Arquivo Técnico

Sweet proteins Sweet proteinS – potentiaL repLaCement for artifiCiaL Low CaLorie SweetenerS

Exponential growth in the number of patients suffering from diseases caused by the consumption of sugar has become a threat to mankind’s health. Artificial low calorie sweeteners available in the market may have severe side effects. It takes time to figure out the long term side effects and by the time these are established, they are replaced by a new low calorie sweetener. Saccharine has been used for centuries to sweeten foods and beverages without calories or carbohydrate. It was also used on a large scale during the sugar shortage of the two world wars but was abandoned as soon as it was linked with development of bladder cancer. Naturally occurring sweet and taste modifying proteins are being seen as potential replacements for the currently available artificial low calorie sweeteners. Interaction aspects of sweet proteins and the human sweet taste receptor are being investigated.

16 FOOD INGREDIENTS BRASIL Nº 8 - 2009 www.revista-fi .com Sweet and taste modifying T1R class [8-10] of taste-specific tions of these proteins are the low calo- proteins proteins hypothesized to function rie sweetener industry and the cola, in combination as a heterodimer. snacks, food and chocolate industries. The prevalence of obesity and The human T1R2-T1R3 receptor diabetes has increased dramatically recognizes natural and synthetic Brazzein in recent years in the United States, sweetness and T1R1-T1R3 rec- Brazzein is the smallest, most with similar patterns seen in sev- ognizes umami taste [11,12]. So heat-stable [13] and pH-stable eral other countries including India far there are seven known sweet member of the set of proteins known [1] as well. Diabetes mellitus is a and taste-modifying proteins, to have intrinsic sweetness. The chronic disease caused by inherited namely Brazzein [13], Thaumatin protein, consisting of 54 amino acid or acquired deficiency in production [14], Monelin [15], Curculin [16], residues, is reported to be between of insulin by the pancreas or by the Mabinlin [17], Miraculin [18] and 500 and 2000 times sweeter than ineffectiveness of the insulin pro- Pentadin [19]. Properties and sucrose [22] and represents an ex- duced [2]. Artificial sweeteners like characteristics of these proteins cellent alternative to available low Saccharin, Aspartame, Cyclamate are illustrated in Table 1. calorie sweeteners. It was originally and AcesulfameK are used world- The key residues on the protein isolated from the fruit of an African wide as low calorie sweeteners by surface responsible for biological plant Pentadiplandra brazzeana patients affected by diseases linked activity have not yet been identi- Baillon [23]. Heat and pH stability to the consumption of sugar, e.g. fied with certainty for any of these of the protein make it an ideal sys- diabetes, hyperlipemia, caries, obe- proteins [20]. Monellin was found tem for investigating the chemical sity, etc., but they have side effects to be 100000 times sweeter than su- and structural requirements of a such as psychological problems, crose on a molar basis [21], followed sweet-tasting protein. Based on the mental disorders, bladder cancer, by Brazzein and Thaumatin which wild-type brazzein, 25 mutants were heart failure and brain tumors [3-7]. are 500 times [13] and 3000 times produced to identify critical regions Sweet proteins have the potential sweeter then sucrose [14] respec- important for sweetness. To assess to replace these artificial sweeten- tively (the latter two on a weight their sweetness, psychophysical ex- ers, by acting as natural, good, low basis). All of these proteins have periments were carried out with 14 calorie sweeteners, as we know that been isolated from plants that grow human subjects. First, the results proteins do not trigger a demand for in tropical rainforests. Although suggest that residues 29–33 and insulin in these patients whereas most of them share no sequence 39–43, plus residue 36 between these sucrose does. homology or structural similarity, stretches, as well as the C-terminus In humans, the sweet taste is Thaumatin shares extensive homol- are involved in the sweetness [24]. mainly due to the recently discov- ogy with certain non-sweet proteins Second, charge plays an important ered T1R2-T1R3 receptor [8-10], found in other plants [15]. role in its interaction with the sweet two of the three members of the The potential industrial applica- taste receptor [24].

Table 1: Comparison of thaumatin, monellin, mabinlin, pentadin, brazzein, curculin and miraculin.

Thaumatin Monellin Mabinlin Pentadin Brazzein Curculin Miraculin

Pentadiplandra Pentadiplandra Thaumatococcus Dioscoreophyllum Capparis Curculingo l Richadella Source brazzeana brazzeana danielli Benth cumminsii Diels masakai Levl atifolia dulcifica Baillon Baillon Geographic West Africa West Africa China West Africa West Africa Malaysia West Africa distribution

Variants I, II, a, b, cª –— I, II - a, III, IVª -–— –— –— –—

Sweetness factor 3000 3000 100 500 2000 550 –— (weight basis) Molecular mass 22.2 10.7 12.4 12.0 6.5 24.9 98.4 (active form, kDa) b 45 (A chain) 33 (A chain) Amino acids 207 ? 54 114 191 50 (B chain) 72 (B chain) Dimer ( Tetramer Active form Monomer Dimer (A + B) Dimer (A + B) ? Monomer A +A) (A+A+A+A) Source: Adapted from Kurihara (1994). ªAt least five different forms of thaumatin (Lee et al., 1988) and four different forms of mabinlin (Nirasawa et al., 1994) have been identified.b A chromatographic fraction containing a 12-kDa protein was sweet. This same fraction, when subjected to electrophoresis under non-reducing condi- tions showed bands in the region between 22 and 41 kDa, suggesting the presence of subunits. www.revista-fi.com FOOD INGREDIENTS BRASIL Nº 8 - 2009 17 Sweet proteins

tHaUmatin may cause separation of the sub- sional model has been built from units and denaturation of the pro- the X-ray coordinates of GNA, a The thaumatins are a class of tein. Despite misgivings about the mannose-binding lectin from snow- intensely sweet proteins isolated stability of the protein to heat and drop (Galanthus nivalis) [38]. The from the fruit of the tropical plant acid, downstream processes have three mannosebinding sites pres- Thaumatococcus danielli. The protein been established. Its D-enantiomer ent in GNA were found in curculin crystallizes in a hexagonal lattice after has been crystallized and analyzed but were not functional. Some well a temperature shift from 293 to 277 K. by X-ray crystallography at 1.8 Å exposed regions on the surface of The structure has been solved at 1.6 resolution. Two crystal forms (I the three-dimensional model of the Å resolution. Its fold was found to be and II) were found under crystal- said protein could act as epitopes identical to that found in three other lization conditions similar, but not responsible for the sweet-tasting crystal forms grown in the presence identical, to the crystallization properties of the protein [40]. The of crystallizing agents of differing conditions of natural L-monellin protein can be crystallized by the chemical natures [25]. It consists of [34]. One NMR study of a non- vapor diffusion method using poly- 207 amino acid residues with eight sweet analog in which the AspB7 ethylene glycol 400 as a precipitant. intramolecular disulfide bonds and of protein was replaced by AbuB7 The crystals belong to orthorhombic contains no free cysteine residues. (L-2-Aminobutylicacid), showed space group P2(1)2(1)2(1) with unit It aggregates upon heating at Ph 7.0 similar 3-dimensional structures of cell dimensions: a = 105 Å, b = 271 above 70 degrees C, whereupon its these two proteins, indicating that Å, c = 48.7 Å. The crystals diffract sweetness disappears [26,27]. The the lack of the beta-carboxyl group X-rays to resolution of 3.0 Å and are protein is approximately 10000 times in the AbuB7 analog is responsible suitable for Xray crystallographic sweeter than sugar on a molar basis for the loss of sweetness [35]. Recent studies [41]. [28]. It is a protein that tastes intense- research on identifying binding sites ly sweet only to Old World monkeys on the receptor by means of struc- maBinLin and to higher primates, including ture-taste relationships, found that Mabinlin is a sweet protein with man [29], as it has been found that four monellin analogues, [AsnA16]-, the highest known thermostablil- the protein binds to certain elements [AsnA22]-, [GlnA25]-, and [AsnA26]- ity [42]. It is derived from Capparis in taste pores of Rhesus monkey foli- monellin were 7500, 750, 2500, and masaikai and its sweetness was es- ate papillae [30]. Thaumatin has been 5500 times as sweet as sucrose on timated to be around 400 times that approved for use in many countries a weight basis, respectively. Thus, of sucrose on weight basis. It consists as both a flavor enhancer and a high- among them, [AsnA22]-monellin and of an A chain with 33 amino acid intensity sweetener [31]. [GlnA25]-monellin were less sweet residues and a B chain composed of than the native monellin [36]. 72 residues. The B chain contains two moneLLin intramolecular disulfide bonds and is Monellin, a sweet protein, con- CUrCULin connected to the A chain through two sists of two noncovalently associated Curculin which is extracted from intermolecular disulfide bridges [43]. polypeptide chains, an A chain of 44 Curculigo latifolia acts as a good Its heat stability is due to the pres- amino acid residues and a B chain low calorie sweetener. Its maximum ence of these four disulfide bridges of 50 amino acid residues [32]. The sweetness is equal to 0.35 M of su- [44]. The sweetness of Mabinlin-2 is protein can be purified from the fruit crose. It has taste modifying abili- unchanged after 48 hr incubation at of Dioscoreophyllum cumminsii ties since water and sour substances boiling point [17]and of Mabinlin-3 grown in West Africa and is approxi- elicit a sweet taste after consumption and -4 are unchanged after 1 hr at mately 100,000 times sweeter than of curculin [37]. There is no other 80°C [45]. sugar on a molar basis and several protein currently available with both thousand times sweeter on a weight sweet taste and taste modifying miraCULin basis [28]. Single-chain monellin abilities [38]. The taste modifying Miraculin is a taste-modifying (SCM), which is an engineered activity of the protein (discussed protein that belongs to the class of 94-residue polypeptide, has proven below) remains unchanged when it sweet proteins. It is extracted from to be as sweet as native two-chain is incubated at 50°C for 1 hr between Richadella dulcifica an evergreen monellin, and is more stable than the pH 3 and 11 [39]. shrub native of West Africa. The native monellin at high temperature The molecular weight of Cur- proteinis a single polypeptide with and in acidic environments [33]. Na- culin was determined by low angle 191 amino acid residues [46]. It tive monellin is relatively sensitive laser light scattering and was found modifies the sweet receptor in such to heat or acid treatment, which to be 27800 [38]. Its three-dimen- a way that it can be stimulated by

18 FOOD INGREDIENTS BRASIL Nº 8 - 2009 www.revista-fi .com acid [47]. Thus, miraculin has the interaCtion of Sweet pro- al. [52] solving the crystal structure unusual property of modifying sour teinS witH tHeir reCeptor of the N-terminal active site region taste into sweet taste [46]. of the subtype 1 of mGluR both free Taste-modifying protein modifies Humans detect taste with taste and complexed with glutamate has the sweet taste receptor on binding receptor cells. These are clustered helped a lot in understanding the and this behavior of these proteins is in taste buds. Each taste bud has a mechanism of interaction between responsible for modification in taste pore that opens out to the surface of ligand and T1R2-T1R3 receptor. of sour substance [46,47]. All acids the tongue enabling molecules and Their structural work on mGluR and (which are normally sour) taste sweet ions taken into the mouth to reach its N-terminal domain [52,53] show- after consumption of these proteins. the receptor cells inside. There are ing considerable conformational The effects of these proteins exist for five primary taste sensations salty, change induced by the glutamate around half an hour after consump- sour, sweet, bitter and umami. Sweet complexation. The ‘Active’ and ‘rest- tion and intake of any sour substance and umami (the taste of monosodium ing’ conformations of m1-LBR, an will therefore taste sweet during this glutamate) are the main pleasant extracellular ligand binding region period of time. The taste buds come to tastes in humans. T1Rs are mam- of mGluR, is modulated by the dimer there normal state with time. malian taste receptors that assemble interface. The protomer can form two heteromeric G-protein-coupled ‘open’ or ‘closed’ confirmations and is pentadin receptor complexes T1R1+T1R3, made up of two domains namely LB1 Pentadin is a sweet protein ex- an umami sensor, and T1R2+T1R3, and LB2. The population of active tracted from the plant Pentadip- a sweet receptor [50]. conformers depends on ligand bind- landra brazzeana, a shrub found Sweet and taste-modifying pro- ing, i.e. the so called ‘closed-open_A’. in tropical forests of a few African teins interact with the T1R2-T1R3 The ligandfree receptor exists as countries. Not much information is receptor with a different mechanism two different structures, free form available about the protein despite compared to small molecular weight I (open-open_R), the ‘resting’ con- its isolation several years ago, in compounds [51]. Recently, it has been formation with two open protomers 1989 [48]. The protein was reported shown that the T1R2-T1R3 receptor and free form II (closed-open_A), to be around 500 times sweeter then has many characteristics similar to the nearly identical to the complexed sucrose on a weight basis. It also con- mGluR [52], apart from some minor form (Figure 1, references 52, 54). sists of subunits coupled by disulfide differences in the active site region. The mechanism suggested by bonds [49]. The major work by Kunishima et these structures is that the receptor is in dynamic equilibrium, and that figUre 1 ligand binding stabilizes the ‘active’ dimer. There are thus two ways, in principle, to activate the receptor: Free form I Free form II first, to complexate form I with the Opem-open_R Closed-open_A proper ligand (glutamate for the (resting state) (resting state) mGluR, aspartame or any other small molecular weight sweetener for the T1R2-T1R3 receptor) and second, +Sweet Protein by shift the equilibrium between free (-Sweeterner) form I and free form II in favor of free form II. The exact mechanism of interac- tion of sweet proteins with the T1R2- T1R3 sweet taste receptor has not yet been elucidated [51]. Low molecular mass sweeteners and sweet proteins interact with the same receptor, the human T1R2-T1R3 receptor[52]. Studies have shown that the T1R3 Diagrammatic representation of the T1R2-T1R3 receptor showing possible sta- bilization by binding of a sweet protein to a secondary binding site on the surface receptor protein is encoded by the of free form II. The sweet protein is represented in red on the left part of free form Tas1r3 gene involved in transduction II, preventing it reverting to free form I. (adapted from references 52 and 54). of sweet taste [55]. Recently it has been found that www.revista-fi .com FOOD INGREDIENTS BRASIL Nº 8 - 2009 19 Sweet proteins

T1R3-independent sweetand umami- sweeter than sucrose and are of low taste receptor. Also mutations can be responsive receptors and/or path- calorie value, these proteins can be induced in candidate sweet proteins ways also exist in taste cells [56]. used as natural low calorie sweeten- to analyze changes in their physical, ers by people suffering from diseases chemical and biological properties. The ConCLUSion and SCope of linked to consumption of sugar e.g. work can be taken forward by solving fUrtHer worK obesity, diabetes and hyperlipemia. the structures of the proteins and taste As it has been found that sweet Candidate proteins can be checked receptors with a view to increasing the proteins are thousands of times for biological activity with the human efficiency of these sweeteners.

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