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Two dimensional electrophoresis and nano-LC–MS were performed in order to identify alterations in protein abundance that correlate with maturation of cacao zygotic and somatic embryos. The cacao pod proteome was also characterized during development. The recently published cacao genome sequence was used to create a predicted proteolytic fragment database. Several hundred protein spots were resolved on each tissue analysis, of which 72 variable spots were subjected to MS analysis, resulting in 49 identifications. The identified proteins represent an array of functional categories, including seed storage, stress response, photosynthesis and translation factors. The seed storage protein was strongly accumulated in cacao zygotic embryos compared to their somatic counterpart. However, sucrose treatment (60 g L −1 ) allows up-regulation of storage protein in SE. A high similarity in the profiles of acidic proteins was observed in mature zygotic and somatic embryos. Differential expression in both tissues was observed in proteins having high p I . Several proteins were detected exclusively in fruit tissues, including a chitinase and a 14-3-3 protein. We also identified a novel cacao protein related to known mabinlin type sweet storage proteins. Moreover, the specific presence of thaumatin-like protein, another sweet protein, was also detected in fruit tissue. We discuss our observed correlations between protein expression profiles, developmental stage and stress responses.

Sweet plant proteins, which are safe, natural, low-calorie sweeteners, may be suitable replacements for sugars in the food and beverage industries. Mabinlin II, a sweet plant protein, shows the most pronounced heat stability and acid resistance of any of the six known types of plant sweet proteins. However, mabinlin II is difficult to extract from the Capparis masaikai plant, which is itself becoming increasingly scarce. This limits the use of naturally acquired mabinlin II. In this study, recombinant mabinlin II proteins were expressed and purified in Escherichia coli and in food-grade Lactococcus lactis. Recombinant mabinlin II proteins MBL-BH (containing the B-chains of mabinlin II downstream fused with His-tag) and MBL-ABH (containing the A- and B-chains of mabinlin II downstream fused with His-tag) were expressed in E. coli in the form of inclusion bodies. They were then purified and renatured. The refolded MBL-BH was found to be 100 times sweeter than sucrose by weight, but it was not heat-stable. Refolded MBL-ABH was neither sweet nor heat-stable. Recombinant mabinlin II proteins were secreted and expressed intracellularly in food-grade L. lactis, in which the concentrated cell samples and culture medium samples were detected using enzyme-linked immunosorbent assay and Western blotting analysis with anti-mabinlin II polyclonal antibody. This study demonstrated that the single B chain of mabinlin II has a sweet taste. The recombinant mabinlin II proteins have been successfully expressed in food-grade L. lactis, which is a crucial step in the production of mabinlin II through microorganism expression systems.

International audience; PlanÉdulcorants d’origine végétaleAllergénicité de la thaumatine et des protéines thaumatin-likeAllergénicité des autres édulcorantsConclusionDéclaration de liens d’intérêts

The quality of fruits and vegetables is mainly dependant on the sweetness determined by the level of soluble sugars such as glucose, fructose and sucrose. Other fruit quality parameters include Brix content, acidity, aroma, color, size and shape. Total sugar content in fruits and vegetables is a function of genetic, nutritional, environmental and developmental factors. Understanding the factors controlling sweetness is important to design strategies for enhancing quality of fruits and vegetables. Modifying the activity of enzymes in carbohydrate metabolism such as sucrose synthase (SuSy), acid invertase, ADP-glucose pyrophosphorylase (AGPase), sucrose phosphate synthase (SPS) and sucrose transporters were found to influence carbohydrate partitioning and sucrose accumulation in sink tissues of several food crops. Plant based taste-modifying sweet proteins such as brazzein, cucurmin, mabinlin, monellin, miraculin, neoculin and thaumatin have potential application for developing transgenic plants to improve the sweetness and quality of fruits and vegetables. The present review envisages various cultural, breeding and molecular approaches used for enhancing sugar content and sweetness in fruits and vegetables.

Mabinlin II is one of the major sweet proteins stored in the seeds of Capparis masaikai Lévl. Its promoter region (779 bp) located 5' upstream of the mabinlin II gene has been isolated and named as MBL-779 (GenBank accession number, EU014073). This promoter contains two typical TATA box regions and a series of motifs related to seed-specific promoters, such as ACGT motifs, RY motif, napin motif, and G box. The MBL-779 promoter drove GUS gene to transiently express in the embryos of bean, maize, and rice seeds or to constantly express in the embryos and anthers of the transgenic Arabidopsis. The MBL-779 promoter regulated gene expression from approximately the 12th day and peaked on approximately the 16th day after flowering in Arabidopsis. The -300-bp promoter region is a minimal sequence required to functionally regulate gene expression. The CAATs at -325 to -322 bp and -419 to -416 bp and the region at -485 to -770 bp play a role in the quantitative regulation of gene expression. The RY motif, CATGAC, at -117 to -112 bp and the ACGT within the G box (CACGTG) at -126 to -123 bp positively regulate gene expression.

The crystal structure of a sweet protein Mabinlin II (Mab II) isolated from the mature seeds of Capparis masaikai Levl. grown in Southern China has been determined at 1.7A resolution by the SIRAS method. The Mab II 3D structure features in an "all alpha" fold mode consisting of A- and B-chains crosslinked by four disulfide bridges, which is distinct from all known sweet protein structures. The Mabinlin II molecule shows an amphiphilic surface, a cationic face (Face A) and a neutral face (Face B). A unique structural motif consisting of B54-B64 was found in Face B, which adopts a special sequence, NL-P-NI-C-NI-P-NI, featuring four [Asn-Leu/Ile] units connected by three conformational-constrained residues, thus is called the [NL/I] tetralet motif. The experiments for testing the possible interactions of separated A-chain and B-chain and the native Mabinlin II to the sweet-taste receptor were performed through the calcium imaging experiments with the HEK293E cells coexpressed hT1R2/T1R3. The result shows that hT1R2/T1R3 responds to both the integrated Mabinlin II and the individual B-chain in the same scale, but not to A-chain. The sweetness evaluation further identified that the separated B-chain can elicit the sweetness alone, but A-chain does not. All data in combination revealed that the sweet protein Mabinlin II can interact with the sweet-taste receptor hT1R2/T1R3 to elicit its sweet taste, and the B-chain with a unique [NL/I] tetralet motif is the essential structural element for the interaction with sweet-taste receptor to elicit the sweetness, while the A-chain may play a role in gaining a long aftertaste for the integrate Mabinlin II. The findings reported in this paper will be advantage for understanding the diversity of sweet proteins and engineering research for development of a unique sweetener for the food and agriculture based on the Mabinlin II structure as a native model.

International audience; Plant proteins with sweet-tasting properties are increasingly used as substitutes for low-calorie sweeteners in the food industry. Thaumatin, the sweet protein isolated from the aril of the katemfe fruit (Thaumatococcus daniellii), is widely used as a natural sweetener. Two other low-calorie sweeteners with enhanced sweet-tasting properties, brazzein and monellin, which are isolated from the fruits of Pentadiplandra brazzeana and Dioscoreophyllum cumminsii, respectively, are still waiting to be produced on a large scale as recombinant sweet proteins for food industry. Although these sweet-tasting proteins are constitutively expressed in fruits, most of them consist of PR-proteins whose synthesis is strongly enhanced as a result of infection of the plant by phytopathogenic micro-organisms, fungi or molds. Both the sequence and structural similarities which the sweet proteins share with allergens, e.g., Art v 1 for brazzein, Mus a 4 and Ole a 13 for thaumatin, Ana o 3 and Pis v 1 for mabinlin, and Gly m Kunitz trypsin-inhibitor for monellin, suggest some possible allergenic propensity for these plant proteins. However, their allergenic potential following ingestion still remains to be demonstrated unambiguously.; Des protéines végétales à propriétés édulcorantes sont utilisées dans différents secteurs de l’alimentation humaine où elles remplacent les édulcorants de synthèse. C’est le cas de la thaumatine, protéine édulcorante extraite de l’arille des fruits du katemfe (Thaumatococcus daniellii). Deux autres protéines édulcorantes à pouvoir sucrant élevé, la brazzéine des fruits de Pentadiplandra brazzeana et la monelline des fruits de Dioscoreophyllum cumminsii, sont également disponibles, mais leur production industrielle n’est pas encore programmée. Bien qu’elles soient exprimées de façon constitutive dans les fruits, la plupart de ces protéines correspondent à des protéines de défense de la plante ou protéines PR, dont la synthèse est exacerbée lorsque la plante est en conditions de stress, lors d’une attaque fongique, par exemple. Les homologies de séquence, et surtout de structure, que ces protéines édulcorantes partagent avec des allergènes avérés, avec Art v 1 pour la brazzéine, avec Mus a 4 ou Ole e 13 pour la thaumatine, avec Ana o 3 ou Pis v 1 pour la mabinline, avec l’inhibiteur de Kunitz du soja pour la monelline, suggèrent une possible allergénicité de ces protéines végétales. Néanmoins, leur potentiel allergénique reste à démontrer lors de leur consommation alimentaire.

Most proteins are tasteless and flavorless, while some proteins elicit a sweet-taste response on the human palate. Six proteins, thaumatin, monellin, mabinlin, brazzein, egg lysozyme, and neoculin (previously considered as curculin) have been identified as sweet-tasting proteins. However, no common features among them have been observed. Herein, recent advances in the research of sweet-tasting proteins and the production of such proteins by biotechnological approaches are reviewed. Information on the structure-sweetness relationship for these proteins would help not only in the clarification of the mechanism of interaction of sweet-tasting proteins with their receptors, but also in the design of more effective low-calorie sweeteners.

The sweet protein mabinlin II isolated from the seeds of Capparis masaikai consists of the A chain with 33 amino acid residues and the B chain composed of 72 residues. The B chain contains two intramolecular disulfide bonds and is connected to the A chain through two intermolecular disulfide bridges. The A chain was synthesized by the stepwise fluoren-9-ylmethoxycarbonyl (Fmoc) solid-phase method in a yield of 5.9%, while the B chain was synthesized by a combination of the stepwise Fmoc solid-phase method and fragment condensation in a yield of 6.0%. Disulfide formation and combination of the A and B chains followed by purification by ion-exchange high-performance liquid chromatography (HPLC) gave mabinlin II in a yield of 47.4%. The characterization of the synthetic mabinlin II by HPLC, electrospray ionization mass spectrometry, amino acid analysis, and disulfide bond determination fully supported the expected structure. A 0.1% solution of the synthetic mabinlin II had an astringent-sweet taste. © 1998 John Wiley & Sons, Inc. Biopoly 46: 215–223, 1998

The search for non-carbohydrate sweeteners from natural sources has led to the discovery of many intensely sweet-tasting substances. The occurrence of sweet-tasting proteins such as thaumatin, monellin, mabinlin and pentadin in the pulp of fruits of various rain forest species has provided a new approach to the potential treatment of diabetes, obesity and other metabolic disorders. These proteins can also be used as low calorie sweeteners to enhance and modify the taste of existing foods. Thaumatin is currently commercially available as a sweetener, flavor enhancer, additive to pharmaceuticals, chewing gum and animal feeds. It can also be secreted extracellularly by genetic modification of the yeast Saccharomyces cerevisiae. The taste-modifying proteins, miraculin and curculin, can be utilized in controlling the palatability of foods or in the development of new food products. This review discusses some properties of sweet and taste-modifying proteins discovered to date.

There are several analogues of the sweet protein mabinlin. In previous studies, we purified the heat-stable analogue, mabinlin II, from the seeds of Capparis masaikai Levl. and determined its amino acid sequence [Liu, X., Maeda, S., Hu, Z., Aiuchi, T., Nakaya, K. & Kurihara, Y. (1993) Eur. J. Biochem. 211, 281–287] and the disulfide structure [Nirasawa, S., Liu, X., Nishino, T. & Kurihara, Y. (1993) Biochim. Biophys. Acta 1202, 277–280]. We have now purified four additional homologues of mabinlin. The sweet activities of mabinlin III and mabinlin IV were unchanged by incubation for 1 h at 80°C, as was found previously for mabinlin II, while the sweet activity of mabinlin I-1 was completely abolished by a 1-h incubation at 80°C. The circular dichroic spectrum showed that α-helical structures of mabinlins II–IV were unchanged by the 1-h incubation at 80°C, while the α-helical structures of mabinlin I-1 were completely destroyed by the 1-h incubation in parallel with the decrease of the sweet activity. To compare the structures of the heat-stable and unstable homologues, we determined their amino acid sequences and the disulfide array. The positions of four disulfide bridges of mabinlin I-1 were the same as those of mabinlin II, suggesting that the disulfide bridges do not contribute to the difference in the heat stability among the homologues. There was a high similarity among amino acid sequences of the homologues. Only three amino acid residues (A-chain residues at positions 22 and 32 and B-chain residue at position 47) were different between mabinlin I-1 and mabinlin III. A-chain residue at position 32 was lacking in mabinlin IV and the A-chain residue at position 22 was identical in both mabinlin I-1 and mabinlin II. The B-chain residue at position 47 was the only residue present in all three heat-stable homologues (mabinlins II–IV) and is not present in the unstable homologue (mabinlin I-1). This suggests that the difference in the heat stability of mabinlin is due to the difference in a B-chain residue at position 47; the difference in the heat-stable homologues is due to the presence of an arginine residue and the difference of the unstable homologue is due to the presence of glutamine. It is possible that a salt bridge, formed between a at B-chain arginine residue at position 47 and the caroxylic group of an A-chain or B-chain C-terminal residue contributes to the heat stability.

Recent progress in studies of antisweet substances, sweet proteins and sweetness-inducing proteins is reviewed. Gymnemic acid, ziziphin and hodulcin, which are triterpene glycosides, suppress the sweet taste sensation in humans, and gurmarin, which is a peptide, suppresses the sweet taste response in the rat. Five sweet proteins, monellin, thaumatin, pentadin, curculin and mabinlin, are known. There are two sweetness-inducing proteins. Miraculin has the ability to modify a sour taste into a sweet taste. Curculin, which is itself a sweet protein, induces a sweet taste in response to water, as well as having a taste-modifying activity similar to that of miraculin.

The heat-stable sweet protein mabinlin was composed of a A-chain of 33 amino-acid residues and a B-chain of 72 amino-acid residues (Liu, X., Maeda, S., Hu, Z., Aiuchi, T., Nakaya, K. and Kurihara, Y. (1993) Eur. J. Biochem. 211, 281–287). A-chain and B-chain contain two and six cysteine residues, respectively. The formation of two interchain disulfide bridges at Cys(A5)–Cys(B21) and Cys(A18)–Cys(B10), and two intrachain disulfide bridges at Cys(B11)–Cys(B59) and Cys(B23)–Cys(B67) were determined by amino-acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. Cleavage of the disulfide bridges with dithiothreitol results in complete loss of the sweet activity of mabinlin II. It was suggested that the structure fixed by four disulfide bridges contributes to heat stability of mabinlin II.

A new taste-modifying protein named curculin was extracted with 0.5 M NaCl from the fruits of Curculigo latifolia and purified by ammonium sulfate fractionation, CM-Sepharose ion-exchange chromatography, and gel filtration. Purified curculin thus obtained gave a single band having a Mr of 12,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 8 M urea. The molecular weight determined by low-angle laser light scattering was 27,800. These results suggest that native curculin is a dimer of a 12,000-Da polypeptide. The complete amino acid sequence of curculin was determined by automatic Edman degradation. Curculin consists of 114 residues. Curculin itself elicits a sweet taste. After curculin, water elicits a sweet taste, and sour substances induce a stronger sense of sweetness. No protein with both sweet-tasting and taste-modifying activities has ever been found. There are five sets of tripeptides common to miraculin (a taste-modifying protein), six sets of tripeptides common to thaumatin (a sweet protein), and two sets of tripeptides common to monellin (a sweet protein). Anti-miraculin serum was not immunologically reactive with curculin. The mechanism of the taste-modifying action of curculin is discussed.

Mabinlin 11 is a thermostable sweet protein isolated from the mature seeds of Capparis masaikai Levl. grown in the subtropical region of the South of China. The Mabinlin 11 has been crystallized and diffraction data were collected to 1.7 angstrom resolution. The crystal belongs to space group C2 with unit cell parameters a = 80.11 angstrom, b = 51.08 angstrom, c = 47.34 angstrom, beta = 122.77 degrees.

A cDNA clone encoding a heat-stable sweet protein, mabinlin II (MAB), was isolated and sequenced. The encoded precursor to MAB was composed of 155 amino acid (aa) residues, including a signal sequence of 20 aa, an N-terminal extension peptide of 15 aa, a linker peptide of 14 aa and one residue of C-terminal extension. Comparison of the proteolytic cleavage sites during post-translational processing of MAB precursor with those of like 2S seed-storage proteins of Arabidopsis thaliana, Brassica napus and Bertholletia excelsa shows that the three individual cleavage sites between respective species are conserved.

Introduction Historical Perspectives Thaumatin Monellin Mabinlin Pentadin Brazzein Curculin Miraculin Comparison of Different Sweet-tasting Proteins: The Thaumatin-like Proteins Patents Outlook and Perspectives Keywords: thaumatin; monellin; mabinlin; pentadin; brazzein; curculin; miraculin; sweet phenotype; recombinant DNA technology; transgenic plants; expression vector

The amine pool of cocoa is known to be an essential component for the development of the typical cocoa flavor. To better understand and to produce an intense in vitro cocoa flavor, identification of the polypeptides that are the source of the amine flavor precursor pool is essential. Chromatographic analysis of the polypeptide profile of unfermented cocoa resulted in identification of a novel storage polypeptide of M(r) 8515. The N-terminal sequence of the first 34 residues of the purified polypeptide shows similarity to 2S storage albumins of cotton and Brazil nut and sweet protein, Mabinlin. To identify the corresponding cDNA of the putative cocoa 2S albumin, 18 randomly chosen clones from the cDNA library of immature Theobroma cacao seed mRNA were sequenced, and a full-length cDNA clone encoding a protein harboring the N-terminal sequence of the novel polypeptide was selected. The open reading frame of the clone encodes a polypeptide of M(r) 17125. Comparison of the translated amino acid sequence of the precursor protein or the mature polypeptide against the Swiss-Prot and TrEMBL databases shows high sequence similarity (52%) and identity (38%) to many plant 2S albumins. Tryptic peptide mass fingerprinting of the purified polypeptide by high-performance liquid chromatography-electrospray ionization mass spectrometry shows 10 masses that match the expected tryptic peptides of the deduced sequence. Together with the published work on plant 2S albumin processing, the results presented here suggest that post-translational processing yields a 73-residue polypeptide (residue positions 78-150) corresponding to the 9 kDa subunit of the mature cocoa 2S albumin protein.

The state of the art regarding the six known sweet-tasting proteins (thaumatin, monellin, mabinlin, pentadin, brazzein and curculin) and the taste-modifying protein miraculin is reviewed. Their biochemical properties, molecular genetics and biotechnological production are assessed. All of these proteins have been isolated from plants that grow in tropical rainforests. They share no sequence homology or structural similarities. Nonetheless, one of them, thaumatin, shares extensive homology with certain non-sweet proteins found in other plants. The potential industrial applications of the sweet-tasting proteins are also discussed, placing special emphasis on the barriers that a recombinant product of these characteristics will have to overcome before it reaches the market.

Mabinlin II is a sweet protein with the highest known thermostablility and is isolated from the seeds of Capparis masaikai Levl. grown in south China. Two crystal forms of mabinlin II were obtained using the hanging-drop vapour-diffusion method. One of them diffracts to 2.8 A resolution and belongs to space group P2, with unit-cell parameters a = 50.16, b = 50.17, c = 76.60 A, beta = 99.6 degrees. There are four molecules per asymmetric unit, with a solvent content of 35.3%.

A 10-kDa protein was isolated from resting seeds of hemp (Cannabis sativa) by buffer extraction, gel filtration, ion-exchange chromatography, and reversed-phase high-pressure liquid chromatography. The protein did not inhibit bovine trypsin. It consisted of subunits composed of 27 and 61 residues and was held together by two disulfide bonds. The complete amino acid sequence was identified by protein analysis, and had 20 mole% of amino acids containing sulfur. The protein was most similar to a methionine-rich protein of Brazil nut (Bertholletia excelsa) and to Mabinlin IV, a sweetness-inducing protein of Capparis masaikai. The high methionine content and the absence of trypsin inhibitory activity suggested that the seed protein can be used to improve the nutritional quality of plant food-stuffs.

1968; Kurihara and Beidler, 1968) amol. wt of 24 600.The search for sweeteners was no longer limited to small moleculesand resulted within a few years in the discovery of monellin andthaumatin (Morris and Cagan, 1972; van der Wel and Loeve, 1972).Later, mabinlin and curculin were discovered (Hu and He, 1983;Yamashita

The plants, Capparis masaikai Levl. (local name mabinlang), which grows in the subtropical region of Yunnan province in China, bears fruit of tennis-ball size. The mature seed is used as traditional Chinese medicine and elicits a sweet taste. The seeds of C. masaikai contain the heat-stable sweet protein named mabinlin.

This chapter reviews the progress in exploring the structures and functions of antisweet substances (gymnemic acid, ziziphin), sweet proteins (mabinlin, curculin), and sweetness-inducing substances (strogin, miraculin, curculin). Table 1 summarizes the sources and functions of these substances.

A new sweet protein. named mabinlin II, was extracted with 0.5 M NaCl solution from the seeds of Capparis masaikai Levl. and purified by ammonium sulfate fractionation, carboxymethylcellulose-Sepharose ion-exchange chromatography and gel filtration. The sweetness of mabinlin II was unchanged by at least 48 h incubation at nearly boiling temperature. Purified mabinlin II thus obtained gave a single band having a molecular mass of 14 kDa on SDS/PAGE. In the presence of dithiothreitol, mabinlin II gave two bands having molecular masses of 4.6 kDa and 5.2 kDa on SDS/PAGE. Two peptides (A chain and B chain) were separated from reduced and S-carboxamidomethylated mabinlin II by HPLC. The amino acid sequences of the A chain and B chain were determined by the automatic Edman-degradation method. The A chain and B chain consist of 33-amino-acid and 72-amino-acid residues, respectively. The A chain is mostly composed of hydrophilic amino acid residues and the B chain also contains many hydrophilic residues. High similarity was found between the amino acid sequences of mabinlin II and 2S seed storage proteins, especially 2S albumin AT2S3 in Arabidopsis thaliana (mouse-ear cress).

Recent studies on structures and functions of sweetness-inhibiting substances (gymnemic acid, ziziphin, and gurmarin); sweet proteins (monellin, thaumatin and mabinlin); and taste-modifying proteins (miraculin and curculin) were reviewed. Several gymnemic acid homologues and gurmarin were purified from the leaves of Gymnema sylvestre and their structures were determined. Ziziphin was also purified from leaves of Ziziphus jujuba. Gymnemic acid and ziziphin are glycoside of triterpenes that suppress sweetness in human, while gurmarin is a peptide having antisweet activity in rat. Mabinlin is a heat-stable sweet protein. The whole amino acid sequence and the position of disulfide bridges of mabinlin were determined. Miraculin has the unusual property of modifying a sour taste into a sweet taste. Curculin elicits a sweet taste. In addition, water and sour substance elicit a sweet taste after curculin. Their amino acid sequences and subunit structures were determined. These proteins are expected to be used as low-calorie sweeteners.

The taste-modifying protein, miraculin, has the unusual property of modifying sour taste into sweet taste. The complete amino acid sequence of miraculin purified from miracle fruits by a newly developed method (Theerasilp, S., and Kurihara, Y. (1988) J. Biol. Chem. 263, 11536–11539) was determined by an automatic Edman degradation method. Miraculin was a single polypeptide with 191 amino acid residues. The calculated molecular weight based on the amino acid sequence and the carbohydrate content (13.9%) was 24,600. Asn-42 and Asn-186 were linked N-glycosidically to carbohydrate chains. High homology was found between the amino acid sequences of miraculin and soybean trypsin inhibitor.

1. 1. The intensely sweet princile from the fruit of the tropical plant Dioscoreo-pyllum cumminsii Diels has been isolated. The sweet principle, herein named monellin, is purified from aqueous extracts by (NH4)2SO4 fractionation and ion-exchange chromatography, first on DEAE-cellulose, and then on CM-cellulose. 2. 2. The yield is over 50% and the material is homogeneous by disc gel electrophoresis. 3. 3. Monellin is non-dialyzable, shows a strong absorption in the ultraviolet (λmax = 277 nm), and reacts with common protein reagents. Purified monellin is free of carbohydrate (< 5 βg/mg protein). This evidence indicates that monellin is a protein.

From an aqueous extract of the fruit of the tropical plant Thaumatococcus daniellii Benth, two sweet-tasting basic proteins, here named thaumatin I and thaumatin II, were isolated by ultrafiltration, gel nitration on Sephadex G-50 and ion-exchange chromatography on SE-Sephadex C-25, using a sodium chloride concentration gradient. The proteinaceous character of the two sweet-tasting compounds was proven by the characteristic ultraviolet absorption spectrum, the presence of almost 100% polypeptide material as determined by the biuret method, the yield of 100% amino acids on hydrolysis with hydrochlorid acid, the positive reaction with amido black colouring agent (as used in the analysis of the fractions by polyacrylamide gel and starch gel electrophoresis) and by the disappearance of the sweet taste of the compounds after incubation with trypsin. The basic character of the two proteins appears from their isoelectric points of almost 12, as estimated by starch gel electrophoresis at different pH values, and from the precipitation of the proteins at pH 12. The molecules of thaumatin I and II are very similar as shown from their amino acid compositions, which are practically equal; they also have the same N-terminal amino acid alanine and molecular weights of 21000 ± 600 and 20400 ± 600, respectively (calculated from ultracentrifugal data). The proteins lose their sweetness on heating, on splitting of the disulphide bridges in the molecule and at pH values below 2.5. Thaumatin I and II have an intensely sweet taste, the degree of sweetness being about 1600 times higher than that of sucrose on a Weight basis or 105 times on a molar basis. The threshold values are near 10−4% or 48 nM.

The prevalence of obesity and diabetes has increased exponentially in recent years around the globe, especially in India. Sweet proteins have the potential to substitute the sugars, by acting as natural, good and low calorie sweeteners. They also do not trigger a demand for insulin in diabetic patients unlike sucrose. In humans, the sweet taste perception is mainly due to taste-specific G protein-coupled heterodimeric receptors T1R2-T1R3. These receptors recognize diverse natural and synthetic sweeteners such as monelin, brazzein, thaumatin, curculin, mabinlin, miraculin and pentadin. Structural modeling of new sweetener proteins will be a great leap in further advancement of knowledge and their utility as sweeteners. We have explored the fingerprints of sweetness by studying the aminoacid composition and structure properties of the above proteins. The structural analysis of monellin revealed that the individual A or B chains of monellin are not contributing to its sweetness. However, the native conformation and ionic interaction between AspB7 of monellin with active site of T1R2-T1R3 receptor, along with hydrogen bonding stability of IleB6 and IleB8 are responsible for the sweet taste. Based on structural similarity search, we found a new hypothetical protein from Shewanella loihica, which has the presence of Asp(32) with adjacent isoleucine residues. Further, we examined the lead protein by two-step docking for the study of interaction of functionally conserved residues with receptors. The identified protein showed similar ionic and hydrophobic interactions with monelin. This gives a promising opportunity to explore this protein for potential health application in the low calorie sweetener industry viz., soft drinks, snacks, food, chocolate industries etc.