of the same pyrophosphate buffer in order to eliminate the enzyme which has not been fi~ed.
Half the complex obtained is added to a solution consisting of 2 ml of a 0.005 M solution of sodium borohydride in distilled water and of 18 ml of 0.005 M pyrophosphate buffer at pH 8.6, and let in contact for 4 hours a~ ambient tempera-ture O
T~hen it ~as been separated, the complex is washed -6~

~63765~2 twice with 20 ml o~ the same Dy~ophosphate bufrer an~ 6 ti~es with ~0 ml of distilled water.
- Enzyme activity is then measuxed, as in Example 5, ~ on treated and non-treated complexes. Determinations were made, - 5 a~ter 5 days and after 8 days, in complexes kept at 4C.

Enzyme activity in mic~ omoles/g of ccmplex After 5 days After 8 days ; Complex 263 125 93 Hydrogenated complex 230 218 _ .
The results obtained show the stability of the hydro-genated complex compared with the non-hydrogenated one.
~ Exam~le 7 - Fixing glucoamylase - 15 ~xample 6 is repeated, but with glucoamylase substi-~ tuted for trypsin, but enzyme activity is measured as follows A mixture of 10 ml of a 3% by weight solution o~
~tarch in 0.01 M acetate buffer at pH 4.5, and 500 mg of silico~glucoamylase complex in 2 ml of distilled water, is k~pt a.t 40C for 10 minutes.
The quantity of glucose llberated is measured in the liquid phase by the dinitrosalicylate method~ Activity is expressed in grams of glucose liberated per liter.

~ Enzyme Activity _ = 1. ter; ~lys l~ltGr ~3 Complex O.LS0 0.070 0.050 Hydrogenated complex 0.23D 0.100 0.100 3~

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After a certain loss of activity, activity is found to remain stable when the complex is hydrogenated.
Example 8 - Fixing of invertase.
The same procedure as in Example 6 is followed, with invertase substituted for trypsin, and enzyme activity is meas- -i; ured as follows:

500 mg of carrier-invertase complex in 5 ml of distilled water is put into contact, for 30 minutes at 40C, with 10 ml of a 0.0585 M solution of saccharose in distilled water and 5 ml of 0.2 M acetate buffer at pH 5.2.
The reaction is stopped by filtering and the liquid separated is brought to the boil for 5 minutes.
The quantity of reducing sugars liberated is measured by the dinitrosalicylate method7 and activity is expressed in grams of reducing sugars liberated per liter. The results ob-tained are as follows:
. _ _ . .
Enzyme e ctivity _ _ After 5 days Complex 0.250 0.130 Hydrogenated complex 0.140 0.110 Example 9 - Fixing papain.
A 1 g/l solution of papain in 0.05 M phosphate-citrate buffer at pH 4 containing 5 x 10 M cysteine and ethylene dia-mine tetracetic acid, kept at 4C, is put into contact with 500 mg of the grafted silica from Example 3 for 2 hours. When it has been separated, the carrier is washed with plenty of the same buffer to eliminate the papain which has not been fixed.

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The activity of the carrier-papain complex is measured.

A mixture of 9 ml of a 3 g/l solution of casein in 0.05 M phos-,~.
phate-citrate buffer at pH 7, and o~ 500 mg of complex in one ml of an aqueous solution or 10 3 ~ hydxochloric acid and

2 x 10-3 M ethylene diamine tetracetic acid, is kept at 37C
for 10 minutes.
After cooling and separation, 4 ml of solution is precipitated by adding 4 ml of a 10% aoueous solution of tri-chloracetic acid (T.C.A.),then flltered. The qua~tity of pep-tides liberated is measured in the filtrate by the Lowry method.
600 ~g of peptides is f~und to have been lihsrated.
The complex is kept at 4C in an queous solution of 10-3 ~ hydrochloric acid containing 5 x 10-3 M cysteine and 2 x 10-3 M ethylene diamine tetracetic acid and has the same degree of activity a month later. This demonstrates the very great stability of the complex.
Example 10 - Fixing glucoamylase.
The glucoamylase is fixed firstly (Test A) to the grafted silica rom Example 3 and secondly (Test B) to that from Example 4.
Each of the two silicas is suspended in acetonitrile, separated, then washed with water; this improves the wettability of the carrier.
500 mg of carrier is added to 100 ml of a 0.1 m ace-tate buffer solution at pH 6, containing 250 mg/l of gluco-amylase, and the dispersion obtained is stirred for 18 hours at 4~C.
;~ When the carriers have been decanted they are washe~
with the 0.1 M acetate buffer at pH 6, and enzyme activity is determined as described in Example 7.

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,~ Their activity consists of liberating 0.200 g of glucose per liter in the case of complex A and 0.500 g per : liter i~ the case of Complex B.
In t~e case of glucoamylase, a more active complex is found to be obtained if it is fixed on a carrier grafted by a silane carrying an aldehyde group.
Complexes which have been kept ~or 1 month at 4C
have the same degree of activity; this demonstrates their great stability.

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Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. Grafted mineral carriers suitable as supports for fixing enzymes, comprising an insoluble porous material having a part-icle size of from 40 µm to 5 mm, a specific surface area of 2 to 600 m2/g, a pore diameter of 600 to 10,000 .ANG. and a pore volume of 0.5 to 1.8 ml/g, said insoluble porous material having hydroxy groups to which have been grafted silicon containing radicals bearing an acetal or aldehyde substituent from a silane having the general formula:

where each of A, B and C, which are similar or different, is a methoxy, ethoxy, methyl or ethyl group, provided that at least one of A, B and C is capable of reacting with a hydroxyl group in the carrier; n is 2 or 3, and Y is an acetal or aldehyde group.

2. A method of preparing carriers with an acetal group as claimed in claim 1, comprising reacting a mineral carrier having hydroxyl groups, a particle size of 40 µm to 5 mm, a specific surface area of 2 to 600 m2/g, a pore diameter of 600 to 10,000 .ANG. and a pore volume of 0.5 to 1.8 ml/g with a silane having the general formula:

where each of A, B and C, which are similar or different, is a methoxy, ethoxy, methyl or ethyl group, provided that at least one of A, B and C is capable of reacting with a hydroxyl group in the carrier; n is 2 or 3, and Y represents a -CH-(OR)2 or -CH2-O-C6H4-CH-(OR)2 group in which R represents a methyl or ethyl group.

3. The method as claimed in claim 2 in which the carrier is selected from the group consisting of brick, glass, mineral silicates and metallic oxides.

4. The method as claimed in claim 2 in which the carrier is selected from the group consisting of alumina and silica.

5. A method of preparing grafted carriers with an aldehyde group comprising hydrolyzing the grafted carrier of claim 2 in hydrochloric acid medium.

6. Mineral carriers grafted in accordance with claim 1 and enzymes fixed thereon.

7. Mineral carriers of claim 6 in which the carrier enzyme complex is hydrogenated.