EXTRACTION AND PURIFICATION OF HYALURONIDASE FROM CRUSTACEAN WASTE

Abstract: The invention relates to a method for the extraction and purification of proteins, such as beta (1}4) glycanohydrolase (hyaluronidase) particularly from the solid waste generated during the processing of marine crustaceans, such as Decapods, which include the Natantian and Reptantian groups such as stomatopods, shrimps, lobsters and crabs. The enzyme is extracted into solution by homogenising the head-waste with 0.25 M sucrose and then precipitating it by the addition of an organic solvent such as acetone or other precipitating agents such as high molecular weight polyethylene glycol. This enzyme extract dissolved in a suitable volume of 10 mM sodium-acetate buffer, pH 5.4 containing 10 mM potassium chloride (or sodium chloride) is chromatographed on Amberlite<TM> IRA 420 anion exchange resin and selectively eluted by subjecting the loaded column to a continuously increasing gradient of chloride ions. The eluent is 10 mM sodium acetate buffer at pH 5.4, containing a starting concentration of 10 mM potassium chloride (or sodium chloride) and rising continuously to 500 mM. Eluted fractions from the column showing enzyme activity are pooled and concentrated by anti-dialysis to yield a highly purified and concentrated form of hyaluronidase suitable for food and/or biomedical applications. This purified enzyme preparation is then subjected to a final polishing stage on Sephacryl<TM> S-300-HR size exclusion column with 10 mM sodium-acetate buffer at pH 5.4 containing 10 mM potassium chloride (or sodium chloride) and 1 % glycerol. The resulting eluate containing the enzyme is pooled and concentrated by anti-dialysis against 50 % polyethylene glycol (PEG) 6000 in deionised water to yield a highly purified and concentrated form of the enzyme suitable for therapeutic or food application.

Description: Title of Invention: EXTRACTION AND PURIFICATION OF HYALURONIDASE FROM CRUSTACEAN WASTE Technical Field This invention relates to a relatively cheap and easy technique with minimum handling steps for the extraction and purification of proteins, particularly hyaluronidase specifically β(l-*4) glycanohydrolase which may be extracted from the solid waste generated during the processing of crustaceans such as Decapods. This purified enzyme is suited for food and/or biomedical applications. Background Hyaluronidase refers to an endo-enzyme that depolymerises the hetero- polysaccharide hyaluronic acid present in all animal connective tissues by cleaving the internal β(l→4) or β(l-»3) linkages between N-acetylglucosamine and D-glucuronic acid residues or D-glucuronic acid and N-acetylglucosamine residues respectively of the said polysaccharide. The current major commercial source of this enzyme is the bovine and sheep testes. However, extraction and purification procedures are complicated with several handling steps, relatively expensive and time consuming. Moreover, recovery during purification is also very low and in all provides low purification levels, high running costs and thereby an expensive product. The use of various ion-exchange materials for the purification of protein is well documented. However, purification of hyaluronidase (4-glycaπohydrolase or endohexosaminidase) from Crustacean waste on such conventional ion-exchange media had proved expensive, inadequate and time consuming with recoveries of 20 to 25% in most cases. Also experiments conducted by the inventor have shown that it is impossible to use the two Amberlite~ resins CG 50 and IRC 50 commonly used for the purification of hyaluronidase by several previously documented authors. This is possibly thought to be due to loss of cations on the resin during purification since both Amberlite 1 * CG 50 and IRC 50 are reported by Rohm and Haas', the manufacturers of the said resin, to split the alkaline salts of monovalent cation as well as those of multivalent cations that are suspected to be essential for retention of biological activity of hyaluronidase extracted from the Crustacean waste. This makes both Amberlite CG 50 and IRC 50 totally unsuitable for the purification of this enzyme. Crustacean processing waters were known to contain this enzyme. But there has been no clear extraction and purification procedure for a commercial exploitation of this enzyme from the solid waste itself. However, an enzyme showing endoglucuronidase-like activity, i.e., an enzyme capable of depolymerising hyaluronic acid by the degradation of the internal β(l→3) linkage between the D-glucuronic acid and N-acetylglucosamine residues of hyaluronic acid (hence the name endoglucuronidase or 3-glycanohydrolase) has been reportedly purified from Euphausia superba. This enzyme prepared through several complicated stages from the Euphausiid group were shown to have a maximum activity of only 295 Units per mg protein. Further from the reported enzyme reaction studies, the inventor has concluded that this was an endoglucuronidase and not an endohexosaminidase. There are also reports on the purification of hyaluronidase from human placenta that again involves several complex handling stages. Another report also provides a process for the production of an enzyme preparation from crude hyaluronidase suitable for injection. Thus the survey has revealed that there is no clear existing method for the purification of this enzyme from the solid Crustacean waste involving a relatively simple and inexpensive technique with a high degree of recovery and purity. The applications of this enzyme in food and biomedical fields and the preparation of specific dosage units for therapeutic applications from a crude preparation of this enzyme are all well documented. Objects and Summary of the Invention On the basis of the available technical background it is the object of the present invention to provide a simple and inexpensive technique to extract or purify a protein. It is a particular object of the present invention to provide improved techniques for obtaining enzymes such as hyaluronidase. In the case of hyaluronidase it is a further object to provide a method of extracting the enzyme from solid waste from the processing of Crustaceans, such as Decapods. A related object of the invention is to provide a partially purified hyaluronidase from the solid Crustacean processing waste suited for further purification. According to the present invention there is provided the method of purifying and/or extracting protein comprising contacting a sample contaimng said protein with a basic gel-type I resin having a matrix, which matrix does not contain a β linkage. The present invention is particularly directed towards the extraction and/or purification of enzymes, notably enzymes which depolymerise hetero-polysaccharides, such as hyaluronidase. The sample may be any sample containing the required protein. In the case of hyaluronidase, the sample may be, for example, extracts from animal testicles or human placental material or, preferably, solid waste generated during the process of crustaceans such as Decapods. It is preferred that the resin is a strongly basic gel, and it is particularly preferred that the matrix is a styrene-DNB matrix. In particularly preferred embodiments of the present invention, the resin may be one of the Amberlite™ 4000 series, the Amberlite 400 series, Ambeηet 4200 or a dowex resin. It is particularly preferred that the sample is contacted with the resin in column chromatography. The invention describes the use of Amberlite~ IRA 420 which is a strongly basic gel type I resin with styrene-DNB matrix having the functional group -Ν + (CH 3 ) 3 supplied in the ionic form with Cl " as the exchange ion for the purification of hyaluronidase from solid Crustacean processing waste. Neither Amberlite"' IRA 420 nor the related resins of this category (such as IRA 400, IRA 416, Amberjet 4200, etc.) have been hitherto reported for the purification of any protein. Whilst extensive applications of these resins have been reported for water conditioning (deionisation including silica reduction, deoxygenation, silica removal, etc.); removal of weakly acid contaminants from processing streams, etc. Amberlite'" IRA 420 and the related resins referred to above are therefore, a unique material for the purification of proteins amd have not previously been considered suitable for this use. It has suφrisingly been found that the basic gel type I resins, which have a matrbc which does not contain a Q 1 "* linkage, are suitable for extraction and/or purification of proteins, contrary to the teaching of the prior art. These resins have been found to be particularly useful for the extraction of enzymes from samples, notably hyaluronidase from a wide range of samples, particularly solid waste generated during processing of crustaceans. In this respect, the present invention further provides a method of purifying and/or extracting hyaluronidase from waste generated by processing of crustaceans, which method comprises contacting said waste with a basic gel type I resin having a matrix, which matrix does not contain a β linkage. It is preferred that, prior to contacting the waste with the resin, the enzyme is extracted from the waste, for example in an isotonic solution such as 0.25 M sucrose. More preferably, subsequent to the extraction step prior to the contacting of the waste with the resin, unwanted proteinaceous material is precipitated from the waste, for example by straining under pressure not exceeding 51b per square inch through a metallic or nylon screen of a pore size less than 100 microns or by centrifugation. Most preferably, subsequent to the extraction and/or precipitation of unwanted protein but prior to the contacting of the waste with the resin, bulk purification takes place, either by precipitation of the enzyme or in a fluidised bed column. If bulk purification is to take place by precipitation of the enzyme, this preferably takes place by centrifugation of the enzyme preferably in a pH range of 4 to 6.5 or, alternatively, by merely standing the sample to attain sedimentation and subsequently decanting the supernatant fluid for further processing. If bulk purification takes place on a fluidised bed column, it is preferred that the fluidised bed column uses a resin such as an ionic exchange media or a hydrophobic interaction resin. It is particularly preferred that subsequent to contacting the waste with the resin, further purification of the extracted protein is effected, preferably by anti-dialysis. In one embodiment of the present invention, the Amberlite"' IRA 420 resin is equilibrated before use by washing it repeatedly in 50 mM sodium-acetate buffer at pH 5.4 containing 10 mM potassium chloride (or sodium-chloride) until the pH registers a steady value of pH 5.4. The resin is then packed into a suitable column and further equilibrated with the same buffer until the bed is completely packed. The object of partial purification is accomplished by solubilising the enzyme present in the solid Crustacean processing waste by homogenising and/or turbulent mixing of the said waste with isotonic sucrose (0.25 M sucrose) at 4°C for a suitable period of time. The crude solution obtained is centrifuged or strained under pressure with a pore size less than 100 microns to remove cell debris and solid wastes. The enzyme present in the fluid component is extracted by organic solvent precipitation using acetone or by using high molecular weight polyethylene glycol in the molecular weight range of 400 to 6000. The suitable precipitate containing the enzyme is dissolved in a small volume of 10 mM sodium-acetate buffer at pH 5.4 containing 10 mM potassium chloride (or sodium-chloride). The crude enzyme preparation can now be purified by passing it through the said column packed with Amberlite"' IRA 420 type I anion exchange resin. The enzyme is bound on to the resin under the said equilibrating conditions and is selectively eluted from the resin in the column using a continuously increasing gradient of chloride ions generated by the use of potassium chloride (or sodium chloride) in the limiting eluting buffer of the same molarity and pH. Those fractions eluting out of the column showing enzyme activity are pooled and concentrated to a desired volume by anti-dialysis using a pharmaceutical grade dialysis bag for a suitable length of time against 30 to 50% polyethylene glycol 6000 or polyethylene glycol 8000 in 10 mM sodium-acetate buffer at pH 5.4 containing 10 mM sodium chloride (or sodium chloride) and 1% glycerol to a desired volume. The resulting concentrate is a partially purified form of hyaluronidase suited for the final polishing step on Sephacryl™ S-300-HR resin for biomedical application or may be used as such without further purification for food application. Sephacryl 1 " S-300-HR is a composite gel formed by covalently cross-linked allyl dextran with N,N'-methylene disacrylamide to form a hydrophillic matrix of high mechanical strength. The resin, as was done for Amberlite™, was washed several times at +4°C in the acidic buffer 10 mM sodium-acetate containing 10 mM potassium chloride (or sodium chloride) and 1% glycerol. The gel is then packed into two suitable columns in series and further equilibrated with the same buffer until the bed is completely packed. The two columns are connected in series so as to obtain an ascending flow in the first column and a descending flow in the next. The concentrated enzyme from the Amberlite 1 " column was applied on the said column set up containing Sephacryl™ S-300- HR and eluted using the said acidic buffer system used for equilibration. The enzyme fractions obtained from this polishing step on Sephacryl™ S-300-HR is again concentrated by anti-dialysis against 30 to 50% polyethylene glycol 6000 or polyethylene glycol 8000 solution in deionised water to a suitable volume. The complete method according to the present invention is performed in the following manner. Detailed Description of the Invention The present invention will now be further described with reference to a particular processing step. In this respect, reference is made to accompanying Figure 1 which shows a flow diagram illustrating this method. In producing the enzyme hyaluronidase described in this invention, the preferred source is the solid Crustacean processing waste, preferably the head waste containing the hepatopancreas. The material should be as fresh as possible and if stored should have been kept at conditions maintained for fresh Crustaceans which is normally below -18°C since higher storage temperatures can result in microbiological contamination and possible destruction of the enzyme. The head waste is homogenised with 0.25 M cold sucrose solution at +4°C at a solid Crustacean processing waste to sucrose ratio of 1:2.5 (w/v) to cause dissolution of the desired enzyme. The resulting mixture contains besides the desired enzyme several unwanted and undissolved proteinaceous material, cell debris and shell fragments that are removed by centrifugation at 1000 x g for 30 minutes at +4°C or by straining under pressure through a nylon or metallic mesh with pore size less than 100 microns. The fluid so obtained contains hyaluronidase which is removed from the solution by fractionation using an organic solvent or by using high molecular weight polyethylene glycol in the molecular weight range of from 400 to 6000. The precipitate from this step containing the enzyme is dissolved in a minimum volume of 50 mM sodium-acetate buffer at pH 5.4 containing 10 mM potassium chloride (or sodium chloride) and 0.25 M sucrose. It is essential that the enzyme be dissolved in an acidic buffer to stabilise it and prevent denaturation during subsequent stages of purification. Although 10 mM Tris-HCl, citrate and phosphate buffers were found compatible, the enzyme was found to exhibit up to approximately 70% or even lower activities in these buffer systems as compared to the acetate buffer. Further, any contact with NaOH or HCl often used for pH adjustments of buffer systems caused irreversible loss of biological activity of the enzyme. The crude enzyme preparation thus obtained by the above procedure is filtered through a 10 micron mesh size nylon filter under pressure (not exceeding 5 psi) for the removal of undissolved and suspended proteinaceous or other similar undissolved particles. This solution can now be applied on the equilibrated Amberlite™ IRA 420 resin packed in to a suitable column. The preparation of the Amberlite™ IRA 420 anion exchange resin is performed as follows. All operations are carried out at the column running temperature of +4°C. The Amberlite™ IRA 420 anion exchange resin is washed several times using several volumes of 50 mM sodium-acetate buffer at pH 5.4 containing 10 mM potassium chloride (or sodium-chloride) and finally suspended in the same buffer. The wet slurry of 75% Amberlite™ IRA 420 resin in the said buffer is now packed into a suitable column fitted with suitable filters at both ends that permits flow of liquid but not the resin itself from the column. Further equilibration is continued with 10 mM sodium- acetate buffer at pH 5.4 containing 10 mM potassium chloride (or sodium chloride) until the bed is completely packed without any air bubbles in it and the eluting buffer shows a steady reading of pH 5.4. The column is now ready for the actual purification scheme that is performed as detailed below. The enzyme obtained from the solid Crustacean processing waste dissolved in the buffer as described earlier in this invention is loaded on to the column containing the equilibrated Amberlite™ IRA 420 anion exchange resin using a peristaltic pump connected to it. The column is now washed with 3 to 4 column volumes of 10 mM sodium-acetate buffer, pH 5.4 containing 10 mM potassium chloride (or sodium- chloride). Consequently, the column is selectively eluted using a continuously increasing strength of chloride ions generated by using a potassium chloride (or sodium chloride) gradient from 10 mM to 500 mM in four to five column volumes of 10 mM sodium- acetate buffer at pH 5.4. The continuous ionic strength gradient of chloride is achieved by the following method. The required amount of 10 mM sodium acetate buffer at pH 5.4 is prepared. To one-half of this buffer is added exactly calculated quantity of potassium chloride (or sodium-chloride) to obtain an ionic strength of 10 mM and to the other half is also added an exactly calculated quantity of potassium chloride (or sodium-chloride) to obtain an ionic strength of 500 mM. The pH of the buffer is maintained at 5.4 and the two exactly measured and equal volumes of buffer placed in suitable containers that are connected from the base in series by a suitable tubing. The first container containing the 10 mM potassium chloride (or sodium chloride) buffer is continuously stirred without introducing air bubbles in to it and connected to the column via a peristaltic pump. As the buffer level in this container drop, the buffer from the second container flows in to maintain equal levels in both containers which results in a continuous gradient of chloride ion being produced. A flow rate of up to 82 ml per hour was easily attainable without any adverse effect on recovery and purity of the enzyme preparation in the elute from the Amberlite™ IRA 420 resin under the set up described above. The elute from the column during gradient elution are monitored by the UV monitor at 280 nm and collected in suitable fractions which are subjected to analysis for enzyme activity. The appropriate fractions containing enzyme activity are pooled and concentrated by anti-dialysis against 50% polyethylene glycol 6000 in 10 mM sodium-acetate buffer containing 10 mM potassium chloride (or sodium chloride) and 1% glycerol at +4°C followed by equilibration with 10 mM sodium acetate buffer at pH 5.4 containing 10 mM potassium chloride and 1% glycerol (for example by dialysis) The enzyme may also be concentrated by accelerated freeze drying after the addition of a protein stabiliser (or biological extender) and the removal of all buffer salts (for example by repeated dialysis against deionised water). This concentrated product is a highly purified form of hyaluronidase suitable for food applications and biomedical applications. It was also found suitable far an extremely high purification/polishing step by size exclusion chromatography using Sephacryl™ S-300-HR resin in two columns connected in series. The size exclusion chromatography on Sephacryl™ S-300-HR is performed as detailed below. Sephacryl™ S-300-HR is equilibrated by washing it several times at +4°C in the acidic buffer 10 mM sodium-acetate at pH 5.4 containing 10 mM potassium chloride (or sodium chloride) and 1% glycerol. Suspended in the same buffer to obtain a 70 to 80% slurry, the resin is poured into two upright columns without air bubbles being trapped in the gel. Equilibration with the same acidic buffer {10 mM sodium-acetate at pH 5.4 containing 10 mM potassium chloride (or sodium chloride) and 1% glycerol} is performed in the same manner as for the Amberlite™ column. The two columns were connected in series so as to obtain an ascending flow in the first column and a descending flow in the next. A flow rate of up to 94 ml per hour could easily be obtained by this set up without loss of recovery or purity of the eluting enzyme. The concentrated enzyme following Amberlite™ purification was applied on the said column set up and eluted using the same acidic buffer system as used for equilibration. The eluate as before is monitored by the UV monitor at 280 nm and suitable fractions collected which are analysed for enzyme activity. Fractions with high enzyme activity are pooled and concentrated as before by anti-dialysis against 30 to 50% polyethylene glycol 6000 or polyethylene glycol 8000 in deionised water. Illustrating specifically a laboratory model for the extraction of hyaluronidase from the Reptantian Crustacean, scampi (Nephrops notvegicus) waste is described here. About lOOg (wet weight) of head waste from scampi containing the hepatopancreas, excluding the external shell component, is homogenised in 250 ml cold 0.25 M sucrose at 4°C and the resulting mixture centrifuged at 1000 x g for 30 minutes at 4°C. The supernatant fluid which is referred to as the crude extract is made up to 250 ml with 0.25M sucrose. At this stage the enzyme extraction can be carried out either by (1) direct fractionation using the organic solvent acetone; or (2) by adjusting the fluid to an acidic pH of 5.0 with IM acetic acid with constant stirring and leaving the solution at 4°C for 15 to 30 minutes to attain equilibrium. Subsequently the resultant flocculates are removed by centrifugation at 1000 x g for 30 minutes at 4°C. This clear supernatant called the pH precipitated supernatant contains the enzyme in solution and can be used for fractionation using the organic solvent acetone; or (3) by polyethylene glycol fractionation of the crude extract with or without pH adjustment {to pH 5.0 as under (2), above}. The organic solvent used is acetone. The said solvent is cooled to a temperature range within -15 to -20°C and the said aqueous solutions containing the enzyme are cooled to a temperature range of +2 to +4°C. The extraction of hyaluronidase from the solutions are carried out in the following manner. In the direct acetone fractionation technique (hitherto referred to as Procedure I for convenience), the volumetric ratio of said solution and crude extract is brought to 1:0.6 by adding the solvent in a slow thin stream to the constantly stirred crude extract and mbced without frothing. The process is carried out at a temperature between -15°C to -20°C. After the addition of the required quantity of acetone, the resulting solution is left at -20°C for 10 minutes to achieve equilibration. The precipitant so formed is separated from the solution by centrifuging at 3000 x g for 15 minutes at -20°C. Further acetone at -20°C is added to this supernatant extract until a final volumetric ratio of 1:0.9 is obtained between the original crude extract and solvent. After the addition of all the acetone, the resulting solution is once again left at -20°C for 10 minutes to achieve equilibration. The resulting precipitate obtained between a volumetric ratio of said solution and solvent from between 1:0.6 to 1:0.9 is now collected by centrifugation at 3000 x g for 15 minutes at -20°C and dissolved in approximately 50 to 60 ml volume of 10 mM sodium-acetate buffer at pH 5.4 containing 10 mM potassium chloride (or sodium-chloride) and 0.25 M sucrose. In the second technique referred to above (hitherto referred to as Procedure II), the pH precipitated supernatant is used. The enzyme is fractionated from the pH precipitated supernatant solution by the addition of acetone at -20°C at a volumetric ratio between said solution and said solvent from 1:0.3 to 1:0.7 under the same conditions of time, temperature and centrifugation speeds as described for the direct acetone fractionation technique, above. The resulting precipitate is also dissolved in approximately 50 to 60 ml volume of 10 mM sodium-acetate buffer at pH 5.4 containing 10 mM potassium chloride (or sodium-chloride) and 0.25 M sucrose. In the third technique fractionation of hyaluronidase from the crude extract is achieved by the use of polyethylene glycol 6000. The fraction obtained between 1:0.2 to 0.325 (v/w of crude extract and polyethylene glycol) contains the desired enzyme which is extracted from the solution by centrifugation. Polyethylene glycol fractionation is carried out by the addition of accurately weighed quantity of polyethylene glycol to the crude extract and dissolving it by slow stirring to avoid frothing at +4°C. Equilibration is achieved after the addition and dissolution of polyethylene glycol in the crude extract by leaving the solution undisturbed for 15 to 30 minutes at +4°C. The desired precipitate is harvested by centrifugation at 3000 x g for 60 minutes at +2 to +4°C and dissolved in approximately 50 to 60 ml volume of 10 M sodium-acetate buffer at pH 5.4 containing 10 mM potassium chloride (or sodium-chloride) and 0.25 M sucrose. The purification of the crude extracts of hyaluronidase obtained by either of the three procedures described above can now be carried out on the Amberlite ™ column in the following manner. The Amberlite™ IRA 420 anion exchange resin is washed several times using several volumes of 50 mM sodium-acetate buffer containing 10 mM potassium chloride (or sodium-chloride). The wet slurry of 75% Amberlite™ IRA 420 resin is now packed into a conventional chromatographic laboratory column of 65 cm x 2.6 cm (length x internal diameter) fitted at both ends with suitable adapters with nylon mesh net filters that permit flow of liquid but not the resin itself from the column. The column is fixed vertically in a cold room at + 4°C and the top end-piece adapter connected to a peristaltic pump. Further equilibration is continued for 3 to 4 hours by pumping in 10 mM sodium-acetate buffer at pH 5.4 containing 10 mM potassium chloride (or sodium-chloride) and 0.25 M sucrose until the bed is completely and evenly packed without any air bubbles in it. The flow rate is maintained at 0.75 to 1.0 ml per minute. The column is now ready for the purification scheme which is performed as detailed below. The suitable precipitate containing the enzyme from the solvent fractionation step above dissolved in 50 to 60 ml of 10 M sodium-acetate buffer at pH 5.4 containing 10 mM potassium chloride (or sodium-chloride) and 0.25 M sucrose is loaded on the Amberlite™ IRA 420 resin using the peristaltic pump. The resin in the column is now washed by pumping 10 mM sodium-acetate buffer at pH 5.4 containing 10 mM potassium chloride (or sodium-chloride) at a flow rate of one ml per minute and the elute from the column monitored by a UV monitor at 280 nm. When as detected by the UV monitor there is no more protein coming out of the column which normally requires three column volumes of the buffer the column is connected to an apparatus that produces a continuous gradient of chloride ions by using 10 mM to 500 mM concentration of potassium chloride (or sodium-chloride) in 500 ml of 10 mM sodium acetate buffer at pH 5.4. The elute from the column at a flow rate of 82 ml per hour are collected at 10 minute intervals and analysed for enzyme activity. Normally, under the conditions described here, the fractions obtained between 1.6 to 2.8 column volume are found to contain the enzyme. Those fractions showing enzyme activity are pooled and concentrated by anti-dialysis at +2 to +4°C against 50% polyethylene glycol 6000 in 10 mM sodium-acetate buffer containing 10 mM sodium chloride and 1% glycerol to a final volume of 5 to 20 ml. A typical result obtained by the above procedure is 294 fold purification over the crude extract and over 87% recovery of the chromatographed enzyme (see Table 1). The resulting product is, therefore, a highly purified form of hyaluronidase and is found to be suitable without further treatment for the final extreme high purification step/polishing stage by size exclusion chromatography (as for example on Sephacryl™ S-300-HR gel filtration columns). The concentrated enzyme preparation from the Amberlite™ column may be used as such for food applications. A 10 ml volume of the concentrated and partly purified enzyme preparation obtained after Amberlite™ IRA 420 purification (base material from Procedure I) was applied on the Sephacryl™ S-300-HR size exclusion column and eluted at a flow rate of 60 ml per hour using 10 mM sodium acetate buffer at pH 5.4 containing 10 mM potassium chloride (or sodium chloride) and 1% glycerol. The column elute was again monitored at 280 nm using the UV monitor and fractions collected at 10 minute intervals. Suitable fractions showing high enzyme activity were pooled and dialysed against 30% polyethylene glycol 6000 in deionised water to obtained an extremely highly purified form of the hyaluronidase enzyme capable of depolymerising hyaluronic acid at it's β(l→4) linkage and hence the name β(l→4) glycanohydrolase (or β- endohexosaminidase or hexosaminidase or hyaluronoglucosidase). A 15 ml volume of the concentrated and partly purified enzyme preparation obtained after Amberlite™ IRA 420 purification (base material from Procedure II) was also applied on the Sephacryl™ S-300-HR size exclusion column and eluted as described above but at a higher flow rate of 94 ml per hour. Once again fractions showing high enzyme activity were pooled and dialysed against 50% polyethylene glycol 6000 in deionised water to obtained an extremely highly purified form of the hyaluronidase enzyme capable of depolymerising hyaluronic acid at it's β(l→4) linkage. The higher flow rate showed no adverse effect on recovery and purity of the enzyme from the column. Anti-dialysis against both 30% and 50% polyethylene glycol also gave similar results, with the only advantage of using 50% polyethylene glycol being a reduction in anti-dialysis time from about 6 hours for the former experiment to about 4 hours for the latter. The activity of hyaluronidase enzyme and protein content in the samples were measured as described below. The actual hyaluronidase activity was measured by a modification of the method given by Linker (1974) 1 .The method is based on the principle that, hyaluronidase being an endohexosaminidase catalyse the degradation of hyaluronic acid to form oligosaccharides with N-acetylglucosamine terminal groups which can be measured in a colorimetric assay developed by Reissig et al (1985) ? . The amount of oligosaccharides with N-acetylglucosamine terminal groups liberated per unit time is, therefore, a measure of the hyaluronidase activity. The substrate was used at a concentration 1.5 mg/ml in 50 mM sodium-acetate buffer, pH 5.4 containing 0.15M sodium chloride. For the actual assay, 400μl of hyaluronate solution (1.5 mg/ml) and lOOμl of enzyme sample were pipetted into test tubes, mixed and incubated at 25°C for 10 minutes, unless otherwise stated. Following incubation, lOOμl of 0.8M potassium-tetra-borate solution was added, mixed and the test tube heated for 3 minutes in boiling water bath and cooled immediately in cold water. To this was added 3 ml p-dimethylaminobenzaldehyde reagent (10% p-DMAB in glacial acetic acid containing 12.5% HCl). The solution was mixed, incubated for 20 minutes in 37°C water bath, cooled in cold water and the absorbance measured at 584 nm. If the solutions were turbid it was centrifuged to clear before reading. Protein concentrations were determined by the Protein Assay Procedure No. P 5656 (Revised Oct. 1989) provided by the Sigma ® Chemical Co., with crystalline bovine serum albumin as standard. The method is essentially the Lowry et al (1951) 2 method as modified by Peterson (1977)-. For chromatographic works, the absorbance (E) of the protein solution to be determined was measured at 280 and 260 nm using a 1 cm silica cell. The Ε^ S0 /B 260 ratio was calculated and the appropriate correction factor given by Warburg and Christian (1941)≥ was applied to obtain the protein concentration from the formula: Protein concentration (mg/ml) = Factor X E^. A set of typical results obtained by the above two purification schemes described earlier in this invention from Nephrops norvegicus are shown in Tables 1 and 2. As can be seen the recovery and purification fold achieved by the second method which involved an acidic pH precipitation step is superior to that obtained by the first method which did not involve an acidic precipitation step. For comparative purposes bovine testicular hyaluronidase obtained from Sigma® Chemical Company (Sigma ® Product No. H-3631 shown in Sigma ® 1994 Catalogue) was used as standard. Results obtained showed that the commercially available highly purified bovine testicular hyaluronidase had a specific activity of 3000 Units/mg protein. However, the highly purified hyaluronidase obtained from scampi through Procedure I and II after the polishing up stage showed a specific activity of 5420 and 6300 units/mg protein respectively. Activity measurements were made during a twenty minute incubation study at 20°C at the optimum assay conditions of sodium chloride and pH arrived at for the bovine testicular hyaluronidase and scampi hyaluronidase respectively in 50 mM acetate buffer. Hyaluronidase obtained by the procedure described in this invention from Nephrops norvegicus was found to have a molecular weight of 320 KDa, a pH optimum of 5.4 in sodium-acetate buffer and an absolute requirement of 0.12 M sodium chloride concentration in the final incubation mixture. The enzyme also exhibited a substrate specificity towards hyaluronic acid with no activity detected towards the other closely related polysaccharides such as Chondroitin sulphate A Chondroitin sulphate B, Chondroitin sulphate C, Heparin and even chitin and chitosan. Also, the presence of Chondroitin sulphate A or B or C or heparin in the incubation mixture caused moderately low to complete inhibition of the enzyme towards hyaluronic acid depending on their relative concentration in the medium- While most embodiments of the invention have been described herein, it will be understood that variations and modifications are possible without departing from the spirit of the invention. For example, the enzyme may be extracted from the solid waste in any isotonic solution such as a glucose or fructose solution and precipitated from the crude extract by using other the organic solvents such as methanol, ethanol, propanol etc., or the salt or solutions of polyethylene glycol in the molecular weight range of 400 to 6000, ammonium sulphate, potassium sulphate, etc., or also by aqueous two-phase separation systems, etc., which can all be further purified on the Amberlite™ IRA 420 resin under the specific conditions arrived at in this invention. The closely related resins Amberlite™ IRA 400 and Amberjet™ IRA 4200 would also be suitable for this purification scheme. The ionic strength and pH of the buffer may also be modified within limits. This purified enzyme preparation can be further subjected to a polishing stage by size exclusion chromatography on two Sephacryl™ S-300-HR columns connected in series using 10 mM sodium acetate buffer system at pH 5.4 containing 10 mM potassium chloride (or sodium chloride) and 1% glycerol at +4°C. Concentration of the dilute enzyme is achieved by anti-dialysis against 30 to 50% polyethylene glycol 6000 or polyethylene glycol 8000. Other concentration steps that may be applicable include standard techniques such as ultrafiltration and freeze drying. But freeze drying must be conducted only after the addition of a biological extender since the enzyme looses all biological activity on freezing. Table 1 : Summary of results obtained on the purification of hyaluronidase from scampi processing waste by Procedure I. Sample Volume Protein A584/ml A584/mg/ Purification Recovery (mg/ml) / 20 min 20 min fold (ml) A280/ Lowry A280/ Lowry A280/ Lowry % % 260 260 260 original chromat graphed Crude extract 120 45 7.83 1.85 0.04 0.2 1.0 1.0 100.0 - 0.65-0.9 acetone fraction 34.3 9.24 4.3 3.22 0.35 0.75 8.5 3.2 50 - 30 ml of the acetone fraction chromatographed on Amberlite™ IRA 420 column and concentrated by anti-dialysis against polyethylene glycol 6000. Cone, samples after purification on Amberlite™ 17.8 0.685 0.25 4.3 6.3 17.1 153.2 73 39 79 10 ml of concentrated Amberlite™ purified fraction chromatographed on Sephacryl™ S-200-HR column and concentrated by anti-dialysis against polyethylene glycol 6000. Concentrated fractions after gel filtration 19.6 0.036 0.04 2.14 59.4 54.0 1440 230 26 98 1 Note: All values are rounded off to the nearest two or one decimal points after the final calculations. Sample weight used for the extraction of the enzyme: 48g 0 Table 2 : Summary of results obtained on the puπfication of hyaluronidase from scampi processing waste by Procedure II. Note: All values are rounded off to the nearest two or one decimal points after the final calculations. Sample weight used for the extraction of the enzyme: 44g REFERENCES 1. LINKER, A. (1974). Hyaluronidase. In: Methods of enzymatic analysis: Vol. II. 2nd Edition, Ed by Bergmeyer, H.U., Academic Press, Inc., New York: 944- 948p. 2. REISSIG, L.J.; STROMINGER, LJ. and LELOIR, F.L. (1955). A modified colorimetric method for the estimation of N-acetylamino sugars. The Journal of Biological Chemistry, 217(5): 959-966p. 3. LOWRY, O.H, ROSEBROUGH, N.J., FARR, A.L. and RANDALL, K.J. (1951). Protein measurement with the Folin-phenol reagent. Journal of Biological Chemistry, 193: 265-275p. 4. PETERSON, G.L. (1979). Review of the Folin Phenol quantitation method of Lowry, Rosenburg, Farr and Randall. Analytical Biochemistry, 100: 201-220p. 5. WARBURG, O and CHRISTIAN, W. (1941). Iso Lierung und kristallisation des Garungsferments, Enolase. Biochem. Z., 310: 384-421p.

Claims: CLAIMS 1. A method of purifying and/or extracting protein comprising contacting a sample containing said protein with a basic gel type I resin having a matrix, which matrix does not contain a β linkage. 2. A method according to Claim 1 wherein the protein is an enzyme. 3. A method according to Claim 2 wherein the protein depolymerises heteropolysaccharides. 4. A method according to Claim 3 wherein the protein is hyaluronidase. 5. A method according to any one of Claims 1 to 4 wherein the sample is solid waste generated during processing of crustaceans. 6. A method according to Claim 5 wherein the crustaceans are Decapods. 7. A method according to any one of Claims 1 to 6 wherein the resin is strongly basic. 8. A method according to any one of Claims 1 to 7 wherein the matrix is a styrene-DVB matrix. 9. A method according to any one of Claims 1 to 8 wherein the resin is one the Amberlite™ 400 series, the Amberlite ™ 4000 series, the Amberlite™ 900 series, Amerjet 2400 or a dowex resin. 10. A method according to any one of Claims 1 to 9 wherein the sample is contacted with the resin in column chromatography. 11. A method of purifying and/or extracting hyaluronidase from crustaceans comprising contacting waste material obtained from solid waste generated during processing of crustaceans with a basic gel type I resin having a matrix, which matrbc doe snot contain a β linkage. 12. A method according to Claim 11 wherein the resin is strongly basic. 13. A method according to Claim 11 or 12 wherein the matrbc is a styrene- DNB matrix. 14. A method according to any one of Claims 11, 12 or 13 wherein the resin is one of the Ameriite™ 400 series, the Amberlite™ 4000 series, the Amberlite™ 900 series, Amberjet™ 4200 or a dowex resin. 15. A method according to any one of Claims 11 to 14 wherein the sample is contacted with the resin in column chromatography. 16. A method according to any one of Claims 11 to 15 further comprising the step of extraction of the sample from said solid waste prior to contacting the protein with the resin. 17. A method according to any one of Claims 1 to 16 further comprising the step of precipitating unwanted protein from the sample prior to . contacting the sample with the resin. 18. A method according to Claim 17 wherein precipitation takes places by straining. 19. A method according to Claim 17 wherein precipitation takes place by centrifigation. 20. A method according to any one of Claims 11 to 19 further comprising bulk purification of the sample prior to contacting the sample with the resin. 21. A method according to Claim 20 wherein bulk purification is effected by precipitation of the enzyme. 22. A method according to Claim 21 wherein said precipitation takes place by centrifugation. 23. A method according to Claim 22 wherein during centrifugation, the sample is at the pH range from 4 to 6.5. 24. A method according to Claim 21 wherein precipitation takes place by leaving the sample to stand and subsequently decanting the supernatant. 25. A method according to Claim 20 wherein bulk purification takes place by contact with a fluidised bed column. 26. A method according to any one of Claims 11 to 25 further comprising the step of further purification of the protein subsequent to contacting the sample with the resin. 27. A method according to Claim 26 wherein said further purification is effected by anti-dialysis. 28. A method for the extraction and purification of hyaluronidase from solid Crustacean processing waste comprising: (A) Extraction of the enzyme hyaluronidase from the solid Crustacean processing waste in an isotonic solution such as 0.25 M sucrose (B) Precipitating the unwanted proteinaceous material by subsequent centrifugation or straining under pressure not exceeding 5 psi through a metallic or nylon screen of pore size less than 100 microns. (C) Adjustment of the crude extract solution to pH 5.0 using IM acetic acid (D) Cooling the enzyme solution to +2 to +4 degrees Celsius (E) Precipitating the desired enzyme by centrifugation from the cooled extract containing the enzyme of Claim 1(B) by means of an organic solvent cooled to a temperature of from -15 to -20°C present in a volumetric ratio between the said crude extract and solvent of from 1:0.6 to 1:0.9 maintained at a temperature range of -15 to -20°C. (F) Precipitating the desired enzyme by centrifugation from the cooled extract containing the enzyme of Claim 1(C) by means of an organic solvent cooled to a temperature of from -15 to -20°C present in a volumetric ratio between the said aqueous solution with pH adjustment and solvent of from 1:0.3 to 1:0.7 maintained at a temperature range of -15 to -20°C. (G) Precipitating the desired enzyme by centrifugation from the cooled extract containing the enzyme of Claim 1(B) or Claim 1(C) by the addition of polyethylene glycol having a molecular weight of from 400 to 6000 in a ratio between the said aqueous solution of crude enzyme extract and polyethylene glycol of from 1:0.2 to 0.325 (v/w) and maintained at a temperature range of +2 to +4°C. (H) Dissolution of the crude enzyme precipitate from Claim (E) or (F) or (G) in 10 mM sodium-acetate buffer at pH 5.4 containing 10 mM potassium chloride (or sodium-chloride). (I) The preparation of Amberlite™ IRA 420 anion exchange resin by repeated washing with 50 mM sodium-acetate buffer containing 10 mM potassium chloride (or sodium-chloride) and its equilibration in the column by 10 mM sodium-acetate buffer containing 10 mM potassium chloride (or sodium-chloride). (J) The selective elution and thereby the purification of hyaluronidase on the Amberlite™ IRA 420 column using a continuous gradient of chloride ions obtained by the use of acetate buffer containing 10 mM to 500 mM potassium chloride (or sodium chloride) in 4 to 5 column volumes of 10 mM sodium-acetate buffer at pH 5.4 . (K) Concentration of the pooled fractions showing enzyme activity under Claim 1(J) by anti-dialysis against 30-50% polyethylene glycol 6000 or 8000 in 10 mM sodium-acetate buffer containing 10 mM sodium chloride and 1% glycerol. (L) Polishing of the concentrated and partially purified enzyme from Claim 1(L) and/or (J) by size exclusion chromatography on Sephacryl™ S-300-HR gel filtration media. (M) Final concentration of the highly purified enzyme from Claim 1(L) by anti- dialysis against 30-50% polyethylene glycol 6000 or 8000 in deionised water.

Inventors: KRISHNAPILLAI ASHOK MELUTTUKEZ, KRISHNAPILLAI, ASHOK, MELUTTUKEZHAKETHIEL
Applicants: KRISHNAPILLAI ASHOK MELUTTUKEZ, WILCHAP I LIMITED, KRISHNAPILLAI, ASHOK, MELUTTUKEZHAKETHIEL
Application year: 1996
Main classification: C07K1
IPC codes: C12N9