Abstract:
The present invention relates to a novel carboxypeptidase gene and the polypeptide encoded thereby. In particular, the present invention relates to the use of the present carboxypeptidase and polypeptide in the manufacture of cocoa flavor and/or chocolate.

Description:
PRIORITY CLAIM 
     This application is a divisional of and claims the benefit of U.S. patent application Ser. No. 10/382,239 filed Mar. 5, 2003, which is a continuation-in-part of the US national phase designation of International application PCT/EP02/07162 filed Jun. 28, 2002, the contents of which are expressly incorporated herein by reference thereto. 
    
    
     BACKGROUND 
     The present invention relates to a novel carboxypeptidase gene and the polypeptide encoded thereby. In particular, the present invention relates to the use of the present carboxypeptidase in the manufacture of cocoa flavor and/or chocolate. 
     It is known that in processing  cacao  beans the generation of the typical cocoa flavor requires two steps: a fermentation step, which includes air-drying of the fermented material, and a roasting step. 
     During fermentation, two major activities may be observed. First, the pulp surrounding the beans is degraded by micro-organisms with the sugars contained in the pulp being largely transformed to acids, especially acetic acid (Quesnel et al., J. Sci. Food. Agric. 16 (1965), 441-447; Ostovar and Keeney, J. Food. Sci. 39 (1973), 611-617). The acids then slowly diffuse into the beans and eventually cause an acidification of the cellular material. Second, fermentation also results in a release of peptides exhibiting differing sizes and a generation of a high level of hydrophobic free amino acids. This latter finding led to the hypothesis that proteolysis occurring during the fermentation step is not due to a random protein hydrolysis, but seems to be rather based on the activity of specific endoproteinases (Kirchhoff et al., Food Chem 31 (1989), 295-311). This specific mixture of peptides and hydrophobic amino acids is deemed to represent cocoa-specific flavor precursors. 
     Until now several proteolytic enzyme activities have been investigated in  cacao  beans and studied for their putative role in the generation of cocoa flavor precursors during fermentation. 
     An aspartic endoproteinase activity, which is optimal at a very low pH (pH 3.5) and inhibited by pepstatin A, has been identified. A polypeptide described to have this activity has been isolated and is described to consist of two peptides (29 and 13 kDa) which are deemed to be derived by self-digestion from a 42 kDa pro-peptide (Voigt et al., J. Plant Physiol. 145 (1995), 299-307). The enzyme cleaves protein substrates between hydrophobic amino acid residues to produce oligopeptides with hydrophobic amino acid residues at the ends (Voigt et al., Food Chem. 49 (1994), 173-180). The enzyme accumulates with the vicilin-class (7S) globulin during bean ripening. Its activity remains constant during the first days of germination and does not decrease before the onset of globulin degradation (Voigt et al., J. Plant Physiol. 145 (1995), 299-307). 
     Also, a cysteine endoproteinase activity had been isolated which is optimal at a pH of about 5. This enzymatic activity is believed not to split native storage proteins in ungerminated seeds. Cysteine endoproteinase activity increases during the germination process when degradation of globular storage protein occurs. To date, no significant role for this enzyme in the generation of cocoa flavor has been reported (Biehl et al., Cocoa Research Conference, Salvador, Bahia, Brasil, 17-23 Nov. 1996). 
     Moreover, a carboxypeptidase activity has been identified which is inhibited by PMSF, and thus belongs to the class of serine proteases. It is stable over a broad pH range with a maximum activity at pH 5.8. This enzyme does not degrade native proteins, but preferentially splits hydrophobic amino acids from the carboxy-terminus of peptides (Bytofet at., Food Chem. 54 (1995), 15-21). 
     During the second step of cocoa flavor production, the roasting step, the oligopeptides and amino acids generated at the stage of fermentation are obviously subjected to a Maillard reaction with reducing sugars present in fermented beans, eventually yielding substances responsible for the cocoa flavor as such. 
     In the art, there have been many attempts to artificially produce cocoa flavor. 
     Cocoa-specific aroma has been obtained in experiments wherein acetone dry powder (AcDP) prepared from unfermented ripe  cacao  beans was subjected to autolysis at a pH of 5.2 followed by roasting in the presence of reducing sugars. It was conceived that under these conditions preferentially free hydrophobic amino acids and hydrophilic peptides should be generated and the peptide pattern thus obtained was found to be similar to that of extracts from fermented  cacao  beans. An 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 aroma could) be detected when AcDP was subjected to autolysis at a pH of as low as 3.5 (optimum pH for the aspartic endoproteinase). Only few free amino acids were found to be released, but a large number of hydrophobic peptides were formed. When peptides obtained after the autolysis of AcDP at a pH of 3.5 were treated with carboxypeptidase A from porcine pancreas at pH 7.5, hydrophobic amino acids were preferentially released. The pattern of free amino acids and peptides was similar to that found in fermented  cacao  beans and to the proteolysis products obtained by autolysis of AcDP at pH 5.2. After roasting of the amino acids and peptides mixture as above, a cocoa aroma could be generated. 
     It has also been shown that a synthetic mixture of free amino acids alone, with a similar composition to that of the spectrum found in fermented beans, was incapable of generating cocoa aroma after roasting, indicating that both the peptides and the amino acids are important for this purpose (Voigt et al., Food Chem. 49 (1994), 173-180. 
     In view of the above data, a hypothetical model for the generation, during fermentation, of the said mixture of peptides and amino acids, i.e. the  cocoa  flavor precursors, had been devised ( FIG. 1 ), where in a first step peptides having a hydrophobic amino acid at their end, are formed from storage proteins, which peptides are then further degraded to smaller peptides and free amino acids. To produce the said peptides having C-terminal hydrophobic amino acids, an aspartic endoproteinase activity related to that mentioned above seems to be involved. Yet, for splitting off hydrophobic amino acids from peptides formed in the preceding step the only known enzymatic activity, which might be considered in this respect, is that of a carboxypeptidase. However, such enzyme has not been isolated and studied in detail in  cacao,  and it is therefore still questionable which  cacao  enzyme might be responsible for the generation of hydrophobic amino acids required for cocoa flavor. 
     Though some aspects of cocoa flavor production have been elucidated, so far there is still a need in the art to fully understand the processes involved, so that the manufacture of cocoa flavor may eventually be optimized. 
     SUMMARY 
     The present invention provides means for further elucidating the processes involved in the formation of cocoa-specific aroma precursors during the fermentation of  cacao  seeds, to improve the formation of cocoa flavor during processing and manufacturing and eventually providing means assisting in the artificial production of cocoa flavor. 
     This problem has been solved by providing a nucleotide sequence encoding a novel carboxypeptidase from  cacao  beans (termed  cacao  CP-III), which is identified by SEQ. ID. No. 1, or functional derivatives thereof having a degree of homology that is greater than 80%, preferably greater than 90% and more preferably greater than 95%. 
     It will be appreciated by the skilled person that a gene encoding a specific polypeptide may differ from a given sequence according to the Wobble hypothesis, in that nucleotides are exchanged that do not lead to an alteration in the amino acid sequence. Yet, according to the present invention, also nucleotide sequences shall be embraced which exhibit a nucleotide exchange leading to an alteration of the amino acid sequence such that the functionality of the resulting polypeptide is not essentially disturbed. 
     This nucleotide sequence may be used to synthesize a corresponding polypeptide by means of recombinant gene technology, in particular, a polypeptide as identified by SEQ. ID. No. 2. 
     As has been shown in a comparison with other carboxypeptidases from other plants, the present enzyme does not show a substantial homology to any of the carboxypeptidases known so far. Since it is assumed, that cocoa may furthermore contain additional carboxypeptidases that might exhibit a higher homology to the carboxypeptidases known so far, it must be considered as a surprising fact that this very enzyme has been detected. 
     For producing the polypeptide by recombinant means, the nucleotide of the present invention is included in an expression vector downstream of a suitable promoter and is subsequently incorporated into a suitable cell which may be cultured to yield the polypeptide of interest. Suitable cells for expressing the present polypeptide include bacterial cells, such as e.g.  E. coli , or yeast, insect, mammalian or plant cells. 
     The present DNA sequence may also be incorporated directly into the genome of the corresponding cell by techniques well known in the art, such as e.g. homologous recombination. Proceeding accordingly will provide a higher stability of the system and may include integration of a number of said DNA-sequences into a cell&#39;s genome. 
     The cells thus obtained may in consequence be utilized to produce the polypeptide in batch culture or using continuous procedures, with the resulting polypeptide being isolated according to conventional methods. 
     The recombinant carboxypeptidase obtained may be used for the manufacture of cocoa flavor. To this end, the enzyme described herein may be utilized in an artificial trial run wherein a mixture of different proteins, such as  cacao  storage proteins, or protein hydrolysates of other resources, are subjected to enzymatic degradation by means of enzymes known to be involved in proteolytic degradation to eventually assist in the production of flavor precursors. The enzyme may likewise also be utilized in the production of cocoa liquor and in the manufacture of chocolate. 
     Yet, the present invention also provides plants, in particular  cacao  plants, comprising a recombinant cell containing one or more additional copies of the carboxypeptidase of the present invention. Such a  cacao  plant will produce beans, which will exhibit a modified degradation of storage proteins when subjected to the fermentation process, allowing a more rapid degradation or a pattern of hydrolysis that yields a higher level of cocoa flavor precursor since a higher amount of carboxypeptidase will be present. 
     The carboxypeptidase of the present invention may also be used to produce other transgenic plants, such as soybean and rice, producing seeds with this new protein modifying enzyme. 
     Additional features and advantages are described herein, and will be apparent from, the following Detailed Description and the figures. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  shows a scheme illustrating a potential process for the proteolytic formation of cocoa-specific aroma; 
         FIG. 2  shows the cloning strategy used for the isolation of a cDNA encoding a carboxypeptidase from  Theobroma cacao;    
         FIG. 3  shows a comparison of the hydrophilicity Plot-Kyte-Doolittle for the  cacao  CP-III sequence with Barley CP-MI, CP-MII and CP-MIII; and 
         FIGS. 4A ,  4 B and  4 C shows a Northern blot analysis of  cacao  CP-III. 
     
    
    
     DETAILED DESCRIPTION 
     As described above, it was suggested that a carboxypeptidase could be involved in the production of cocoa flavor precursors during  cacao  fermentation. However, it was not known in the art which cacao carboxypeptidase carried out this function considering that five classes of carboxypeptidases (Type I-V) have been identified in different plants by references to differences in substrate specificities, molecular weights and chromatographic profiles. Furthermore, 50 sequences having homologies with serine carboxypeptidases exist in the completed  Arabidopsis  genome. 
     The proteoleytic formation of cocoa-specific aroma according to the invention is illustrated in  FIG. 1 . 
     The following examples illustrate the invention further without limiting it thereto. In the examples, the following abbreviations have been used: 
     PCR: Polymerase Chain Reaction 
     RACE: Rapid Amplification cDNA Ends 
     cDNA: complementary deoxyribonucleic acid 
     mRNA: messenger ribonucleic acid 
     DEPC: Diethyl pyrocarbonate 
     3,4-DCI: 3,4-dichloroisocoumarin 
     EXAMPLES 
     Materials 
       Cacao  ( Theobroma cacao  L.) seeds (male parent unknown) from ripe pods of clone ICS 95 were provided by Nestlé ex-R&amp;D Center Quito (Ecuador). The seeds were taken from the pods immediately after arrival at Nestle Research Center Tours (4-5 days after harvesting). The pulp and the seed coat were eliminated, and the cotyledons were frozen in liquid nitrogen and stored at −80° C. until use. 
     Preparation of mRNA 
     Total RNA was prepared using the following method. Two seeds were ground in liquid nitrogen to a fine powder and extraction was directly performed with a lysis buffer containing 25 mM Tris HCl pH8, 25 mM EDTA, 75 mM NaCl, 1% SDS and 1M 3-mercaptoethanol. RNA was extracted with one volume of phenol/chloroform/isoamylalcohol (25/24/1) and centrifuged at 8000 rpm, 10 min at 4° C. The aqueous phase was extracted a second time with one volume of phenol/chloroform/isoamylalcohol (25/24/1). RNA was precipitated with 2M lithium chloride at 4° C. overnight. The RNA pellet obtained after centrifugation was resuspended in DEPC-treated water, and a second precipitation with 3M sodium acetate pH 5.2 was performed in presence of two volumes of ethanol. The RNA pellet was washed with 70% ethanol and resuspended in DEPC-treated water. Total RNA was further purified using the Rneasy Mini kit from Qiagen®. 
     Cloning of a Carboxypeptidase cDNA 
     Cloning Strategy 
     A 1.5 kb 5′-end fragment of a carboxypeptidase from  cacao  seed was amplified by RT-PCR using a degenerate oligonucleotide. Based on the sequence of this fragment, a primer was designed to amplify a 3′-end fragment. Finally, a full-length cDNA ( cacao  CP-III) was amplified using primers specific to both extremities. The cloning strategy used for isolation of a cDNA encoding a carboxypeptidase from  Theobroma cacao , clone ICS 95 is shown in  FIG. 2 . 
     Primer Design 
     A search for carboxypeptidase sequences in the GenBank database led to the identification of several plant sequences. A multiple alignment of these sequences revealed the presence of conserved regions. The conserved sequence MVPMDQP located near the histidine catalytic site has been used to design a degenerate oligonucleotide in the antisense orientation: pCP2r (5′-GGYTGRTCCATNGGNACCAT) (SEQ ID No. 3). 
     Synthesis of cDNA 
     Total RNA (see above) was used to synthesize first strand 3′ and 5′ cDNAs with the SMART™ RACE cDNA Amplification Kit (Clontech, USA). Synthesis has been performed exactly as described in the kit instructions using 1 μg of total RNA and the Superscript™ II MMLV reverse transcriptase (Gibco BRL, USA). After synthesis, cDNAs were used directly for PCR or kept at −20° C. 
     5′ RACE Amplification 
     Specific cDNA amplification was performed with 2.5 μl of the first strand 5′ cDNA in 50 μl buffer containing: 40 mM Tricine-KOH, pH 8.7, 15 mM KOAc, 3.5 mM Mg(OAc) 2 , 3.75 μg/ml BSA, 0.005% Tween-20, 0.005% Noninet-P40, 0.2 mM dNTP&#39;s, 14 pmoles of pCP2r primer, 5 μl of 10× Universal primer Mix (UPM) and 1 μl 50× Advantage 2 polymerase Mix (Clontech, USA). Amplification was performed in a Bio-med thermocycler 60 (B. Braun). A first denaturation step (94° C., 2 min) was followed by 35 cycles of denaturation (94° C., 1 min), primer annealing (55° C., 1.5 min) and extension (72° C., 2 min). The extension time was increased by 3 sec at each cycle. Amplification was ended by a final extension step (72° C., 10 min). The amplified fragment was cloned in pGEM®-T vector and sequenced. 
     3′ RACE PCR 
     The sequence information obtained after the sequencing of the 5′ end fragment was used to design a specific oligonucleotide pCP5 (5′-GCTTTTGCTGCCCGAGTCCACC) (SEQ ID No. 4), which was used for 3′-RACE amplification. 3′-RACE PCR was performed with 2.5 μl of SMART single strand 3′ cDNA in 50 μl buffer containing 40 mM Tricine-KOH pH 8.7, 15 mM KOAc, 3.5 mM Mg(OAc) 2 , 3.75 μg/ml BSA, 0.005% Tween-20, 0.005% Nonidet-P40, 0.2 mM dNTP&#39;s, 10 pmoles of pCP5 primer, 10 μl of 10× Universal primer Mix (UPM) and 1 μl 50× Advantage 2 polymerase Mix (Clontech, USA). Amplification was performed via touchdown PCR, in a Bio-med thermocycler 60 (B. Braun). 
     A first denaturation step (94° C., 1 min) was followed by:
         5 cycles including denaturation at 94° C. for 30 sec and annealing/extension at 72° C. for 3 min   5 cycles including denaturation at 94° C. for 30 sec and annealing/extension at 70° C. for 30 sec and 72° C. for 3 min   30 cycles including denaturation at 94° C. for 30 sec and annealing/extension at 68° C. for 30 sec and 72° C. for 3 min.       

     The amplified fragment was cloned in pGEM®-T vector and sequenced. 
     Full Length cDNA 
     The sequence information obtained after the sequencing of 5′-and 3′-RACE fragments was used to design two specific oligonucleotides. 
     
       
         
               
               
               
               
             
           
               
                 pCP8: 
                 A sense primer 
                 (SEQ ID No. 5) 
                   
               
               
                   
                 (5′-CAAAGAGAAAAAGAAAAGATGGC) 
               
               
                   
               
               
                 pCP7r: 
                 A reverse primer 
                 (SEQ ID No. 6) 
               
               
                   
                 (5′-CCCCAGAGCTTTACGATACGG). 
               
             
          
         
       
     
     PCR reaction was performed with 2.5 μl first strand cDNA in 50 pl buffer containing: 40 mM Tricine-KOH pH 8.7, 15 mM KOAc, 3.5 mM Mg(OAc) 2 , 3.75 μg/ml BSA, 0.005% Tween-20, 0.005% Noninet-P40, 0.2 mM dNTP&#39;s, 10 pmoles of pCP8 primer, 10 pmoles of pCP7r primer and 1 μl 50× Advantage 2 polymerase Mix (Clontech, USA). Amplification was performed in a Bio-med thermocycler 60 (B. Braun). A first denaturation step (94° C., 1 min) was followed by 35 cycles of denaturation (94° C., 30 sec), primer annealing (63° C., 1 min) and extension (72° C., 2 min). The extension time was increased by 3 sec at each cycle. Amplification was ended by a final extension step (72° C., 10 min). The amplified fragment was cloned in pGEM®-T Easy vector and sequenced. 
     Sequencing and Analysis of DNA Sequences 
     cDNA sequencing has been performed by Eurogentech (Belgium) and ESGS (France). Sequence analysis and comparison were performed with Lion&#39;s software bioScout, Lasergene software (DNAStar) and Genedoc programme. 
     The  cacao  CP-III cDNA sequence is 1768 bp long. A putative initiation start codon was assigned by comparison with other carboxypeptidase sequences. It is located 25 bp from the 5′ end. The open reading frame is broken by a stop codon (TGA) at position 1549, followed by a putative polyadenylation signal (TATAAA) at position 1725. 
       Cacao  CP-III encodes a 508 amino acid type III carboxypeptidase C with a predicted molecular weight of 56 kDa and a pH of 5.04. The catalytic amino acids are present at position Ser 228 , Asp 416  and His 473 . A hydrophilicity analysis was performed using a Lasergene program (DNASTAR) and a window of 9. The results of a comparison of the hydrophiliccity Plot-Kyte-Doolittle for the  cacao  CP-III sequence with Barley CP-MI, CP-MII AND CP-MIII ( FIG. 3 ) reveals that  cacao  CP-III encodes a hydrophilic protein with a very hydrophobic N-terminal end, indicating the presence of a signal peptide. 
     Northern Blot Analysis 
     Total RNA samples were separated on 1.5% agarose gel containing 6% formaldehyde. RNA was separated on agarose gels, then transferred to a nylon membrane and probed with radiolabelled  cacao  CP-III cDNA under stringent hybridization conditions. An equal loading of the RNA samples was confirmed by ethidium bromide staining of ribosomal RNA in the gel before transfer to the membrane ( FIG. 4 ). After electrophoresis, RNA was blotted onto nylon membranes (Appligene) and hybridized with  32 P-labeled  cacao  CP-III probe at 65° C. in 250 mM Na-phosphate buffer pH 7.2, 6.6% SDS, 1 mM EDTA and 1% BSA. Cacao CP-III cDNA fragment was amplified by PCR using pCP8 and pCP7R primers and labelled by the random priming procedure (rediprime™ II, Amersham Pharmacia Biotech). Membranes were washed three times at 65° C. for 30 min in 2×SSC, 0.1% SDS, in 1×SSC, 0.1% SDS and in 0.5×SSC, 0.1% SDS.  FIG. 4A  illustrates the total RNA (15 μg per lane) from seed and leaf.  FIG. 4B  illustrates the total mature seed (15 μg per lane) from different  T. cacao  clones while  FIG. 4C  illustrates total RNA (15 μg per lane) from seed at different stages of germination. 
     It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.