U.S. patent application number 11/619420 was filed with the patent office on 2007-08-30 for cocoa polypeptides and their use in the production of cocoa and chocolate flavor.
This patent application is currently assigned to Nestec S.A.. Invention is credited to Carl Erik Hansen, Marcel Alexandre Juillerat, Sunil Kochhar.
Application Number | 20070204365 11/619420 |
Document ID | / |
Family ID | 8177253 |
Filed Date | 2007-08-30 |
United States Patent
Application |
20070204365 |
Kind Code |
A1 |
Kochhar; Sunil ; et
al. |
August 30, 2007 |
COCOA POLYPEPTIDES AND THEIR USE IN THE PRODUCTION OF COCOA AND
CHOCOLATE FLAVOR
Abstract
The present invention pertains to novel cocoa polypeptides
having a molecular weight of about 10 and 14 kDa and being derived
from a 69 kDa precursor. In particular, the present invention
relates to the production of the polypeptides via recombinant means
and the use of the polypeptides or fragments thereof for the
production of cocoa/chocolate flavor.
Inventors: |
Kochhar; Sunil; (Savigny,
CH) ; Hansen; Carl Erik; (Epalinges, CH) ;
Juillerat; Marcel Alexandre; (Lausanne, CH) |
Correspondence
Address: |
BELL, BOYD & LLOYD LLP
P.O. Box 1135
CHICAGO
IL
60690
US
|
Assignee: |
Nestec S.A.
Vevey
CH
|
Family ID: |
8177253 |
Appl. No.: |
11/619420 |
Filed: |
January 3, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10691590 |
Oct 24, 2003 |
7176348 |
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11619420 |
Jan 3, 2007 |
|
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PCT/EP02/04258 |
Apr 17, 2002 |
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10691590 |
Oct 24, 2003 |
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Current U.S.
Class: |
800/278 ;
426/660; 435/193; 435/212; 435/252.3; 435/254.2; 435/325; 435/348;
435/419; 435/468; 536/23.2 |
Current CPC
Class: |
C12N 15/8242 20130101;
C12N 15/8251 20130101; C07K 14/415 20130101 |
Class at
Publication: |
800/278 ;
426/660; 435/193; 435/468; 435/419; 435/212; 536/023.2; 435/252.3;
435/254.2; 435/325; 435/348 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C07H 21/04 20060101 C07H021/04; C12N 9/10 20060101
C12N009/10; C12N 9/48 20060101 C12N009/48; C12N 15/82 20060101
C12N015/82; C12N 5/04 20060101 C12N005/04; A23G 3/00 20060101
A23G003/00; C12N 5/06 20060101 C12N005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 25, 2001 |
EP |
011110251.4 |
Claims
1. An isolated or synthesized cocoa polypeptide identified by SEQ
ID NO:1, SEQ ID NO:2, or a fragment thereof comprising SEQ ID NO:3
or SEQ ID NO:4.
2. The fragment of claim 1, obtained by enzymatic degradation
involving one or more of the following enzymes: aspartic
endoproteinase, cysteine endoproteinase and carboxypeptidase.
3. The fragment according to claim 2, wherein the enzyme is derived
from cocoa.
4. The polypeptide according to claim 1, which is further reacted
with a reducing sugar.
5-11. (canceled)
12. A method of producing cocoa or chocolate flavor comprising
isolating, synthesizing or producing a polypeptide according to
claim 1 and reacting the peptide with a reducing sugar.
13. A method of enhancing the cocoa or chocolate flavor of a
composition, comprising supplementing the composition with one or
more of the peptides of claim 1.
14. A method of producing cocoa or chocolate flavor comprising the
steps of isolating, synthesizing or producing one or more cocoa
polypeptides identified by SEQ ID NO:1 and SEQ ID NO:2 or a
fragment thereof comprising SEQ ID NO:3 or SEQ ID NO:4.
15. The method of claim 14, further comprising the step of reacting
the peptide with a reducing sugar.
16. The method of claim 14, wherein the fragment thereof is
obtained by subjecting a peptide identified by SEQ ID NO: 1 or SEQ
ID NO:2 to enzymatic degradation.
17. The method of claim 16, wherein the step of enzymatic
degradation involves one or more of the following enzymes: aspartic
endoproteinase, cysteine endoproteinase or carboxypeptidase.
18. The method of claim 17, wherein the enzyme is derived from
cocoa.
19-20. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International
Application PCT/EP02/04258 filed Apr. 17, 2002, the content of
which is expressly incorporated herein by reference thereto, which
claims priority to EP Application No. 011110251.4 filed Apr. 25,
2001.
FIELD OF THE INVENTION
[0002] The present invention relates to novel cocoa polypeptides
having a molecular weight of about 10 and 14 kDa. The newly
identified peptides were first derived from a 69 kDa precursor. In
particular, the present invention relates to the production of the
polypeptides via recombinant means and the use of the polypeptides
or fragments thereof for the production of cocoa/chocolate
flavor.
BACKGROUND OF THE INVENTION
[0003] The traditional processing of coca beans to generate cocoa
flavor requires two steps--a fermentation step, which includes
air-drying of the fermented material, and a roasting step.
[0004] During fermentation the pulp surrounding the beans is
degraded by micro-organisms and the sugars contained in the pulp
are mainly transformed to acids. In the course of the fermentative
process these acids slowly diffuse into the bean eventually causing
an acidification of the cellular material. Furthermore, during
fermentation peptides of different sizes are generated as well as
high levels of hydrophobic free amino acids, which are mainly
attributed to the activity of specific proteinases. This specific
mixture of peptides and hydrophobic amino acids is thought to be
the cocoa-specific flavor precursors.
[0005] Research to date has focused on the different proteolytic
enzymes involved in these reactions. A number of different types of
enzymes, such as an aspartic endoproteinase, a cysteine
endoproteinase or a carboxypeptidase have been found to participate
in these degradative reactions leading to the formation of the
cocoa flavor peptide/amino acid precursor pool.
[0006] During the second step of cocoa flavor production--the
roasting step--the oligopeptides and amino acids generated during
the fermentation stage are subjected to a Maillard reaction in the
presence of reducing sugars in the mixture, yielding substances
thought to be responsible for the typical cocoa flavor.
[0007] There have been attempts to artificially produce cocoa
flavor in the past, such as, by subjecting acetone dried powder
prepared from unfermented ripe cocoa beans to autolysis at a pH of
5.2 followed by roasting in the presence of reducing sugars. It was
taught that under these conditions preferentially free hydrophobic
amino acids and hydrophilic peptides would be generated. The
peptide pattern obtained from this process was found to be similar
to that of extracts from fermented cocoa beans.
[0008] Analysis of free amino acids revealed that Leu, Ala, Phe and
Val were the predominant amino acids liberated in fermented beans
or autolysis (Voigt et al., Food Chem. 49 (1994), 173-180). In
contrast to these findings no cocoa-specific flavor could be
detected when the above powder was subjected to autolysis at a pH
of 3.5. Few free amino acids were found in the by product of the
autolysis, but there were a large number of hydrophobic peptides
formed.
[0009] Synthetic mixture of free amino acids whose composition
resembles that found in fermented beans also have been found to not
produce the cocoa flavor desired. These findings indicate that both
the peptides and the amino acids are important in producing cocoa
flavor (Voigt et al., Food Chem. 49 (1994), 173-180.
[0010] To date, little attention has been paid to the protein pool
from which the peptide/amino acid flavor precursor pool is
generated from, since cocoa proteins are often difficult to
isolate. One of the major reasons is because that coca seeds
contain a high amount of polyphenols and fat. Separating the
polyphenols and fat traditionally requires the use of lipophilic
organic liquids, such as acetone. The use of these liquids often
result in the removal of lipophilic flavor precursors and active
substances. Another reason is because of the poor solubilization of
proteins purified with acetone, resulting in a poor recovery of the
total proteins.
[0011] To date, four major proteins with an apparent molecular
weight of 14.5, 21, 31 and 47 kDa, have been identified before
fermentation in cocoa bens. These proteins are thought to give rise
to the peptide/amino acid pool responsible for producing the cocoa
flavor.
[0012] It is an object of the present invention to further
elucidate and identify other protein responsible for producing the
cocoa flavor in sufficient detail to eventually provide means for
improving the preparation of cocoa flavor and cocoa flavor
substitutes.
SUMMARY OF THE INVENTION
[0013] The present invention is directed to an isolated or
synthesized cocoa polypeptide identified by SEQ ID NO: 1, SEQ ID
NO:2, or a fragment thereof comprising SEQ ID NO:3 or SEQ ID NO:4.
These newly identified peptides have a molecular weight of about 10
and 14 kDa, respectively, and were originally derived from the 69
kDa cocoa bean precursor proteins.
[0014] Typically fragments of the SEQ ID NO:1 and SEQ ID NO:2 are
obtained by enzymatic degradation, preferable using one or more of
the following enzymes: aspartic endoproteinase, cysteine
endoproteinase and carboxypeptidase. In one embodiment the enzymes
used are derived from cocoa plants.
[0015] The present invention is also directed to an isolated or
synthesized nucleotide sequence that encodes SEQ ID NO:1, SEQ ID
NO:2 or fragments thereof. In those embodiments wherein the
nucleotide sequence of the invention encodes a fragment of SEQ ID
NO:1 and/or SEQ ID NO:2. The peptides encoded by these nucleotides
preferably comprises SEQ ID NO:3 and/or SEQ ID NO:4.
[0016] The present invention encompasses recombinant cells,
vectors, and cells comprising vectors containing one or more copies
of the nucleotide sequence described above.
[0017] Typically the recombinant cell is a bacterial cell, a yeast
cell, an insect cell, a mammalian cell or a plant cell. Preferably
the cell is a plant cell and most preferably are part of a
plant.
[0018] The present invention is further directed to a method of
producing cocoa or chocolate flavor comprising isolating,
synthesising or producing a polypeptide of the invention. In one
embodiment, the method further comprises reacting such a peptide
with a reducing sugar.
[0019] In yet another embodiment of the invention, the newly
identified peptides are used to enhance the cocoa or chocolate
flavor of a composition. The method typically comprises
supplementing a food composition with one or more of the
peptides.
[0020] The method can further comprise subjecting the peptide to
enzymatic degradation, preferably involving one or more of the
following enzymes: aspartic endoproteinase, cysteine endoproteinase
or carboxypeptidase, followed by reacting the fragments with
reducing sugars.
[0021] Still further, the present invention also encompasses a
method of producing cocoa beans with increased cocoa flavor
proteins. The method typically comprises transforming a cocoa cell
with one or more of the nucleotide sequences of the invention
followed by generating at least one cocoa plant from the
transformed cell. Preferably the transformed cell comprises at
least 40 copies of the nucleotide sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows photograph of a two-dimensional SDS-PAGE
proteins isolated from unfermented cocoa beans;
[0023] FIG. 2 shows the result of a LC_ESI-MS analysis of a GndHCl
extract of unfermented cocoa beans.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0024] During the studies leading to the invention the present
inventors have designed novel methods for an improved isolation of
cocoa proteins by using denaturing agents, such as e.g. SDS (1%),
urea or GndHCl, which resulted in an about 3-fold increase in
solubility of proteins as compared to conventional methods. In
particular, the use of 6 M GndHCl provided good results. GndHCl
showed up to be easily removable by RP-HPLC and no reaction with
the proteins occurred. Moreover, it could also be shown that even
crude bean powder, after subjection to a treatment with a
solubilization buffer including a denaturing agent could be
successfully analysed, which made no special care necessary to
remove polyphenols.
[0025] During the above-described studies directed to provide a
better total recovery of cocoa proteins, the inventors ran a crude
coca bean powder on a two-dimension SDS-PAGE gel, whereby a cluster
of several polypeptides exhibiting a molecular weight of about 9-16
kDa, were detected. The polypeptides contained in the acidic
cluster was further isolated by making use of RP-HPLC.
[0026] Finally two polypeptides were isolated having a molecular
weight of about 10 and about 14 kDa. These polypeptides were
N-terminally sequenced and are identified as SEQ ID NO:3 (10 kDa
protein; AA residues 1-26 of SEQ ID NO:1) and SEQ ID NO:4 (14 kDa
protein; AA residues 1-10 of SEQ ID NO:2), respectively.
[0027] Upon comparison with known protein sequences it was shown
that these polypeptides were derived from the 69 kDa precursor
protein. Upon processing the 69 kDa cocoa beans precursor, it gave
rise to the above mentioned 47 and 31 kDa proteins and also to the
newly identified 10 and 14 kDa proteins, also representing part of
the protein/peptide pool of cocoa beans.
[0028] The present invention is directed to an isolated or
synthesized cocoa polypeptide identified by SEQ ID NO: 1, SEQ ID
NO:2. According to another embodiment the present invention, the
polypeptides are subjected to enzymatic degradation, preferably
with aspartic endoproteinases, cystein endoproteinases and/or
carboxypeptidases. In this embodiment it is preferable that the
fragments comprise SEQ ID NO:3 or SEQ ID NO:4.
[0029] According to yet another embodiment the invention the
polypeptides obtainable by the enzyrnatic degradation are
subsequently reacted with reducing sugars.
[0030] The present invention also provides for a recombinant
nucleotide encoding the polypeptides of the invention, preferably a
nucleotide sequence encoding at least one of the new polypeptides
or fragments thereof. Such nucleotides may be easily derived from
the given polypeptide sequence by translating the amino acid
according to the genetic code into corresponding triplets. Such a
nucleotide sequence may well be expressed in a suitable cell by
means well known in the art, such as e.g. in a bacterial cell, e.g.
in E. coli, or in yeast, insect cells, mammalian or plant
cells.
[0031] To this end, a nucleotide sequence encoding a polypeptide of
the present invention is inserted into a suitable vehicle, such as
an expression vector, and is incorporated into a cell of choice.
With respect to plant cells, the nucleotides encoding the
polypeptides of the present invention may also be incorporated into
any of the plant cell's chromosome by using e.g. the phenomenon of
homologous recombination. In this respect, at least one copy,
preferably more than 40 copies of a nucleotide sequence, encoding
any of the present polypeptides may be present on the DNA sequence
to be inserted into a plants cell's chromosome.
[0032] The present invention further encompasses the generation of
plants comprising the recombinant cells. Preferably the transformed
plant is a cocoa plant.
[0033] Furthermore, the invention provides for the use of the
polypeptides for the manufacture of cocoa flavor. To this end it is
conceived that the present polypeptides may be added to a
fermentation mixture of cocoa beans, in order to provide a higher
amount of the polypeptides for degradation. When using cocoa plants
that have been modified by recombinant means and contain a high
number of copies of nucleotide sequences encoding the polypeptides
of the present invention, the plants will inherently contain a
higher concentration of the polypeptides and eventually will result
in the production of a stronger cocoa flavor after the
processing.
EXAMPLES
[0034] These and other aspects of the present invention may be more
fully understood with reference to the following non-limiting
examples, which are merely illustrative of the preferred
embodiments of the present invention, and are not to be construed
as limiting the invention, the scope of which is defined by the
appended claims.
[0035] The following examples illustrate the invention in a more
detailed manner. It is, however, understood that the present
invention is not limited to the examples but is rather embraced by
the scope of the appended claims.
Example 1
[0036] Separation of Proteins
[0037] Preparation of Crude Cocoa Bean Powder
[0038] Unfermented cocoa beans were obtained from Ivory Coast and
unless stated otherwise all studies were carried out using West
African Amelonado cocoa beans. Dried cocoa beans were passed
through a bean crusher, followed by a winnover to remove shells.
The nibs were kept in a brown bottle at -20.degree. C. Cocoa nibs
were milled for few seconds in an universal mill. The nib powder
was passed through 0.8-mm sieve and kept at 4.degree. C.
[0039] Two-Dimensional SDS-PAGE Electrophoretic Analysis of
Unfermented Cocoa Bean
[0040] Crude (unfermented) cocoa bean powder (100 mg) was dissolved
in 1 ml of solubilization buffer [8 M urea, 3% (w/v) CHAPS, 2.8%
(v/v) carrier ampholytes (ampholine pH range 4-6.5, 5-8, and 3-10
(2:4:1) and 10 mM DTT (dithiothreitol)]. The clear supernatant was
subjected to first dimension of separation on an immobilized
pH-gradient (IPG) from 4-7, and second dimension on a 10% T
SDS-PAGE gel. Proteins were visualized by silver staining.
[0041] The resulting electrophoretic profile of proteins in a
typical unfermented cocoa bean on a two-dimensional SDS-PAGE is
shown in FIG. 1. The 47, 31 and 21 kDa proteins were represented by
several subforms and in addition two distinct clusters (acidic and
basic) were clearly identified in a molecular weight range of about
9-16 kDa. All of the protein spots could be shown to gradually
disappear upon fermenation of beans.
[0042] A Tricine-SDS-PAGE of unfermented cocoa bean genotypes
showed up that the clusters in the molecular weight region 9-16 kDa
were present in all of the 21 different genotypes representing
three cocoa groups, namely Criollo, Forastero and Trinitario.
[0043] The acidic cluster has been selected for further
investigation.
Example 2
[0044] Isolation of Proteins Having a Molecular Weight of about
9-16 Kda
[0045] Preparation of an GndHCl Extract of Cap
[0046] Cocoa nib powder (10 g) (supra) was suspended in 200 ml 80%
(v/v) aqueous acetone and stirred for 1 hr at 4.degree. C. The
resulting suspension was centrifuged at 15.000 rpm for 15 min at
4.degree. C. The residue was extracted 5-times with 200 ml 80%
(v/v) aqueous acetone and followed by 3-times washing with 100%
acetone. The resulting acetone powder was dried under reduced
pressure.
[0047] Subsequently a GndHCl extract and a pyridine-ethylated
GndHCl extract of CAP from unfermented cocoa beans was prepared. 1
g CAP was suspended with 10 ml GndHCl buffer (100 mM ammonium
phosphate, 66.7 mM potassium hydroxide, 3 mM EDTA and 6 M GndHCl)
and sonicated for 1 min. The suspension was cooled on ice for 15-30
min and centrifuged at 15000 rpm at 4.degree. C. for 15 min. The
clear supernatant was carefully removed. In order to obtain a
pyridine-ethylated GndHCl extract, the CAP extract (2 ml) was
sparged with argon and mixed with 50 .mu.l of reducing solution
(0.8 M DTT in 3 M tripotassium phosphate/3 mM EDTA). The solution
was kept at room temperature in dark for 60 min.
Pyridine-ethylation at cysteine residues of the reduced CAP was
carried out by mixing vigorously 40 .mu.l of 4-vinyl pyridine and
further incubation for 30 min at room temperature (Lundell and
Schreitmuller, Anal. Biochem. 266 (1999) 31-47). The reaction
mixture was dialyzed against 500 ml of the extraction buffer for
overnight at room temperature. The dialyzed sample was centrifuged
and the clear supernatant passed through 0.22 .mu.m filter disc and
kept at 4.degree. C. until analyzation.
[0048] LC-ESI-MS Analysis of the Reduced and Pyridylethylated
GndHCl-Extract
[0049] A LC-ESI-MS analysis of the reduced and pyridylethylated
extract was performed, as may be seen from FIG. 2. To this end,
reduced and pyridylethylated GndHCl extracts of CAP were injected
onto reverse phase HPLC columns [Bio-Rad HRLC series 800 system;
columns C4 and C8 from Aquapore RP butyl (7 .mu.m, 4.6.times.220
mm), Aquapore RP 300 (7 .mu.m, 4.6.times.220 mm), Perkin-Elmer;
Spherisorb 80-5C8 (220.times.4 mm); Marchery Nagel and Vydac
protein C4 (4.6.times.220 mm)) pre-equilibrated with solvent A
(0.1% v/v TFA in water) and eluted with a linear gradient of
increasing concentration of solvent B (80% v/v acetonitrile and
0.1% v/v TFA): 0-15% B in 5 min, 15-27% B in 40 min, 27-35% B in 2
min, isocratic at 35% B for 3 min, 35-43% B in 25 min, 43-56% B in
50 min, 56-70% B in 5 min, 70-100% B in 10 min and isocratic at
100% B for 5 min].
[0050] Fractions containing proteins were found to elute at a
retention time of about 41, 52, 68, 78 and 87 min, were of about
M.sub.r 10,425 (marked as CSP10), 9,010 (marked as CSP9), 20,540
(marked as CSP22) and 12,500 (marked as CSP12), respectively, as
can be seen in Table 1 and FIG. 6. In the case of proteins eluting
at 41 min and 97 min, no molecular mass could be identified.
TABLE-US-00001 TABLE 1 Retention time, min Sample code Average
M.sub.r Comments 41.4 CSP14 Not detected 52.5 CSP10 10,425 67.7
CSP9 9,010 78.5 CSP22 20,540 Albumin CSP 86.9 CSP12 12,245 97.3
CSP67 Not detected Vicilin type CSP 132.2 CSPAgg Not detected
[0051] Since the average Mw of the protein designated CSP14 could
not be assigned with the above method, the peak fraction was
dissolved in 500 .mu.l of 25% solvent B (0.05% (v/v) TFA/80%, v/v
ACN). For SDS-PAGE, a 10 .mu.l aliquot was dried in speedvac and
dissolved in 20 .mu.l SDS-sample buffer and analyzed on gradient
10-20% T ready Tris-Tricine acrylamide gels using the miniprotean 3
system from Bio-Rad. Protein bands were visualized by staining the
gels in the staining solution [0.5% (w/v) Commassie Brilliant Blue
R250 in 30% (v/v) methanol and 10% (v/v) acetic acid] for 1 hr
followed by destaining [30% (v/v) methanol plus 10% (v/v) acetic
acid] until bands were visible against the clear background
(Graffin, Methods Enzymol. 182 (1990) 425-477). Accordingly it
could be observed that CSP 14 corresponds to a protein having a
molecular weight of about 14 kDa.
[0052] Purification/Collection by Repetitve Rp-Hplc
[0053] Subsequently, the cocoa proteins, reduced and
pyridine-ethylated were isolated/collected by repetitive injections
and automatic fraction pooling and collection from the GndHCl
extracts of unfermented CAP, as described above. The pooled
fractions of each proteins were dried under reduced pressure and
dissolved in 400 .mu.l solvent A and rechromatographed [column
Aquapore RP 300 (7 .mu.m, 4.6.times.220 mm), solvent TFA/ACN
system; injection volume 400 .mu.l; detection at 215 nm; gradients:
1. FIG. 7a (CSP14) and FIG. 7b (CSP9): 0-15% B in 5 min, isocratic
at 15% B for 5 min, 15-35% B in 60 min, 35-50% B in 10 min, 50-100%
B in 5 min and isocratic at 100% B for 5 min; 2. FIG. 7c (CSP12):
0-35% B in 5 min, isocratic at 15% B for 5 min, 35-60% B in 60 min,
60-75% B in 10 min, 75-100% B in 10 min and isocratic at 100% B for
10 min;]. Fractions 1 ml each were automatically collected and
those containing the peak fractions for each of CSP14 and CSP10
were pooled, dried and kept at -20.degree. C. until used.
Example 3
[0054] Characterization of purified proteins
[0055] The purified cocoa seed proteins CSP10 and CSP14 were
subjected to N-terminal amino acid sequencing by automated Edman
degradation protein sequencer. The initial and repetitive yield of
Edman cycle was between 80 to 90%. The results obtained are shown
in table 2 below. TABLE-US-00002 TABLE 2 Initial Initial amount,
yield, Protein pmol pmol Sequence CSP10 400 120 RREQE EESEE ETFGE
FXQVX APLXP G (SEQ ID NO: 3) CSP14 200 100 GRKQY ERDPR (SEQ ID
NO4)
[0056] The above listed N-terminal sequence of CSP 10 and 14 has
been found to be a part of the 67 kDa vicillin type cocoa storage
protein (WO 91/19801, supra). Thus, both of CSP 10 and 14 are so
far not identified fragments of the 67 kDa vicilin type cocoa
storage protein produced during the normal processing of the
protein in cacao beans. By aligning the 47 and 31 kDa proteins,
known to be derived from the 67 kDa vicillin protein, to the
protein the remaining sequence for the CSP 10 and 14 was derived,
which yielded the sequences as identified by SEQ ID NO: 1 and SEQ
ID NO:2.
[0057] A calculation of the molecular weights of the amino acids
contained in the polypeptides according to SEQ ID NO: 1 and 2
confirmed the approximate molecular weights of the resulting
polypeptides were about 10 and 14 kDa.
[0058] Consequently the peptides also seem to be excised during the
normal processing of the 67 kDa protein and represent a part of the
protein/polypeptide pool of cacao beans.
Sequence CWU 1
1
4 1 89 PRT Theobroma cacao 1 Arg Arg Glu Gln Glu Glu Glu Ser Glu
Glu Glu Thr Phe Gly Glu Phe 1 5 10 15 Gln Gln Val Lys Ala Pro Leu
Ser Pro Gly Asp Val Phe Val Ala Pro 20 25 30 Ala Gly His Ala Val
Thr Phe Phe Ala Ser Lys Asp Gln Pro Leu Asn 35 40 45 Ala Val Ala
Phe Gly Leu Asn Ala Gln Asn Asn Gln Arg Ile Phe Leu 50 55 60 Ala
Gly Arg Pro Phe Phe Leu Asn His Lys Gln Asn Thr Asn Val Ile 65 70
75 80 Lys Phe Thr Val Lys Ala Ser Ala Tyr 85 2 101 PRT Theobroma
cacao 2 Gly Arg Lys Gln Tyr Glu Arg Asp Pro Arg Gln Gln Tyr Glu Gln
Cys 1 5 10 15 Gln Arg Arg Cys Glu Ser Glu Ala Thr Glu Glu Arg Glu
Gln Glu Gln 20 25 30 Cys Glu Gln Arg Cys Glu Arg Glu Tyr Lys Glu
Gln Gln Arg Gln Gln 35 40 45 Glu Glu Glu Leu Gln Arg Gln Tyr Gln
Gln Cys Gln Gly Arg Cys Gln 50 55 60 Glu Gln Gln Gln Gly Gln Arg
Glu Gln Gln Gln Cys Gln Arg Lys Cys 65 70 75 80 Trp Glu Gln Tyr Lys
Glu Gln Glu Arg Gly Glu His Glu Asn Tyr His 85 90 95 Asn His Lys
Lys Asn 100 3 26 PRT Theobroma cacao misc_feature (17)..(17) Xaa
can be any naturally occurring amino acid misc_feature (20)..(20)
Xaa can be any naturally occurring amino acid misc_feature
(24)..(24) Xaa can be any naturally occurring amino acid 3 Arg Arg
Glu Gln Glu Glu Glu Ser Glu Glu Glu Thr Phe Gly Glu Phe 1 5 10 15
Xaa Gln Val Xaa Ala Pro Leu Xaa Pro Gly 20 25 4 10 PRT Theobroma
cacao 4 Gly Arg Lys Gln Tyr Glu Arg Asp Pro Arg 1 5 10
* * * * *