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Method of producing non-bovine chymosin and use hereof

Method of producing non-bovine chymosin and use hereof description/claims

The present invention relates generally to the field of cheese manufacturing. In particular, novel recombinant means of providing milk-clotting enzymes of non-bovine, ie non-Bos taurus, animal origin are provided. Specifically, the invention pertains to a process of recombinantly providing pre-prochymosin, prochymosin and chymosin of non-bovine origin including such enzymes that are derived from camels.

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) and pepsin (EC 3.4.23.1), the milk clotting enzymes of the mammalian stomach, are aspartic proteases belonging to a broad class of peptidases (Kappeler, 1998). Aspartic proteases are found in eukaryotes, retroviruses and some plant viruses. Eukaryotic aspartic proteases 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 Å. 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 proteases are embedded, with low accessibility, in the middle of a cleft, about 40 Å in length, which separates the two domains, and which is covered by a flap that, in bovine and camel chymosin, extends from about Leu73 to Ile85 in the N-terminal domain.

When produced in the gastric mucosal cells, chymosin and pepsin occur as enzymatically inactive pre-prochymosin and pre-pepsinogen, respectively. 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 substantially inactive form of the enzyme which, however, becomes activated under acidic conditions to the active chymosin by autocatalytic removal of the pro-fragment. This activation occurs in vivo in the gastric lumen under appropriate pH conditions or in vitro under acidic conditions.

The structural and functional characteristics of bovine, ie Bos taurus, pre-prochymosin, prochymosin and chymosin have been studied extensively (Foltman et al. 1977). 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. Foltman 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 290 in chymosin A and a glycine residue at that position in chymosin B.

Whereas chymosin is produced naturally in mammalian species including ruminant species such as bovines, caprines, buffaloes and ovines; pigs (Houen et al., 1996); Camelidae species; primates including humans and monkeys; and rats, bovine chymosin and (to a lesser extent) caprine chymosin are presently the only of these animal chymosin species that are commercially available to the dairy industry. Bovine chymosin, in particular calf chymosin, is commercially available both as stomach enzyme extracts (rennets) comprising the natively produced chymosin and as recombinantly produced chymosin which is expressed in bacterial, yeast or fungal host cells (see e.g. WO 95/29999, Ward et al. 1990).

Recently, studies on functional characteristics of rennet extracted from the stomach of Camelus dromedarius chymosin have been reported (Wangoh et al., 1993; Elagamy, 2000) and it has been found that the clotting time of camel's milk is significantly reduced when camel rennet is used instead of bovine calf rennet. Fractions of crude camel and calf rennets, which were isolated by anion-exchange chromatography, have been tested for their respective capabilities to clot camel's milk and cow's milk and it was found that the main clotting activity of calf rennet (i.e. an extract containing both chymosin and pepsin) resides in the pepsin fraction, i.e. bovine chymosin is substantially inactive in respect of clotting camel's milk, whereas the main clotting activity of camel rennet extracts on camel's milk resided in a first fraction that, compared to calf chymosin, eluted at a somewhat lower NaCl concentration. The active enzyme of this fraction has not yet been characterised, but it is assumingly chymosin. It has also been demonstrated that this camel rennet fraction has a clotting activity on cow's milk that is similar to that of bovine chymosin (Wangoh et al., 1993). It is evident, therefore, that more effective clotting of camel's milk could be achieved at an industrial level were camel chymosin commercially available and it is also conceivable that camel chymosin is highly suitable as a cow's milk clotting enzyme as well.

The primary structure of chymosin isolated from gastric mucosa of camels has been determined (Kappeler, 1998). The mature and active form of camel chymosin is 323 aa residues long and it has a molecular weight of 35.6 kDa and an isoelectric point at pH 4.71. It shows 85.1% aa sequence identity with bovine chymosin.

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 of bovine origin, the industry has not yet explored the possibility of providing effective chymosin preparations based on non-bovine, ie non-Bos taurus, chymosin species. However, the present inventors have surprisingly found that it is possible to provide industrially highly useful non-bovine chymosin using recombinant DNA technology at a production yield level which, relative to that which can be obtained in current, optimised bovine chymosin production systems, is significantly improved.

In addition to the potential of significantly improved chymosin production cost-effectiveness, the provision of such non-bovine chymosin species at a commercial level makes available chymosin products that are not only capable of clotting cow's milk at least as effectively as chymosin of bovine origin, but which, additionally, are capable of more effectively clotting milk from other animal species including milk of the source species. Specifically, the invention has made it possible to provide, for the first time, camel chymosin in sufficient quantities to render an industrial, cost-effective and high quality production of cheese based on camel's milk possible, which, due to the scarcity of camel calf stomach material, has not hitherto been possible.

Additionally, it has been discovered that camel chymosin has a high clotting activity on cow's milk, which renders the enzyme useful for manufacturing cheese based on cow's milk. It was a surprising finding of the present inventors that camel chymosin has a specific κ-casein hydrolysing activity (Phe-Met 105/106), i.e. C/P ratio as defined hereinbelow, which is superior to that of bovine chymosin. A higher C/P ratio implies generally that the loss of protein during cheese manufacturing due to non-specific protein degradation is reduced, i.e. the yield of cheese is improved, and that the development of bitter taste in the cheese during maturation is reduced.

The invention relates in one aspect to a method of producing a non-bovine pre-prochymosin, prochymosin or chymosin, the method comprising the steps of (i) isolating or constructing a nucleic acid sequence coding for the pre-prochymosin, prochymosin or chymosin, (ii) constructing an expression vector comprising said coding sequence and, operably linked thereto, appropriate expression signals permitting the pre-prochymosin, prochymosin or chymosin to be expressed in a host cell, (iii) transforming said host cell with the expression vector, (iv) cultivating the thus transformed host cell under conditions where the coding sequence is expressed and (v) harvesting the pre-prochymosin, prochymosin or chymosin. As used herein the expression “non-bovine pre-prochymosin, pro-chymosin or chymosin” refers to such enzymes or precursors herefor that are derived from a mammalian species other than Bos taurus.

In further aspects, the invention pertains to a DNA construct capable of expressing non-bovine pre-prochymosin, prochymosin or chymosin, said construct comprising an expression vector comprising a nucleic acid sequence comprising a gene coding for the pre-prochymosin, prochymosin or chymosin and, operably linked thereto, appropriate expression signals permitting the pre-prochymosin, prochymosin or chymosin to be expressed in a host cell, and to a host cell transformed with such a DNA construct.

In still further aspects a composition is provided comprising a non-bovine pre-prochymosin, prochymosin or chymosin produced by the above method including such an enzyme that is in a substantially deglycosylated form and a method of manufacturing cheese, comprising adding a milk clotting effective amount of such a composition to milk and carrying out appropriate further cheese manufacturing steps.

In yet another aspect, the invention relates to a method of manufacturing cheese, comprising adding a milk clotting effective amount of a non-bovine prochymosin or chymosin to the milk and carrying out appropriate further cheese manufacturing steps, the non-bovine prochymosin or chymosin having in said milk a C/P ratio as determined herein which is in the range of 2-20.

In other aspects the invention provides a milk clotting composition comprising a bovine milk clotting enzyme selected from prochymosin, chymosin and pepsin and a non-bovine milk clotting enzyme selected from prochymosin, chymosin, pepsin and a microbial aspartic protease and a method of manufacturing cheese from milk, comprising adding to milk a milk clotting effective amount of such a composition, and carrying out appropriate further cheese manufacturing steps.

In accordance with the invention, there is, in one aspect of the invention, provided a method of recombinantly producing pre-prochymosin, prochymosin or chymosin of non-bovine origin.

For the purposes of this application, the expression “non-bovine origin” refers to any non-Bos taurus mammalian species where pre-prochymosin is produced naturally in the gastrointestinal tract. Such species include any of those mentioned above, e.g. ovine species, caprine species and Camelidae species comprising the genus Camelus with the two species Camelus dromedarius and Camelus bactrianus; buffalo species including water buffaloes, Indian buffaloes and Cape buffaloes, the genus Lama including Lama glama, Lama guanicoe and Lama paco; and the genus Vicugna. Camels are ruminating, but do not belong to the suborder Ruminantia as do e.g. bovine, ovine and caprine species, but they belong to the suborder Tylopoda.

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