WO1997032894A1

WO1997032894A1 - Crystallization of a protein with a sulphur salt

Info

Publication number: WO1997032894A1
Application number: PCT/DK1997/000094
Authority: WO
Inventors: Stig Nielsson; Mads Aage Laustsen
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 1996-03-08
Filing date: 1997-03-03
Publication date: 1997-09-12
Anticipated expiration: 1998-09-08
Also published as: AU2021797A; US6066481A; DK0892810T3; JP2000506145A; CN1213375A; ATE310745T1; EP0892810B1; DE69734708D1; EP0892810A1; DE69734708T2

Abstract

The invention deals with a method for crystallization of a protein obtained from a protein solution comprising: (a) treatment of the protein solution with a salt containing a sulphur atom having an oxidation state less than 6; (b) recovering of the protein on crystalline form.

Description

Crystallization of a Protein with a Sulphur Salt

TECHNICAL FIELD

The present invention relates to a simple, inexpensive and very effective method for crystallization of a protein with a salt.

BACKGROUND ART

Enzymes are usually provided as liquids or amorphous materials for industrial purposes. When not provided as liquids, they are usually provided as amorphous materials, because the known methods for crystallization of enzymes are usually regarded as too expensive to be used in an industrial scale.

There is an abundance of literature concerning crystallization of enzymes. It is difficult to generalize in respect to the outcome of specific crystallization procedures, as the art of enzyme crystallization is highly empirical.

Characteristic features of most of the hitherto known protein crystallization processes are: Pure and con¬ centrated initial solutions, very long crystallization time, high consumption of chemicals such as salts, for reference see, e.g., Biotechnology and Bioenαineering 48, 1995, p. 316- 323.

Industrial enzyme crystallization processes using polyethylene glycol have also been described, for reference see WO 95/01989.

It has also been described that it is possible to crystallize enzymes by leaching out salts from the solution, followed by adjustment of the pH of the solution to a level around the pi of the enzyme, for reference see WO 94/22903. SUMMARY OF THE INVENTION

In this invention it is surprisingly found that oxidizable sulphur salts are very effective as crystallization salts: They can be used in low amounts on impure solutions giving short crystallization times and high yields in a simple, inexpensive and environmentally friendly process.

Accordingly, the present invention provides a method for crystallization of a protein obtained from a protein solution comprising:

(a) treatment of the protein solution with a salt containing a sulphur atom having an oxidation state less than 6;

(b) recovering of the protein on crystalline form.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to the accompanying drawing, in which

Fig. 1 shows the temperature ramp: the relation between temperature and time which is used to control the primary nucleation rate as described in Example 1.

DETAILED DISCLOSURE OF THE INVENTION

The present invention provides a method for crystallizing a protein or a polypeptide from a protein solution, in particular from a fermentation (culture) broth.

A fermentation broth contains besides the protein of interest many other compounds such as substrate compounds, e.g., carbohydrates and salts, cells, and other metabolites such as nucleic acids and other proteins than the one of interest.

Preferably the method of the invention is applied to a fermentation broth that has first been purified by, e.g., flocculation, centrifugation, filtration, micro filtration, ultrafiltration, diafiltration, electrodialyse, adsorption, precipitation, evaporation, or any combination thereof. As the method of the invention works very well on relatively impure solutions (see Example 1) , it will normally not be necessary to purify the protein solution obtained from the fermentation broth by use of chromatographic methods before crystallization.

In a more specific embodiment, the method of invention comprises concentration of the protein containing solution by methods known per se . Such methods include con¬ centration by ultrafiltration, by diafiltration, by dialysa- tion, or by evaporation.

Concentration of the protein containing solution, although not essential for carrying out crystallization, is convenient from a handling and a yield perspective. For practical reasons the protein containing solution may be concentrated to a content of proteins of from 0.1 to 25% w/w, preferably of from 0.5 to 20% w/w, more preferably of from 1 to 15% w/w, even more preferably 5-12% w/w.

In a preferred embodiment, the method of the invention is applied to crystallization of an enzyme, in particular an enzyme selected from the group consisting of proteases, peptidases, lipases, a ylases, cellulases, xylanases, isomerases and oxidoreductases.

Proteases Suitable proteases to be crystallized according to the present invention include any protease which may be a fermentation product from a cell such as a microorganism. Bacterial or fungal origin is preferred. Chemically or genetically modified mutants are included. The protease may be a serine protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279) . Examples of trypsin-like proteases are trypsin (e.g. of por¬ cine or bovine origin) and the Fusarium protease described in WO 89/06270. Preferred commercially available protease enzymes include those sold under the tradenames Alcalase, Savinase, Primase, Durazym, and Esperase by Novo Nordisk A/S (Denmark) , those sold under the tradename Maxatase, Maxacal, Maxapem and Properase by Gist-Brocades, those sold under the tradename Purafect and Purafect OXP by Genencor International, and those sold under the tradename Opticlean and Optimase by Solvay Enzymes.

Lipases

Suitable lipases to be crystallized according to the present invention include any lipase which may be a fermenta¬ tion product from a cell such as a microorganism. Bacterial or fungal origin is preferred. Chemically or genetically modified mutants are included.

Examples of useful lipases include a Humicola lanucrinosa lipase, e.g., as described in EP 258 068 and EP 305 216, a Rhizomucor miehei lipase, e.g., as described in EP 238 023, a Candida lipase, such as a C. antarctica lipase, e.g., the C. antarctica lipase A or B described in EP 214 761, a Pseudomonas lipase such as a P. alcalicrenes and P. pseudoalcaliqenes lipase, e.g., as described in EP 218 272, a P. cepacia lipase, e.g., as described in EP 331 376, a P. stutzeri lipase, e.g., as disclosed in BP 1,372,034, a P. fluorescens lipase, a Bacillus lipase, e.g., a B. subtilis lipase (Dartois et al. , (1993), Biochemica et Biophysica acta 1131, 253-260), a B. stearothermophilus lipase (JP 64/744992) and a B. pumilus lipase (WO 91/16422) .

Furthermore, a number of cloned lipases may be useful, including the Penicillium camembertii lipase described by Yamaguchi et al. , (1991) , Gene 103, 61-67) , the Geotricum candidum lipase (Schimada, Y. et al . , (1989), J. Biochem., 106, 383-388) , and various Rhizopus lipases such as a R. deletnar lipase (Hass, M.J et al. , (1991), Gene 109, 117-113), a R. niveus lipase (Kugimiya et al., (1992), Biosci. Biotech. Biochem. 56, 716-719) and a R. oryzae lipase.

Other types of lipolytic enzymes such as cutinases may also be crystallized, e.g., a cutinase derived from Pseudomonas mendocina as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (e.g. described in WO 90/09446) . Especially suitable lipases are lipases such as Ml

Lipase™, Luma fast™ and Lipomax™ (Gist-Brocades) , Lipolase™ and Lipolase Ultra™ (Novo Nordisk A/S) , and Lipase P "Amano" (Amano Pharmaceutical Co. Ltd.) .

Amylases

Suitable amylases (α or β) to be crystallized according to the present invention include any amylase which may be a fermentation product from a cell such as a microorganism. Bacterial or fungal origin is preferred. Chemically or genetically modified mutants are included.

Amylases include, for example, α-amylases obtained from Bacillus. in particular a special strain of B. licheniformis, described in more detail in British Patent Specification No. 1,296,839. Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ and BAN™ (available from Novo Nordisk A/S) and Rapidase™ and Maxamyl P™ (available from (Gist-Brocades) .

Other useful enzymes are the CGTases (cyclodextrin glucanotransferases, EC 2.4.1.19) obtainable from e.g. Bacil- lus, Thermoanaerobactor or Thermoanaerobacteriu .

Cellulases

In the present context, the term "cellulase" refers to an enzyme which catalyses the degradation of cellulose to glucose, cellobiose, triose and other cello-oligosaccharides.

In a preferred embodiment of the invention, the cellulase to be crystallized is an endoglucanase (EC 3.2.1.4) , preferably a monocomponent (recombinant) endoglucanase.

Preferably, the cellulase is a microbial cellulase, more preferably a bacterial or fungal cellulase.

Useful examples of bacterial cellulases are cellul¬ ases derived from or producible by bacteria from the group consisting of Pseudo onas. Bacillus. Cellulomonas, Clostridium, Microspora, Thermotoga, Caldocellum and Actinomycets such as Streptomyces, Termomonospora and Acidothemus, in particular from the group consisting of Pseudomonas cellulolvticus. Bacillus lautus. Cellulomonas fimi, Microspora bispora, Termomonospora fusca, Termomonospora cellulolyticum and Acidothemus cellulolvticus.

A useful cellulase to be crystallized is an acid cellulase, which is derived from or producible by fungi from the group of genera consisting of Trichoderma, Myrothecium. Asperqillus, Phanaerochaete, Neurospora, Neocallimastix and Botrytis, in particular from the group consisting of Tricho¬ derma viride, Trichoderma reesei, Trichoderma longibrachiatum. Myrothecium verrucaria. Asperqillus niqer, Aspergillus oryzae, Phanaerochaete chrysosporium. Neurospora crassa,

Neocallimastix partriciarum and Botrytis cinerea.

Another useful cellulase to be crystallized is a neutral or alkaline cellulase, preferably a fungal neutral or alkaline cellulase, which is derived from or producible by fungi from the group of genera consisting of Asperqillus, Penicillium, Myceliophthora. Humicola, Irpex, Fusarium, Sta- chybotrys, Scopulariopsis, Chaetomium, Mycogone. VerticiIlium, Myrothecium, Papulospora. Gliocladium, Cephalosporium and Acremonium, in particular from the group consisting of Humicola insolens. Fusarium oxysporum, Myceliopthora ther- mophila, Penicillium ianthinellum and Cephalosporium sp. , preferably from the group of species consisting of Humicola insolens, DSM 1800, Fusarium oxysporum, DSM 2672, Myceliopt¬ hora ther ophila, CBS 117.65, and Cephalosporium sp.. RYM-202. Other examples of useful cellulases to be crystallized are variants having, as a parent cellulase, a cellulase of fungal or bacterial origin, e.g. a cellulase derivable from a strain of the fungal genus Humicola, Trichoderma or Fusarium. Oxidoreductases

Oxidoreductases which may be crystallized according to the invention include peroxidases, and oxidases such as laccases.

Peroxidases

An enzyme exhibiting peroxidase activity may be any peroxidase enzyme comprised by the enzyme classification (EC 1.11.1.7) , or any fragment derived therefrom, exhibiting peroxidase activity.

Preferably, the peroxidase employed in the method of the invention is producible by microorganisms such as fungi or bacteria. Some preferred fungi include strains belonging to the subdivision Deuteromvcotina, class Hvphomycetes, e.g., Fu- sarium, Humicola. Tricoderma, Myrothecium, Verticillum. Arthromvces. Caldariomyces. Ulocladium, Embellisia. Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672) , Humicola insolens, Trichoderma resii. Myrothecium verrucana (IFO 6113) , Verticillum alboatrum, Verticillum dahlie, Arthromvces ra osus (FERM P-7754) , Caldariomyces fumago, Ulocladium chartarum, Embellisia alii or Dreschlera halodes.

Other preferred fungi include strains belonging to the subdivision Basidiomycotina. class Basidiomycetes, e.g. Coprinus, Phanerochaete, Coriolus or Trametes. in particular Coprinus cinereus f. microsporus (IFO 8371) , Coprinus macror- hizus, Phanerochaete chrysosporium (e.g. NA-12) or Trametes (previously called Polyporus) , e.g. T. versicolor (e.g. PR4 28-A) . Further preferred fungi include strains belonging to the subdivision Zygomycotina, class Mycoraceae, e.g. Rhizopus or Mucor, in particular Mucor hiemalis.

Some preferred bacteria include strains of the order Actinomycetales, e.g., Streptomyces spheroides (ATTC 23965) , Streptomyces thermoviolaceus (IFO 12382) or Streptoverticillum verticillium ssp. verticillium.

Other preferred bacteria include Bacillus pumilus (ATCC 12905) , Bacillus stearothermophilus, Rhodobacter sphae- roides. Rhodomonas palustri. Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B- 11) . 5 Further preferred bacteria include strains belonging to Myxococcus, e.g., M. virescens.

Particularly, a recombinantly produced peroxidase is preferred, e.g., a peroxidase derived from a Coprinus sp. , in particular C. macrorhizus or C. cinereus according to WO

1092/16634, or a variant thereof, e.g., a variant as described in WO 93/24618 and WO 95/10602.

Laccases and Laccase Related Enzymes

In the context of this invention, laccases and

15 laccase related enzymes contemplate any laccase enzyme com¬ prised by the enzyme classification (EC 1.10.3.2) , any cha- techol oxidase enzyme comprised by the enzyme classification (EC 1.10.3.1), any bilirubin oxidase enzyme comprised by the enzyme classification (EC 1.3.3.5) or any monophenol mono-

20 oxygenase enzyme comprised by the enzyme classification (EC 1.14.18.1) .

The microbial laccase enzyme may be derived from bacteria or fungi (including filamentous fungi and yeasts) and suitable examples include a laccase derivable from a strain of

25 Aspergillus, Neurospora. e.g., N. crassa, Podospora. Botrytis, Collybia, Fomes. Lentinus. Pleurotus, Trametes, e.g., T. vil- losa and T. versicolor. Rhizoctonia, e.g., R. solani, Copri- nus, e.g. C. plicatilis and C. cinereus. Psatyrella, Myceliophthora, e.g. M. thermophila, Schytalidium, Polvporus,

30 e.g., P. pinsitus, Phlebia, e.g., P. radita (WO 92/01046), or Coriolus, e.g., C. hirsutus (JP 2-238885) , in particular laccases obtainable from Trametes, Myceliophthora, Schytalidium or Polvporus.

35 Salts

We have surprisingly found that low concentrations of an oxidizable sulphur salt, i.e., a salt containing at least one sulphur atom having an oxidation state less than 6, in particular a salt containing at least one sulphur atom having an oxidation state of from 2 to 4 , may produce enzyme crystals from impure solutions, wherein the purity of the crystals and the yield of crystals are extraordinary good. Furthermore the morphology of the crystals may be improved compared to crystallization with other salts as the crystals are relatively large (see Example 1) .

The crystallization process according to the invention passes so quickly that normally it is not necessary to add stabilizing agents or inhibitors to the protein solution.

Preferred sulphur salts according to the invention are salts of alkali or ammonium as these salts are normally easily dissolved in water, e.g. :

Na₂S₂0₃, Na₂S0₃, NaHS0₃, Na₂S₂0₄, Na₂S₂0_€x2H₂0, Na₂S₃0₆, Na₂S₄0₆, Na₂S₂O_s, HSCH₂COONa, K₂S₂0₃xl/3H₂0, K₂S0₃x2H₂0, KHS0₂, K₂S₂0_€/ K₂S₃0₆, K₂S₃0₆, K₂S₄0₆, K₂S₂0₄, Li₂S0₃xH₂0, Li₂S₂0₆x2H₂0, (NH₄)₂S0₃, LiHS0₃, KHS0₃, (NH₄)HS0₃, NaSCN or KSCN. In particular a sulphite salt or a thiosulfate salt is preferred.

The sulphur salt according to the invention may be the only salt added or it may be part of a mixture which besides the sulphur salt also comprises other salts, examples of such mixtures are:

1) a mixture of a sulphur salt according to the invention and a sulphate salt;

2) a mixture of a sulphur salt according to the invention and an acetate salt;

3) a mixture of a sulphur salt according to the invention and a carbonate salt;

4) a mixture of a sulphite salt and a hydrogen sulphite salt;

5) a mixture of a sulphite salt, a hydrogen sulphite salt and an acetate salt; or

6) a mixture of a sulphite salt, a hydrogen sulphite salt and a carbonate salt . A salt containing a halogen such as a chloride may also be included in the salt mixtures disclosed above.

In a particular embodiment of the invention the salt according to the invention is added to the protein solution at a concentration of 0.02-1.2 moles per liter, preferably at a concentration of 0.04-1.0 moles per liter, more preferably at a concentration of 0.04-0.7 moles per liter, most preferably at a concentration of 0.04-0.5 moles per liter. The salt according to the invention may be added in one step, or it may be added in more than one step; normally the crystallization will perform satisfactorily when the salt is added in one step.

In crystallizations it may often be an advantage to reduce the agitation after the salt addition and any other adjustment to ensure against breakage of the crystals formed, this will facilitate the subsequent crystal harvest.

Adjustment of pH The pH of the protein solution to which the salt according to the invention has been added, may be adjusted in order to find the optimum wherein the crystallization is maximized and at the same time the protein is stable. A way of finding this optimum is to run a trial, typically starting at pH 10, then perform the crystallization at pH 9, then at pH 8, then at pH 7, and so on down to pH 3, and then if it is found, e.g., that the optimum is between pH 4 and pH 5, then do a trial within this range whereby the optimum pH is found; the optimum will normally be in the range between pH 4 and pH 9. In some cases it may also be an advantage to perform the crystallization process by ramping the pH; i.e. perform the crystallization process at various pH.

For adjustment of pH virtually any acid or base can be used. The acid may be inorganic or organic. Some examples are hydrochloric acid, sulphuric acid, sulphurous acid, nitrous acid, phosphoric acid, acetic acid, citric acid, and formic acid. Preferred acids are phosphoric acid, formic acid, citric acid, and acetic acid. Preferred bases are sodium hydroxide, potassium hydroxide, and ammonium hydroxide, in particular sodium hydroxide.

Crystallization

By using the method of the invention it may be possible to make the crystallization with a crystallization time of less than 48 hours, in particular to make the crystallization with a crystallization time of less than 36 hours, preferably to make the crystallization with a crystallization time of less than 24 hours, more preferably to make the crystallization with a crystallization time of less than 12 hours, most preferably to make the crystallization with a crystallization time of less than 6 hours, without adding any seeds to start the crystallization.

The temperature of the protein solution to be crystallized is preferably of from 5°C to 40°C, in particular of from 10°C to 30°C; preferably the temperature is increased stepwise as demonstrated in Example 1, i.e., the ramp may be started at e.g. 10°C, then increased to e.g. 20°C for 1 hour, then to e.g. 23°C for 1 hour, then to e.g. 25°C for 1 hour and ending at e.g. 28°C at which temperature it may be kept for about 24 hours.

Recovery after Crystallization

The method of the invention causes the protein, in particular the enzyme, to crystallize. Recovery of the crystalline protein may be accomplished by conventional methods, e.g. by centrifugation and/or filtration and optionally drying. By using the method of the present invention the harvest properties of the crystals may be improved compared with the use of other salts such as KAc (see Example 1) ; because the crystals are larger this feature makes the centrifugation and/or filtration following the crystallization much easier.

If crystalline products of a very high purity are desirable, the process of the invention may be repeated, i.e. the crystalline end product of the process of the invention is redissolved and subjected to one or more additional crystalli¬ zation processes. The end product may be a crystal product or the crystals may be redissolved in order to produce e.g. a liquid protein product.

Another advantage of the present invention is that the end product contains an oxidizable sulphur salt which means that the protein, e.g. the enzyme, is stabilized against oxidations.

The invention is further illustrated in the following examples which are not intended to be in any way limiting to the scope of the invention as claimed.

EXAMPLE 1

Crystallization of a Protease using Sulphites

200 kg culture broth containing a Bacillus lentus protease, fermented e.g. as described in US 3,723, 250, was divided into two fractions of each 100 kg named Fraction A and Fraction B.

Each fraction was pretreated as described in Table 1 below:

Fraction A Fraction B 1

Broth 100 kg 100 kg

Water 200 kg 100 kg

CaCl₂x2H₂0 1 kg 3 kg

NaA10₂ (53-55%Al₂0₃) 4 kg -

PH pH adjusted pH adjusted to to pH=5.9 pH=7.5

Cationic flocculant 1 kg 1 kg (20% C521)

Anionic flocculant 2 kg 1 kg (0.1% A130) Table 1. Pretreatment and flocculation of Broth Fraction A and Fraction B

Al-compounds significantly reduce the amount of

CD* _™ P^er activity unit, so Fraction A is more pure than

Fraction B.

From each fraction the production strain was removed by centrifugation yielding two supernatants which thereafter were filtered using Seitz K900 pads. The resulting filtrates were concentrated on Asahi (6 kD) membranes. Fraction A was further diafiltered using 2 volumes of water.

In this way Concentrate B contained relatively more than Concentrate A of low molecular fermentation compounds (carbohydrates, salts, nucleic acids, other proteins, etc.) .

This is illustrated by measurements of the (OD_2B0nm/gram active protease) ratio, of the (OD_440n_,/gram active enzyme) as well as

(g active protease/gram Mettler drymatter) as shown in Table 2 below:

'440m./ g active g active g active protease/ protease protease g drymatter

Concentrate A 2081 233 0.56 concentrate B 5411 821 0.36

Table 2. Purity of Concentrate A and Concentrate B

The concentrates were subjected to various amounts of Na₂S0₃ or KAc and adjusted to pH=4.9 using a 20 w/w % phosphoric acid. A discrete temperature ramp was used to control the primary nucleation rate. The ramp started at 10°C, then increased to 20°C for 1 hour, then to 23°C for 1 hour, then to 25°C for 1 hour and ended at 28°C at which temperature it was kept for 24 hours as illustrated in Fig. 1. The crystal suspensions were centrifuged and the crystals washed using a 5.5 % CaCl₂ (pH=4.9) solution. The crystals were harvested using centrifugation and dissolved in

10 fold 100 % MPG and the resulting products were analyzed for OD_440nm and protease activity.

The crystallization yields and purities of the final products A and B are shown in Table 3 and Table 4, respectively. mMol / mMol / 1 Yield of Ω_t*0um/ Morphology

1 anion anion, active g active of

Ac^" P0₄ ^3", protease protease crystals or S0₃ ^_ from in final in final formed from phos¬ product product salt phoric acid

0%

Na₂S0₃ 0 0 0 % - -

0% KAc 0 0 0 % - -

1% 15μx4μ

Na₂S0₃ 79 105 39 % 29 rods

1% KAc 102 63 < 5 % - -

2% 5-25μx4μ

Na₂S0₃ 158 180 82 % 16 rods

2% KAc 205 96 51 % 23 5-25μ needles

4% 5-25μx4μ

Na₂SO, 317 336 87 % 10 rods

4% KAc 408 175 79 % 17 5-25μ needles

Table 3. Concentrate A crystallized using increasing amounts of Na₂S0₃ and KAc

We find that yield and purity is increased using Na₂S0₃ instead of KAc. Additionally we find increasing thickness of the crystals produced (rods instead of needles) .

This is important when using filtration equipment for harvest in order to avoid penetration of the finer needles through the filter plates.

Table 4. Concentrate B crystallized using increasing amounts of Na₂S0₃ and KAc

On this much more impure concentrate we find that yield and purity is indeed increased using Na₂S0₃ instead of KAc. We need as high as 10% KAc in order to crystallize but only 4% of Na₂S0₃. Also we find increased thickness of the crystals.

The cost difference using KAc or Na₂S0₃ is neglectable regarding the price per Kg of raw material, however, since less salt is needed in the case of Na₂S0₃ than in the case of KAc this favours Na₂S0₃.

EXAMPLE 2

Crystallization of an Amylase using Thiosulphate

100 Kg culture broth containing a Bacillus licheniformis amylase, fermented e.g. as described in GB 1,296,839, was subjected to the following treatment:

Fraction

Broth 100 Kg

Water 100 Kg

CaCl₂x2H₂0 5 Kg

Na₃P0₄ 1 Kg

NaAl₂0₃ 2 Kg pH adjusted using 20 % pH=8.0 Formic acid

Cationic flocculant 2 Kg (20% C521)

Anionic flocculant 3 Kg (0.1% A130)

Table 5. Pretreatment of Bacillus licheniformis broth

The production strain was removed by drum filtration yielding a filtrate which successively was germfiltered using Seitz EK1 filter pads. The resulting filtrate was concentrated using evaporation to a drymatter contents of 15% (measured as refractive index) .

The purity of the resulting concentrate is illustrated by the ratio of (OD_440nm/g active amylase) and the ratio of active enzyme/drymatter (g enz/g drymatter) .

OΩ_ii0jm/g g enz/ active g drymatter amylase

Concentrate 70 0 . 54

Table 6. Purity of Concentrate before crystallization starts.

The concentrate was further subjected to various amounts of potassium acetate, sodium thiosulphate and sodium sulphate and adjusted to pH=7.3 using a 13 w/w % NaOH solution and left for 24 hours of crystallization time at 28°C. The crystal suspensions were harvested by centrifugation. The crystal cakes were dissolved and formulated in 10 fold by weight of a 50% MPG solution. The resulting formulated products were analyzed for OD_440ratl and amylase activity. Amylase activity analysis is based on the digestion of a p-nitrophenyl-alfa, D-maltoheptaoside (pNP-G₇) by the α-amylase. The substrate, pNP-G₇, is degraded by the amylase to pNP-G₃ and pNP-G₄. pNP-G₃ is further degraded by an excess amount of α-glucosidase to glucose and the yellow p- nitrophenol. The reaction is followed in situ whereby the change in absorbance per time unit can be calculated. This figure is a measure of the reaction rate and the enzyme activity. In order to avoid interference from the protease, PMSF (Phenyl Methyl Sulfonyl Floride) is added to the test solution.

The yields and purities of the final products are shown in Table 7 .

Amount Yield of active amylase ^OD44_θι» 9 active amylase of salt in formulated product in formulated product (w/w%)

KAc Na₂S0₄ Na₂s₂o₃ Kac Na₂S0₄ Na₂s₂o₃

0 . 25 % 0% 0% 24% - - 31

0 . 5% 0% 0% 72% - - 26

1% 0% 11% 80% - 39 25

3% 44% 72% - 30 26 -

Table 7. Concentrate subjected to increasing amounts of three different salts and crystallized at pH=7.3 and 28°C showing yield and purity.

We find, surprisingly, that yield is increased using sodium thiosulphate (Na₂S₂0₃) as crystallization agent. In other words, we can increase yield substantially and even gain improved purity using thiosulphate.

The cost difference using either KAc or Na₂S₂0₃ is neglectable regarding the price per Kg of raw material, however since less salt is used in the case of Na₂S₂0₃ than in the case of KAc this favours Na₂S₂0₃ for crystallization of this amylase.

EXAMPLE 3

Crystallization of a Protease using Sodium Thiosulphate

100 Kg culture broth containing a Bacillus lentus protease, fermented e.g. as described in US 3,723,250, was subjected to the following treatment: Amount

Broth 100 Kg

Water 200 Kg

CaCl₂x2H₂0 1 Kg

NaA10₂ (53-55%Al₂0₃) 1 Kg pH 5.9

Cationic flocculant 1 Kg (20 % C521)

Anionic flocculant (0.1% 5 Kg A130 )

The production strain was removed by drum filtration yielding a filtrate which was further filtered using Seitz K900 filter plates and subsequently germfiltered using Seitz EK1 filter pads. The resulting filtrate was concentrated on Asahi (6 kD) membranes.

The purity of the resulting concentrate is illustrated by the ratio of (OD_440nm/g active protease) and the ratio of (OD_2β0nm/g active protease) in Table 8 below.

ΓOT ':440o 9^' '2β0mn/ SF accttiive aaccttive protease protease concentrate 179 2498

Table 8. Purity of Concentrate before crystallization starts,

The concentrate was further subjected to various amounts of potassium acetate and sodium thiosulphate and adjusted to pH=4.9 using a 20 w/w% HCOOH solution and left for 24 hours of crystallization time at 28°C.

The crystal suspensions were harvested by centrifugation. The crystal cakes were washed using 1 fold by weight of a 5.5% CaCl₂ solution. The resulting crystal cakes were dissolved and formulated in 10 fold by weight of a 70 % MPG solution.

The resulting formulated products were analyzed 5 for protease activity. Protease activity was measured using a Kinetic Dimethyl Casein assay.

The yields of the final products are shown in Table 9.

Table 9. Yield and morphology of concentrate subjected to increasing amounts of two different salts and crystallized at pH=4. at 28°C.

15

We find surprisingly that yield is increased significantly by using sodium thiosulphate (Na₂S₂0₃) as crystallization agent at low salt concentrations. Furthermore we gain improved crystal morphology as can be seen from Table

209.

Claims

1. A method for crystallization of a protein obtained from a 5 protein solution comprising:

(b) recovering of the protein on crystalline form.

102. A method according to claim 1, wherein the sulphur atom has an oxidation state of from 2 to .

3. A method according to claim 1, wherein the salt is a sulphite salt or a thiosulfate salt.

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4. A method according to claim 1, wherein the salt is selected from the group consisting of Na₂S₂0₃, Na₂S0₃, NaHS0₃, Na₂S₂0₄, Na₂S₂0₆x2H₂0, Na₂S₃0₆, Na₂S₄0₆, Na₂S₂0₅, HSCH₂COONa, K₂S₂0₃xl/3H₂0, K₂S0₃x2H₂0, KHS0_2/ IC-S^, K₂S₃0₆, K₂S₄0₆, K₂S₂0₄, Li₂S0₃xH₂0,

20 Li₂S₂0₆x2H₂0, (NH₄)₂S0₃, LiHS0₃, KHS0₃ and (NH₄)HS0₃.

5. A method according to claim 1, wherein the protein solution comprises more than one protein.

256. A method according to claim 1, wherein the protein solution is obtained from a fermentation broth.

7. A method according to claim 6, wherein the fermentation broth prior to the salt treatment is purified by

30 centrifugation, filtration, microfiltration, ultrafiltration, diafiltration, electrodialyse, precipitation, evaporation, or a combination thereof.

8. A method according to claim 6, wherein one or more 35 flocculating agents are added to the fermentation broth prior to the salt treatment.

9. A method according to claim 1, wherein the salt is added to the protein solution at a concentration of 0.02-1.2 moles per liter protein solution.

510. A method according to claim 1, wherein the protein solution is concentrated to a content of proteins of from 0.1 to 25% w/w.

11. A method according to claim 1, wherein the protein is an 0 enzyme.

12. A method according to claim 11, wherein the enzyme is a protease, a lipase, a cellulase, an amylase or an oxidoreductase. 5

13. A method according to claim 1, wherein the pH of the protein solution is between 4 and 9.

14. A protein product obtainable from the method according to 0 claim 1.