IL43573A - Enzymes fixed on a solid cellulose support - Google Patents

Description

32574/3- Z 43573/2 ENZYMESFIXED ON A SOLID CELLULOSE SUPPORT 43573/2 The invention concerns new enzyme complexes wherein the latter are fixed in a stable manner on a solid support and more particularly on a cel lulosic carrier. The invention thus relates to so linked enzymes and to a process of their preparation.

The fixation of enzymes on sol id carriers has been the subject matter of research work during the last decade in order to make possible enzymatic reactions in a heterogenous phase. In fact it would be interesting to recover the enzyme once its effect in the reaction has been concl uded.

On the one hand the purity of the final product would be higher if the enzyme would not remain in the reaction mixture - as generally is the case; on the other hand the operation would be more economic, especial ly in the case of costly enzymes. This problem is of particular importance when we deal with rennet which becomes less and less available at present, while this substance i s very much needed in the manufacture of cheese, the demand growing steadi ly. It has also been suggested to fix enzymes on solid carriers especially on agarose and cellulose so as to serve in continuous systems, however these efforts did not lead to practical results; even after the acti vati on of the carrier by agents such as cyanogen-hal ides, glutaraldehyde and the l ke the complexes with the enzymes did not have the necessary stabil ity. Further Green and Crutchfield (Biochem J. (1969) , p. 183-190) , who have worked on this problem and tried to obtain the coagulation of milk in the presence of rennet fixed on pure cellulose -activated by glutaraldehyde did not succeed in their efforts, as far as industrial use thereof was concerned. On the other hand, R. Axen and S. Ernback (Eur.J.Bichem. 18 (1971 ) , p. 351-360) , having cellulose activated with cyanuryl- 43573/2 hal ides in order to fix thereon proteases did find that the obtained compound did not have any proteolytic acti vity.

In contradistincti on to the abo«e, the present Invention departing from the known art, permits the attachment of different enzymes, selected from rennet, chymotrypsin, trypsin and beta-gal actosi dose, to a cell ulose carrier and in that way to obtain sol id, water-insoluble complexes which are stable and of good enzymatic activity. The invention therefore makes possible its application to industri al processes , particularly 1n devices with enzymatic beds , both stationary and mobile ones. According to one form of the invention the new enzyme complexes have the advantage that they can be used in much greater granules, than it was possible hitherto. In one particular application - which is of great importance - namely coagulation of mi lk, the new complexes according to the Inventi on lead to considerable economy of rennet used and further therein that the cheese thus produced is free of rennet.

The invention uses no longer pure cel l ulose which has been activated by known means, as indicated above, but instead resides in the unknown idea of using most profoundly modified cel l ulose, particularly chlorinated, or sul furyl ated cell ulose. There are known different processes for the preparation of cel l ulose derivatives, such as esters, ethers , halides , sulfonates and the l ike. Amongst them, chlori nation and sul fochlori nation have been widely used wi th cel luloses. These modified cel l uloses are adapted to the attachment of enzymes so as to result in a solid cell ulose-enzyme complex whi ch is both very active and stable, provided that this chlorination or sulfochlori nation process 1s performed In a l iquid permitting a good and thorough dispersion of the cell ulose.

Thus^ process according to the present invention for the fixation of an enzyme on a cellulose carrier is characterized thereby that the cellulose used contains in its molecule chlotine -and/or sulfur atoms. This sulfo- and/or chloro-cellulose is contacted with an aoqueous solution of an enzyme in the presencd of an appropriate pH buffer during a period of time sufficient for the attachment of a substantial quantity of enzyme on the cellulose carrier.

Roughly, the contents of CI and S in the molecule of the cellulose may vary between zero and 4$ and zero and 7$> respectively, it being understood that there is always CI and or S present in the molecule. The contents of chlojlne generally is 0.5$ to 4$ by weight, preferably between 1 and 1.6#* there being practically no sulfur present in the cellulose.

In the caee of cellulose containing sulfur- normally in the form of oxides of sulfur, which are very well suited or the purposes of the present invention, the contents of sulfur may be e.g. between 1$ to 7$ by weight. Whenever S and CI are both present the contents of CI should always be less than than . that of S and preferably less Jay- a.hnuft l#-»that it is to say between 0 and 1$. As to sulfur the preferred proportions are approximately between 3$ and $.

The sulfo- and/ or chloro-eelluloses adapted for the use in the present invention may be products obtained by the action of known chlorinating agents, such as PCI , PCl^, oxalyl chloride, succinyl chloride or any other suitable organic acid and especially sulfochlorlnating agents, such as thionyl chloride (SODlg) or sulfuryl chloride (SOgClg). The cellulose is preferably treated by an alkaline solution and is subsequently washed and dispersed in an organic liquid which is not affectdd by the chlorinating whi ch i s i ntroduced i nto rth'e. ce l l ulose di spers on may be l eft : to react duri ng- a period of time which is not more than a few hours, at moderate termperature - not exceeding 60°C. Normally -according to the kind and nature of the cellulose used -the temperature will be betxireen 10°C and 55°C. After separation of the thus treated cellulose from the reactimmixture, it is washed and dried before the attachment of the enzyme. PreferaBly the addition of the chlorinating agent to the cellulose dispersion is effected during the course of the reaction. m prepared The p-2ap.a_eafcie.a--.Qf sulfochorinated cellulose may e/by the method described by Carre and aucleres (Comptes Rendus de l'Academie des Sciences, (1931 ) 192-1567 )$ and by R.L.Boehm (J* Org* Chem. (1958 ) 23-1716 ) * that is to say by means of thionyl chloride in pyridine. However, this methodr leads to with a the celluloses whieh-has too high-a-content of chlorine and containing relatively little sulfur and do not lend themselves well to the conditions of this invention, as set ut above* Thus, had the reaction conditions have to be adjusted in order to obtain satisfactory chlooo- and/or sulfochlorinated celluloses. The c omposition of the treated product depends also on the nature of the cellulose used as starting material.

As to the proportion of chlorinating agents to be used, this depends on the factors indicated above and on the nature of the organic liquid used in which the cellulose is dispersed. complexes The preparation of eorapounds accordin to the invat4on is effected by contacting treated cellulose with a buffered enzymatic solution. The pH of the Buffer is between 5 and 10 and best results are obtained at values between 6 and 9. The temperature of the reaction medium is preferably below 45°C and in any case below the limits of tohermal stability of the enzyme. This ° the enzyme to the activated cellulose requires a period of contact of between 24 to 48 hours.

The proportion of enzyme which is reacted with the cellulose carrier varies in wide limits. It is between 0.1 and 100 by weight, preferably between 1% and 6 hy ©eight of the cellulose used.

Amongst enzymes suitable for attachment, to activated cellulose are: hydrolases of sugar metabolista, such as invertase, amylase, raaltase, pectinase; hydrolases of protein metabolism such as proteases, peptidases, trypsine, chymotrypsine, lysozyme and other hydrolases auch as phosphatases and ribonucleases. comprises The invention is-eepeeially-suited-for complexes of cellulose with rennet, with beta-galactosidase and chymotrypsine.

One preferred embodiment of the present inventioniis based on fce the unexpected discovery that cellulose^ containing a certain proportion of lignine" constitutes - after chlorination or sulfo- the chlorination - a much better carrier for enzymes than corresponding has been derivatives of pure cellulose. Hitherto it was always tried to prepare cellulose as pure as possible in GEder to serve as an enzyme carrier since ife was believed that the presence of lignin and other vegetable matter which might accompany the cellQlose would preveHfc the attaehement of enzymes. This is correct in principle Insofar as/too high content of lignin is of the enzyme. feature harmful to fixation/and- the unexpected element of the present invention besides therein that while too much of lignin is harmful, a certain proposition is very favourable, provided that in""ttte"-StractW?€"Of~€h"e "chosen"cellulose the 1lgnin^ is dispersed in the cellulosic mass and is not concentrated on therefore the surface thereof, ensuring a-strthe cohesion of granulates or fibres.

The new enzyme complexes attached on cellulose according to the present invention is characterized in that the cellulo^ic carrier contains between 5 and 25$ of lignin by weight of dry material the preferred proportion being between 10$ and 20$.

Cellulose containing moderate quantities of lignin and itfhich is adapted for use in the present invention may be derived from any of knonn natural sources, such as from wood ^whereof waste may be used say, saw-dust or chips, straw or hay and the like. Paper pulp may also be utilised, provided that a lignin contents- as indicated above is present. A special source which is highly recommended in view of its quality of attachment of enzymes is stalks of cereals and particularly of com.

Lignifled .cellulose treated in accordance with the invention is obtained in the form of pieces or particles of varying dimensions, from between 0.2 mm up to several centimetres, according to the origin of the material and the kind of apparatus used inhe preparation. Generally, the best dimensions are 0.3 to lOmmillimetres, and preferably between ob 0.5 and 5 rani. With such dimensions there are attained efficiently surfaces of aontact sufficient for/utilizing the activity of the cellulose/ enzyme complex. In the knonra art - that is to say with pure cellulose it was impossible to use fibres of a diametre above 0.05 mm which of course did not permit the use of a stationary bed.*ii^x-ims--a c_a^^ •and--l-o&s--of- - r<) Abit-i-z--dimension..

A cellulose which gives particularly advantageous results is obtained from corn stalks reduced to pieces of 0.5 - 10 ram. previously activated by the action of thionyl^ chloride in the presence of an excess of pyridine so that the final product contains papproxlraately between 2 to 6% of sulfur- preferably between 3 and 4 , the contents of chlorine being alvays less than that of sulfur, pre e&bly by 1J&, The process of the invention for the preparation of iiqui-lignif ied lea cellulose enzyme completes comprises comminuting a cellulose containing an adequate quantity of lignin, to pieces or particles of the size indicated above, treating normality these with an alkaline solution of 3 - 6 ttormai preferably .5N, so as to dissolve the lignin present on the surface of the pieces. This treatment is preferably effected at room temp pwrature, during 10 to 30 minutes. The cellulose grains are separated from the liquid and washed with plenty of cold water until the filtrate is no longer colored.

Subsequently the pieces are dehydrated with an alcohol and rinsed with pyridine. Into suspension of these grains there is successively added SOCl^, o possibly another chlorinating agent, the temperature is adjusted to about up to 60°, preferably at or in the vicinity of 55°C. The obtention of a containing temperature control influences the resul ing carrier jjhich comprises an optimum conten of S and 01, as indicated above.

The ratio of S/Cl is as shown in the graph of French Patent Application No.

After standing for about one hour the reaction is stopped by chilling e.g. by introducing the reaction mixture into a bath of ice water. The grains are separated and the thoroughly rinsed with water and then pl-i with an acid so¾¾ ion of^about pH 2, up to the complete elimination of pyridine. Finally the grains are rinsed with alcohol and dried at a moderate temperature - preferably belovT 50°.

The attachment of the enzyme to the grains thus prepardd takes place in an aoqueous buffer the pH of which is adjusted νσ¾e particular enzyme. As example, for rennet there is advantageously used a phosphate buffer having a pH of 5.9 to 6· . The material is agitated at a moderate temperature, normally of about 50°C and for a period of time required for the attachment of the enzyme, normally in the order of about 24 hours.

Afteto separation of the grains thus bonded with the enzyme, the remaining enzyme which is not cotalently bonded is removed by rinsing with a buffer having a properly adjusted pH and with water. It is recommended to keep the thus obtained' rains at a relatively low temperature in a buffer of a pH at which the enzyme is stable.

The following examples are by way of examples not Intended to restrict the scope of the invention.

Example 1.

Sulfo-Chlorination of cellulose in the presence of pyridine, a. 32 grams of cellulose (chromatographic grade) were treated with an a<£queous solution of 18$ of NaOH during two hours. The so obtained alkali-cellulose wash- washed on a sintered glass with absolute methanol until the filtrate ceased to be basic. It was repeatedly washed with pyridine and suspended in 800 ml. pyridine.

To this suspension were added 146 ml. of thionyl chloride S¾312 of 9 ^ purity and were reacted during 4 hours at o 28 C. At the end of that period of time the reaction mixture was placed in a bath of ice water†4&e and agitated during one hour. The cellulose was then separated by filtra- s tion and suspended overjni^ht in a .demi-saturated sodium-_ solution of sediura bicarbonate. Next morning the cellulose was washed until the filtrate became clear. Then the cellulose powder which had been so treated was dried during 24 hours at 50°C. It contains 3.5$ of chlorine and 4.4$ of sulfur. were b.c.d. ¥he above operations had-been repeated three times, twice with different reaction times with SOClg and once at a different temperature. In each case the quantity of chlorine and sulfur in the cellulose was determined.

The following results were obtained: temperature duration g (a) - 28°C 4 h 4.40 3.57 (b) - 4°C 11 6.48 3.85 These results show that due to the pyridine the sulfur is bonded to the cellulose during the first hour of the reactinn and its holding capacity decreases if the operation is extended at a temperature of 28°C; on the other hand the bonding of chlorine increases after the first hour. Thus it is possible , „ „ , _ „ . and .the time of the reaction by a proper control of temperature during the reaction /to obtain a desired content of chlorine and of sulfur.

Example 2 Sulfo- Chlorlnation of cellulose in the presence of dioxane; The procedure is the sameaas in Example 1, starting witha a suspension of 60 grams of cellulose in 50 m 1. of dioxane.

There are added 30 ml. of S©C12 which corresponds to the maximum quantity of chlorine which the cellulose can bind in dioxane.

After one hour of reaction the reaction mixture is filtered, rinsed with water, and dried. The contents of chlorine in the cellulose is only 1.51$ and it does not increase even if the amount of su £0chlorinating agent is increased or the time of reaction is prolonged; contrarily to what occurs in pyridine where the proportion of chlorine increases with time.

Example 3» Treatment with sulfuryl chloride.

Prodedure according to Example 1 but the concentration~in" the ing not to heat too chlalnatea c&^ xon._pr.Qdu.at. is reduced in order to.-prevent._taa_ much starting , , , , . ^ , ■¾ , « hig -heating._up_o£ the^ixtrsre which could bring about a degradation of the cellulose; thus there are added only 44 ml. of sulfuryl chloride, i.e. 0.55 mole.

Example 4 Chlorlnatio by phosphorus trichloride: Procedure as in Example 3 with 50 ml., i.e. 0.6 mole of Example 5.

Chlorination by phosphorus pentachloride: The PClg of Example 4 (carried out in pyridine) is replaced by 62.5 grams of PCl^, i.e. 0.3 mole which.-had--been- dissolved 3.X 6 in dioxane. Thus in this treatment there present both pyridine and dioxane.

Example 6» Preparation of cellulose-rennet complex; 16 grams of sulfo-chlorlnated cellulose of Example la are brought into contact with 40 ml. of a rennet solution diluted H five times in a phosphate buffer of /β.2 pH, during 48 hours.

Temperature is maintained between 4 and 6°C with agitation. ¾nzyme complex At the end of the period of contact the cellulose /is washwd with 250 ml. of a phosphate buffer ofH6.2 pH, 250 ml. of an aqueous solution of sodium ctiorlde (5 moles per litre) and 250 ml. of water. Then the com ex is suspended in 250 ml. of an acetate buffer of pH 4.4, i.e. near the pH of milk at a temperature of 4 to 6°0, with magnetic agitation during one remove hour. This treatment is carried out in order to e¾©sepfe- that fraction of enzyme which had anot been covalently bonded. After a further filtration the enzyme which had been rendered insolublized is placed under same conditions in 250 ml. of acetate rennet complex ©o obtained is dispersed in 100 ml of the said buffer acetate of ' . pH and is kept in refrigeration at 4 to 6°C. The activity of 16 grams of this complex is equivalent to 0.12 ml. of industrial rennet.

Example 7« Cellulose-chymotrypsin^ complex: i Into tubes of plastic material used for centrvtfugation are introduced 500 ml. of cellulose which have been treated in accordance with Example 2, to this are added 10 ml. of a buffered anzymatlc solution of pH 9, the buffer being boric acid Cl¾fa0H/ which is commercially available under the name of Eitrisol. The tubes are then placed on a vibration for ten minutes at ambient temperature. Then they are subjected to centrifugation during 20 min utes at 2500 r.p.m. The supernatant portion is removed whe2¾¾on- the sediment is rinsed in order to remove the part of enzyme which had not been bonded, the washing being effected with 10 ml of a solution of sodium chloride containing 5 m¼es of NaCl per litre. After agitation, further centrifufeation is effected for 20 minutes. The sediment is treated with 10 ml of a buffer of pH 8.5 commercially avail* able under the name of "Tris" which is 2-amino 2- hydroxymethyl- 1,3-propane-diol ¾¾jh 10 ml of "Tr-itrisol" at pH/as indicated enzyme , above. Two further rinsings are made after which the trjaa.te.4 complex cellulose /is dried. The proteolytic acitivity of the dry at complex is determined on aaseineat 8.5 pH and 37 . In a conical vessel 300 ml, ofte13mplex and 20 ml. of a caselne solution (or de-creamed milk) are put together in order to hydrolise the caselne. At the end of 10 minutes of incubation 10 ml. of the solution are taken out and placed in a test tube containing already 10 ml. of trichloroacetic acid in order to in a bath maintained at 37° in order to precipitate the whole Of casein which had not been hydrollzed. The liquid is filtered and the quantity of liberated tyrosine is determined with a spect6meter by measurin the optical density of the 8 it _ . Thus is has been found mg that the activity of 500 ml. of cellulose-chymotrypsine complex is equal to 0.8 ¾!. of free, chrymotrypsine. against 10 ml. of de-creamed milk under same conditions.

Example 9» Complex of beta-galactosldase-cellulose To 13 grams of modified cellulose according to Exammple 1, 40 ml. are added of a enzyme solution diluted in 100 ml. of a buffer pH 7.2 constituted by 10~2 mole per litre of potassium phospkate and 10"^ mole per litre of magnesium chloride. The (O.N.P.G. ) per ml. The mixture is agitated during 43 hours at 4o°C. After first filtration the complex is cas ed and suspended in the following solutions:- 1. Water 150 ml.

Phosphate Buffer pH 6.2 150 ml. 2. NaCl, 5 moles per litre- 150 ml.

Water - 150 ml. 3. Phosphate buffer,pH - 150 ml.

Follows filtration. The enzyme ixed on the cellulose carrier is kept in the phosphate buffer pH 6.2.

The activity of the complex obtained in this way, starting from 13 gram of modified cellulose is estimated at . free 1000 units ofVbeta-g^Lactosidase. The activity of beta- galactosidase is determined by its hydrolysing action on pyranoside o-mitrophenyl-beta-D-galactos-icia-se (O.N.P.G.) which causes a yellow color of the O.N.P.G. solution, this bein due to liberated o-nitrophenol. The quantity of hydrolized O.N.P.G. is measured by determination of optical density at 4200 S.

Example 10.

Llgnified - cellulose rennet complex Corn stalks containing approximately 80$ of cellulose and 15$ of lignine is comminuted to grains of 1.5 up to 2.5 mm. 50 grams of these grains are placed in a solution of-soda-- of aOH of 18$ which is agitated during 15 minutes at normal temperature The grains are separated and thoroughly washed with cold water until the filtrate ceases to be colored. Washing is ¾ terminated with 1 litre of methanol, followed by 400 ml, of pyridine.

The grains are poured into 800 ml. of pyridine whereafter 100 ml. of S0Q12 are added drop-wise to the agitated pyridine during 5 minutes. The temperature of the reaction mixture rises to 55°C and the material colors intensely and suddenly.

One hour after the introduction of thionyl chloride the mixture s poured into a bath of iced water; the grains are then separated and rinsed with several litres of water and subsequently by a solution of HC1 at pH 2, until total elimination of absorbed pyridine occurs* Rinsing is concluded with 1 litre of methanol.

The so-obtained grains are dried at 40°C, It is found that so treated cellulose contains about 3.5$ of sulfur and only traces of chlorine.

I to 160 ml. of a buffer solution of phosphates of pH 6·2 there are introduced 16 grams of the prepared grains and 40 ml of rennet commercially known under the name of Carlln. The whole is maintained at 4o°C with agitation during 24 hours.

The grains are now separated and the chemically not bound rennet is removed by rinsing; - first with 200 ml. of the same Suffer solution and then By 2 litres of distilled water and finally by 250 ml. of a acetate buffer solution of pH 4.4. The mother solutions of these treatcssnts may be re-used. The grains of the rennet cellulose complex from corn stalks which have so been obtained are kept at 4°C in 250 ml. of acetate buffer. This complex has given excellent results in the coagulation of milk; 8 grams of that complax (including 0.04 geam of fixed rennet did coagulate in a continuous operation 35 litres of milk in 55.5 hours while by conventional coagulation with disolution of rennet JLn the. milk the same quantity of 0.04 grams can coagulate only 0.4 litres, the rennet bein definitely lost.

Enzyme complexes according to the invention retain an enzymatic activity for a long period of time if submitted to a lyophylisation treatment and when kept at low temperature. The activity may be maintained for two years and more .

The invention extends also to ether applications for cellulose enzyme corapleaes as described and to enzymatic industrial processes, particularly to continuous coagulation of milk.

Claims (16)

43573/2 'WHAT IS CLAIMED IS :

1. Process for the preparation of a complex^ covalently bonding -to- an enzyme -^selected from rennet , chymotrypsln » tryps1n :., beta-gal actos1dOSe to cell ulose , characterized In that a cellulose derivative containing chl or ne and/or a sulfur oxide 1nV e moledule 1s used, which derivative is dispersed 1n an aqueous solution of the enzyme, In the presence of a buffer whi ch maintains the pH of the solution at a value between 5 and 10, leaving the sol uti on In contact at a temperature of between 0 to 45°C during a period of time necessary for the bonding of the enzyme to the cel lulose.

2. Process as claimed in claim 1, characterized 1n that the cellulose derivative results from a treatment of cellulose with a chlorinating or suifochlorinatlng agent, preferably 1n the form of an al kal i -cellulose dispersed 1n an organic liquid which Is not affected by the said chlorinating agent.

3. Process according to claims 1 or 2 characterized 1n that the organic liquid Is pyridine.

4. Process according to claim 1 or 2 characterized In that the organi c liquid Is dioxane.

5. Process according to cl aim 2,3 or 4 characterized in that the chlorinating agent 1s a phosphorus chloride or an acyl -chloride.

6. Process as cl aimed in claims 2 ,3 or 4 characterized In that the suifochlorinatlng agent 1s thlonyl chloride or sulfuryl chloride.

7. Process according to any one of claims 1 to 6 characterized 1n that the cel lulose used as a starting material 1s llgnlfted and contains from 5¾ to 25% by weight or Hgnln, the said Hgnlne being dispersed 1n the mass of cellulose. 43573/2

8. Process as claimed 1n claim 7 characterized in that the lignine cor nt e in the cellulose used as starting material in between 10% and 20% by weight.

9. Process as claimed in claims 7 and 8, characterized in that the Hgnified cellulose originates from wood, straw and preferably from stalks of cereals especially corn.

10. Process according to any one of the claims 1 to 9 characterized 1n that the chlorine content of the cellulose derivative 1s between 0% and 4% by weight, that of sulfur between 0% and 7%, the said derivative containing always at least one of the elements CI and S.

11. Process as claimed in claim 10 characterized thereby that the sulfur content of the said derivative is between 1 and 7% , the proporation of chlorine always being below that of sulfur and less than 1¾.

12. Process according to claim 11 characterized thereby that the sulfur content is between 3 and 4% by weight.

13. Cellulose-enzyme complexes, whenever obtained by a process as claimed in any of claims 1 to 12.

14. Enzyme complexes comprising enzymes defined 1n claim 1 covalently bonded to chlorinated or sulfochlorlnated cellulose.

15. Enzyme complexes as claimed 1n claim 17, wherein the cellulose 1s lignified cellulose.

16. Enzyme complexes as defined 1n claim 17 substantially as herein before described and with reference to any of