KR102058483B1 - Method for preparing novel multi-mutated Brazzein having higher sweetness - Google Patents

Abstract

The present invention relates to a novel brazein multiple variant with increased sweetness, a nucleic acid molecule encoding said variant, a recombinant vector comprising said nucleic acid molecule, a host cell transformed with said recombinant vector, and a method of producing said variant. . Brazein A1 ins. Of the present invention. Multiple variants are about 29.96 million times more sweet than comparable amounts of sucrose (sugar). Compared with the wild type brazein and the conventionally developed brazein variants, the increase in sweet taste is excellent. Thus, the brazein variants of the present invention can be used in very small amounts to achieve the desired sweetness and can be used in place of other sweeteners, including sugar, in food.

Description

Method for preparing novel multi-mutated Brazzein having higher sweetness}

The present invention relates to a novel brazein multiple variant having a high sweet taste and a method for preparing the same.

Refined sugar added to foods and beverages has no nutritional value and only contributes to increased energy intake. This excessive consumption of sugar has led to global obesity rates. Therefore, as one of the methods to solve the problem of obesity, it has been devised to replace the sugar with high-intensity sweeteners to reduce energy intake. Alternative sweeteners currently used to replace sugar can be broadly divided into artificial sweeteners including aspartame and saccharin, and natural sweeteners including sweet proteins, sugar alcohols and oligosaccharides. Among them, sweet protein is a natural substance derived from the fruit of the plant, and its types include taumatin, monellin, mabinlin, curculin, miraculin and brazein. (brazzein) and the like. Sweet protein has attracted attention as a substitute for synthetic artificial sweeteners because it has a relatively low calorie and has a much sweeter taste than sugar.

As a ligand for sweet receptors, brazein is a sweet protein consisting of 54 amino acid residues, the smallest sweet protein known to date, and is known to have a sugar-like sweetness. Wild-type brazein can be classified into major form and minor form based on the presence of pyro-glutamate at the N-terminus. The main form with pyro-glutamate at the N-terminus (pyrE) accounts for 80% of the wild type. The other 20% of the minor forms lack pyro-glutamate at the N-terminus (des-pyrE) and are twice as sweet as the main forms. Brazein has three antiparallel beta chains (strand I, residues 5-7; strand II, residues 44-50; strand III, residues 34-39) and one small alpha helix (residues 21-29). These three beta chains are linked to a central hydrogen bond and four disulfide bonds to maintain a stable structure (FIG. 1). Because of this structural stability, brazein is stable over a wide range of temperatures and pH compared to other sweet proteins.

While maintaining such structural stability, the present inventors are continuously studying the variants exhibiting higher sweetness than wild type brazein (Patent Documents 1 to 3).

Korean Patent Application No. 2007-0117013 Korean Patent Application No. 2008-0019008 Korean Patent Application No. 2010-0016660

The present inventors have tried to develop a new type of brazein variant protein with increased sweetness as compared to the conventional wild type brazein. As a result, the present inventors have found that the multivariate having amino acid mutations at four different sites of the wild type brazein at the same time. The present invention was completed successfully by experimentally confirming that this variant has a much higher sweetness than wild type brazein and previously developed variants.

Accordingly, it is an object of the present invention to provide multiple variants of Brazein with increased sweetness.

Another object of the present invention is to provide a nucleic acid molecule encoding the brazein multiple variants.

Still another object of the present invention is to provide a recombinant vector comprising the nucleic acid molecule.

Another object of the present invention to provide a host cell transformed with the recombinant vector.

It is another object of the present invention to provide a method for producing the brazein multiple variants.

Still another object of the present invention is to provide a sugar composition for improving sugar content comprising the multiple variants of brazein as an active ingredient.

The objects and advantages of the invention will become apparent from the following detailed description, claims and drawings.

The present invention relates to a brazein multiple variant [A1 ins_H30R_E35D_E40A] having an amino acid sequence represented by SEQ ID NO: 5.

In the brazein multiple variant of the present invention, the histidine residue, which is the 30th amino acid of the brazein tertiary variant H30R_E35D_E40A (SEQ ID NO: 1) having the amino acid sequence of SEQ ID NO: 3, is an arginine residue. Brazein tertiary variant wherein the 35th amino acid glutamic acid residue is replaced with an aspartic acid residue (SEQ ID NO: 3) and the 40th glutamic acid residue is replaced with an alanine residue. 981087, which is a multiple variant having an alanine inserted in the first position, is represented by SEQ ID NO: 5.

In particular, the brazein multiple variant proteins of the present invention have an improved sweetness of about 2038 times compared to wild type brazein.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the brazein multiple variants.

As used herein, the term “nucleic acid molecule” is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides, which are the basic structural units in nucleic acid molecules, are modified from sugar or base sites, as well as natural nucleotides. Analogues (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

According to a preferred embodiment of the present invention, the nucleic acid molecule has a nucleotide sequence represented by SEQ ID NO: 6.

The invention also includes a recombinant vector comprising (i) a promoter and (ii) said brazein multiple variant coding nucleic acid molecule operably linked with said promoter.

According to a preferred embodiment of the present invention, the nucleic acid molecule encoding the brazein multiple variant in the recombinant vector is Kluyveromyces lactis ( Kluyveromyces) lactis ) can be linked to a nucleic acid molecule encoding an alpha-mating signal sequence.

Kluyveromyces the Kluyveromyces lactis ) alpha-mating signal sequence may have a nucleotide sequence of SEQ ID NO: 7. The alpha-mating sequence is linked to have the same frame upon translation into a protein on the 5 'top of the brazein nucleotide sequence of the present invention.

In addition, the recombinant expression vector may further comprise an effector gene.

In the present invention, a "recombinant expression vector" is a vector capable of transcription of a target protein or a target RNA in a host cell, and refers to a gene construct including an essential regulatory element operably linked to express a gene insert. Say.

The term "promoter" means a DNA sequence that regulates the expression of a protein coding sequence or functional RNA.

The 'operator gene' refers to a DNA sequence that controls the expression of a nucleic acid sequence operably linked in a particular host cell, 'operably linked' means that one nucleic acid fragment is combined with another nucleic acid fragment The function or expression thereof is influenced by other nucleic acid fragments. In addition, it may further comprise any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation. The effector gene may be a promoter (constitutive promoter) to induce the expression of the target gene at all times at all times or a promoter (inducible promoter) to induce the expression of the target gene at a specific position, time, for example, yeast LAC Promoter, GAL promoter (Rosaura Rodicio et al ., Microbiology 152 (2006), 2635-2649), KlADH4 promoter (Michele Saliola et al ., Appl Environ Microbiol. 1999 January; 65 (1): 53-60), maltase / maltose permease bi-directional promoter (US Pat. No. 6,596,513), PGK1 promoter (V. Melvydas et al ., BIOLOGIJA, 4: 1-4, 2006). In addition, all of the promoters disclosed in US Pat. No. 227,326 and the like can be used. Preferably, the promoter may use a LAC4 promoter.

The recombinant expression vector for expression of the brazein variant may be pKLAC2-brazein variant, pKLAC2-brazein variant :: LAC4, but is not limited thereto. In addition, the recombinant expression vector containing the PDI gene derived from Kluyveromyces lactis may be pKLAC2-PDI.

The present invention also provides a host cell transformed with the recombinant expression vector. In the present invention, as the host cell, Kluyveromyces lactis , a kind of yeast, was used. The yeast is transformed with the recombinant expression vector according to a conventional transformation method, wherein the transformation includes any method for introducing a nucleic acid into a host cell, a standard technique suitable for the host cell as known in the art This can be done by selecting. Such methods include electroporation, lithium acetate (LiCH ₃ COO), lithium chloride (LiCl), PEG-mediated fusion, and carrier-DNA mediated. fusion), and the like.

According to another aspect of the present invention, the present invention provides a method for producing a brazein multiple variant comprising the steps of: (a) a host cell transformed with a recombinant vector expressing the brazein multiple variant described above Culturing; And (b) isolating brazein multiple variant proteins from the cultured host cell.

Host cells transformed with the vector expressing the brazein multiple variant of the present invention are cultured under suitable culture conditions using an appropriate medium capable of inducing the expression of the brazein multiple variant. Media and culture conditions for host cell culture are known to those skilled in the art, and those skilled in the art can modify and use known media and culture conditions as appropriate for the present invention.

On the other hand, the present invention includes a method for mass production of brazein comprising the step of culturing Kluyveromyces lactis transformed with the recombinant expression vector ( Kluyveromyces lactis ).

The expression of brazein in this invention is made by compounds that promote the expression of conventional inducible promoters, such as lactose, galactose, glucose, starch. The brazein containing the expressed alpha-Mating signal sequence is transferred to the yeast endoplasmic reticulum by the signal sequence, and the signal peptidase and Kex peptidase of the yeast are transferred to the yeast endoplasmic reticulum. This removes the signal sequence and synthesizes brazein. Thus, the yeast transformed to contain the brazein expression recombinant expression vector of the present invention can be cultured under appropriate media and conditions such that the polynucleotides encoding brazein are expressed, which is 0.5 to 5% yeast extract as a nitrogen source. , Peptone is used alone or in a mixture of 0.5 to 5%, one selected from the group consisting of 1-4% galactose, 1-4% glucose, 1-4% lactose and 1-4% starch as a carbon source and expression inducer The above can be used. In particular, it is more preferable to culture in a medium containing 0.5 to 5% of yeast extract, 0.5 to 5% of peptone and 1 to 4% of galactose.

According to another aspect of the present invention, the present invention provides a food composition for enhancing sugar content, comprising the multiple variants of brazein as an active ingredient.

The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art. For example, beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruit, canned foods, jams, marmalade, etc.), fish, meat and processed foods (e.g. ham, sausage cornbeans, etc.) , Breads and noodles (e.g. udon, soba, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, malts, dairy products (e.g. butter, cheese, etc.), edible vegetable oils, margarine, vegetable protein, retort food , Frozen foods, various seasonings (e.g. miso, soy sauce, sauce, etc.).

In order to use the food composition containing the brazein multiple variant of the present invention in the form of a food additive, it may be prepared and used in powder or concentrate form.

The brazein multiple variant of the present invention in the food composition of the present invention may be included in the content range of 0.01 to 10% by weight relative to the total composition weight.

The present invention provides a novel braze multiple variant with increased sweetness, a nucleic acid molecule encoding said variant, a recombinant vector comprising said nucleic acid molecule, a host cell transformed with said recombinant vector, a method of producing said variant and said variant It relates to a food composition for enhancing sugar content as an active ingredient. The brazein multiple variants of the invention are about 2 million times more sweet than comparable amounts of sucrose (sugar). Compared with the wild type brazein and the conventionally developed brazein variants, the increase in sweet taste is excellent. Thus, the brazein variants of the present invention can be used in very small amounts to achieve the desired sweetness and can be used in place of other sweeteners, including sugar, in food.

Figure 1 shows the structure of wild type brazein (four intramolecular disulfide bonds visualized in yellow).
2 shows a test sheet used for a SIAM yes-no task.
Figure 3 shows the sequence of the brazein variants.
Figure 4 shows the electrophoresis of purified brazein multivariate [Lane M, Polypeptide SDS-PAGE molecular weight marker; Lane 1, des-pE1M (des-pE1MBRZ); Lane 2, H30R / E35D / E40A (3M); Lane 3, A1 ins / H30R / E35D / E40A (3M-A1 ins)].
FIG. 5 shows homology based modeled extracellular domain structure of T1R2 and T1R3 subunits ((a) extracellular domain of T1R2 subunit, (b) extracellular domain of T1R3 subunit).
Figure 6 is a model of homology-based brazein multivariate [(a) des-pE1M brazein; (b) H30R / E35D / E40A; (c) A1 ins / H30R / E35D / E40A].
FIG. 7 shows the seven best docking directions found in brazein des-pE1M for the T1R2-T1R3 heterodimeric complex [all structures are represented by backbone ribbons. Orange: T1R2 subunit; Blue: T1R3 subunit; Green, brazein, (a) complex model # 1 of brazein des-pE1M, (b) complex model # 2 of brazein des-pE1M, (c) complex model # 3 of brazein des-pE1M, (d) Complex model # 4 of brazein des-pE1M, (e) Complex model # 5 of brazein des-pE1M, (f) Complex model # 6 of brazein des-pE1M, (g) Complex model of brazein des-pE1M # 7].
FIG. 8 shows the seven best docking directions found in Brazein H30R / E35D / E40A for the T1R2-T1R3 heterodimeric complex [all structures are represented by backbone ribbons. Orange: T1R2 subunit; Blue: T1R3 subunit; Green, brazein, (a) complex model # 1 of brazein H30R / E35D / E40A, (b) complex model # 2 of brazein H30R / E35D / E40A, (c) complex model of brazein H30R / E35D / E40A # 3, (d) complex model # 4 of brazein H30R / E35D / E40A, (e) complex model # 5 of brazein H30R / E35D / E40A, (f) complex model # 6 of brazein H30R / E35D / E40A , (g) complex model # 7 of brazein H30R / E35D / E40A].
FIG. 9 shows the seven best docking directions found in Brazein A1 ins / H30R / E35D / E40A for the T1R2-T1R3 heterodimeric complex [all structures are represented by backbone ribbons. Orange: T1R2 subunit; Blue: T1R3 subunit; Green, brazein, (a) complex model # 1 of brazein A1 ins / H30R / E35D / E40A, (b) complex model # 2 of brazein A1 ins / H30R / E35D / E40A, (c) brazein A1 ins Complex Model # 3 of / H30R / E35D / E40A, (d) Complex Model # 4 of Brazein A1 ins / H30R / E35D / E40A, (e) Complex Model # 5 of Brazein A1 ins / H30R / E35D / E40A, (f) complex model # 6 of brazein A1 ins / H30R / E35D / E40A, (g) complex model # 7 of brazein A1 ins / H30R / E35D / E40A].
10 shows a schematic representation of hydrophobic and hydrophilic interactions between des-pE1M brazein and the T1Rs subunit [chain A, T1R2 subunits; Chain B, T1R3 subunit; Chain C, brazein; Green dotted line: hydrogen bond, red dotted line: hydrophobic interaction, (a) hydrogen bond between Lys14 and Asn24, (b) hydrogen bond between Tyr23 and Leu112, (c) hydrogen bond between Arg32 and Arg123, ( d) hydrogen bonding between Tyr53 and Met162].
11 shows a schematic representation of hydrophobic and hydrophilic interactions between H30R / E35D / E40A brazein and T1Rs subunits [Chain A, T1R2 subunits; Chain B, T1R3 subunit; Chain C, brazein; Green dotted line: hydrogen bond, red dotted line: hydrophobic interaction, (a) hydrogen bond between Lys2 and Asn130, (b) hydrogen bond between Arg32 and Tyr28, (c) hydrogen bond between Arg30 and Asp26, ( d) hydrogen bonding between Asn43 and Asp419, (e) hydrogen bonding between Asp39 and Ser122].
12 is Schematic representation of hydrophobic and hydrophilic interactions between A1 ins / H30R / E35D / E40A brazein and T1Rs subunits [chain A, T1R2 subunits; Chain B, T1R3 subunit; Chain C, brazein; Green dotted line: hydrogen bond, red dotted line: hydrophobic interaction, (a) hydrogen bond between Arg33 and Asn24, (b) hydrogen bond between Gln53 and Ile132, (c) hydrogen bond between Glu9 and Trp424].
FIG. 13 shows mean residue ellipticity ( MRE) of T1R2, T1R3 subunit and T1R2-T1R3 heterodimer by circular dichroism [dotted line: T1R2 subunit, dashed line: T1R3 subunit, solid line, T1R2- T1R3 heterodimer].
FIG. 14 shows the mean residual ellipticity (MRE) of brazein multivariates due to circular dichroism (solid line: des-pE1M brazein; Dashed line: H30R / E35D / E40A brazein; Short dashed line: A1 ins / H30R / E35D / E40A brazzein)
FIG. 15 compares the mean residual ellipticity (MRE) between complexes of Brazain multiple variants docked in T1R2-T1R3 heterodimers by circular dichroism [solid line: T1R2-T1R3 heterodimer; Dashed line: T1R2-T1R3-des-pGlu brazein complex; Dashed line: T1R2-T1R3-H30R / E35D / E40A brazein complex].
FIG. 16 compares the mean residual ellipticity (MRE) between complexes of Brazain multiple variants docked in T1R2-T1R3 heterodimers by circular dichroism [solid line: T1R2-T1R3 heterodimer; Dashed line: T1R2-T1R3-A1 ins / H30R / E35D / E40A brazein complex].
FIG. 17 shows the mean heat duration of brazein multivariates (solid line: des-pE1M brazein, dashed line: A1 ins / H30R / E35D / E40A).
FIG. 18 is an ultraviolet-ray circular polarization dichroism spectrum result of thermally modified brazein multivariate [blue: brazein, red: 80 ° C brazein, green, 83 ° C brazein, purple: 86 ° C brazein, light blue, 89 ° C. braze, orange, 92 ° C. braze, dark blue, 95 ° C. braze].
Figure 19 shows pKLAC2-brazein variant expression vectors.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

[ Example ]

I. Brazein  Structural research

1.1 Brazein Variant  And stability studies

The sweetness of braze is largely influenced by two factors. The first is the structure of the protein. If the bonds supporting the structure of the braze are broken or the folding of the brazein occurs, the structure of the protein is greatly denatured, resulting in a significant decrease or loss of sweet activity. For example, the four disulfide bonds of brazein are present in Cys3-Cys51, Cys15-Cys36, Cys21-Cys46, and Cys25-Cys48 (Kohmura et al., 1996), all variants that remove or transfer these disulfide bonds are sweet. The disappearance of activity could be observed (Assadi-Porter et al., 2010). In addition, it was confirmed that sweet taste disappears or changes even when the residues of the N- or C-terminus are changed or new residues are added or removed (Table 1) (Assadi-Porter et al., 2000). And when the structure of the flexible loop containing arginine, residue 42 of brazein, was disrupted, the sweetness activity was greatly reduced. Through this result, the flexible loop composed of residues 37-44 of brazein was sweet. It was predicted that they would be involved in the interaction of receptors with brazein (Assadi-Porter et al., 2000). The second factor in determining the sweetness of braze is the surface charge of the protein. Brazein contains five aspartic acids, two arginine, four glutamic acid, one histidine, seven lysine, six tyrosine, two It has a serine residue, which makes up about 50% of brazein. Changing these residues to amino acids with no opposite charge or no charge changed the total charge of brazein, resulting in a decrease or increase in sweetness. For example, aspartic acid residue 28 of brazein was converted to a neutral residue or a positive charge of opposite charge, resulting in an increase in sweet taste activity, whereas glutamate residue 35 was neutralized to alanine, a neutral residue. It was confirmed that sweetness activity disappeared as a result of mutating with glutamine or lysine with opposite charge. This suggests that for residues 28 and 35, the charge of the residue plays an important role in the sweetness activity, rather than the direction or length of the side chain (Jin et al., 2003).

In the previous study, we studied the activity of brazein variants based on these studies. First, a variant in which the histidine residue 30 of the flexible loop including residues 28-31 of brazein was transformed into an arginine with a greater positive charge increased sweetness activity, but the neutral amino acid alanine. The variant that removed the positive charge of residue No. 30 and reduced to sweet taste decreased. Therefore, it was confirmed that the positive charge of residue 30 has a significant effect on the sweet taste activity of brazein (Yoon et al., 2011). In addition, the glutamate residue located at β-strand III including residues 33-38 of brazein was transformed into aspartate to enhance negative charge, resulting in an increase in sweet activity (Do et al., 2011). Glutamic acid, which is residue 40 of the flexible loop containing residues 37-44 of brazein, was converted to neutral amino acids alanine, basic amino acids lysine and arginine, respectively. Interaction modeling studies of brazein and sweet receptors have shown that when braze and sweet receptor bind, glutamic acid residues of brazein are in close proximity to glutamic acid residues 252 of T1R2. It is believed to be repulsive (Walters & Hellekant, 2006). Therefore, the increased sweetness activity of the variant of residue 40 of brazein is expected to be due to the removal of this electrical repulsion by changing the charge of residue 40 of brazein to neutral or opposite charge (Yoon et al., 2011). . Based on the above findings, a variant was made by multiple mutations of histidine 30 to arginine, glutamate 35 to aspartate, and glutamate 40 to alanine. Phosphorus H30R / E35D, H30R / E40A, E35D / E40A, and H30R / E35D / E40A were prepared respectively and the sweet taste activity was compared. As a result, H30R / E35D / E40A showed the strongest sweet activity (Lee et al., 2013). Recently, experiments were carried out to create variants of the 52-mutated glutamic acid residue in the C-terminal portion of brazein, indicating that the positive charge of residue 52 will play an important role in the interaction with the receptor. Conclusions were obtained (Lim et al., 2015). It was also demonstrated that the lysine residue 4, belonging to the N-terminal β-strand I, influences the sweetness activity of brazein and plays an important role in maintaining structural stability (Lim et al., 2016).

Based on the results of the above study, the study was based on the H30R / E35D / E40A variant to study the brazein multivariate. To find out the action of the brazein's N-terminus, alanine was inserted into the N-terminus Extended variants were created.

2.1 computer modelling  And docking simulation

Higher order structures of proteins whose crystal structures are not known can be predicted through homology-based structure modeling. Among the programs for predicting the structure of proteins, the SWISS-MODEL EXPASY (http://swissmodel.expasy.org/) program predicts the tertiary structure of proteins based on the homology of amino acid sequences. Overall structural modeling with SWISS-MODEL is a four step process of template identification, proper template selection, protein model building and quality measurement of the built model (Biasini et al. al . , 2014).

The SWISS_MODEL template library (SMTL) contains information on 33,000 proteins that were experimentally identified (Biasini et. al . , 2014). As a first step in predicting the higher order structure of the desired protein, the appropriate template candidates are first searched for in SMTL. In this case, HHblits by hidden markov model (HMM) and basic local alignment search tool (BLAST) are compared and used together (Arnold et. al . , 2006). After selecting the template to build the protein model, the modeling results of the finished template protein are used to generate all the elemental structural models of the protein to be modeled using ProMod-II (Guex et. al . , 1997). In response, we build predictive protein models based on oligomeric structures estimated from other protein models. The quality of the finally constructed protein models is assessed through qualitative model energy analysis (QMEAN) (Biasini et. al . , 2014).

Protein-protein docking simulations are used to measure the docking direction or binding behavior of two different proteins and can be performed using a variety of software. Among them, GRAMM-X, a full range of molecular matching software, is characterized by a complete search through relative translation and orientation of protein molecules (Vakser et al . , 1995). Because the quality of the binding results is strongly influenced by the accuracy of the input protein, docking simulations between high resolution structures with minimal structural changes yield accurate predictions, while docking simulations between proteins with low resolution structures have only overall characteristics. Only protein complexes can be obtained (Vakser et. al . , 1995). However, if there are large structural changes in protein binding, a low-accuracy structure can be used that only knows the total structure information. GRAMM-X can predict molecular pairs, such as protein-subunits, as well as protein-protein docking results.

2.2 Circularly polarized light Dichroic

Circular dichroism (CD) is the difference in absorbance for left circularly polarized light and right circularly polarized light when a molecule is structurally asymmetric or is chiral. This is what happens. The CD measuring instrument measures the absorbance difference between left-turning circularly polarized light and right-turning circularly polarized light, and this value is expressed as ellipticity (θ). If the absorbance A _L of the left rotating circularly polarized light is higher than the absorbance A _R of the right rotating circularly polarized light, the positive CD spectrum appears and vice versa. If the absorbance of the two polarizations is the same, it means that the molecule is a symmetric chromophore, and the spectrum does not appear because there is no CD effect (Kelly et al., 2005). There is a wide range of protein information available from CD measurements. For example, aromatic amino acids absorb wavelengths from 250 to 350 nm, which correspond to the near UV area. By replacing these aromatic amino acids with other residues, one can characterize the CD spectrum of a specific residue, By providing useful information to identify tea structures, structural differences between wild-type proteins and variants can be analyzed (Kelly et al., 2005). In addition, the amide backbone between the peptide bonds of the proteins absorbs wavelengths of 260-178 nm, corresponding to the far UV area. In this region there are two types of CD-like electron transitions: the first is n → π ^* transitions around 222 nm and the second is π → π ^* transitions in the 190 to 208 nm range (Miles and Wallace). 2016).

The secondary structure of a protein absorbs a different range of wavelengths depending on its shape, and consequently each has its own CD spectral shape (Miles and Wallace, 2016). These CD spectral results can be analyzed using commercial calculation software such as non-constrained multilinear regression (MLR) or K2D3 (http://kal-el.ugr.es/k2d/) to estimate components of protein secondary structure. Andrade et al ., 1993). Absorbance analysis of the far ultraviolet region provides useful information for determining the secondary structure content of the protein as well as for observing ligand-protein interactions and protein folding (Miles and Wallace, 2016). Since CD is a quantitative method for protein analysis, spectral changes can be directly related to protein structural changes. Therefore, the pattern of protein interaction can be estimated by spectral change (Kelly et. al ., 2005). In addition, spectral changes between single proteins and complexes can be used to calculate dissociation and binding constants. When a protein binds to other proteins or ligands, intrinsic CD changes may appear due to structural changes in the protein or optical activity of the ligand (Greenfield, 1996). The binding constant can be determined by four methods: direct titration, serial dilution, change in sensitivity to denaturant, and change in thermal stability. Among them, direct titration calculates the saturation fraction (S) by titrating the region where the A-B complex formed by the combination of A and B using another protein B is in equilibrium with free proteins A and B. As a result, an approximation of the dissociation constant for protein B used in the titration can be obtained (Greenfield, 1996).

The sweetness activity of the brazein variants was measured by the SIAM yes-no task method, which was verified by sugar threshold experiments, and compared with the sweetness activity of the previously established brazein multivariates (Hautus et al., 2010). In addition, the thermal and pH stability of wild-type brazein including brazein multivariates were compared to determine the effect of each residue on the structural stability.

In this study, we studied the multivariants of sweet protein brazein and obtained important results regarding the structural change of sweet receptors, the beginning of taste signal transduction process, and the residues or bindings that significantly influence the sweetness activity of sweet protein brazein. It is expected to be.

Ⅱ. Instrument and reagents

1. Appliance

A sterile workbench for handling bacteria is Vision Biotech. The VB-700H1 Clean bench (Incheon, Korea) was used, and the VB-125A60 Autoclave was used for autoclaving. In order to cultivate the bacteria were used VS-1203P3N incubator of Vision Scientific (Daejeon, Korea) and SI-220R Shaking incubator of LAB house (Gyeonggi, Korea). The centrifuge for cell aggregation was Supra 22K, Micro-12, Smart-R17 from Hanil (Seoul, Korea), and the ultracentrifuge for membrane protein purification was Optima L- from Beckman Coulter (Indianapolis, IN, USA). 100K was used. pH measurement was performed using an ORION STAR A211 pH meter from Thermo Scientific (Pittsburgh, PA, USA). Vortex Mixer was used Maxi Mix II of Barnsted-Thermolyne (Dubuque, IA, USA), the stirrer was used MS300 Hotplate & Magnetic Stirrer of MTOPS. The balance used an Analytical Balance CPA224S from Sartorius (Goettingen, Germany). Peristaltic pumps for protein purification include Pharmacia Biotech. (Uppsala, Sweden) Pump P-1 was used and the fractionation collector was Fraction Collector FRAC-100. The cold chamber used KMC-1302L from Vision Scientific (Daejeon, Korea). The freeze dryer and cryogenic freezer were Ilshin Lab Co. Ltd. The TFD5505 Freeze Dryer and DF8503S Ultra Low Temperature Freezer from (Gyeonggi, Korea) were used. Agarose gel electrophoresis apparatus of the gene was used mupid-2plus of Takara (Tokyo, Japan), and EIDO (Tokyo, Japan) NA-1010 Protein Electrophoresis was used as the protein electrophoresis apparatus. Dry bath heating block for protein thermal stability experiment was used MaXtableTM H10 of Korea Science (Seoul, Korea). PCR and real time PCR devices were used Bio-Rad (Berkeley, CA, USA) Gene Cycler ^TM and CFX96, gene synthesis was commissioned by GenScript (Piscataway, NJ, USA). UV / VIS Spectrophotometer was used for OPTIZEN POP of MECASYS (Daejeon, Korea) for protein quantification and cell number. Circular Dichroism for protein secondary structure analysis was Jasco (Easton, MD, USA). J-815 was used. The thermostat used DH.WB000106 of Seoul, Korea.

2. Reagent

Plasmid miniprep kit and gel extraction kit for DNA purification were performed using CMG 0112 and CMP 0112 from Cosmo Genetech (Seoul, Korea). DNA cleavage was performed using buffers provided by restriction enzymes Sac I, Sac II and Takara (Shiga, Japan), and the DNA marker for checking the gene size was 1kb Plus DNA Ladder Marker from Doctor protein (Seoul, Korea). Was used. Yeast extract, Bacto peptone, Bacto tryptone, and Bacto agar were used by Difco Laboratories (Sparks, NV, USA) to culture E. coli and yeast. As a reagent for transformation of P. pastoris yeast strain, Pichia EasyComp ^™ Kit, G418 used the product of Invitrogen (Carlsbad, CA, USA). Yeast nitrogen base, Tris-base, agarose, ampicillin, TEMED, sodium dodecyl sulfate (SDS), glass beads, sucrose, dimethyl sulfoxide (DMSO), etc. are used by Sigma Chemical Co. (St. Louis, USA) It was. 30%, 40% -Acryl amide solution, polypeptide protein marker, and coomassie brilliant blue R-250 are manufactured by Bio-Rad (CA, USA) .SYBR used for real-time PCR is Thermo Fisher Scientific Inc. (Waltham, SYBR® Green Real-Time PCR Master Mixes from MA USA) was used. CM Sepharose Fast flow used for protein purification was a product of GE healthcare (Buckinghamshire, UK). Butanol, acetic acid and sodium chloride were used by Duksan Pure Chemical Co. (Gyeonggi, Korea). Potassium chloride and L-ascorbic acid were produced by Daejung (Incheon, Korea), sodium phosphate, sodium acetate, sodium hydroxide, D-galactose, methanol, ethanol, glycine, and tricine were used by Samchun (Seoul, Korea). Reagents for making other buffers and all reagents used were tested using first-class and premium reagents.

3. Strains and Expression Vectors Used

Escherichia for Gene Cloning coli strain TOP10 was purchased from Novagen (Madison, WI, USA). Strains Kluyveromyces lactis GG799 and pKLAC2 vectors for expressing sweet protein brazein are described in New England Biolab. (Ipswich, MA, USA). Pichia expressing strains of sweet receptors T1R2, T1R3 pastoris GS115 was purchased from Invitrogen (Carlsbad, Calif., USA) and pPIC9K vector was purchased from GenScript (Piscataway, NJ, USA).

III. Experiment method

1. K. lactis Expression system  Used Brazein Multivariate  Expression and Characterization Studies

1.1 Brazein Multivariate  Fabrication, Expression, and Purification

3M A1 ins (A1 ins / H30R / E35D / E40A) were constructed with 3M brazein variants (H30R / E35D / E40A) as templates to derive key residues affecting the sweetness activity of brazein. The oligonucleotide primer (Table 1) used for site-specific mutagenesis having a complementary sequence was synthesized by Cosmo Genetech.

Kluyveromyces in a previous study (Domestic Registration No. 1536683) In pKLAC2 used as the expression vector of lactis , the Kex cleavage site recognized by Kex protease is located after the α-mating factor signal sequence that induces secretion expression. A brazein variant sequence was inserted at the Xho I and Stu I restriction enzymes contained in the multi cloning site of the vector. The pKLAC2 vector with the brazein sequence inserted was K. lactis Sac II restriction enzyme treatment was performed to have homology to the ends of the LAC4 promoter present in genomic DNA of (FIG. 19). By inserting the brazein gene into K. lactis on the basis of homologous recombination, strains containing the most inserted brazein gene are selected by real-time PCR (Jo et al., 2013; Yun et al. 2016).

K. lactis with brazein gene was cultured by the established method.

First, a small amount was taken from 20% glycerol stock of K. lactis cell transformed with the gene of brazein mutant using Platinum YPD-Agar (1% Yeast extract, 2% Peptone, 2% Glucose, 2% Agar). Plated in solid medium and incubated at 30 ℃ for about 72 hours. A single colony formed from YPD-Agar was inoculated 1 per 5 mL of YPD (1% Yeast extract, 2% Peptone, 2% Glucose) at 30 ° C and 200 rpm until the value of OD ₆₀₀ reached about 20-22. Shake culture was performed. This culture was inoculated to 2% in YPGal (1% Yeast extract, 2% Peptone, 2% Galactose at pH 5.0, using acetic acid) medium containing Galactose as an inducer and incubated for 96 hours at 30 rpm at 200 rpm.

When the incubation is completed, the supernatant is taken by centrifugation for 30 minutes under conditions of 8650g, 4 ℃ to separate the bacteria and the culture supernatant. Brazein was purified using CM Sepharose Fast flow, a cation exchange resin. Before loading the culture supernatant, equilibrate the resin by filling the column with a balance buffer solution (50 mM sodium acetate, pH 4.0, using acetic acid), which is about 20 times the volume of the filled resin. I set it in a state. Since the isoelectric point (pI) of brazein is 5.4, the acetic acid is used to adjust the supernatant to 4.0, which is sufficiently lower than the isoelectric point of brazein within the stable pH range of cation exchange resin, and 1 mL / min to the equilibrium resin. Loaded at a flow rate of. When loading is complete, flush the wash buffer solution (50 mM sodium acetate, 40 mM sodium chloride, pH 4.0, using acetic acid) until the OD ₂₈₀ value is 0, then dissolve buffer solution (50 mM sodium acetate, 400 5 mL fractions were eluted with mM sodium chloride, pH 4.0, using acetic acid). The fraction eluate OD ₂₈₀ measurement, 2 mL for about taking was then dialyzed against distilled water and then measuring the OD ₂₈₀ value was lyophilized unify the protein total amount on the basis of OD ₂₈₀ value after dialysis 16.5% Tris-Tricine Gel for each predetermined period after the bra Jane was eluted and separated. The brazein eluate was dialyzed in distilled water, and the elution buffer was exchanged with distilled water and lyophilized. Finally, the obtained brazein powder was put in a vial to seal the inlet and stored at -80 ° C.

1.2 Modified SIAM yes - no to task  by Brazilian  Sweetness absolute threshold measurement

In order to objectively evaluate the sweetness of brazein, we introduced a SIAM yes-no task method that proved its usefulness in sugar threshold experiments (Hautus et. al ., 2010). The main feature of this experiment is the evaluation of blindness, and half of the 30 tests are blank. The experiment uses 10 dilution samples. The concentration of the thickest solution that needs to be diluted should be properly adjusted to obtain the proper experimental result in the next step, so that the absolute threshold value can be accurately determined. Therefore, a minimum of 3 to 5 participants was used to determine the upper limit of protein solution concentration for the experiment. Initially, the gap was widened with 10, 100, and 1000-fold dilutions, and the direction was gradually narrowed down according to the participants' responses. The protein solution of this concentration is diluted 10-fold using a volumetric flask from the original protein solution (Stock) to prepare a protein solution corresponding to 100 mL. This solution was 10 samples of the sweetness measurement experiment, and the solution was uniformly diluted to prepare 10 mL of protein solution corresponding to Nos. 1 to 10, respectively.

Participants in the experiment consisted of non-smokers and strictly limited the experimental conditions such as fasting from the day before the experiment and 2-3 hours before the experiment. Absolute threshold experiments are run a total of 30 times per set, 15 of which are tertiary distilled water. The experimenter randomly placed distilled water in the terms except for the beginning and the end, so as not to taste distilled water or protein solution three times or more consecutively. The starting concentration was designated as sample 1 and participants were asked whether they felt sweet after administering 200 μL of the sample. At this time, if the answer is sweet (Yes) because the participant properly recognized the protein solution was considered as Hit, and the experimenter prepared a sample of the lower concentration (-1) at the corresponding concentration. On the contrary, if the answer is no sweetness (No), the participant was regarded as Miss because the participant was not aware of the protein solution, and the experimenter prepared a sample of a higher concentration (+1) at the corresponding concentration. If a sample of distilled water was administered, the result of the sample that had been tasted until the last time was maintained. If the participant responded that the sample tastes sweet (Yes), false recognition occurred in this case. Therefore, the false alarm was regarded as a false alarm, and the experimenter prepared a sample of two levels (+2) higher in the number. On the contrary, if the answer was no sweetness (No), the participant answered correctly and considered the result as correct rejection, and the experimenter reflected the result of the sample that had been tasted until the last time in the next number of experiments. At this time, the number of samples at the point where the concentration increases (+) and decreases (-) or decreases and then increases is defined as a turnaround number. After the experiment was completed, the experimenter excluded the first two or three turnaround numbers so that the number of turnaround numbers was even. The average value of this turnaround number is the absolute threshold of the participant.

1.3 Brazein Multivariate  Thermal stability studies

The thermal and pH stability of the H30R / E35D / E40A, A1 ins / H30R / E35D / E40A variants, including the brazein wild type subtype, were performed and compared respectively. The concentrations were unified based on the wild type subtype with the weakest sweet activity to directly measure the decrease in sweet activity.

First, each variant was prepared at a concentration of 40 μM by 15 mL for CD measurement and sweetness activity measurement. A 4 μM sample of 10-fold dilutions of this sample corresponds to 0.025 mg / mL on a wild type subtype, which is similar to the concentration of Stock (0.022 mg / mL) in actual sweetness testing. 5 mL in 15 mL of solution was used for pH stability experiments and 9 mL was aliquoted and diluted to 20 μM for use in thermal stability experiments. The remaining 1 mL of solution was used for protein quantification.

For the thermal stability test of brazein, the experiment was conducted by subdividing the temperature range of 80 ~ 95 ℃ by 3 ℃ interval. First, a 20 μM brazein sample was aliquoted in 3 mL aliquots and incubated for 6 hours in a dry bath maintaining the temperature. Sweet activity was measured at 30 minute intervals during the incubation time, 240 μL of brazein samples were taken and mixed with 960 μL of tertiary distilled water and diluted to 4 μM. Diluted this solution in the prepared vials with 0 μM, 0.8 μM, 1.6 μM, 2.4 μM, 3.2 μM of brazein dilution solution. Three subjects are fixed and the diluted solution is administered at a low concentration of 200 μL to respond whether it is sweet or not. Incubated brazein samples were stored at 4 ° C. after sealing for circular dichroism spectroscopic measurements.

2. Protein Quantitation

All purified sweet acceptor monomers and brazein variants were quantified by measuring absorbance at 205 nm. Most proteins have an intrinsic extinction coefficient at 205 nm on a 1 mg / mL solution, which allows the optimal wavelength for protein measurement to be 205 nm (Scopes 1974).

The reference absorbance of the UV / Vis absorbance photometer was measured and fixed with a solvent in which the sweet receptor monomer and the brazein sample were dissolved. Each protein sample was gradually diluted until the absorbance value at 205 nm approached 0.3 to minimize the stray effect. The diluted protein solution was measured three times by absorbance at 205 nm and 280 nm, respectively, to obtain an average value. Then, the intrinsic molar extinction coefficient of each protein sample was calculated | required using following formula (1). The original protein sample concentration was obtained by multiplying the dilution factor by the value obtained using Equation 2 below.

[Equation 1]

ε ₂₀₅ ^{1mg / mL} = 120 × (A ₂₈₀ / A ₂₀₅ ) +27

[Equation 2]

Concentration (mg / mL) = A ₂₀₅ / ε ₂₀₅ ^{1mg / mL}

3. Protein-protein interaction studies

3.1 Computer modelling  And docking simulation

The amino acid sequences of brazein (NCBI Reference Sequence: ACK76425.1) and sweet receptors T1R2 (NCBI Reference Sequence: NP_689418.2) and T1R3 (NCBI Reference Sequence: NP_689414.1) are described in National Center for Biotechnology Information (NCBI: http: / /www.ncbi.nlm.nih.gov). T1R2 and T1R3 used only the sequence of extracellular domain (T1R2: # 1 ~ 566, T1R3: 1 ~ 570) except for the membrane penetration site. Computer structural modeling of the brazein wild type subtypes and multivariants and sweet receptors was performed using the SWISS-MODEL EXPASY (http://swissmodel.expasy.org) program, an automated protein modeling software based on homology of amino acid sequences. Optimal models are compared by comparing each protein model with criteria such as sequence indentity, qualitative model energy analysis (QMEAN) values, torsion factor, and solvation factor. Selected. The first protein model was selected to give hydrogen and gasteiger charges using the Autodock Tools software. This compensates for the structural elements lacking in the implemented protein model by modifying the rotatable bonds within the protein structure and removing water molecules that may be present in the surroundings. The final protein model after modification was saved as a protein data bank (pdb) file.

The computer-implemented T1R2 and T1R3 were analyzed for protein-protein binding using "GRAMM-X" docking simulation software. Ten combined models were implemented with UCSF Chimera software and each stored as a pdb file. For each binding model, we analyzed the interactions between amino acids involved in the binding of T1R2 and T1R3 with DIMPLOT and calculated the dissociation constant and affinity energy using the SEQMOL software. Based on this analysis, the optimal T1R2-T1R3 binding model was selected.

The protein models of the brazein wild type subtypes and variants were analyzed for binding with the T1R2-T1R3 heterodimeric binding model using the "GRAMM-X" docking simulation software. The 10 combined models predicted by 'GRAMM-X' are stored as pdb files, as when selecting the T1R2-T1R3 combined models. Interactions between the T1R2-T1R3 heterodimers and the amino acids involved in the interaction of brazein were analyzed using "DIMPLOT" and "SEQMOL" software to calculate dissociation constants and affinity energy. Finally, the selected binding model was identified using the 'UCSF Chimera' to confirm the tertiary structure.

3.2 Circularly polarized light Dichroic  Spectroscopy

Circular dichroism spectra measurements of T1R2-T1R3 sweet receptors, brazein multivariates and sweet receptor-brazein complexes were measured at J-815 spectrometer (Jasco co., Ltd.), a standard analytical laboratory at Inha University. Was used. A 150 W xenon lamp was used as a light source and measured using a high performance UV_Vis_IR avalache photo-diode fluorescence monochro detector. Real-time measured data was recorded and analyzed with Spectra Manager ™ II software. Before the measurement, the scan range was set from 180 nm to 260 nm and the scan rate was set to 100 nm / min, and then the absorbance of the baseline was measured without the sample. Brazein samples were then measured at 20 μM concentration in TDW solvent and T1R2, T1R3 monomers and dimers were measured at 5 μM concentration in 0.9 mM Tween 20, 20 mM Tris-HCl (pH 8.0) solvent.

Ⅳ. result

1. K. lactis Expression system  Used Brazein Multivariate  Expression and Purification

3M A1 ins. Was prepared by introducing alanine at the first position using the 3M brazein variant (H30R / E35D / E40A) as a template (FIG. 3).

YPGlu was prepared using the K. lactis GG799 strain in which the genes of the produced brazein wild type subtype (des-pE1MBRZ) and the brazein variants H30R / E35D / E40A (3M) and A1 ins / H30R / E35D / E40A (3M A1 ins) were inserted. The cells were grown to cells, inoculated and cultured in YPGal medium containing Galactose, which is an inducer, to induce the expression of brazein variants to produce 15 mg / L of brazein on average. After the culture was completed, the cells were separated by low speed centrifugation, and the supernatant was purified by carboxymethyl sepharose cation exchange resin. Brazein sample of fractional elution was completed by dialysis to remove the salt contained in the elution equilibrium solution and lyophilized and the purity was determined through SDS-PAGE (Fig. 4). When the purity was low because other proteins were included, brazein was isolated using Sephacryl S-100 size exclusion chromatography. Brazein after final purification was obtained in a yield of about 7 mg / L.

2. Sweetness Activity and Interaction Analysis

2.1 computer modelling  And docking simulation

2.1.1 Protein Structure modelling

T1R2 and T1R3 monomers of the sweet receptor can be classified into three structural regions based on amino acid sequence similarity with the metabotropic glutamate receptor (mGluR). The Venus Flytrap Module (VFTM), located outside the cell membrane, consists of lobes 1 and 2, with 'open' and 'closed' structures depending on the location of the two lobes, followed by nine Cystein-rich domains (CRDs). It has a conserved cysteine residue and the transmembrane domain (TMD) consists of seven helixes across the membrane (Assadi-Porter et. al ., 2010). Homology-based modeling of the extracellular regions of T1R2 and T1R3 used mGluR3 as a template structure. The shortcomings of homology-based structural modeling indicate a lack of a small number of residues at the N- and C-terminus of the modeled structure, which can be neglected since they do not significantly affect the interaction. In addition, T1R2 and T1R3, which were modeled, selected an optimal prediction model in consideration of sequence homology, sequence coverage, QMEAN value, lysis factor, and torsion factor (FIG. 6, Table 3). The predicted models of T1R2 and T1R3 showed 26.5% average sequence homology with mGluR3, which is the highest among other proteins of known structure. It also showed high sequence coverage of an average of 90%. Like the sweet receptor, the multivariate of brazein was selected for the optimal prediction model in consideration of sequence homology, sequence coverage, QMEAN value, solubility factor and torsion factor (FIG. 5, Table 2).

Evaluation of the best predictive model of the T1R2 and T1R3 subunits Subunit Seq . queried Seq . modeled Seq . identity Seq . coverage QMEAN Solvation  factor Torsion  factor T1R2 # 1-566 # 24 ~ 556 27.08 0.89 -12.82 -8.84 -8.27 T1R3 # 1-570 # 26 ~ 559 25.97 0.91 -9.91 -7.58 -5.90

Evaluation of Optimal Prediction Model for Brazein Multivariants Brazzein mutant Seq . queried Seq . modeled Seq . similarity Seq . coverage QMEAN Solvation  factor Torsion  factor des -pE1M # 1-53 # 1-53 93.26 1.00 -1.25 -0.75 -1.39 H30R / E35D / E40A # 1-53 # 1-53 94.34 1.00 -2.67 -1.26 -2.48 A1 ins / H30R / E35D / E40A # 1-54 # 1-54 92.59 1.00 -5.62 -2.01 -7.20

2.1.2. T1R2 - With T1R3 Brazein Multivariate  Docking simulation

In order to form the T1R2-T1R3 heterodimer model, a predictive model of binding between T1R2 and T1R3 was prepared using 'GRAMM-X'. Of the 10 predicted orientations, the most likely model was selected based on the previous results. According to the crystal structure study of the metabotropic glutamate receptor (mGluR), mRluR has an orientation where VFTM and CRD are arranged side by side outside the cell membrane when dimers are formed, and TMD of both monomers leads to the cell membrane (Kunishima et al. al ., 2000). Since T1R2 and T1R3 modeled homology based on mGluR as a template, this result can be applied to sweet receptor modeling (Assadi-Porter et. al ., 2010). Based on this, the Gibbs free energy and dissociation constant between T1R2 and T1R2 of the optimal heterodimer model selected were predicted by 'SEQMOL' software. The Gibbs free energy of the heterodimer coupling model is -2.69 kcal / mol and the dissociation constant is 1.09E-02 M.

To find amino acids that play an important role in the interaction of brazein with the receptor, the multivariate brazeins H30R / E35D / E40A and A1 ins / H30R / E35D / E40A, including the wild-type subtype des-pE1M brazein, were respectively identified as T1R2- The complex model was created by binding to the T1R3 heterodimer (FIGS. 7-9). Each complex model was then derived from Gibbs free energy and dissociation constants via the SEQMOL software (Table 4). In addition, the 'DIMPLOT' software was used to analyze the amino acids involved in the interaction of the heterozygotes with brazein variants (Figs. 10-12, Tables 5-7). As a result, it was predicted that the amino acid residues of Ser13, Gln16, Tyr38, Arg42, and Leu44 of brazein would play an important role in the interaction with the sweet receptor by strong hydrogen bonding or multiple hydrophobic interactions with the sweet receptor.

Evaluation of models docked with T1R2-T1R3 heterodimers of brazein multivariants. Brazzein
variant Complex
model Gibbs  binding energy
( kcal Of mol ) Dissociation constant
( μM ) des - pE1M #One -5.46 1.04E + 02 #2 -5.30 1.36E + 02 # 3 -7.17 5.87E + 00 #4 -8.49 6.39E-01 # 5 -10.84 1.23E + 02 # 6 -12.88 4.00E-04 # 7 -4.91 2.62E + 02 H30R Of E35D Of E40A #One -2.93 7.28E + 03 #2 -3.48 3.48E + 02 # 3 -5.31 1.34E + 02 #4 -5.82 5.67E + 01 # 5 -7.44 3.73E + 00 # 6 -15.32 6.64E + 06 # 7 -2.77 9.53E + 03 A1 ins Of H30R Of E35D Of E40A #One -16.01 1.15E-06 #2 -8.12 1.19E + 00 # 3 -15.05 1.05E-05 #4 -13.88 7.46E-05 # 5 -14.15 4.74E-05 # 6 -8.64 4.97E-01 # 7 -8.85 3.49E-01

Representative amino acid residues involved in hydrophilic and hydrophobic interactions between the T1R2-T1R3 heterodimer and des-pE1M brazein Model Type T1R2 subunit T1R3 subunit Brazzein Distance #One Hydrophilic Ile424 Leu17 3.28 Å Ser155 Tyr38 2.43 Å Leu158 2.79 Å Ser59 Glu8 2.54 Å Tyr10 3.26 Å Lys111 Lys41 2.54 Å Hydrophobic Lys174, Pro178, Pro417, Tyr418, Leu421, Glu422 Leu51, Val61 Ser13, Cys15, Gln16, Ala18, Glu35, Cys36, Leu44 within 5 Å #2 Hydrophilic Leu521 Asn43 2.88 Å Gln536 Ser13 1.93 Å Ala537 2.78 Å Glu525 Lys4 2.70 Å Ser528 2.60 Å Arg530 Glu40 2.77 Å Trp546 Lys2 2.49 Å Hydrophobic Cys517, Asp519, Pro522, Thr524 Gln507, Ala526, Glu549, Asp520, Val522, Asp523 Val6, Glu8, Pro11, Val12, Tyr38, Asp39, Arg42 within 5 Å # 3 Hydrophilic Leu156 Ser13 3.24 Å Hydrophobic Phe157, Tyr416, Trp418, Leu158 Leu51 Pro11, Val12, Phe37 within 5 Å #4 Hydrophilic Ser155 Ser13 3.30 Å Glu422 Asn43 2.82 Å Ala49 Asn19 2.94 Å Ser59 Gln16 3.19 Å Hydrophobic Val175, Pro178, Trp418, Ile424, Trp425 Glu48, Gly50, Leu51 Pro11, Gln16, Leu17, Lys41, Arg42 within 5 Å # 5 Hydrophilic Asn24 Tyr7 2.66 Å Ala120 Tyr53 2.52 Å Ala417 Lys2 3.15 Å Hydrophobic Ala116 Phe159, Pro420, Val421, Lys422, Pro423, Trp424 Cys3, Tyr50, Cys51, Gln52 within 5 Å # 6 Hydrophilic Asn24 Lys14 2.29 Å, 3.06 Å Leu112 Tyr23 2.96 Å Arg123 Arg32 2.80 Å Met162 Tyr53 2.51 Å Hydrophobic Tyr113, Ala116 Phe159, Ala120, Ser122 Asp28, His39, Cys51, Glu52 within 5 Å # 7 Hydrophilic Lys174 Gln16 3.33 Å Leu421 Ser13 3.33 Å Hydrophobic Asp173, Val175, Ile424, Pro440, Gln441 Ser58, Pro246, Arg247 Val12, Tyr38, Glu40, Arg42, Asn43 within 5 Å

Representative amino acid residues involved in hydrophilic and hydrophobic interactions between T1R2-T1R3 heterodimer and H30R / E35D / E40A brazein Model Type T1R2  subunit T1R3  subunit Brazzein Distance #One Hydrophilic Ser155 Gln16 3.09 Å Ser59 Lys14 2.13 Å Hydrophobic Leu156, Leu158, Pro178, Tyr416, Trp418 Glu48, Leu51, Val61, Lys111, Leu114 Asn9, Tyr10, Val12, Ser13, Try38, Asn43 within 5 Å #2 Hydrophilic Asn24 Val6 3.12 Å Ser122 Lys26 1.87 Å 3.01 Å Arg32 2.67 Å Hydrophobic Tyr113, Ile132, Ala116, Tyr131 Phe159, Val421, Lys422, Pro420, Pro423, Trp424 Asn9, Leu27, Asp28, Tyr50, Cys51, Glu52 within 5 Å # 3 Hydrophilic Tyr28 Tyr38 3.19 Å Asn83 Ser13 3.33 Å Asn84 Lys14 2.96 Å Hydrophobic Leu29, Pro30, Arg76, Glu80, Leu93, Phe338, Trp341 Val12, Gln16, Leu17, Asn19 within 5 Å #4 Hydrophilic Ser59 Tyr38 3.22 Å Hydrophobic Pro178, Trp418, Leu421, Ile424, Pro440 Arg52 Lys4, Val6, Leu17, Arg42, Asp39, Ala40, Lys41 within 5 Å # 5 Hydrophilic Asp124 Lys41 3.28 Å Ser413 Gln16 2.72 Å Cys415 Tyr38 2.97 Å Hydrophobic Arg123, Ala126, Tyr128, Arg137, Pro416, Asp419 Cys15, Asp39, Ala40, Arg42, Leu44 within 5 Å # 6 Hydrophilic Asp26 Arg30 3.32 Å Tyr28 Arg32 2.85 Å Asn130 Lys2 3.09 Å Ser122 Asp39 3.12 Å Asp419 Asn43 2.99 Å Hydrophobic Asn42, Tyr113, Tyr131, Ile132 Phe159, Pro420, Lys422, Pro423, Trp424 Lys5, Asp9, Pro11, Ser13, Lys41, Tyr50 within 5 Å # 7 Hydrophilic Arg30 Lys4 3.30 Å Glu52 2.87 Å Ile83 Tyr38 3.15 Å Lys86 2.23 Å Thr346 Arg32 2.74 Å Asp347 Ser33 2.00 Å Hydrophobic Lys32, Pro91, Gly92, Arg94, Ala345, Pro348, Ala439 Cys36, Phe37, Leu44, Arg42, within 5 Å

Representative amino acid residues involved in hydrophilic and hydrophobic interactions between the T1R2-T1R3 heterodimer and the A1 ins / H30R / E35D / E40A brazein variants Model Type T1R2  subunit T1R3  subunit Brazzein Distance #One Hydrophilic Asn24 Arg33 2.92 Å Ile132 Glu53 2.93 Å Trp424 Glu9 3.33 Å Hydrophobic Tyr113, Ala116 Phe159, Trp424 Ala1, Lys3, Glu9, Arg33, Glu53, Tyr54 within 5 Å #2 Hydrophilic Asn24 Lys5 3.05 Å Tyr96 Tyr54 3.11 Å Ser122 Ala19 3.27 Å Pro181 Arg43 2.91 Å Ser182 2.50 Å Val421 Gln17 3.08 Å Hydrophobic Tyr28, Tyr113 Ala120, Ser122, Leu161, Pro420, Val421, Trp424 Ser14, Gln17, Leu18, Asn20, Tyr39, Ala41, Leu45, Glu53, Tyr54 within 5 Å # 3 Hydrophilic Ser66 Ala1 3.11 Å Asp249 Tyr54 3.16 Å Gly314 Tyr39 3.30 Å Gln368 Tyr51 3.11 Å Hydrophobic Leu308, Leu312, Met315, Ala316, Arg357, Leu361, Asp2, Arg33, Asp40, Ala41, Cys52 within 5 Å #4 Hydrophilic Asn24 Asp40 3.28 Å Tyr357 Arg43 3.18 Å Thr358 Ser14 2.86 Å Gln17 3.25 Å Ser182 Asp2 2.23 Å Ser446 Arg33 3.03 Å Hydrophobic Ser25, Val56, Arg177, Pro181, Gln425, Leu426, Met430, Ala1, Cys4, Tyr30, Phe38, Ala41, Lys42, Cys52, Gln53 within 5 Å # 5 Hydrophilic Gln265 Asp50 3.28 Å Asn292 Tyr39 2.57 Å Hydrophobic Asp262, Thr268, Arg270, Thr294, Thr485, Val486, Met492 Lys512, Gly513, Phe514 Glu17, Leu18, Asn20, Tyr24, Phe38, Arg43, Leu45, Glu53 within 5 Å # 6 Hydrophilic Asp307 Tyr54 2.96 Å Arg33 3.18 Å Met310 1.96 Å Glu358 Lys5 2.01 Å Gln379 Asn23 2.99 Å Hydrophobic Leu312, Arg369, Ser464, Ala383, Val405 Lys27, Tyr39, Glu53 within 5 Å # 7 Hydrophilic Tyr128 Tyr39 2.83 Å Cys129 Gln17 3.18 Å Gly414 Gln46 2.20 Å Hydrophobic Tyr35, Gly92, Ile125, Ala127, Leu408, Cys415, Pro416, Asp419 Lys5, Val7, Leu18, Ala19, Asp40, Ala41, Lys42, Asn44, Ile48 within 5 Å

3.2 Determination of sweetness activity of brazein by modified SIAM yes-no task

High purity purified brazein variants were diluted so that the absorbance at 205 nm (A205) was close to 0.3 in order to accurately determine the molar extinction coefficient. The diluted protein solution was then measured for absorbance at 280 nm (A280). All absorbance measurements were repeated at least three times and averaged, and the intrinsic molar extinction coefficients of the brazein variants were determined using the A205 and A280 values (Table 8).

The sweetness activity of the Brazain multivariate was measured and compared by obtaining at least two SIAM yes-no task threshold experiments for 15 fixed participants (Table 9). As a result, it was confirmed that the H30R / E35D / E40A variant was 10.2 times higher and the A1 ins / H30R / E35D / E40A variant was 2038 times higher than the wild-type brazein.

Estimated molar extinction coefficient (ε205) Brazzein mutants Abs205 Abs280 ε205 des-pE1M 0.322 0.023 35.57 H30R / E35D / E40A 0.314 0.024 36.17 A1 ins / H30R / E35D / E40A 0.304 0.011 31.34

Comparison of sweetness intensity of brazein multivariate Sweet tasting molecule Molecular mass (Da) Experimental taste threshold Sweetness in comparison to sucrose Sweetness in comparison to wild type brazzein (g (100 mL) ^-1 ) (g / g) Sucrose 342.3 2.0 One des-pE1M-
brazzein 6370 0.0013720 1458 One H30R / E35D / E40A 6317 0.0000133 15,000 10.2 A1ins / H30R / E35D / E40A 6388 0.00000067 2,971,428 2038

From the above Table 9, the brazein multiple variants A1ins / H30R / E35D / E40A were about 2,970,000 times higher in sweetness compared to the same amount of sucrose (sugar). These sweetness can be measured by the SIAM yes-no task method verified by the sugar threshold experiments to derive the results.

In addition, the brazein multiple variant A1ins / H30R / E35D / E40A increased about 2038 times sweeter than wild type brazein. Such sweetness activity was derived from the brazein variant sweetness activity compared to the brazein wild type with reference to the prior patent (10-1795573, 10-1416892).

3.3 Circularly polarized light Dichroic  Spectroscopy

3.3.1 Sweet receptors and Brazilian  Interaction analysis

Secondary structures of sweet receptors T1R2, T1R3 and brazein variants were analyzed by circular dichroism spectroscopy. In addition, the structural changes caused by protein-protein interactions with brazein of T1R2-T1R3 heterodimer were measured.

First, the mean residue ellipticity (MRE), [θ] (deg · cm ^{2 ·} dmol ⁻¹ ) of T1R2 and T1R3 monomers, T1R2-T1R3 heterodimers, and brazein variants were measured in the range of 250 to 190 nm. (FIG. 13). The content of α-helices and β-strands was determined by K2D3 software. T1R2 contains 69.43% α-helix and 11.26% β-strands. T1R3 contains 65.87% α-helix and It was found to contain 12.13% of the β-stranded structure. In addition, each brazein variant had a 10.27% α-helix structure and a 28.93% β-strand structure for the wild type subtype, and a 10.11% α-helix structure and 31.89% β-strand for the H30R / E35D / E40A variant. The strand structure was found to contain 8.46% α-helix structure and 32.1% β-strand structure, respectively, for the A1 ins / H30R / E35D / E40A variant (FIG. 14, Table 10).

The T1R2-T1R3 heterodimer and the brazein variant were mixed at a 1: 1 molar ratio to form a complex, and circular dichroism spectroscopy was performed (FIGS. 15 and 16). The process of calculating the dissociation constant between T1R2-T1R3 and Brazein is as follows.

The dissociation constant (K _d ) when the T1R2-T1R3 heterodimer is defined as R, the brazein variant as B, and the heterodimer and brazain complex as RB can be represented by [R] [B] / [RB] ( Martin and Schilstra 2008). At this time, the spectra signal of the circular dichroic dispersion appearing according to the concentration of the T1R2-T1R3 heterodimer and the brazein variants can be expressed as Equation 3 below (Martin and Schilstra 2008).

[Equation 3]

S = Δε _R [R] + Δε _B [B] + Δε _RB [RB]

= Δε _R [R ₀ ] + Δε _B [B ₀ ] + (Δε _RB -Δε _R -Δε _B ) [RB]

([X ₀ ] = initial concentration of X, Δε = extinction coefficient)

In Equation _{3 (Δε RB - Δε R -} Δε B) [RB] refers to the amount of change in the circular dichroism signal that appears from the interaction of T1R2-T1R3 and bra agent conjugate (Greenfield 2004). Thus, (Δε _RB -Δε _R -Δε _B ) [RB] = Δ [θ] = ε [RB], and the maximum change in signal Δ [θ] _max is the T1R2-T1R3 heterodimer and all Brazein variants in solution Is reacted to form a complex, that is, when the complex concentrations of the T1R2-T1R3 and the brazein variant are equal to the initial concentration of the brazein variant (Greenfield 2004). At this time, the saturation fraction can be obtained from Equation 4 below, and the dissociation constant was derived (Greenfield 2004) (Table 11).

[Equation 4]

Prediction of Secondary Structural Composition of T1R2-T1R3 and Brazein Multiple Variants by K2D3. Protein
sample Wavelength
Range (nm) α-helix β-sheet T1R2 subunit 240-190 69.43% 11.26% T1R3 subunit 65.87% 12.13% des -pGlu-brazzein 10.27% 28.93% H30R / E35D / E40A 10.11% 31.89% A1 ins / H30R / E35D / E40A 8.46% 32.1%

Estimated Dissociation Constants of Brazein Multiple Variants for T1R2-T1R3 Heterodimer Brazzein
variant Dissociation constant by
docking simulation (μM) Dissociation constant
by CD (μM) des -pGlu-brazzein 4.00E-04 3.81E-02 H30R / E35D / E40A 6.64E-06 6.44E-03 A1 ins / H30R / E35D / E40A 1.15E-06 2.69E-03

3.3.2 by temperature Brazilian  Structural change analysis

The thermal stability of brazein was analyzed by confirming the activity change through the measurement of sweetness activity and the secondary structure change through spectroscopic measurement using circular polarization dichroism. Determination of sweetness activity at 30-minute intervals at each temperature showed that all variants, including the wild-type brazein, averaged 5.5 hours at 80 ° C, 5 hours at 83 ° C, 4.7 hours at 86 ° C, 3.6 hours at 89 ° C, and 92 ° C. It was confirmed that sweet activity was maintained for 2.7 hours at 95 ° C. for 2.4 hours (FIG. 17). In addition, thermal stability was measured by observing the secondary structure change of brazein through circular dichroism spectroscopy (Table 12, Figure 18). As a result, all the variants were found to maintain the sweetness activity on average, even if heat was applied at 80 ℃ for 5.5 hours at 95 ℃ 2.4 hours.

The multivariate H30R / E35D / E40A had somewhat lower structural stability than wild-type brazein but retained a somewhat similar ratio of secondary structure. That is, it was confirmed that the multivariate H30R / E35D / E40A had structurally high stability as the wild type brazein. In particular, among the amino acid residues that are subject to mutation in this multivariate, glutamic acid residue No. 36 is located in β-strand including residues 33-38 of brazein, and it is transformed into aspartic acid to enhance negative charge. It can be interpreted that it has a structure that enhances sweet taste activity (structure that binds more strongly to sweet receptors) without significantly affecting structural stability. Comparing the N-terminal variants mutated based on the H30R / E35D / E40A variant, the residual content of β-sheet is relatively high and the residual content of α-helix is relatively large compared to H30R / E35D / E40A. appear.

Secondary Structural Composition Prediction of Thermodenatured Brazein Multiple Variants by K2D3 Brazzein mutant Temperature ( ° C ) α- helix β- sheet des -pGlu-brazzein control 10.27 34.41 80 8.95 32.87 83 7.48 32.35 86 7.45 31.93 89 6.47 28.93 92 5.16 22.47 95 3.01 22.34 H30R / E35D / E40A control 8.11 31.89 80 6.93 30.84 83 6.84 31.46 86 6.44 19.69 89 6.22 18.85 92 3.86 18.63 95 2.11 18.43 A1 ins / H30R / E35D / E40A control 8.46 32.1 80 5.91 30.39 83 5.42 30.17 86 4.94 30.07 89 3.21 29.05 92 3.14 28.84 95 2.98 21.75

Ⅴ. Review

One. Brazein Multivariate  Sweet activity and stability studies

    According to the study of X-ray crystal structure of brazein, the part composed of brazein's N-terminus and C-terminus is one of the central elements of the brazein structure, and the brazein's N- and C-terminus and the center There are many interactions, suggesting that these interactions will contribute significantly to the activity and stability of brazein (Nagata et al., 2013).

As a result of the sweetness activity test in the present invention, the A1 ins / H30R / E35D / E40A variant increased about 198 times sweeter than the H30R / E35D / E40A variant. This suggests that amino acid residues belonging to the N-terminus and C-terminus of brazein play an important role in the sweetness activity of brazein.

The threshold results of variant brazeins obtained by measuring the sweetness threshold show the same tendency as the dissociation constant values between brazein and sweet receptors derived from circular dichroism measurement and computer docking simulation. In other words, the lower the threshold value, the stronger the sweetened activity of the brazein variant, the lower the dissociation constant with the sweet receptor was. Therefore, the intensity of the sweetness activity of brazein was determined by how strongly it interacts with the sweet receptor.

2. Brazein Multivariate and T1R2 - Of T1R3  Interaction research

Three theories have been suggested to date on the interaction between sweet substances and sweet receptors that initiate sweet signal transduction. The first is the AH-B theory, which is based on the assumption that the taste units of all sweet substances are bifunctional groups containing hydrogen bond donors and hydrogen bond acceptors (Khan, 1979). In this theory, it is assumed that sweetness begins with intermolecular hydrogen bonding with AH and B and similar groups of sweet receptors located on the tongue (Khan, 1979). The second theory is the Wedge model. The metabotropic glutamate receptor (mGluR), which is the basis of sweet receptor structure prediction, showed that VFTM is composed of lobe1 and lobe2 in the crystal structure studies. Could be distinguished (Kunishima et al., 2000). When this property is applied to the sweet receptor, the sweet receptor without ligand is labeled as 'free form I (resting open-open form, Roo)', 'active active complex consisting of two open protomers'. Ideally, it was assumed that a mixture of two ligand-free forms of the near free form II (active open-closed form, Aoc) 'was in equilibrium (Temussi, 2009). Stabilization of the active form can occur when complex moieties of small molar mass sweeteners, such as glutamic acid, or when the sweet protein is attached to secondary binding sites on the surface of free form II. This was confirmed by docking calculations of elin, taumartin and model receptors (Temussi, 2002). These three sweet proteins are believed to bind a large margin of sweet receptors with wedge-shaped surfaces in their structure. Finally, the Sweet finger model is based on the assumption that the sweetness of sweet proteins is due to the fact that certain protein lobes interact structurally with sweet receptors (Tancredi et al. al ., 2004). A sweet stimulus occurs as any overhanging part of the sweet protein enters the structurally appropriate portion of the sweet receptor. Recently, among these three theories, wedge model and sweet finger model have been suggested as potential candidates.

In the present invention, in order to identify the initiation process of sweet signal transduction, an experiment was conducted to analyze the interaction between the sweet receptor T1R2-T1R3 and the substrate, the zebrain multivariate. Interaction studies carried out computer-based structural modeling and docking simulations, as well as measurement of actual protein-protein interactions using circular polarization dichroism spectroscopy.

The structure of T1R2 and T1R3 was constructed by referring to the structure of metabotropic glutamate receptor (mGluR), which is known to have similar sequence, through homology-based structural modeling. According to the crystal structure studies of mGluR, mRluR has been reported to have a side-by-side arrangement of VFTM and CRD outside of the cell membrane, and TMD of both monomers to the cell membrane when forming dimers (Kunishima et al. al ., 2000). The heterodimer of T1R2-T1R3 obtained through computer docking simulation first selects a model consisting of side-by-side connected orientation based on these previous studies, and then selects the binding model with the lowest value by comparing the dissociation constants derived from each model. It was. Thereafter, docking simulations of T1R2-T1R3 heterodimers and brazein variants were performed. As a result, it was confirmed that the brazine variants have multiple interactions with the VFTM of T1R2-T1R3. In addition, the dissociation constant values obtained as a result of the docking simulation showed a very large difference from the dissociation constant values calculated through circular dichroism spectroscopy, but it was confirmed that similar tendency was observed for each of the multiple variants of Brazein. When the dissociation constants derived from each experimental method are compared with the absolute threshold values of the brazein variants determined by the SIAM yes-no task sweetness measurement method, the variants with relatively strong sweetness activity have low dissociation constants with the sweet receptors. I could confirm it. The results thus demonstrate that the lower the absolute threshold value of brazein, i.e., the stronger the sweetening activity of the brazein variant, the stronger the interaction with the sweet receptor. The results also suggest that the sweetness of sweet proteins can be measured by spectroscopic measurements.

In previous studies on the interaction between brazein and receptors, the variants of brazein and receptor were prepared based on the Wedge model to derive key amino acids that contribute to the multiple interactions of brazein and the receptor (Assadi-Porter et. al ., 2010). On the other hand, the brazein multivariates in this study mainly shifted the residues of the flexible loop based on the sweet finger model. For example, histidine residue 30 belongs to flexible loop II, leading from residues 29 to 32. The results of this study, which are obtained from multiple variants of the residues of the flexible loop, are good data to support the sweet finger model, which is one of the interaction models of sweet substances and sweet receptors.

Total Comparison of Sweetness Receptor-Brazin Interaction Studies Brazzein mutants Sweetness in comparison to sucrose Circular dichroism Computer docking simulation (g / g) (molecular) Dissociation constant (M) Gibbs binding energy
(kcal / mol) Dissociation constant
(μM) des-pE1M-brazzein 693.38 1.29E + 04 3.81E-02 -12.88 4.00E-04 H30R / E35D / E40A 2638.84 4.85.E + 04 6.44E-03 -15.32 6.64E-06 A1ins / H30R / E35D / E40A 3217.03 6.00E + 04 2.69E-03 -16.01 1.15E-06

Ⅵ. conclusion

In the present invention, the effect of amino acid residues on the sweet taste activity and structural stability of the sweet protein brazein multivariate and protein-protein interaction with the sweet receptor was analyzed. First, the sweetness threshold experiments were conducted using the SIAM yes-no task method to compare the sweetness activity of the Brazein multivariate. As a result, it was confirmed that the amino acid residues of the N-terminal and C-terminal have a greater influence on the sweetness activity of brazein than the amino acid residues of other positions.

In order to predict the interaction between sweet receptors and brazein, we conducted docking simulations of the multiple variants and sweet receptors through computer modeling to analyze the binding patterns of the variants and the sweet receptors and predict the dissociation constants. In addition, after expressing and purifying brazein multivariants and sweet acceptors using a yeast expression system, complexes were formed and measured by circular dichroism spectroscopy. And the dissociation constant was calculated using the elliptic ratio derived through this. As a result, the dissociation constants between Brazein multivariants and sweet receptors predicted by computer modeling were different from those calculated by circular dichroism spectroscopy. However, the dissociation constants derived by each method showed similar tendency according to the type of multivariate, and as a result, it was confirmed that the brazein multivariate had an average lower dissociation constant than the brazein wild type subtype. This suggests that stronger sweet signal signaling leads to stronger interaction with sweet receptors, resulting in stronger sweet activity.

On the other hand, brazein multivariate maintained the sweet taste for a long time under high temperature conditions as well as brazein wild type. Through this, it was confirmed that the structural change caused by multiple mutations did not significantly affect the framework structure maintaining the stability of brazein.

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<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Method for preparing novel multi-mutated Brazzein having higher          sweetness <130> P17U21C0353 <150> KR 10-2017-0025516 <151> 2017-02-27 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> brazzein minor type <400> 1 Asp Lys Cys Lys Lys Val Tyr Glu Asn Tyr Pro Val Ser Lys Cys Gln   1 5 10 15 Leu Ala Asn Gln Cys Asn Tyr Asp Cys Lys Leu Asp Lys His Ala Arg              20 25 30 Ser Gly Glu Cys Phe Tyr Asp Glu Lys Arg Asn Leu Gln Cys Ile Cys          35 40 45 Asp Tyr Cys Glu Tyr      50 <210> 2 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> brazzein minor type gene <400> 2 gacaaatgca aaaaagttta cgaaaattac ccagtttcta agtgccaact ggccaatcaa 60 tgcaattacg attgcaagct tgataagcat gctcgatctg gagaatgctt ttacgatgaa 120 aagagaaatc ttcaatgcat ttgcgattac tgcgaatac 159 <210> 3 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> brazzein multiple mutated variant H30R_E35D_E40A <400> 3 Asp Lys Cys Lys Lys Val Tyr Glu Asn Tyr Pro Val Ser Lys Cys Gln   1 5 10 15 Leu Ala Asn Gln Cys Asn Tyr Asp Cys Lys Leu Asp Lys Arg Ala Arg              20 25 30 Ser Gly Asp Cys Phe Tyr Asp Ala Lys Arg Asn Leu Gln Cys Ile Cys          35 40 45 Asp Tyr Cys Glu Tyr      50 <210> 4 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> brazzein multiple mutated variant H30R_E35D_E40A gene <400> 4 gacaagtgta agaaggtcta cgaaaactac ccagtctcta agtgtcaatt ggctaaccaa 60 tgtaactacg actgtaagtt ggacaagaga gctagatctg gtgactgttt ctacgacgcc 120 aagagaaact tgcaatgtat ctgtgactac tgtgaatac 159 <210> 5 <211> 54 <212> PRT <213> Artificial Sequence <220> <223> brazzein multiple mutated variant A1 ins_H30R_E35D_E40A <400> 5 Ala Asp Lys Cys Lys Lys Val Tyr Glu Asn Tyr Pro Val Ser Lys Cys   1 5 10 15 Gln Leu Ala Asn Gln Cys Asn Tyr Asp Cys Lys Leu Asp Lys Arg Ala              20 25 30 Arg Ser Gly Asp Cys Phe Tyr Asp Ala Lys Arg Asn Leu Gln Cys Ile          35 40 45 Cys Asp Tyr Cys Glu Tyr      50 <210> 6 <211> 162 <212> DNA <213> Artificial Sequence <220> <223> brazzein multiple mutated variant A1 ins_H30R_E35D_E40A gene <400> 6 gccgacaagt gtaagaaggt ctacgaaaac tacccagtct ctaagtgtca attggctaac 60 caatgtaact acgactgtaa gttggacaag agagctagat ctggtgactg tttctacgac 120 gccaagagaa acttgcaatg tatctgtgac tactgtgaat ac 162 <210> 7 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> a-Mating signal sequence <400> 7 atgaaattct ctactatatt agccgcatct actgctttaa tttccgttgt tatggctgct 60 ccagtttcta ccgaaactga catcgacgat cttccaatat cggttccaga agaagccttg 120 attggattca ttgacttaac cggggatgaa gtttccttgt tgcctgttaa taacggaacc 180 cacactggta ttctattctt aaacaccacc atcgctgaag ctgctttcgc tgacaaggat 240 gatctcgaga aaagagaggc tgaagctaga agagctagat ctcctagggg taccgtcgac 300 ggcgcgcctg cggccgctta a 321

Claims (5)

Brazein multiple variant represented by amino acid sequence of SEQ ID NO: 5.
A nucleic acid molecule encoding the brazein multiple variant of claim 1.
A recombinant vector comprising the nucleic acid molecule of claim 2 operably linked with (i) a promoter and (ii) said promoter.
A host cell transformed with the recombinant vector of claim 3.
Method for producing brazein multiple variants comprising the following steps:
(a) culturing the host cell of claim 4; And
(b) separating the brazein multiple variant protein from the cultured host cell.

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