IL34055A - Water insoluble pullulanase,carboxypeptidase,dextranase and papain - Google Patents

Description

WATER INSOLUBLE to on particular rofcrenco to the nodi of enzymes by attachment to matrices this invention to tho water of enzymes attaching to cellulose derivatives and haa the provision of enzyme preparations in a form they can reused repeatedly and be stable to heat than the corresponding soluble It io well known that when is attached to an insoluble support of tho markedly affects features of tho carrier tend enhance tho stability of tho attached enzyme whereas hydrophobic features the effec Polysaccharide carriers such as fibrous cellulose and Lilly and E 1966 and linked dextran Axen and have been shown to be particularly effective in conferring stability to tho attached Commercial samples of water insoluble forms of glucose oxidaso and ficin became available in Those were obtained by reaction of tho appropriate with carboxyncthyl cellulose and marketed by Seravac Ltd l coupled with the hydroxypropyl and isothiocyanato phenoxy hydroxy propyl ethers derived from It is an object of the present invention to provide active water insoluble preparations of other enzymes wherein the enzyme is chemically coupled with the ether derived from The present invention provides a water insoluble or papain coupled to a solid More speci the present invention provides a or papain water insoluble carboxypeptidase dextranase chemically to According to the present invention there is also provided a process for preparation of a water of ether groups i e cellulose can he 13 and microequivalents of ether groups per gram of Active water insoluble preparations 0 of dextranase and papain were by this process which were more heat stable when suspended in an aqueous than the corresponding soluble The fungus fillul r synthesises a gl e n 5 designated pullulan containing and linkages in the proportions 2 2 1 and Acta 17 and in the regular sequence Jn where n is and Acta 17 797 sinilar polysaccharide designated which differs only from pullulan in certain fine details of structure has been shewn to be completely degraded to a 3 of much potential industrial by the pullulanase obtained from an Acta Since pullulanase is specific for the cleavage of 6 is useful for the debranching of the amylopectin of starches to produce dextrino of industrial i and Cereal 111 The role of sucrose in cariogenesis has recently British Dental implicates those oral such as streptococci which producre extracellular polysaccharides of the dextran hese saccharides of high viscosity and high molecular weight have found and Caries to form a major of the extracellular matrix of dental pla Early work and in vitro established that sucrose is the specific substrate by the dex ransucrase for dextran synthesis whether the was derived Leuconostoc or streptococci However considerable variation in the type of dextran synthes was among the various strains of these Whereas they all had in short on long chains units most of dextrsns were either by or and Polysaccharides of and Ba ker type and the synthesis of linked therefrom enzymes present in Acc or Claims have made that dental plaque has already formed is apparently dissipated addition of dextranase to the diet of experimental The use of dextranase attached to a water insoluble matrix should he even more The solid would not so quickly dissipated from the mouth as in the liquid It would have the further advantage that the enzyme would have an enhanced stability if it was required to he incorporated into foods or Example V hereafter describes the attachment of the enzyme Dextranase to the cellulose ether described The proteolytic enzyme carboxypeptidase A is an amino acid hydrolase derived from It hydrolyses peptides from the carboxylic acid splitting off the acid residue unless this is a basic residue or The general source is from bovine 20 1937 603 and Putnam and J 1 9 60 It is useful in research determinat on of end groups in peptides and Such a preparation purchased from Sigma Chemical Company from bovine pancreas was bound to the cellulose ether as hereinafter described in Example I II The part of the invention for providing water insoluble enzymes is that it provide a product with a retention of activity when as a of the activity that protein to the cellulose derivative display in its original The second advantage is the use of cellulose in the preparation of the ether affords a dense hydrophiii carrier available in a fine particulate for maximum surface exposure yet easily recoverable after use cr The third advantage is the much heat of the insoluble enzymes which may obtained the process of the present invention compared with the corresponding soluble enzyme giving a greater shelf a greater retention of activity at operating temperatures and enabling maximum repetitive use to be made of the The water insoluble papain may be used treat beer on a continuous for an extended period of time for the reduction of of mcthodd of carrying tho invention into ef A of 10 Sigracel type purchased from Chemical was placed in a stoppered which was subsequently evacuatedj left for one hour and filled with The flask was warned to and an aliquot 20 ml of a oolution of ether 2 of the ether in 20 added followed by an aliquot 10 of 0 aqueous eodiuia Tho solutions of ether and the sodium hydroxide were deoxygenated before being added The contents of tho flask were then well nixed and All separations were performed under The flask was then at for The reaction was transferred to a mortar and ground lightly with 2 acetic acid before being suspended and stirred for ou s with the same solvent The colluloso ether was then washed9 and ground lightly with acetone After five washings with distilled water 1 and one further washing with acetone yellow ether was collected on a filter and Reduction of the colluloso was effected by suspending the 3 a solution of tltanous in 6 acid for at Tho until excess titanous After grinding lightly in a with distilled water tho times with distilled water 1 and finally with acetone collected on a filter and yield of cellulose ether 03 A 1 00 of propyl cellulose ether hydrochloride was placed in a stoppered tube together with an aliquot 5 of hydrochloric acid 1 The tube placed in an ice and magnetically stirred 1 5 when of nitrite was After a further 1 5 minutes the was tho natant 15 of phosphate 7 added and the contents stirred magnetically for 1 5 The washing cycle was repeated three times at Tho centrifuged cellulose derivative was pullulanase 20 in acetate 5 1 was and the tube stirred magnetically for 1 8 5 in saturated sodium acetate After stirring a further 1 5 minutes o tho pullulanase derivative was subjected to five cycles of washing with 1 5 acetate and sodium chlorido 1 5 in the The pullulanase derivative wao finally with acctato buffer Tho insoluble pullulanase was finally suspended in tho ouopenoion pullulan solution One pullulanaso unit is that which liberates 1 of naltotrioso pe mg protein in 1 ni uto at and 25 A sample of pullulanaoo suspended in buffer and incubated at at 1 3 5 7 clays and o 30 pipetted directly into a magnetically stirred pullulan solution in aco buffer 5 pullulanaoo wao then determined and of tho with reagent the percentago of the original activity in tho preparation A control incubation wao performed in which the pullulanaoe wao replaced a oolution iO of an equivalent amount of free pullulanaso in acetate pH RESULTS Mode of Retention of activity day days 7 Pullulanaoo 0 None 3 0 III A sample 100 of the propyl ccllulooe ether hydrochloride in Example wao placed in a stoppered teot tube together with of hydrochloric acid 1 The tube placed in an ice and Dtirred for 15 when of sodium nitrite wao added After a further 15 minuteo the tube tho oupcrnatant 15 of phosphate uffer 075 added and the stirred magnetically for 15 The washing cycle wao repeated three at derivative wao in phosphate 075 wao tho tube was After stirring a further 15 minutes at the carboxypeptida e derivative subjected to five cycles washing with 15 phosphate buffer pH and sodium chloride in the same The carboxypeptidase derivative finally washed three times with phosphate buffer pH and the stirrer bars removed and phosphate buffer pH Carboxy peptidase activity was assayed by stirring the suspension with phenyl alanine solution 10 in phosphate buf pH at Samples were removed at times 15 minutes and pipetted directly into assay tubes containing ninhydrin reagent The Moore and Stein ninhydrin reagent always prepared 20 immediately before 2 was dissolved in citrate buffer pH ninhydrin stock solution added and white spot nitrogen passed through the solution for 15 Assay tubes were then heated at for 5 cooled rapidly on a water centrifuged and the optical density read at 570 against a blank prepared containing phosphate buffer instead of RESULTS Functional group bound Enzyme Units ac active in protein 100 derivative bound retained by binding protein enzyme after coupling phenoxy 1 hydrox ropyl tidaee unit is that which liberates 1 o o u o 20 in phosphate buffer pH 2 incubated a Aliquo 25 at ono 3 7 days and pipetted directly into a magneticall stirred solution in phoGphate buffer pH at 10 The activity of the water insoluble then determined by periodic sampling and of the digests with ninhydrin reagent ao described j Hence the percentage of the original activity in the 15 preparation was A control incubation performed in which the replaced by a solution of an equivalent amount of eo in phosphate buffer pH 2 Retention of activity coupling 3 1 day days days 7 d 79 5k 26 peptidase None A sample 100 of the propyl cellulose other hydrochloride in was placed in a test tube 25 an aliquot of hydrochloric acid 1 Tho tubo in an ice bath and magnetically stirred o 1 minutes when of sodium ni i e phosphate added tho contento stirred magnetically Tho washing cycle wao rcpoated three tinoo at Tho centrifuged cellulose dorivotivo was dextranase in phocphato buffer added and tho tubo stirred magnetically at for hours when in sodium acetate was After stirring a further 15 at tho water insoluble dextranase dcrivativo was subjected to cycl03 of washing with 15 phosphate buffe pH and chloride 15 in tho s The insoluble dextranase derivative was finally washed three with citrate buffer pH the stirrer bars removed and 10 citrate buffer pH wao A of tho water insoluble dextranase suspension in Example in citrate buffer 5 incubated at Aliquo of wero removed after tines 1 2 and 3 days pipetted directly in a stirred dextran solution The activity of tho water insoluble dextranase was then by periodic 1 9 30 and 60 and assay of tho digests with the reagent as described in Hence tho percentage of tho activity in tho dextranase was A control incubation wao of of Activity coupling 3 1 day 2 3 cxtranase o 53 Hone days 5 days 7 days After at for 3 the insoluble diazo coupled dextranase still retained of its original enzyraic Samples of phenoxy hydroxypropy cellulose ether in Example Ϊ were placed in a centrifuge Ali of hydrpchloric acid were added and the slurry stirred magnetically for 15 minutes sodium solution were added after a further minutes the tubes were centrifuged and the supernatant The solid washed 3 times with aliquots of phosphate After the final washings had aliquots of a solution of papain activity in phosphate pH containing and BDTA added and the stirred magnetically at After 5 of an solution of in saturated aqueous sodium acetate were Af a tho lc papain dcrivntlvco to cyclcD of washing with pH 15 chloride solution in tho Tho derivatives finally washed with Tho activity of tho water papain was in following suspended dissolved in tho case o in phosphate suffer of activator and wore added and tho allowed to stirred magnetically at 600 at added to 2 of aci in toot agitated and allowed to stand for Tho optical density of the solutions was read at 280 in an spectrophotometer using 1 silica cuvettes Tho activities from a plot of tho optical density change with V pH unite o coupling protein per hound activity protein retention derivative 1 A sample of 1 0 according to was carried out at gave a derivative containing protein pe derivative and with a activity equal to enzyme sample was suspended in phosphate pH mixed with an activator solution 5 containing and and incubated at 1 hour in a stirred test after which the samples were rapidly cooled and the papain activity assayed at A control incubation was performed in which the water insoluble papain was replaced by a solution of an equivalent amount of free in phosphate buffer pH 5 and activator solution Initial Pinal retention activity activity of activity diazo A sample of water insoluble papain as in Example coupling was carried out at pH gave a derivative containing 1 00 carrier and with a specific activity of 105 enzyme units per of in a 1 litre Two 1 Beer gravity 5 chilled and opened poured the centrifuge added and tho then stirred for 72 hour Tho pot for 15 1100 the into which wcro crowned and repas c Further opened and treated in same for of hours and 18 hours using the After the fina treatment the papain washed in distilled water and resuspended in phosphato 100 An aliquot of the suspension assayed as The of treated Bee together with untreated controls were stored for alternate at and to aecelerato tho of token at intervale Activity of papain Activity on Initi 2 5 ally 5 After 10 v JJ o 0 of of incubation 0 1 hours 5 of solid were suspended in phosphate pH of casein prepared phosphate buffer pH taking a 1 casein solution equivalent to The velocity of the initial reaction of the insoluble papain on casein for each dilution determined the method in Example VII Initial casein Initial Reaction concentration Velocity insufficientOCRQuality

Claims (12)

What we claim is:

1. A process for the preparation of a wate insoluble pullulanase, carboxypeptidase, dextranase or papain which process comprises reacting at 0-5°C. the pullulanase, carboxypeptidase, dextranase or papain dissolved in a buffer within a pH range of 6.5 - 8.6 with the p-diazophenoxy-hydroxypropyl ether of cellulose.

2. A process as claimed in claim 1 wherein the pH is between 7.6 and 7.7 for pullulanase, carboxypeptidase and dextranase.

3. · A process as claimed in claim 1 for the preparation of a water insoluble papain which process comprises reacting at 0-5°C. the papain dissolved in a buffer within a pH range of 6.5 - 8.5 and containing L-cysteine and diaminoethane tetra-ecetic acid with the p-diazophenoxy hydroxypropyl ether of cellulose.

4. A process as claimed in claim 3 wherein the pH is between 6.8 and 7.0.

5. · A process as claimed in claim 1 wherein unreacted diazo groups in the cellulose derivative are annealed by reaction with β-naphthol or phenol.

6. A process, as claimed in claim 1 wherein microcrystalline cellulose is used for the preparation of the ether and the degree of substitution of ether groups in the cellulose is between 13 and 56.2 micro-equivalents of j»-diazophenoxyhydroxypropyl ether groups per gram of cellulose, 3405,^2 ;

7. A water insoluble hydrolytie enzyme selected from the group consisting of pullulanase, carboxypeptidase, dextranase and papain chemically coupled to a solid matrix consisting of p-toazophenoxyhydroxypropyl cellulose.

8. Pullulanase chemically coupled to p-diazophenoxyhydroxy-propyl cellulose.

9. Carboxypeptidase chemically coupled to p-diazophenoxy-hydroxypropyl cellulose.

10. Dextranase chemically coupled to p-diazophenoxyhydroxypropyl cellulose*

11. Papain chemically coupled to p-diazophenoxyhydroxypropyl cellulose.

12. A process for the preparation of a water insoluble pullulanase, carboxypeptidase, dextranase or papain as claimed in claim 1 substantially as described with reference to any one of the specific examples herein-b before set forth. 13· A process for the treatment of beer for the reduction of haze which comprises contacting the beer with papain chemically coupled to p-diazo-phenoxyhydroxypropyl cellulose. Attorney for Applicants