Recombinant 21 kD cocoa protein and precursor

Details Recombinant 21 kD cocoa protein and precursor Recombinant 21 kD cocoa protein and precursor

1992-12-11

1997-09-16

Patterson, Jr., Charles L.

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

530370, 530377, 530379, 435 691, 435 711, 435 712, 4351723, 435325, 43525233, 43525421, 4353201, 536 236, 536 231, C07K 14415, C12N 1529, C12N 121, C12N 119

Patent

active

056680074

DESCRIPTION:

BRIEF SUMMARY
This invention relates to proteins and nucleic acids derived from or otherwise related to cocoa.
The beans of the cocoa plant (Theobroma cacao) are the raw material for cocoa, chocolate and natural cocoa and chocolate flavouring. As described by Rohan ("Processing of Raw Cocoa for the Market", FAO/UN (1963)), raw cocoa beans are extracted from the harvested cocoa pod, from which the placenta is normally removed, the beans are then "fermented" for a period of days, during which the beans are killed and a purple pigment is released from the cotyledons. During fermentation "unknown" compounds are formed which on roasting give rise to characteristic cocoa flavour. Robart suggests that polyphenols and theobromine are implicated in the flavour precursor formation. After fermentation, the beans are dried, during which time the characteristic brown pigment forms, and they are then stored and shipped.
Biehl et al, 1982 investigated proteolysis during anaerobic cocoa seed incubation and identified 26 kD and 44 kD proteins which accumulated during seed ripening and degraded during germination. Biehl asserted that there were storage proteins and suggested that they may give rise to flavour-specific peptides.
Biehl et al., 1985 again asserted that amino acids and peptides were important for flavours.
Fritz et al, 1985 identified polypeptides of 20 kD and 28 kD appearing in the cytoplasmic fraction of cocoa seed extracts at about 100 days after pollination. It appears that the 20 kD protein is thought to have glyceryl acyltransferase activity.
Pettipher et al., 1990 suggested that peptides are important for cocoa flavour and refers to 48 kD and 28 kD storage proteins.
In spite of the uncertainties in the art, as summarised above, proteins apparently responsible for flavour production in cocoa beans have now been identified. Further, it has been discovered that, in spite of Fritz's caution that "cocoa seed mRNA levels are notably low compared to other plants" (loc. cit.), it is possible to apply the techniques of recombinant DNA techniques to the production of such proteins.
According to a first aspect of the invention, there is provided a 23 kD protein of Th. cacao or a fragment thereof.
The 23 kD protein may be processed in vivo to form a 21 kD polypeptide.
According to a second aspect of the invention, there is provided a 21 kD protein of Th. cacao or a fragment thereof.
The term "fragment" as used herein and as applied to proteins or peptides indicates a sufficient number of amino acid residues are present for the fragment to be useful. Typically, at least four, five, six or even at least 10 or 20 amino acids may be present in a fragment. Useful fragments include those which are the same as or similar or equivalent to those naturally produced during the fermentation phase of cocoa bean processing. It is believed that such fragments take part in Maillard reactions during roasting, to form at least some of the essential flavour components of cocoa.
Proteins in accordance with the invention may be synthetic; they may be chemically synthesised or, preferably, produced by recombinant DNA techniques. Proteins produced by such techniques can therefore be termed "recombinant proteins". Recombinant proteins may be glycosylated or non-glycosylated; non-glycosylated proteins will result from prokaryotic expression systems. Theobroma cacao has two primary subspecies, Th. cacao cacao and Th. cacao sphaerocarpum. While proteins in accordance with the invention may be derived from these subspecies, the invention is not limited solely to these subspecies. For example, many cocoa varieties are hybrids between different species; an example of such a hybrid is the trinitario variety.
The invention also relates to nucleic acid, particularly DNA, coding for the proteins referred to above (whether the primary translation products, the processed proteins or fragments). The invention therefore also provides, in further aspects:
nucleic acid coding for a 23 kD protein of Th. cacao or for a fragment thereof; and
nucleic acid coding for a 21 k

REFERENCES:
Biehl et al., J. Sci. Food Agric. 33, 1291-1304 (1982).
Fritz et al., Journal of Food Science 50, 946-950 (1985).
Pettipher, Cafe Cacao The, vol. XXXIV, No. 1, 23-26 (1990).
Wilson et al., Abstr. Pap. Am. Chem. Soc., #148 (1984).
Leah et al., Plant Molecular Biology 12, 673-682 (1989).
Spencer et al., Planta 183, 528-535 (1991).
M. P. Deutscher "Guide to Protein Purification" Meth. in Enzymology. vol. 182 pp. 602-613 738-751 (1990).
D.C. Wright et al. "Accumulation of Lipids. Proteins, Alkaloids . . ." J. Am. Oil Chemists Soc. 59(11) 475-479 (Nov. 1982).
P. J. Fritz et al. "Cocoa Butter Biosynthesis" J. Biol Chem. 261(1) 194-199 (Jan. 1986).
S. L.Berger et al. "Guide to Molecular Cloning Techniques" Meth. in Enzymology vol. 152: 393-399, 415-423, 432-447, 661-704 (1987).

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