WO2005089040A2 - Method of producing chymosin c and uses thereof - Google Patents

Abstract

The invention relates to a preparative method of obtaining a highly purified preparation of an aspartic protease, chymosin C. The invention further relates to the use of chymosin C in milk coagulating compositions in cheese manufacturing based on cow's milk and milk from any animal species, which are used in cheese.

Description

METHOD OF PRODUCING CHYMOSIN C AND USES THEREOF

FIELD OF INVENTION

The present invention relates generally to the field of cheese manufacturing. In particular to a method of obtaining a preparation of an aspartic protease, bovine chymosin C and its use for preparing cheese.

TECHNICAL BACKGROUND AND PRIOR ART

Enzymatic coagulation of milk by milk clotting enzymes, such as chymosin and pepsin, is one of the most important processes in the manufacture of cheeses. Enzymatic milk coagulation is a two- phase process: a first phase where a proteolytic enzyme, chymosin or pepsin, attacks κ-casein, resulting in a metastable state of the casein micelle structure and a second phase, where the milk subsequently coagulates and forms a coagulum.

Chymosin (EC 3.4.23.4) is an aspartic protease belonging to a broad class of peptidases. Aspartic protease s are found in eukaryotes, retroviruses and some plant viruses. Eukaryotic aspartic protease s are monomers of about 35 kDa, which are folded into a pair of tandemly arranged domains with a high degree of similarity, i.e. 20% or higher. The overall secondary structure consists almost entirely of pleated sheets and is low in α-helices. Each domain contains an active site centred on a catalytic aspartyl residue with a consensus sequence [hydrophobic]-Asp-Thr-Gly-[Ser/Thr] which aids in maintaining the correct Φ-loop conformation of the site, and with multiple hydrophobic residues near the aspartic residue. The two catalytic sites are arranged face-to-face in the tertiary structure of correctly folded proteins. In bovine chymosin, the distance between the aspartic side chains is about 3.5 A. The residues are reported to be extensively hydrogen bonded, concomitantly with the adjacent threonine residues, to the corresponding residues of the other domain or the neighbouring atoms of the own domain, to stabilise the correct position. Optimum activity of an aspartic protease is achieved when one of the aspartic residues is protonated and the other one is negatively charged. The active sites of chymosin and other aspartic protease s are embedded, with low accessibility, in the middle of a cleft, about 40 A in length, which separates the two domains, and which is covered by a flap that in bovine chymosin extends from about Leu73 to Ile85 in the N- terminal domain.

Bovine stomachs and the commercial extracts thereof, calf rennet and adult bovine (ox) rennet, contain three types of aspartic protease s (EC 3.4.23) chymosin, pepsin and the minor component gastricsin (Szecsi, 1992). By "rennet" is meant an enzymatic substance that makes milk thick and sour and is used in making cheese. Chymosin is the dominating enzyme in calf rennet and pepsin is the main component in adult bovine rennet whereas gastricsin is a minor component accounting for less than 1 % of the total enzymes in a calf rennet up to 5 % in adult bovine rennet. The two main enzymatic components of calf/bovine rennet show heterogeneity in electrophoretical mobility due to differences in their amino acid sequence (chymosin) or different degree of phos- phorylation (pepsins), both resulting in different net charge of each subcomponent (Foltmann, 1979). For bovine chymosin three iso-enzymatic fractions A, B, C, mentioned in the order of de- creasing milk-clotting activity (and in reverse order of elution), has been described (Foltmann, 1966). Other authors (Asato & Rand, 1972 and 1977) have found four forms of (pro)chymosin named A, B, C and D, separated by their electrophoretic mobility. These four components were not well characterised and it is doubtful if they represent different genetic forms. The two major chymosin A and B has been fully characterised incl. amino acid and gene sequence (Foltmann, 1979; Foltmann, 1992). It is generally agreed that chymosin A and B, are allelic variants of a single gene locus (Donnelly, 1984), which differs by only one amino acid : the A form has an Asp in position 244 (according to the natural numbering of the chymosin sequence) where chymosin B has a Gly.

There has been controversial reports about chymosin C. Several papers (Foltmann, 1964 and Foltmann, 1977) conclude that chymosin C originates, at least partly, from autolytic degradation of chymosin A and Danley & Geoghegan (1988) describe the mechanism behind formation of chymosin C.

Donnelly, Carroll, O'Callaghan & Walls (1986) have however proposed, based on extraction of a few single stomachs, that a third allelic chymosin exist. A similar result was reported by Wislinski et al. (1994) and Rampili et al. (2002). Interesting, Foltmann (1992) reported that chymosin C has an activity similar to that of chymosin B, whereas Donnelly et al. (1986) reported that the specific activity of chymosin C is lower that of chymosin B. Besides this, Harboe (1992) has proposed that further microheterogeneity, which has been observed in rennets and fermentation-produced chy- mosin (FPC), is due to easy deamidations of a specific asparagine 160. This observation is supported by the finding of the code for an asparagine in the gene sequence but an aspartic acid in the protein sequence.

Thus, the literature on chymosin C is not clear, but it has generally been believed that the C frac- tion obtained by chromatography of calf rennet mainly was degraded chymosin A.

Lately, Lilla et al. (2001 ) have characterised the chymosin variants in rennets and other chymosin compositions by mass spectrometry. Chymosin A and B were each found in three active forms differing at the N-terminal end, one being three residues longer and one two residues shorter than the published chymosin, indicating that, at least under some conditions, the splitting site between the pro-part and the active chymosin can vary. Further two degradation fragments were found in rennet, one corresponds to the degradation of chymosin A whereas the other seems new. However, neither the genetic variant chymosin C nor the deamidated forms of chymosin were detected in this last study. When produced in the gastric mucosal cells, chymosin occurs as enzymatically inactive pre- prochymosin. When chymosin is excreted, an N-terminal peptide fragment, the pre-fragment (signal peptide) is cleaved off to give prochymosin including a pro-fragment. Prochymosin is a substan- tially inactive form of the enzyme which, however, becomes active under acidic conditions and forms the active chymosin by autocatalytic removal of the pro-fragment. An intermediate pseudo- chymosin, where only part of the propart is removed) is formed under some condition, but psedo- chymosin is at higher pH values such as pH 5 -6, further processed to chymosin. This activation occurs in vivo in the gastric lumen under appropriate pH conditions or in vitro under acidic condi- tions.

The structural and functional characteristics of bovine, ie. Bos taurus, pre-prochymosin, prochymosin and chymosin have been studied extensively (Foltmann, 1977). However, this reference has 2 printing errors in the sequence. The correct sequencefor chymosin is published by Foltmann (1979). The pre-part of the bovine pre-prochymosin molecule comprises 16 aa residues and the pro-part of the corresponding prochymosin has a length of 42 aa residues. Foltmann et al. (1997) have shown that the active bovine chymosin comprising 323 aa is a mixture of two forms, A and B, both of which are active, and sequencing data indicate that the only difference between those two forms is an aspartate residue at position 302 (in accordance with the amino acid positions used for chymosins used herein) in chymosin A and a glycine residue at that position in chymosin B. Nishi- mori et al. (1994) have provided a partial chymosin sequence comprising 241 amino acid residues as well as the cDNA sequence comprising 1261 nucleotides. Although the authors refer to this sequence as chymosin C they specifically note that this gene most likely is a pseudogene or contain errors.

Hitherto there has not been demonstrated any commercial interest in isolating chymosin C in pure form from rennet or extracts of bovine stomachs. One possible reason is that the existence of a third chymosin has not been clearly established. Another reason is the rarity of the gene and the CC phenotype making it difficult to identify proper source animals, cf. Example 1 below. A third reason could be the reported low activity compared to chymosin B and its relative instability, Donnelly et al. (1986).

Bovine chymosin, in particular the major isoforms of the enzyme, chymosin A and chymosin B, is or has been commercially available as recombinantly produced enzymes expressed in bacterial, yeast or fungal host cells (see e.g. WO 95/29999; Ward et al.,1990). There are, however, serious drawbacks connected with each of the presently produced bovine chymosins. Chymosin A is unstable as it will easy undergo autocatalytic degradation, and chymosin B, although more stable than chymosin A, has considerably less enzymatic activity than chymosin A. Typically, the activity of chymosin B measured as International Milk Clotting Units (IMCU) per mg is only about 60% of the chymosin A activity. In the present context IMCU is measured according to International Dairy Federation, IDF, standard 157. Donnelly et al. (1986) reported that also chymosin C undergo autocatalytic degradation during its preparation.

Presently, bovine chymosin is manufactured industrially using recombinant DNA technology, e.g. using filamentous fungi such as Aspergillus species (see e.g. Ward, 1990), yeast strains, e.g. of Klyuveromyces species, or bacterial species, e.g. E. coli, as host organisms. Such recombinant microbial production strains are constructed and continuously improved using DNA technology as well as classical strain improvement measures directed towards optimising the expression and secretion of the heterologous protein, but it is evident that the productivity in terms of overall yield of gene product is an important factor for the cost effectiveness of industrial production of the enzyme. Accordingly, a continued industrial need exists to improve the yield of chymosin in recombinant expression systems. Whereas efforts to improve yields of chymosin activity up till now have exclusively been concerned with chymosin A and chymosin B of bovine origin, the industry has not yet explored the possibility of providing effective bovine chymosin C preparations, probably due to the disadvantageous activity and stability features reported in literature.

SUMMARY OF THE INVENTION The invention relates in one aspect to a method of obtaining a preparation of an aspartic protease, chymosin C, having the following features: (a) it possess milk-clotting activity, (b) when crude preparations thereof are analyzed by ion-exchange HPLC the major peak of chymosin C protein elutes after the peak of the degradation product A2 of bovine chymosin A and before the major peak of bovine chymosin B, (c) chymosin C protein shows an apparent molecular weight similar to bovine chymosin A and B, (d) when purified preparations of chymosin C are analyzed by isoelectric focusing (IEF) and compared with purified bovine chymosin B preparations, chymosin C has a higher isoelectric point than bovine chymosin B, (e) The morphology of the precipitates formed by chymosin C in Rocket immunoelectrophoresis using rabbit polyclonal monospecific antibodies raised against purified calf prochymosin (mainly B variant) is identical to the morphology of the pre- cipitates formed by bovine chymosin A and B, and (f) The specific milk clotting activity of the chymosin C is as least 20 % higher than the specific milk clotting activity of bovine chymosin B. The method comprises the steps: i) obtaining an aqueous rennet preparation which comprises chymosin C, but comprise less than 1% chymosin A and less than 1% chymosin B compared to the amount of chymosin C in the preparation, ii) subjecting said aqueous rennet preparation to either a hydrophobic chromatography followed by a anion chromatography or chromatography on a column containing a mixed-mode, hydrophobic-/anion exchange chromatographic material to obtain a further purified fraction of chymosin C, discarding any other chymosin variant fractions, and Hi) optionally, subject said chymosin C fraction to gel filtration on a suitable resin to obtain an even further purified chymosin C preparation. As used herein "chymosin C" refers to refer to chymosin C derived from Bos taurus.

In further aspects a preparation is provided comprising an at least 90% pure bovine chymosin C preparation obtained through isolation and purification from a natural source, such as calf stomachs or cultivated cells which contains a bovine chymosin C coding sequence and which comprise at least 3 mg chymosin C. One preferred method uses the isolation steps and purification steps described in Example 1 below as well as a chymosin C preparation produced by the above method including such an enzyme that is in a substantially deglycosylated form. By "preparation" is meant any final enzyme containing products which is formulated with stabilising agents such as sodium chloride and preservatives such as sodium benzoate. A chymosin preparation may be in liquid or solid form.

In yet another aspect, the invention relates to a method of manufacturing cheese, comprising add- ing a milk clotting effective amount of an essentially pure preparation of chymosin C to the milk and carrying out appropriate further cheese manufacturing steps.

As used herein "an essentially pure preparation of chymosin C" refers to an enzyme preparation having only insignificant milk clotting activity derived from other milk clotting enzymes, such as pepsin and chymosins A and B. Said essentially pure preparation of chymosin C can be prepared by isolating and purifying the enzyme from bovine stomachs or by harvesting and purifying the enzyme from a recombinant host cell which is capable of expressing the enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates a HPLC ion-exchange chromatographic profile of a commercial calf rennet showing four isoenzymes fractions for chymosin and four-five fractions for pepsin. Two fermentation produced chymosins with known chymosin variant ("Chy Max" for recombinant chymosin A from E. coli, and "Maxiren" for recombinant chymosin B) are included in order to identify the positions of chymsin A and B.

Fig. 2 illustrates a HPLC ion-exchange chromatographic profile showing the six different types of chymosin compositions found in individual stomachs.

Fig. 3 shows the HPLC analysis of an individual stomach extract (chymosin AC) before and after autolysis, in comparison with a commercial calf rennet.

Fig. 4 illustrates a rocket immuno electrophoresis showing immunological identity between the three chymosin variants. Antiserum: Rabbit polyclonal monospecific antibodies raised against purified calf prochymosin (mainly B variant). Sample Nos. 1 , 2 and 3: Chymosin standard containing 8.44, 5.07 and 1.90 IMCU per ml. Sample Nos. 4, 5 and 6: The three purified chymosin variants A, B and C respectively.

Fig. 5 shows a chromatogram of an autolysis test performed according to Danley, DE et al. (1988) on an individual chymosin A extract. The thick curve line corresponds to chymosin A from a homo- zygous animal and the thin curve line corresponds to the autolysis product, A₂.

Fig. 6 shows a chromatogram of an autolysis test performed according to Danley, DE et al. (1988) on an individual chymosin B extract. The thick curve line corresponds to chymosin B from a homo- zygous animal and the thin curve line corresponds to chymosin B after autolysis.

Fig. 7 shows a chromatogram of an autolysis test performed according to Danley, DE et al. (1988) on an individual chymosin C extract. The fat curve line corresponds to chymosin C from a homozy- gous animal and the thin curve line corresponds to chymosin C after autolysis.

Fig. 8 shows the result of the hydrophobic chromatography step of the preparative purification. The dotted curve is the absorption at 280 nm of each fraction, the dashed line is the activity measured as IMCU per ml of separate fractions and the unbroken line indicates fraction no. 9-20 which was pooled and purified further.

Fig. 9 illustrates the ion-exchange chromatographic profile step of the preparative purification. The thick curve is the absorption at 280 nm of each fraction the thin line indicates fraction no. 9-13 which was pooled and purified further.

Fig. 10 illustrates the gel filtration step of the preparative purification. The thick curve is the absorption at 280 nm of each fraction the horizontal line indicates the fraction that contained the purified chymosin C.

Fig. 1 1 illustrates the a chromatography was performed on a column comprising a mixed mode resin (Mimo 1300™, Upfront, Copenhagen). The unbroken curve is the absorption at 280 nm of each fraction, the dotted line is the activity measured as IMCU per ml of separate fractions and the horizontal line indicates the fraction that contained the purified chymosin C

DETAILED DISCLOSURE OF THE INVENTION

In accordance with the invention, there is, in one aspect of the invention, provided a method of obtaining a preparation of chymosin C which comprise at least 3 mg chymosin C. Such a preparation has to the best of our knowledge not been provided before, and allowed the present inventors to perform a detailed characterisation of chymosin C. During the experimentation leading to the inven- tion it was a highly unexpected finding that isolated chymosin C exhibited an enzymatic activity measured as IMCU per mg which was 43% higher than the activity of bovine chymosin B and corresponding to 92% of the activity of bovine chymosin A, cf. Table 2 below. In accordance herewith, the above method of the invention is preferably a method wherein the enzymatic activity of chy- mosin C is at least 20%, 35%, 40%, 45% or 50% higher than the activity of bovine chymosin B, or wherein the enzymatic activity of chymosin C is at least 85%, 90%, 95% or equal to the activity of bovine chymosin A. Another unexpected finding was that chymosin C was not found to be auto- catalytically degraded at acid pH as described for chymosin A (Danley (1988)), cf. Example 1 and Fig. 7. In that respect chymosin C behaved identically to chymosin B and different from chymosin A. Thus as defined herein chymosin C refers to an aspartic protease having the following features: (a) it possess milk-clotting activity, (b) when crude preparations thereof are analyzed by ion-exchange HPLC the major peak of chymosin C protein elutes after the peak of the degradation product A2 of bovine chymosin A and before the major peak of bovine chymosin B, (c) chymosin C protein shows an apparent molecular weight similar to bovine chymosin A and B, (d) when purified preparations of chymosin C are analyzed by isoelectric focusing (IEF) and compared with purified bovine chymosin B preparations, chymosin C has a higher isoelectric point than bovine chymosin B, (e) The morphology of the precipitates formed by chymosin C in Rocket immunoelectrophore- sis using rabbit polyclonal monospecific antibodies raised against purified calf prochymosin (mainly B variant) is identical to the morphology of the precipitates formed by bovine chymosin A and B, and (f) The specific milk clotting activity of the chymosin C is as least 20 % higher than the specific milk clotting activity of an essentially pure preparation of bovine chymosin B.

Crude preparations of rennet containing both chymosin A and B can be resolved into well separated peaks of chymosin A and B by HPLC on a number of different columns, this is not the case with chymosin C. It is in particular difficult to distinguish chymosin C from the degradation product (A₂) of chymosin A. The inventors have however found that chymosin C separates well from A₂ when the ion-exchange HPLC is performed on a PL-SAX column (Polymer Laboratories Inc., Am- herst, USA) or a similar HPLC media containing a hydrophilic, strong anion exchange chromatographic packing material, cf. Example 1 below. Thus in one embodiment of the invention the chymosin C is characterised by an ion-exchange HPLC using such columns and preferably using the assay conditions described by Panari et al. (1990).

The apparent molecular weight may be determined by a number of different methods. In one embodiment the apparent molecular weights of chymosin A, B and C are determined by SDS PAGE under reducing conditions essentially as described by Laemmli (1970) or both by SDS PAGE under reducing conditions and by gelfiltration on Superose 12 HR 10/30 (Amersham Biosciences, Piscataway, USA). In both embodiments chymosin C protein showed an apparent molecular weight similar to bovine chymosin A and B.

5 As described in example 1 the isoelectric points were estimated by isoelectric focusing (IEF) and it was found that chymosin C had a slightly higher isoelectric point than both chymosin A and chymosin B. Accordingly in one embodiment of the invention the chymosin C is characterised by having an isoelectric point which is between 0.01 and 0.2 pH units higher than the isoelectric point of bovine chymosin B.

10 As mentioned it was surprisingly found that chymosin C isolated according to the present invention expressed a higher specific activity than purified bovine chymosin B, cf. Table 2. Thus, in one embodiment of the invention the chymosin C is further characterized by having a higher specific activity measured as IMCU according to IDF standard 157 that is as least 20%, 35%, 40%, 45% or even

15 50% higher than the specific milk clotting activity of an >95% pure preparation of bovine chymosin B and at least 85%, 90%, 95% or equal to the activity of an >95% pure preparation of bovine chymosin A. The purity of enzyme preparations are determined Coomassie stained sels after SDS- PAGE.

20 It was similarly surprising to observe that the chymosin C not was susceptible to autocatalytic degradation during its preparation, therefore in one embodiment chymosin C is further characterized by being more stable than chymosin A with regard its degradation caused by autocatalytic self- degradation, cf. example 1. 5 As chymosin preparations typically is used in the preparation of foodstuff wherein reproducibility is an essential factor it is often considered advantageous to provide costumers with preparations of a known (high) purity. Thus a preferred embodiment of the invention provides a method obtaining a preparation of chymosin C which comprise at least 3 mg chymosin C of a high purity from an aqueous rennet preparation, comprising the method comprises the steps of i) subjecting the aque-

30 ous rennet preparation to hydrophobic chromatography on a suitable resin to obtain an almost pure chymosin eluate, ii) further subjecting this eluate to anion chromatography on a suitable resin to obtain a further purified fraction of chymosin C, discarding any other chymosin variant fractions, and iii) finally, further purify the fraction of chymosin C by subjecting it to gel filtration on a suitable resin to obtain an at least 70%, 80%, 95% or even 99% pure chymosin C preparation. One suit- 5 able resin is the Superose 12 HR 10/30 (Amersham Biosciences, Piscataway, USA). The purity of the chymosin preparations are determined Coomassie stained sels after SDS-PAGE, e.g. by laser densitometric scanning of the gels. In the industrial setting it is advantageous if the number of purification steps in a procedure can be reduced. A number of so-called "mixed-mode" resins, which are resins that incorporates a two or more functionalities, have been described. As shown in example 3 the hydrophobic chromatography step and the anion chromatography step may be substituted with a single chromatographic step on a column containing a mixed-mode resin which incorporates a hydrophobic interaction as well as an anion exchange functionality. Consequently in a preferred embodiment of the present invention the chymosin C is obtained from an aqueous rennet preparation by a method, which comprises chromatography on a column containing a mixed-mode, hydrophobic-affinity/anion exchange chromatographic packing material. In a particularly preferred embodiment the method of obtaining a preparation of chymosin C comprises the use of the mixed-mode, hydrophobic- affinity/anion exchange chromatographic packing material MIMO 1300™ (Upfront A/S, Copenhagen). It is a further advantage of the MIMO 1300™ that it is suitable for standard packed bed column chromatography as well as for expanded bed adsorption chromatography.

Although excellent purification are obtained with mixed-mode, hydrophobic-affinity/anion exchange chromatographic packing material such as the MIMO 1300™ the chymosin C preparation may be further purified by subjecting the chymosin C fractions to gel filtration on a suitable resin, such as Superose 12 HR 10/30 (Amersham Biosciences, Piscataway, USA), to obtain an at least 70%, 80%, 95% or even 99% pure chymosin C preparation.

We have found that it may be advantageous to include into the purification method an activation step which is performed before the first chromatography step in the method. The activation comprises: i) treatment of the crude stomach extract at pH between 1.8 and 2.2, preferably at pH approximately 2, ii) centrifugation and collection of the supernatant, and iii) raising the pH of the su- pernatant was raised to between pH 6.0 and pH 5.6, preferably pH approximately 5.8. During the activation step the pre-prochymosin C and the prochymosin C present in the aqueous rennet preparation is converted to chymosin C. Furthermore a substantial amount of non-chymosin proteins are removed. An even further removal of non-chymosin proteins can be obtained if a clarification step is introduced before the first chromatography step, said clarifying step being a centrifuga- tion step or a chemical clarification procedure comprising the following steps: i) addition of 1/3 M aluminium sulphate to a chymosin comprising preparation until pH is between 3.6 and 4, preferably approximately 3.8, ii) addition of 1 M di-sodium phosphate until pH is between 5.4 and 5.8, preferably approximately 5.6, and iii) obtaining a clear supernatant by centrifugation, and iv) harvesting the chymosin C comprising cleared supernatant.

It is an important step of the method of the invention to obtain an aqueous rennet preparation which comprises chymosin C, but comprise less than 1 % chymosin A and less than 1 % chymosin B compared to the amount of chymosin C in the preparation. In one embodiment the aqueous ren- net preparation is obtained from chymosin C homozygous bovine abomasums, however the chymosin C comprising aqueous rennet preparation may be obtained from other sources.

One possible source of chymosin C comprising aqueous rennets are obtainable by fermentation of a recombinant microorganism expressing chymosin C. With the dissimination of modern gene cloning techniques the skilled artisan will appreciate that when a) preparations of milligram-amounts of highly purified chymosin C, and b) well preserved tissue samples from CC phenotype animals are available, cf. Example 1 ; then it is relative strait forward to obtain a nucleic acid sequence, i.e. a polynucleotide, of that comprises a sequence that codes for chymosin C.

Several approaches for obtaining such a sequence can be used including one based on the isolation of mRNA from calf or ox mucosal cells and using this RNA as template in a nucleotide amplification procedure such as a PCR reaction using suitable sense and anti-sense primers which e.g. may be constructed synthetically based on the known sequences for bovine chymosin A and B. The person of skill in the art will appreciate that other methods for obtaining a coding sequence according to the invention may be used such as hybridisation procedures using as probes fragments of known coding sequences for chymosin that will permit the presence of homologous DNA or RNA to be detected in preparations of bovine. Alternatively, it is possible to construct a coding sequence based on the isolation of chymosin C followed by determining the amino acid sequence of the enzyme or fragments hereof which in turn permits the construction of primer oligonucleotides for detection and construction of coding sequences. The basic techniques that are required in the above procedures of obtaining coding sequences are generally within the common knowledge of the skilled artisan (Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular cloning. A labora- tory manual. 2^nd edition. Cold Spring Harbor Laboratory Press).

Whereas it may appear relative strait forward to obtain a nucleic acid sequence that comprises a sequence that codes for a certain protein, e.g. chymosin C, experience tells us that it may prove rather cumbersome to obtain host cells that express the protein as a polypeptide.

The critical step is often to select a proper host cell organism and in particular to transform said host cells with the DNA construct. In one specific embodiment, the host cell is Aspergillus niger var. awamori and the chymosin coding nucleotide sequence is inserted into pGAMpR as described in Ward et al., 1990 by substituting the coding sequence of that vector for chymosin C. The detailed transformation procedure of Aspergillus niger var. awamori with a pGAMpR variant is described in example 4.

Having obtained suitable host cells, e.g. Aspergillus niger var. awamori , containing a DNA construct which comprise a bovine chymosin C coding sequence operably linked to appropriate expression signals, such as those provided by pGAMpR, permitting said chymosin C comprising cod- ing sequence to be expressed as a polypeptide in the host cell, then the cells are cultured under conditions conducive to expression of the polypeptide having chymosin C activity, e.g. as described in example 4 section 2.1.

The resulting chymosin C preparation is typically recovered or harvested from the cultivation medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, if necessary, after disruption of the cells, followed by precipitating the proteina- ceous components of the supernatant or filtrate e.g. by adding a salt such as ammonium sulphate, followed by the purification according to the present invention.

It is generally known that polypeptides expressed by eukaryotic host organisms may be glycosy- lated when expressed, the degree of glycosylation depending on the type of polypeptide and host organism. It has been found previously that the milk clotting activity of aspartic protease s of micro- bial origin that are glycosylated upon expression may be enhanced by subjecting the proteases to a deglycosylating treatment to at least partially remove the sugar moieties attached to the proteases. Such a deglycosylation treatment may e.g. comprise treating the glycosylated protease with an enzyme having a deglycosylating activity including as examples PNGase and endo-β-N- acetylglucosaminidase (EC 3.2.1.96) (Endo-H). Alternatively, the deglycosylation may be obtained by subjecting the glycosylated protease to a chemical treatment, such as treatment with periodate.

Accordingly, in a specific embodiment, the above mentioned method comprises, as a further step, that the harvested chymosin C is subjected to a deglycosylation treatment, in particular an embodiment wherein the deglycosylation treatment comprise the deglycosylation by enzyme EndoH is preferred.

One useful method of deglycosylating a protein is described in WO 96/19582 which is hereby incorporated by reference.

In a still further aspect a milk clotting preparation is provided comprising a chymosin C as defined herein and produced by the above method including such a chymosin C that is in a substantially deglycosylated form. Such a preparation may, in addition to the active milk clotting enzyme, comprise additives that are conventionally used in rennets of animal origin such as e.g. NaCI.

To the best of our knowledge this is the first disclosure an at least 90% pure preparation of chy- mosin C, comprising at least 3 mg Chymosin C, likevise we believe that this is the first disclosure of an at least 90% pure preparation of chymosin C, produced by the preparative method of the invention, and which comprises at least 3 mg Chymosin C. The preparative method is described in details in Example 1 and 2. The chymosin C as provided herein is useful as a milk coagulant product. Accordingly, an important objective of the invention is to provide a method of manufacturing cheese, comprising adding a milk clotting effective amount of the above preparation to milk and carrying out appropriate further cheese manufacturing steps. The chymosin C of the invention is suitable for cheese manufacturing processes wherein the milk is selected from cow's milk, buffalo milk, goat's milk and sheep's milk.

In addition, the invention relates to a method of obtaining essentially pure chymosin through the following steps: 1 ) Identifying a cow and a bull which are homozygous for chymosin C 2) Using these to make a breeding population of homozygotes 3) Using calves from this population to make 'traditional' rennet which will be a mixture of chymosin C and pepsin.

The invention will now be described in further details in the following, non-limiting examples.

EXAMPLE 1

The chymosin component in calf and adult bovine rennet show heterogeneity with respect to charge and activity. A large number of single stomachs have been extracted and their isoenzymes has been analysed by HPLC. The results showed three genetic forms of chymosin A, B and C and these are present in individual stomachs in the following combinations: AA, BB, CC, AB, AC and BC. The C variant, which can be distinguished from the degradation product (A₂) of chymosin A, has never been characterized before. All three chymosins have been purified and partially characterised.

1.1 Preparation of stomach extract

Ninety frozen bovine abomasa were purchased from different countries and individually ground and extracted, activated and clarified by the following procedure. Mucosa, cut off from whole stomach, were mixed with equal amounts (by weight) of Milli-Q water and minced by blending for 1 minute. The extract was centrifuged 15 min at 5000 rpm and the proenzymes in the supernatant were activated at pH 2.0 by sulphuric acid. After centrifugation 15 min at 5000 rpm, pH in the supernatant was raised to 5.8 and clarified by the following alum clarification procedure : to the supernatant of activated extract, 1/3 M aluminum sulphate was added until pH dropped to 3.8. Immediately after 1 M di-sodium phosphate was added until pH increased to 5.6 and the unclear liquid was centrifuged for 30 min at 5000 rpm. The clarification was repeated if the enzyme extract was not clear after one treatment. The extract was preserved with 0.05 % sodium azide and filtered on paper filter.

1.2 Characterization of the stomach extracts All the individual stomach extracts were tested for activity in IMCU per ml by IDF standard 157 and composition by IDF standard 110. Test for autocatalytic degradation of chymosin was performed on selected samples of extract according to conditions described by Danley & Geoghegan (1988).

1.3 HPLC analysis of stomach extracts

All samples were desalted on Econo-Pac 10 DG columns (Bio Rad) and analysed by ion-exchange HPLC on PL-SAX column (Polymer Lab.) according to Panari (1990). Different amounts of sample solutions (50-150 ml) were loaded to the column with respect to their clotting activity previously tested and the relative amounts (% peak area) of chymosin and pepsin fractions were calculated. Some samples of commercial calf rennet (Chr. Hansen) and fermentation produced chymosin (CHY MAX from Pfizer; MAXIREN from Gist-brocades) have also been analysed by the same HPLC procedure.

1.4 Purification of the chymosin variants and their characterization In order to characterize the chymosin variants further each of the three variants was isolated from three individual stomach extracts, each containg one homozygote-type (AA, BB and CC respectively), and purified to homogeneity on FPLC, (Pharmacia) by the following steps: Hydrophobic chromatography on Phenyl-Superose (Pharmacia), anion exchange chromatography on Mono Q (Pharmacia) and finally by gelfiltration on Superose 12HR 10/30 (Pharmacia). The specific activities of the three purified chymosin variants were expressed as IMCU per mg. The activity were measured in IMCU per mg and the concentration was determined by the absorbance at 280 nm which was converted to mg assuming E1 % = 14.0 (Rothe, Axelsen, Jøhnk & Foltmann, 1976). The identity and the concentration of the purified chymosin variants were further confirmed by rocket immuno electrophoresis (rocket IE) as described by Rothe et al., (1976). The same samples were, in comparison with samples of fermentation-produced chymosin A nad B, analysed by standard electrophoretical methods (SDS-PAGE and IEF) to confirm the molecular weight, purity and charge characteristics of the purified chymosin variants.

Results and discussion In the HPLC ion-exchange chromatographic profile of a commercial calf rennet, as shown in Fig. 1 , four isoenzymes fractions for chymosin and four-five fractions for pepsin can normally be distinguished. The comparison with the HPLC profiles of two fermentation-produced chymosin A and B, allows identifying these two chymosin fractions in the calf rennet (RT= 18.6 min; 17.4 min for A and B respectively). The two minor peaks eluted earlier (RT= 15.9 min ; 15.0 min) could be the chy- mosin named C in the literature and an unidentified fraction. No external reference allows their clear identification at this point. Fig. 2 shows the six different types of chymosin compositions found in individual stomachs. The observed distributions of chymosin isoenzymes suggest a genotypic expression of three different alleles (A, B, C) for the chymosin. From these results it seems that chymosin C could represent a third allelic chymosin variant which is the minor peak eluted at 15.9 min on Fig. 1. The main evidence for this hypothesis is the presence of two allelic expression in each stomach, following a Mendelian distribution model where all six possible combinations can be found: homozygotes AA, BB, CC and heterozygotes AB, AC, BC. In other words, two chymosin alleles seem to be expressed co-dominantly in each stomach.

A specific trial was performed in order to identify the chromatographic position of the degradation product of chymosin A, in particular in relation to that of chymosin C : Fig. 3 shows the HPLC analysis of an individual stomach extract (chymosin AC) before and after autolysis, in comparison with a commercial calf rennet. The HPLC profile of the chymosin AC sample submitted to autolysis shows a consistent loss in A peak height, while a newborn chymosin fraction was eluted earlier than the C peak. We suggest the name A2 for this degradation product of chymosin A in order to emphasize its origin: by auto-catalytic excision of one peptide ( Asp244-Glu245-Phe246) from chymosin A (Danley & Geoghegan, 1988). This degradation product A2 of chymosin A seems to be the unidentified fraction in commercial rennet, which is eluted at 15 min (see Fig. 1 ). By the same procedure on other selected samples we verified that neither chymosin B nor chymosin C were hydrolysed under the same conditions (data not presented). The results obtained agree with previous findings (Foltmann, 1979 and Danley & Geoghegan, 1988) that only chymosin A undergoes autolysis under acid conditions, while the other chymosin fractions were unchanged. The result further shows that now, for the first time, is possible to separate and distinguish between chymosin C and the degradation product A2. Our results on many commercial rennets show that they contain no or very little degradation product A2. What had generally been considered as degradation product in the well known chymosin elution profiles (Foltmann, 1966) was probably mainly chymosin C or, if the separation had not been kept under optimal condition, a mixture of C and A2 fractions. The problems concerning the characterisation of chymosin C in the past were most likely due to a combination of its low amount and poor separation of the chymosin variants.

The overall results of HPLC analysis of extracts from ninety single stomachs ( table 1) indicate that the distribution of chymosin allele (table. 1 ) seems to follow the Mendelian model of three alleles without dominance where six possible combinations are expressed. The combinations of pheno- types AB and BB were more frequently found, while the phenotypes containing the allele C were rarely present. The observed allelic frequencies for each of the three chymosin variants were : 39 % A, 53 % B and only 8 % C. These occurrences of A B and C chymosin are close to the values found by Foltmann (1966): 35%, 50% and 15% respectively and confirm previous data of higher mean values for the B variant in bovine rennet (Donnelly, Carroll, O'Callaghan & Walls, 1986; Collin, Cornee & Besaut, 1990) . Table 1 - Phenotype combinations and allele frequency of Chymosin in bovine stomach extracts

Origin Samples Phenotype combinations Allele frequency calf Adult bovine AA AB AC BB BC CC % A % B % C

USA 8 2 3 2 1 0,31 0,44 0,25

Australia 11 1 5 4 2 1 0,21 0,62 0,17

New Zealand 22 14 1 7 0,34 0,64 0,02

France 12 2 2 5 4 3 0,46 0,39 0,14

Italy 15 2 7 3 1 6 0,53 0,44 0,03

Denmark 8 1 3 4 0,31 0,69 0

Austria 4 1 1 1 1 0,38 0,5 0,12

Brazil 4 2 2 0,75 0,25 0

Germany 1 1 0 1 0

Total 80 10 13 35 9 28 4 1 0,39 0,53 0,08

The three chymosin variants were purified from extracts of individual stomachs ofd homozygote- type (contained only one chymosin variant ) and characterised. The specific activities of chymosin A, B and C were measured and the results are shown in table 2: the activity of chymosin B (237 IMCU mg-1) was close to the value earlier found (Harboe & Budtz, 1999), but the activity of chy- mosin A (368 IMCU mg-1 ) resulted higher than the previous data ( Foltmann, 1992). The most surprising result is that chymosin C has been found to have higher activity (339 IMCU mg-1) than chymosin B, whereas earlier estimates stated it had only 60 % of the activity of chymosin B. These new data on the different activities of chymosin fractions give therefore evidence to the knowledge of the real composition of commercial calf rennets.

Table 2 - Specific activity of chymosin variants

IMCU per mg variant data from this study data from Harboe & Budtz (1999)

Chymosin A 368 291

Chymosin B 237 223

Chymosin C 339 not determined The molecular weight of the three purified chymosins were shown to be Identical and the same as that of chymosin A and B in the FPC samples by SDS-PAGE and gelfiltration on Superose 12. The isoelectric points were estimated by isoelectric focusing (IEF). Identical values, close to those published, were found for each purified chymosin (4.44, 4.57 and 4.60 for chymosin A, B and C, respectively) and the corresponding variant in FPC (4.45 and 4.52 for chymosin A and B, respec- tively). Chymosin C has a slightly higher isoelectric point than chymosin B. The differences in isoelectric points of the chymosin variants are also reflected by the elution time from ion exchange chromatography both on Mono Q and by the HPLC. Rocket immuno electrophoresis in Fig. 4 shows immunological identity between the three chymosin variants, which is seen from the identi- cal morphology of the precipitates). Besides identifying the purified chymosin A, B and C fractions as chymosin, they confirmed the concentration of chymosin determined by absorbance at 280 nm.

The individual stomach extracts were classified as calf (80 samples) or ox (10 samples) in relation to their % chymosin activity by Standard IDF 110B: higher or lower than 75% (corresponding about to 50% peak area) respectively.

Conclusions A consistent and surprising variability has been observed in individual bovine stomachs, either in qualitative or quantitative distribution of chymosin and pepsin fractions. Three allelic forms A, B, C of chymosin exist, not only A and B as earlier believed. Their combinations follow a Mendelian model where six possible phenotypes were found. Two chymosin alleles are co-dominantly expressed in each single stomach. A degradation product of chymosin A, named A2, can be distinguished from the chymosin C by the HPLC method described. Chymosin C was found to have a surprisingly high specific milk-clotting activity (IMCU per mg)

EXAMPLE 2 Preparative purification of chymosin C

Chymosin C was purified from a homozygote (CC) stomach by three different types of chromatography in order to obtain a completely pure preparation.

All chromatography was carried out on a FPLC (Pharmacia/Amersham)

The first purification step was by hydrophobic chromatography on Phenyl suparose™ (Pharmacia) HR 5/5 (1 ml). 5 ml of a stomach extract, prepared as described in example 1 ), containing 118 IMCU/ml was added 9% sodium sulphate and adjusted to pH 5.85 before being applied on the column.

The equilibration and starting buffer were 0.005 M sodium phosphate pH 5.85 containing 9 % sodium sulphate (buffer A). The elution consisted of: 5 ml washing with buffer A, a gradient (5 ml) from buffer A to buffer B (0.005 M sodium phosphate pH 5.85) and further elution by 30 ml buffer B. Flow rate was 0.5 ml per min. 2 ml fractions were collected, the absorbance at 280 nm was recorded and activity as IMCU/ml was followed. Fractions with activity (no 9-20) were pooled and purified further. The result of the first purification step is shown in figure 8. The second purification step was an ion exchange chromatography on Mono Q HR 5/5. 2.5 ml from the previous step was applied. The equilibration and starting buffer (buffer A) was 0.05 M sodium phosphate pH 5.8. Elution took place as a linear gradient from buffer A to 0.5 M sodium phosphate pH 5.5, using a total volume 36 ml. 2 ml fractions were collected. Flow rate was 1 ml per min. Absorbance at 280 nm were recorded and activity in each fraction were measured. Fractions with activity (no 9-13) were pooled and purified further. The result of the second purification step is shown in figure 9.

The third purification step was a separation by molecular size (gelfiltration) on Superose 12 HR 10/30. 0.5 ml from the previous step was applied. The buffer used was 0.05 M sodium phosphate pH 5.8, 0.15 M sodium chloride. A total volume of buffer was 40 ml. 10 ml fractions were collected in order to check for the activity but the main peak was collected in one fraction (2.5 ml). Flow rate was 0.5 ml per min. Absorbance at 280 nm was recorded and activity in each fraction was measured. The main fractions with activity were used for further characterization. The result of the gelfil- tration step is shown in figure 10.

SDS-PAGE

SDS-PAGE was carried out with Novex XCell Mini Cell system (Invitrogen Life Technology) using Bio Rad Power Pac 200 and Mini gel Drying system. NuPAGE Novex high-performance precast (Invitrogen Life Technology) gel 4-12% Bis-Tris was used. Electrophoresis took place for 10 min at 150 V followed by 200 V for 25 min. The gel was stained with Coomassie blue as described (Sam- brook, J., Fritsch, E. F. and Maniatis, T. (1989)).

Isoelectric focusing (IEF) IEF was carried out on Multiphor Electrophoresis System (Pharmacia/Amersham) and Horisontal flat bed electrophoresis - A ready to use kit (Serva). Gel size 125 x 125 mm, thickness 150 μ. Running condition 2000 V, 4 mA, 6 W. pH range 45-6. The conditions recommended by the supplier was followed.

EXAMPLE 3 Isolation of chymosin from a preparation which contains chymosin as the only chymosin variants

This experiment has been done on a calf rennet extract from a stomach containg chymosin C but no chymosin A or B as judge by the HPLC method described in example 1. The chromatography was performed on a FPLC (Pharmacia/Amersham) using a mixed mode resin (Mimo 1300, Up- front) packed in a fixed bed column size HR 5/5 (Pharmacia/Amersham). The column was equilibrated with 0.025 M sodium phosphate pH 5.5 (buffer A). Elution took place by a linear gradient from buffer A to 0.122 M sodium phosphate pH 2.0 using a total of 40 ml. Flow rate was 0.5 ml per min. 1 ml fractions were collected, the absorbance at 280 nm was recorded and activity as IMCU/ml was followed. Fractions with activity (no 12-13) contained the chymosin and were pooled. The result is presented in figure 11. This chromatography does not separate the chymosin variants but is efficient to isolate the chymosin and is thus in particular useful when only one chymosin variant is present. 5 EXAMPLE 4 Transformation of Aspergillus niger var. awamori with pGAMpR-C For these transformation experiments, a derivative of Aspergillus niger var. awamori, strain dgr246pyrG Ward et al. (1993) is used as recipient. This strain is a derivative of Aspergillus niger var. awamori strain GCI-HF1-2dgr246 having a pyrG mutation, rendering the strain incapable of 10 growing in the absence of uridine, and which comprises several copies of the pGAMpR plasmid. The derivative strain, dgr246pyrG used as recipient is derived by curing the pyrG mutant parent strain for all copies of pGAMpR.

An optimised protocol as developed by Chr. Hansen A/S will be applied for transformation of the 15 "cured" Aspergillus strain. This protocol comprises the steps of providing a liquid culture medium, propagation of fungal biomass, generation of protoplasts and transformation including regeneration of protoplasts and selection of transformants.

2.1 Propagation of fungal biomass

20 50 ml of CSL medium [per litre: corn steep liquor, 100 g; NaH₂P0₄.2H₂0, 1 g; MgS0₄, 0.5 g; Mazu antifoaming agent, 2g, maltose, 100 g, glucose, 10 g, fructose, 50 g, water 736.5 g] is added to a sterile 250 ml flask, 0.5 ml penicillin/streptomycin supplement (Gibco-BRL #15140-114) is added and the medium inoculated with 106 spores per ml. The inoculated medium is cultivated overnight at 34-37C at 200-250 rpm to obtain a dense suspension of mycelium. 10 ml of this pre-culture is

25 transferred to 100 ml complete Aspergillus medium in a 500 ml flask without baffles, incubation overnight at 34-37C at 200-250 rpm to obtain a mycelial biomass.

2.2 Generation of protoplasts Mycelium as obtained in the above step is filtered over sterile myracloth, washed with sterile water 30 and subsequently with 1700 mOsmol NaCl/CaCI2 (0.27 M CaCI₂.2 H₂0, 39.7 g/l; 0.58 M NaCI, 33.9 g/l), gently squeezed dry and transferred to a Falcon tube to determine the weight and left to stand on ice.

20 ml 1700 mOsmol NaCI/CaCI₂ per g mycelium is added to resuspend the mycelium followed by 35 adding 50 mg Sigma L-1412 Trichoderma harzianum Lytic Enzyme per g mycelium dissolved in a small volume of 1700 mOsmol NaCI/CaCI₂ , incubation in an Erlenmeyer flask at 100 rpm, 37C for about 4 hrs during which period the mycelium is repeatedly resuspended every 30 minutes. When good protoplasting is obtained, i.e. many free protoplasts occur and with hardly any intact mycelium left, the mixture is filtered on ice, using Mesh sheet or myracloth and an equal volume of ice cold STC1700 (1.2 M sorbitol, 218 g/l; 35 mM NaCI, 2.04 g/l; 10 mM Tris.HCI pH 7.5 and 50 mM CaCI₂.2 H₂0, 7.35 g/l) is added. The number of proto-plasts is counted using a glass Bϋrger- Turk chamber. The protoplast suspension is spun using a bench top centrifuge at 2,000 rpm at 4C. The resulting pellet is resuspended very gently in 20 ml ice cold STC1700. This washing procedure is repeated twice and the final pellet is resuspended in ice cold STC1700 to a final concentration of about 1 x 10⁸ protoplasts per ml followed by adjustment to 1 x 10⁸ protoplasts per ml.

2.3 Transformation

200 I (2x10⁷ protoplasts), 2 I of 0.5 M ATA (0.5 M aurine carboxylic acid (Sigma) in 20% ethanol) and DNA (comprising a marker) up till 15 I, typically 5-10 g of DNA, is mixed in a 12 ml test tube. As control a corresponding mixture, but without DNA is used. The transformation mixtures are incubated on ice for 25 min. followed by adding a first drop of 250 I PTC (60% PEG 4000; 10 mM Tris.HCI pH 7.5; 50 mM CaCI₂) by tipping the tube a couple of times without letting the mixture touch the lid and a second drop of 250 I, mixing and adding 850 I followed by mixing. Each tube is incubated at room temperature exactly 20 min. followed by filling the tubes with ice cold STC1700 and mixing by reverting the tubes. The mixture is centrifuged for 8-10 min. using a bench top centrifuge at 2000 rpm at 4C. The resulting pellet is dissolved gently in about 400-800 I STC1700.

2.4 Regeneration and selection of transformants

The transformation mixture is spread onto solid selective regeneration medium plates containing per I medium: agar, 15 g; sorbitol, 218 g; AspA salts 50x (per litre: 300 g NaN03, 26 g KCI, 76 g KH2P04, 18 ml 10 M KOH, pH about 6.5); glucose 50%, 20 ml; Gibco-BRL #15140-114 Pen- Strep, 10 ml; MgS04, 2 ml; trace elements (2.2 g ZnS0₄, 1.1 g H₃B0₃, 0.5 g MnCI₂.7H₂0, 0.5 g FeS0₄.7H₂0, 0.17 g CoCI₂.6H₂0, 0.16 CuS0₄.5H₂0, 0.15 NaMo0₄.2H₂0, 5 g EDTA, water to 100 ml, pH 6.5), 1 ml. The plates are incubated at 37C for 5-10 days and transformants selected. About 80 transformants were obtained and spores of these transformants were obtained.

EXAMPLE 5 The milk clotting activity of recombinant chymosin C

The milk clotting activity of the chymosin C prepared according to the invention was studied at 32°C using as substrate 10% (w/v) low heat spray-dried bovine skimmed milk was used (according to International Dairy Federation, IDF, standard 157). The milk clotting activity is expressed as In- ternational Milk Clotting Units (IMCU) per mg protein (according to IDF standard 157). References

Asato N, & Rand AG, Jr. (1972). Fractionation and isolation of the multiple forms of prorennin (prochymosin). BiochemicalJournal 129:841-846.

Asato N, & Rand AG, Jr. (1977). Activation studies of the multiple forms of prochymosin (prorennin). Biochemical Journal 167:429-434.

Collin, J. C. Cornee, R. & Besaut J. M. (1990) Quantification des chymosines A et B dans les presures animales. Brief Communications of the .XIII International Dairy Congress, Montreal, Oc- tober 8-12, 1990.

Danley DE, & Geoghegan KF. (1988). Structure and mechanism of formation of recombinant- derived chymosin C. The Journal of Biological Chemistry. 263:9785-9789.

Donnelly WJ, Carroll DP, O'Callaghan DM, & Walls D. (1986). Genetic polymorphism of bovine chymosin. Journal of Dairy Research. 53:657-664.

Donnelly, W. J., O'Callaghan, D. M., & Carroll, D. P. (1984) Multiple forms of calf prochymosin and chymosin. Biochemical Society Transactions. 12, 440-441.

Foltmann B, Pedersen VB, Jacobsen H, Kauffman D, & Wybrandt G. (1977) The complete amino acid sequence of prochymosin. Proceeding of the National Academy of Sciences of the U S A 74:2321-2324.

Foltmann B, Pedersen VB, Kauffman D, & Wybrandt G. (1979) The primary structure of calf chymosin. The Journal of Biological Chemistry. 254: 8447-8456.

Foltmann B. (1992) Chymosin: a short review on foetal and neonatal gastric proteases 3. The Scandinavian Journal of Clinical & Laboratory Invesigation. Supplement 210:65-79.

Foltmann, B. (1964) Studies on rennin. IX: On the limited proteolysis of A-rennin and the proteolytic activity of chromatographically purified fractions of rennin. Comptes Rendus des Travaux du Labo- ratoire Carlsberg 34, 319-325.

Foltmann, B. (1966) A review on prorennin and rennin. Comptes Rendus des Travaux du Labora- toire Carlsberg 35, no 8, 143-231.

Harboe, M. & Budtz, P. (1999) The production, action and application of rennet and coagulants. Technology in cheesemaking , 33-65.. Sheffield Academic Press (ed B.A. Law). Harboe, M. (1992) Chymogen, a chymosin rennet manufactured by fermentation of Aspergillus niger. Bulletin of the International Dairy Federation 269:3-7.

International Dairy Federation. (1997A) Bovine rennets: determination of total milk-clotting activity. IDF standard 157A.

International, Dairy Federation. (1997B) Calf rennet and adult bovine rennet: determination of chymosin and bovine pepsin contents (chromatographic method). IDF standard 110B.

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacterio- phage T4. Nature, : 227, 680-685.

Lilla S., Caira S., Ferranti P., & Addeo F. (2001). Mass spectrometric characterisation of proteins in rennet and in chymosin-based milk-clotting preparations. Rapid Communications in Mass Spec- trometry 15: 1101-1112.

Nishimori et al, GenBank Accession No. AAA30449, February 28, 1994.

Panari, G. Corradini, C, Rampilli, M., & Molinari, P. (1990). Chromatographic characterization by HPLC of enzymes of rennet and milk coagulants. Scienza e Tecnica Lattiero-Casearia 5, 437-444.

Rampilli M., Rossi, N., Harboe M., Eltong, S.C. and Larsen R., "Natural Heterogeneity of Chymosin and Pepsin in Extracts of Bovine Stomachs", poster Congrilait, Paris 2002.

Rothe, G. A. L., Axelsen, N. H., Jøhnk, P., & Foltmann, B. (1976) Immunochemical, chromatographic, and milk-clotting activity measurments for quantification of milk-clotting enzymes in bovine rennets. Journal of Dairy Science 43, 85-95.

Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular cloning. A laboratory manual. 2nd edition. Cold Spring Harbor Laboratory Press.

Szecsi PB. (1992). The aspartic proteases. The Scandinavian Journal of Clinical & Laboratory Invesigation. Supplement 210:5-22.

Ward, M., Wilson, L.J., Kodoma, K.H., Rey, M.W. and Berka, R.M. (1990) Improved production of chymosin in Aspergillus by expression of glycoamylase-chymosin fusion, Bio/Technology 8:435- 440. Ward, M., Wilson, L.J. and Kodama, K.H. (1993) Use of Aspergillus overproducing mutants, cured for integrated plasmid, to overproduce heterologous proteins, Appl. Microbiol. Biotechnol. 39:738- 743.

Claims

1. A method of obtaining a preparation of an aspartic protease, chymosin C, having the following features: (a) it possess milk-clotting activity, (b) when crude preparations thereof are analyzed by ion-exchange HPLC the major peak of chymosin C protein elutes after the peak of the degradation product A2 of bovine chymosin A and before the major peak of bovine chymosin B, (c) chymosin C protein shows an apparent molecular weight similar to bovine chymosin A and B, (d) when purified preparations of chymosin C are analyzed by isoelectric focusing (IEF) and compared with purified bovine chymosin B preparations, chymosin C has a higher isoelectric point than bovine chymosin B, (e) The morphology of the precipitates formed by chymosin C in Rocket immunoelectropho- resis using rabbit polyclonal monospecific antibodies raised against purified calf prochymosin (mainly B variant) is identical to the morphology of the precipitates formed by bovine chymosin A and B, and (f) The specific milk clotting activity of the chymosin C is as least 20 % higher than the specific milk clotting activity of of bovine chymosin B.

said method comprises: i) obtaining an aqueous rennet preparation which comprises chymosin C, but comprise less than 1 % chymosin A and less than 1% chymosin B compared to the amount of chymosin C in the preparation, ii) subjecting said aqueous rennet preparation to either a hydrophobic chromatography followed by a anion chromatography or chromatography on a column containing a mixed- mode, hydrophobic-/anion exchange chromatographic material to obtain a further purified fraction of chymosin C, discarding any other chymosin variant fractions, and iii) optionally, subject said chymosin C fraction to gel filtration on a suitable resin to obtain an even further purified chymosin C preparation.

2. The method of claim 1 , wherein the ion-exchange HPLC is performed on a PL-SAX column (Polymer Laboratories Inc., Amherst, USA) or a similar HPLC media containing a hydrophilic, strong anion exchange chromatographic packing material.

3. The method of claim 1 or 2, wherein the apparent molecular weights of chymosin A, B and C are determined by SDS PAGE under reducing conditions.

4. The method of claim 1 to 3, wherein the apparent molecular weights of chymosin A, B and C are determined by SDS PAGE under reducing conditions and by gelfiltration on Superose 12 HR 10/30 (Amersham Biosciences, Piscataway, USA)

5 5. The method of claim 1 to 4, wherein the isoelectric point of chymosin C is between 0.01 and 0.2 pH units higher than the isoelectric point of bovine chymosin B.

6. The method of claim 1 to 5, wherein chymosin C further is characterized by having higher specific activity measured as IMCU according to IDF standard 157 that is as least 20 % higher than the

10 specific milk clotting activity of an >95% pure preparation of bovine chymosin B and at least 85% equal to the activity of an >95% pure preparation of bovine chymosin A.

7. The method of claim 1 to 6, wherein chymosin C further is characterized by being more stable than chymosin A with regard its degradation caused by autocatalytic self-degradation.

15 8. The method according to any of the preceding claims, wherein said aqueous rennet preparation is: i) subjected to hydrophobic chromatography on a suitable resin to obtain an almost pure chymosin eluate, 20 ii) this eluate is subjected to anion chromatography on a suitable resin to obtain a further purified fraction of chymosin C, discarding any other chymosin variant fractions, and iii) finally, said further purified fraction of chymosin C is subjected to gel filtration on a suitable resin to obtain an at least 70% pure chymosin C.

25 9. The method according to any of the preceding claims, wherein said aqueous rennet preparation which comprises chymosin C is subjected to chromatography on a column containing a mixed- mode, hydrophobic-affinity/anion exchange chromatographic packing material to obtain a further purified fraction of chymosin C.

30 10. The method according to claim 9, wherein said mixed-mode, hydrophobic-affinity/anion exchange chromatographic packing material is MIMO 1300 (Upfront A/S, Copenhagen).

11. The method according to claim 10, wherein said further purified fraction of chymosin C is subjected to gel filtration on a suitable resin to obtain an at least 90% pure chymosin C.

35 12. The method according to any of the preceding claims, wherein the aqueous rennet preparation is obtained from a chymosin C homozygous bovine abomasum.

13. The method according to any of the preceding claims, wherein the aqueous rennet preparation is obtained by: (a) providing host cells containing DNA construct comprising a bovine chymosin C coding sequence operably linked to linked to appropriate expression signals permitting said chy- 5 mosin C comprising coding sequence to be expressed as a polypeptide in said host cell, (b) cultivating said cells under conditions conducive to expression of the polypeptide having chymosin C activity; and (c) harvesting the chymosin C comprising composition.

10 14. The method according to claim 13 wherein, the host cell is a strain of Aspergillus niger var. awamori.

15. The method according to any of the preceding claims further comprising a activation step which is performed before the first chromatography step, said activation step comprises:

15 i) treatment of the crude stomach extract at pH between 1.8 and 2.2, preferably at pH approximately 2, ii) centrifugation and collection of the supernatant, and iii) raising the pH of the supernatant was raised to between pH 6.0 and pH 5.6, preferably pH approximately 5.8.

20 16. The method according to any of the preceding claims further comprising a clarifying step which is performed before the first chromatography step, said clarifying step being a centrifugation step or a chemical clarification procedure comprising the following steps: i) addition of 1/3 M aluminium sulphate to a chymosin comprising preparation until pH is be-

25 tween 3.6 and 4, preferably approximately 3.8, ii) addition of 1 M di-sodium phosphate until pH is between 5.4 and 5.8, preferably approximately 5.6, and iii) obtaining a clear supernatant by centrifugation, and iv) harvesting the chymosin C comprising cleared supernatant.

30

17. The method according to any of preceding claims further comprising as a step wherein the chymosin C is subjected to a deglycosylation treatment.

35 18. The method according to claim 17 wherein the deglycosylation treatment comprise the deglycosylation by the enzyme EndoH (EC 3.2.1.96).

19. An at least 90% pure preparation of chymosin C, comprising at least 3 mg Chymosin C.

20. An at least 90% pure preparation of chymosin C, produced by the method of any of claims 1-17 and which comprises at least 3 mg Chymosin C.

21. Use of the at least 90% pure preparation of chymosin C, prepared according to the method of method of any of claims 1 -17 to manufacture a preparation of chymosin C for the manufacture of food or feed.

22. A method of manufacturing cheese, comprising adding to milk a milk clotting effective amount of chymosin C.