Structure-sweetness Relationship Research Articles

Study on the Thermal Stability of the Sweet-Tasting Protein Brazzein Based on Its Structure-Sweetness Relationship.

Brazzein (Brz) is a sweet-tasting protein composed of 54 amino acids and is considered as a potential sugar substitute. The current methods for obtaining brazzein are complicated, and limited information is available regarding its thermal stability. In this study, we successfully expressed recombinant brazzein, achieving a sweetness threshold of 15.2 μg/mL. Subsequently, we conducted heat treatments at temperatures of 80, 90, 95, and 100 °C for a duration of 2 h to investigate the structural changes in the protein. Furthermore, we employed hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to analyze the effect of heating on the protein structure-sweetness relationships. Our results indicated that the thermal inactivation process primarily affects residues 6-14 and 36-45 of brazzein, especially key residues Tyr8, Tyr11, Ser14, Glu36, and Arg43, which are closely associated with its sweetness. These findings have significant implications for improving the thermal stability of brazzein.

Structure-Sweetness Relationship of Sweet Proteins: A Systematic Review on “Sweet Protein” Studies as a Sub-Group of “Sweetener” Investigations

Structure-Sweetness Relationship of Sweet Proteins: A Systematic Review on “Sweet Protein” Studies as a Sub-Group of “Sweetener” Investigations

甘味タンパク質の構造と機能に関する研究

甘味タンパク質の構造と機能に関する研究

ChemInform Abstract: Stevioside and Related Compounds — Molecules of Pharmaceutical Promise: A Critical Overview

AbstractReview: 117 refs.

Stevioside and Related Compounds – Molecules of Pharmaceutical Promise: A Critical Overview

Stevioside, an ent-kaurene type of diterpenoid glycoside, is a natural sweetener extracted from leaves of Stevia rebaudiana (Bertoni) Bertoni. Stevioside and a few related compounds are regarded as the most common active principles of the plant. Such phytochemicals have not only been established as non-caloric sweeteners, but reported to exhibit some other pharmacological activities also. In this article, natural distribution of stevioside and related compounds, their structural features, plausible biosynthetic pathways along with an insight into the structure-sweetness relationship are presented. Besides, the pharmacokinetics, wide-range of pharmacological potentials, safety evaluation and clinical trials of these ent-kaurene glycosides are revisited.

Developments in biotechnological production of sweet proteins

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.

Structure–Sweetness Relationship in Egg White Lysozyme: Role of Lysine and Arginine Residues on the Elicitation of Lysozyme Sweetness

Lysozyme is one of the sweet-tasting proteins. To clarify the structure-sweetness relationship and the basicity-sweetness relationship in lysozyme, we have generated lysozyme mutants with Pichia systems. Alanine substitution of lysine residues demonstrated that two out of six lysine residues, Lys13 and Lys96, are required for lysozyme sweetness, while the remaining four lysine residues do not play a significant role in the perception of sweetness. Arginine substitution of lysine residues revealed that the basicity, but not the shape, of the side chain plays a significant role in sweetness. Single alanine substitutions of arginine residues showed that three arginine residues, Arg14, Arg21, and Arg73, play significant roles in lysozyme sweetness, whereas Arg45, Arg68, Arg125 and chemical modification by 1,2-cyclohexanedione did not affect sweetness. From investigation of the charge-specific mutations, we found that the basicity of a broad surface region formed by five positively charged residues, Lys13, Lys96, Arg14, Arg21, and Arg73, is required for lysozyme sweetness. Differences in the threshold values among sweet-tasting proteins might be caused by the broadness and/or the density of charged residues on the protein surface.

Structure-sweetness relationship in thaumatin: importance of lysine residues.

To clarify the structural basis for the sweetness of thaumatin I, lysine-modified derivatives and carboxyl-group-modified derivatives were prepared by chemical modification followed by chromatographic purification. The sweetness of derivatives was evaluated by sensory analysis. Phosphopyridoxylation of lysine residues Lys78, Lys97, Lys106, Lys137 and Lys187 markedly reduced sweetness. The intensity of sweetness was returned to that of native thaumatin by dephosphorylation of these phosphopyridoxylated lysine residues except Lys106. Pyridoxamine modification of the carboxyl group of Asp21, Glu42, Asp60, Asp129 or Ala207 (C-terminal) did not markedly change sweetness. Analysis by far-UV circular dichroism spectroscopy indicated that the secondary structure of all derivatives remained unchanged, suggesting that the loss of sweetness was not a result of major disruption in protein structure. The five lysine residues, modification of which affected sweetness, are separate and spread over a broad surface region on one side of the thaumatin I molecule. These lysine residues exist in thaumatin, but not in non-sweet thaumatin-like proteins, suggesting that these lysine residues are required for sweetness. These lysine residues may play an important role in sweetness through a multipoint interaction with a putative thaumatin receptor.

Saponin and sapogenol. XLIX. On the constituents of the roots of Glycyrrhiza inflata Batalin from Xinjiang, China. Characterization of two sweet oleanane-type triterpene oligoglycosides, apioglycyrrhizin and araboglycyrrhizin.

Two sweet oleanane-type triterpene oligoglycosides named apioglycyrrhizin and araboglycyrrhizin were isolated from the air-dried roots of Glycyrrhiza inflata Batalin, collected in Xinjiang province (Shinkyo-Kanzo in Japanese), together with glycyrrhizin (3), licorice-saponins A3 (8), G2 (10), and H2 (11) and known flavonoid glycosides. On the basis of chemical and physicochemical evidence, the structures of apioglycyrrhizin and araboglycyrrhizin have been determined to be expressed as 3-O-[beta-D-apiofuranosyl(1-->2)-beta-D- glucuronopyranosyl]glycyrrhetic acid (1) and 3-O-[alpha-L- arabinopyranosyl(1-->2)-beta-D-glucuronopyranosyl]glycyrrhet ic acid (2), respectively. During the course of these studies, it has been found that the hydroxyl groups in the oligosaccharide moiety of the glucuronide saponins may be partially methylated by prolonged treatment with diazomethane in methanol. The sweetness of the saponins hitherto isolated from various Glycyrrhizae Radix has been examined and a structure-sweetness relationship, as compared with glycyrrhizin, has been found.

Isovanillyl sweeteners. Synthesis and sweet taste of sulfur heterocycles

As part of an investigation into the structure-sweetness relationship in isovanillyl sweeteners, 15 compounds containing sulfur atoms either in the heterocyclic or the isovanillyl ring have been synthesised and tasted. In general the replacement of oxygen by sulfur maintained or improved the sweetness potency when the heteroatom is in the heterocyclic ring, while the same change in the OH or OMe groups eliminated the sweetness. The most effective structure for generating sweetness is that with a sulfur atom occupying position 1 and an oxygen atom position 3 of a chain, in which the isovanillyl ring is linked to C-2. 2-(3-Hydroxy-4-methoxyphenyl)-4H-3, 1-benzoxathiane 10b(sweet potency 9000) is the sweetest isovanillyl sweetener synthesised until now.

Enzymic Production of Sweet Stevioside Derivatives: Transglucosylation by Glucosidases

For the purpose of improving sweetness and a further study on the structure-sweetness relationship of steviol glycosides, transglycosylation of stevioside by a variety of commercial glucosidases was investigated. It was revealed that two alpha-glucosidases gave glucosylated products. Transglucosylation of stevioside by Pullulanase and pullulan exclusively afforded three products, 13-O-[beta-maltotriosyl-(1----2)-beta-D-glucosyl]-19-O-beta-D-g luc osyl- steviol (1), 13-O-[beta-maltosyl-(1----2)-beta-D-glucosyl]-19-O-beta-D-glucosyl- steviol (2) and 13-O-beta-sophorosyl-19-O-beta-maltotriosyl-steviol (3). All of these products have already been obtained by trans-alpha-1,4-glucosylation of stevioside by the cyclodextrin glucanotransferase starch system, and 1 and 2 have been proven to be tasty and potent sweeteners. Transglucosylation of stevioside by Biozyme L and maltose afforded three new products, 4, 5 and 6, the structures of these compounds being elucidated as 13-O-beta-sophorosyl-19-O-beta-isomaltosyl-steviol (4), 13-O-[beta-isomaltosyl(1----2)-beta-D-glucosyl]-19-O-beta-D-glucosyl- steviol (5) and 13-O-[beta-nigerosyl-(1----2)-beta-D-glucosyl]-19-O-beta-D- glucosyl-steviol (6). A significantly high quality of taste was evaluated for 4.

Further study on the 1,4-.ALPHA.-transglucosylation of rubusoside, a sweet steviol-bisglucoside from Rubus suavissimus.

Rubusoside (the beta-D-glucosyl ester of 13-O-beta-D-glucosyl-steviol), which is the major sweet principle of leaves of Rubus suavissimus S. Lee, was subjected to 1,4-alpha-transglucosylation by the cyclodextringlucanotransferase-starch system (the CGTase system). The tri- and tetra-glucosylated products were isolated together with the mono- and di-glucosylated products, which had already been isolated. A prominent increase in intensity of the sweetness was observed for the compounds which were di- and tri-glucosylated at the 13-O-glucosyl moiety. This result further substantiated the structure-sweetness relationship for 1,4-alpha-glucosylated compounds of steviol-glycosides reported previously. For protection of the 19-COO-glucosyl moiety against glucosylation by the CGTase system, the 4-hydroxyl group of the 19-COO-glucosyl moiety was beta-galactosylated by the beta-galactosidase-lactose system. This galactosylated compound was subjected to a regio-selective glucosylation of the 13-O-glucosyl moiety by the CGTase system, which was followed by enzymic elimination of the galactosyl group to furnish an exclusive preparation of the improved sweeteners just mentioned.

Study on Improvement of Sweetness of Steviol Bisglycosides: Selective Enzymic Transglucosylation of the 13-O-Glycosyl Moiety

Based on studies on the acceptor specificity of the transglucosylation with cyclodextringlucosyl-transferase (CGT-ase) as well as on the structure-sweetness relationship of steviol glycosides, selective elongation of the 13-O-glucosyl moiety of steviol bisglycosides was done to improve sweetness. The d-glucosyl ester group at the 19-carboxylic acid of rubusoside and stevioside was chemically replaced by β-d-galactosyl group as a blocker against the glucosylation and both the galactosyl esters were regioselectively 1,4-a-glucosylated on the 13-glycosyl moiety with CGT-ase using soluble starch as a donor. Great improvement in sweetness was observed for the products which have three or four glucosyl units at the 13-position, while the products with five or six glucosyl units had less sweetness than the starting materials. It was also revealed that the sweetness of the products of the stevioside series were better than the corresponding isomers of the rubusoside series.

Study on improvement of sweetness of steviol bisglycosides: Selective enzymic transglucosylation of the 13-O-glycosyl moiety.

Based on studies on the acceptor specificity of the transglucosylation with cyclodextringlucosyl-transferase (CGT-ase) as well as on the structure-sweetness relationship of steviol glycosides, selective elongation of the 13-O-glucosyl moiety of steviol bisglycosides was done to improve sweetness. The d-glucosyl ester group at the 19-carboxylic acid of rubusoside and stevioside was chemically replaced by β-d-galactosyl group as a blocker against the glucosylation and both the galactosyl esters were regioselectively 1,4-a-glucosylated on the 13-glycosyl moiety with CGT-ase using soluble starch as a donor. Great improvement in sweetness was observed for the products which have three or four glucosyl units at the 13-position, while the products with five or six glucosyl units had less sweetness than the starting materials. It was also revealed that the sweetness of the products of the stevioside series were better than the corresponding isomers of the rubusoside series.

Structure–sweetness relationships for fructose analogs. Part II. Synthesis and properties of 1,6-dihydroxy-2-hexanone, a simple analog of the ketohexoses, and X-ray structure determination of the corresponding dianhydrides

Labile 1,6-dihydroxy-2-hexanone (4) was prepared from the corresponding triol by selective di-O-tritylation, Collins oxidation, and careful detritylation of the resulting keto compound 3 under mild, acidic conditions. As shown by its 1Hmr and 13Cmr spectra, 4 exists as a 3:2 mixture of open-chain and cyclic forms, respectively, at equilibrium in deuterium oxide (25 °C). Upon standing, 4 is slowly converted into a hemiacetalic dimer. Under acidic conditions, dehydrative dimerization takes place to give the corresponding tricyclic dianhydride, one or both of the possible isomers (1:1 mixture) of which being formed according to the presence or absence of water in the medium. The structure (trans) of the thermodynamic isomer 5 was firmly established by an X-ray crystallographic analysis.

Enzymic Transglucosylation of Rubusoside and the Structure-Sweetness Relationship of Steviol-Bisglycosides

Rubusoside, the β-D-glucosyl ester of 13-O-D-glucosyl-steviol which was isolated from leaves of Rubus suavissimus collected in China as the major sweet principle (yield: 5.4%), was subjected to α-1 4 transglucosylation with the cyclodextrin glucosyltransferase produced by Bacillus megaterium Strain No. 5 using soluble starch as a donor. A significant improvement in the quality of sweetness was observed for the crude reaction mixture, which was separated into mono-, di-, tri-, tetra-, penta-, and hexa-glucosylated products. All isomers of the mono- and di-glucosylated products were further separated. Evaluation of the sweetness of these products compared with stevioside, rebaudioside A, etc. disclosed that the ratio of the number of glucose units at the 13-hydroxyl group to that at 19-carboxyl group seems to have a significant relationship with the sweetness as well as the quality of taste for glucosides of this type.

Enzymic transglucosylation of rubusoside and the structure-sweetness relationship of steviol-bisglycosides.

Rubusoside, the β-D-glucosyl ester of 13-O-D-glucosyl-steviol which was isolated from leaves of Rubus suavissimus collected in China as the major sweet principle (yield: 5.4%), was subjected to α-1 4 transglucosylation with the cyclodextrin glucosyltransferase produced by Bacillus megaterium Strain No. 5 using soluble starch as a donor. A significant improvement in the quality of sweetness was observed for the crude reaction mixture, which was separated into mono-, di-, tri-, tetra-, penta-, and hexa-glucosylated products. All isomers of the mono- and di-glucosylated products were further separated. Evaluation of the sweetness of these products compared with stevioside, rebaudioside A, etc. disclosed that the ratio of the number of glucose units at the 13-hydroxyl group to that at 19-carboxyl group seems to have a significant relationship with the sweetness as well as the quality of taste for glucosides of this type.

Chemical structure and sweet taste of isocoumarins and related compounds. X. Syntheses of sweet 5-hydroxyflavanones and related dihydrochalcones.

On the basis of the information from our previous studies on structure-sweetness relationships of phyllodulcin and related 3, 4-dihydroisocoumarins, syntheses of sweet flavanones and dihydrochalcones are designed. 5-Hydroxyflavanones, VII, IX, and X, and dihydrochlacone XIII has a sweet taste and the others are tasteless. The structuresweetness relationships of these compounds are discussed. It is considered that the mode of interaction of 5-hydroxyflavanones and Dihydrochalcone (DHC) lacking glycoside group with a sweet receptor seems to be similar to that of 3, 4-dihydroisocoumarins.