FI84835C - A process for the recovery and concentration of polygalacturonase from a solution - Google Patents

Description

84835

METHOD FOR THE RECOVERY OF POLYGALACTURONASE AND

FOR CONCENTRATION OF THE SOLUTION

The present invention relates to a process for recovering and concentrating polygalacturonase from solution by adsorbing the polygalacturonase protein to titanium oxyhydrate.

Adsorption has long been used in the chemical and biochemical industries and in oil refining to remove process solutions and gaseous contaminants and to separate components. As early as 1916, Nelson and Griffen (1) adsorbed the enzyme invertase to activated carbon and alumina. Enzymes and other proteins have since been adsorbed to a number of inorganic adsorbents for the purpose of purifying proteins either from other proteins or from other: impurities, immobilizing enzymes, or separating all proteins from solution.

In his 1953 review, Zittle (2) lists several inorganic adsorbents: calcium phosphate, diatomaceous earth, bauxite, bentonite, alumina, zinc and calcium carbonate, activated carbon, silica gel, and magnesium and aluminum hydroxide. In 1976, Morris and Morris (3) mention titanium dioxide in addition to the above, stating that it has little use in the adsorption of proteins due to its crystalline, non-porous structure. Stability and availability of very pure material are good properties of titanium dioxide for adsorbent use. AERE (Atomic Energy Research.. Establishment, Harwell UK) has developed 2,84835 macroporous, spherical, especially suitable for column chromatography, adsorbents from a variety of inorganic materials. Miles and Thomson (4) mention the following: silica gel, bentonite, porous glass, alumina, calcium phosphate, barium sulfate and magnesium pyrophosphate, and titanium dioxide.

A 1986 review by Green and Wasen (5) identifies the possibility 5 of selectively separating proteins by varying binding and elution conditions.

As a result of AERE's development work, the English Sterling Organics Ltd has launched several macroporous, inorganic adsorbents under the brand name Macrosorb. Macrosorb T is an adsorbent made of titanium dioxide for adsorption of proteins, immobilization of enzymes and cells, extraction of microbial culture solutions, removal of impurities and chemical catalysis (6). Macrosorb T

is chemically titanium dioxide, TiO2.

From the results obtained with titanium dioxide, Miles and Thomson (4) present the separation of tissue homogenate lactate dehydrogenase, phosphoglycerate kinase and malate dehydrogenase as well as the separation of proteins. 15 from wastewater from grain processing and cheese making. Thomson (7) has used

titanium dioxide also in the recovery of bromelain from pineapple press. Wase et al. (8) describe the use of titanium dioxide to separate enzymes produced by Bacillus cereus, cephalosporinase and penicillinase, both directly from the culture medium and from the clarified culture medium. More than 90% of the activity of both enzymes is bound to titanium dioxide. The enzymes are cleaved, e.g., 1 M

with ammonium citrate buffer (pH 7). Upon release, 50-60% of said enzyme activities are released into solution. By using alumina first and then titanium dioxide as adsorbent, the enzymes can be separated.

n 3 84835

Kent et al. (9) refer to the use of inorganic carriers for immobilizing enzymes. Catalase, glucose oxidase, papain and urease have been adsorbed on titanium dioxide as either a porous or non-porous material.

No mention is made of the use of inorganic adsorbents for the separation and purification of endopolygalacturonase. As organic adsorbents, e.g.

cross-linked pectic acid (10), alginate (11) and ion exchange resins (12,13). Kennedy (14) and Kennedy and Kay (15) investigated the use of titanium oxyhydrate mainly for the immobilization of glucose oxidase. The experiments were performed with a commercial enzyme concentrate and were preliminary baseline experiments (scale 10 1.5 ml enzyme solution). In a Japanese patent publication from 1983 (16), titanium oxyhydrate was used as an adsorbent to remove liver fibrinogen from solutions. Also in this application, the scale presented was very small (5.0 ml of the solution to be treated). There is no mention in the literature of the use of titanium oxyhydrate as an adsorbent for polygalacturonase, nor as an adsorbent for other enzymes -.1.5 on a larger scale than the above-mentioned test tube experiments.

Previous studies (17) developed a method for the production of polygalacturonase (PG) by the fungus Aspergillus niger in submerged culture. In addition to polygalacturonase, the finished fermentation broth also contained a significant amount of side activities, including 20 β-glucosidase, hemicellulase (xylanase), and cellulase. Indeed, in some applications, these side activities may be useful (17, 18). Other 4,84835 applications, especially for various types of lignocellulose treatments, require enzyme preparations containing pectinase and / or hemicellulase that do not have significant cellulase activity (e.g., 19, 20).

5 The studies which led to the patent application examined the possibility of using already known mineral adsorbents for concentrating polygalacturonase from A. niger fermentation broth. It was found that bentonite (Na-Al silicate, trade name Berkbond No. 2, Steeley Minerals Division, Milton Keynes, UK) was a potent adsorbent for both polygalacturonase and β-glucosidase (Table 1). 10 Bentonite has previously been used as an adsorbent in the concentration of β-galactosidase (lactase) produced by A. niger (21). However, attempts to release adsorbed polygalacturonase from bentonite with various changes in pH and ionic strength were completely unsuccessful (pH 5.0-9.0; NaCl concentration 5-200 g / l), although β-glucosidase activity was readily and almost quantitatively released into the solution when 15 the enzyme-adsorbent mixture was suspended at pH 5.8 or above.

Another rather promising adsorbent for polygalacturonase based on the results in Table 1 was titanium dioxide. Further studies were performed on samples from Kemira Oy's Pori plant, which were from different process stages of titanium oxide production.

The present invention is based on the surprising finding that amorphous titanium oxyhydrate (TiO 2, 1.9H 2 O, 0.06SO 2) precipitated by neutralization from an acidic solution of titanyl sulfate (or titanyl chloride) is much more effective adsorbent than titanium oxide 5,84835 in polygalacturide.

Titanium oxyhydrate is prepared from a titanyl chloride solution by doping with a base and, when washed as clean as possible with water, contains 0-3% by weight of bound alkali metal corresponding to Na 2 O and 10-20% by weight of hydrate water (H 2 O) after drying at 35 ° C. Alternatively, the titanium oxyhydrate is prepared from a titanyl sulfate solution by doping with a base and contains, when washed as pure as possible with water, bound sulfate in an amount corresponding to 0-4% by weight of sulfur (S) and 10-20% by weight of hydrate water (H 2 O) after drying at 35 ° C. temperature. Up to 1 g / l of amorphous titanium oxyhydrate 10 (TiO 2 * 1.9H 2 O -0.06SO 2) is sufficient to adsorb more than 90% of the polygalacturonase activity from A. niger fermentation broth (initial PG activity about 5000 nkat / ml) when the pH of the enzyme adsorbent suspension is lowered to 4 , 8 or below (Figure 1). The same result is achieved with titanium dioxide only at an adsorbent concentration of about 30 g / l.

15.

After binding, the titanium oxyhydrate to which the polygalacturonase is bound is separated from the solution, preferably by centrifugation. The centrifugation method depends mainly on the scale of the process: a laboratory centrifuge, a pilot ball centrifuge and a pilot separator can be used equally successfully. After centrifugation, the mass is recovered as accurately as possible and suspended in a much smaller amount of water or a suitable buffer (preferably 0.2 M sodium phosphate buffer, pH 7.8-8.0) than the original volume of solution. At this stage, it must be ensured that the mixture is initially strong enough to obtain a homogeneous suspension of s 84835. The pH of this suspension to specify, if necessary, preferably to 7.5 (mainly when the suspension is made in water or buffer) and the suspension is stirred calmly for about half an hour. In this case, the polygalacturonase is released from the adsorbent into the solution.

The desorbed polygalacturonase is recovered by centrifugation and collecting the solution fraction in a suitable container. If desired, the adsorbent mass can be washed with a small amount of water, which is mixed into the mass until a uniform suspension is obtained. This suspension is stirred for a few minutes and centrifuged. The solution fraction is combined with the previous one. The pH of the combined solution is lowered to 5.5-6.0 and the shelf life of this polygalacturonase concentrate is improved by the addition of suitable stabilizers such as sodium and / or calcium salts, glycerol, etc.

It is known from previous studies that the isoelectric point (pI) of Aspergillus nieer polygalacturonase is 5.3 when the LT produced by the same organism. cellulase and β-glucosidase have pI values less than 4.0 (17). Β-glucosidase does not adhere to titanium dioxide or oxyhydrate even at pH 3.0 (Table 1), but the cellulase is at least partially adsorbed when the pH drops below 4.0. To prepare a cellulase-free polygalacturonase concentrate, the polygalacturonase is bound at pH 4.2-4.6, whereby the desired enzyme is efficiently adsorbed, but not the cellulase at all. Thus, the polygalacturonase preparation thus produced contains very little cellulase.

11 1 84835

The polygalacturonase concentration method described above achieves three clear advantages: PG activity can be concentrated in a fast and product-friendly manner.

The results have been obtained using large volumes of culture medium (80 liters, see below), 10 and are thus as such applicable to factory scale.

Example A fermentation broth of 80 l of A. niger strain VTT-D-86267 with a PG-15 activity of 5500 nkat / ml was treated. The levels of major side activities were / 3 * glucosidase (bG) 65 nkat / ml, cellulase (HEC) 60 nkat / ml and xylanase (XYL) 290 nkat / ml and the protein content of the solution was 0.9 g / l. In our experience, the cellulase activity in the fermentation broth of A. niger was sufficient to completely dissolve 5 g / l of pure cellulose, so that it would have caused fiber damage in the handling of 20 cellulosic materials.

To the fermentation solution (80 L) was added 1.0 g / L of titanium oxyhydrate (by dry weight) premixed with about 1.5 liters of water. The pH of the stirred suspension was lowered to 4.4 by the addition of phosphoric acid. The suspension was stirred gently for two hours 25, after which the mass was separated from the solution by centrifugation. The § 84835 mass separated in the separation was recovered as accurately as possible, finally by rinsing with 1.5 liters of 0.2 M sodium phosphate buffer, pH 8.0. From the solution fraction obtained in the separation, β-glucosidase was recovered by adsorption on bentonite.

After the above separation, the mixture of pulp and buffer was stirred into a uniform suspension and its pH was adjusted to 7.4 with 1.0 M sodium hydroxide. After stirring for about 15 minutes, this suspension was centrifuged in a laboratory centrifuge (4500 rpm, 20 min, 10 ° C) and the solution fraction (1460 ml) was collected. The mass was mixed with 200 ml of water and this suspension was centrifuged. The solution fraction (190 ml) was combined with the previous 10. The pH of the combined solution was lowered to 5.5 with hydrochloric acid and the polygalacturonase activity was stabilized by adding 100 g / l NaCl and 20 g / l CaCl2 to the stirred solution in small portions. After the addition of salt, the solution was stored at + 4 ° C overnight, after which the slight precipitate was separated by centrifugation. The volume of the final clear solution was 1800 ml.

13 '.

Activities, specific activities and calculated yields of polygalacturonase - ;;; in concentrate is shown in Table 2. Approximately 80% of the initial PG activity was recovered in the concentrate, which, however, contained less than 1% of the initial cellulase activity (HEC). Polygalacturonase specific activity 20 '. in the concentrate was almost ten times the specific activity of the initial fermentation broth. The PG to cellulase ratio (PG / HEC) was about 100 in the initial solution and more than 10,000 in the concentrate.

2,84835 REFERENCES: 1. Nelson J.M., Griffen E.G., J.Am.Chem.Soc., 38 (1916) 1109.

2. Zittle C.A., Adv.Enzymol. 14 (1953) 319-374.

3. Morris C.J.O.R., Morris P., Separation methods in Biochemistry. 2nd ed., Pitman Pub. Ltd., London 1976, 153-248.

4. Miles B.J., Thomson A.R., Proc.Biochem. 1974 (4) 11-16.

5. Green S., Wase D.A.J., Proc.Biochem. 1986 (12) 200-203.

6. Sterling Organics Ltd., Macrosorb T, Macrosorb C, Macrosorb K Technical data sheet, May 1986.

7. Thomson A.R., J.Chem.Tech.Biotechnol. 34B (1984) 190-198.

8. Wase D.A.J., Green S., Thomson A.R., Proc.Conf.Adv.Fermentation 1983. 54-58.

9. Kent C., Rosevear A., Thomson A.R., Topics in Enzyme and Fermentation Biotechnology vol 2 (Ed. Wiseman A.,) 12-119.

10. Rexova-Benkova L., Biochim.Biophys.Acta 276 (1972) 215-220.

11. Somers M., Rozie H., Bonte A., Rombouts F., Visser J., van't Riet K., Proc.4th Eur.Congr.Biotechnol. 1987 560-563.

12. McColloch R.J., Kertesz Z.I., J.Biol.Chem. 160 (1945) 149-154.

13. Talboys P.W., Nature 166 (1950) 1077.

14. Kennedy J.F., Chem. Soc. Rev. 8 (1979) 221-257 15. Kennedy J.F., Kay I.M. J. C. S. Perkin I (1976), no. 3, 329-335.

16. Japanese Patent 58 70,835 (1983) [83 70,835].

10 84835 17. Bailey M.J., Pessa E. Enzyme Microb Technol 12 (1990) 266-271.

18. Beldman G., Rombouts F.M., Voragen A.G.V., Pilnik W. (1984) Enzyme Microb. Technol. 6, 503-507.

19. Kantelinen A., Viikari L., Linko M., Rättö M., Ranua M., Sundquist J. (1988) International Pulp Bleaching Conference. Tappi Proceedings, pp. 1-5.

20. Bailey M.J., Poutanen K. Appl. Microbiol. Biotechnol. 30 (1989) 5-10.

21. Harvey M.S. (1971) U.S. Patent 3,620,924.

Ii 11 84835

Table 1. Residual activities of polygalacturonase and β-glucosidase in A. niger fermentation broth after 3 h incubation with different adsorbents (mixtures 10 g / l) at pH 3.0. Methods of activity are described in reference (17).

Residual activities in solution Polygalacturonase / 3-Glucosidase nkat / ml% nkat / ml%

Fermentation solution 6000 100 105 100

Bentonite (Steeley, UK) 20 <1 2 2

Diatomaceous earth (Witco, USA) 4200 70 92 88

Amberlite XAD-2 (BDH, UK) 5300 88 85 81

TiO2 (May & Baker, UK) 1600 26 110 100 A1A (Merck, FRG) 2400 40 98 93

Micro-cel C (Manville, USA) 3700 62 92 88

Silica gel (Woelm, FRG) 5300 88 88 84 84835

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Claims (4)

A method for recovering and concentrating polygalacturonase from solution, characterized in that the polygalacturonase is adsorbed on amorphous titanium oxyhydrate (TiO2 * 1.9H2O, 06SO2) by stirring the adsorbent and the solution in a pH range below , recovering the polygalacturonase-containing adsorbent by centrifugation, filtration or the like, then releasing the polygalacturonase by suspending the adsorbent in a much smaller volume of water or buffer solution, the pH of which is adjusted to favor the polygalacturonase release and preferably by centrifugation, filtration or other similar process. Process according to Claim 1, characterized in that the polygalacturonase is recovered from a clarified or non-clarified fermentation broth or from a homogenate of microbial, plant or animal cells. Process according to Claims 1 to 2, characterized in that the polygalacturonase is produced by the fungus Aspergillus niger. Process according to Claims 1 to 3, characterized in that the undesired protein to be separated from the polygalacturonase is cellulase. 14 8 4 8 35