KR20110097042A - Method for preparing novel multi-mutated brazzein having higher sweetness - Google Patents

Abstract

The present invention relates to a novel brazein quaternary variant having a high sweet taste and a method for preparing the same, and more particularly, to a brazein quaternary variant having a higher sweetness than the sweet protein of wild-type brazein, and a method for preparing the same. It relates to a food composition for enhancing the sugar content. The brazain quaternary variant of the present invention has an equivalent thermal stability as compared to conventional brazein and has a sweetness of at least 15 to 50 times higher than that of the conventional brazein. Accordingly, the brazein quaternary variant of the present invention can be used in combination with other sweeteners such as sugar (sucrose) in a small amount and can further replace it, and can be variously used as an additive in food preparation. Can be.

Description

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

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

White sugar (white sugar) is one of the sugars, or more precisely, one of the simple carbohydrates called sucrose (sugar), also called saccharose (chemical term for sugar). For a long time, sugar has been commonly used as a sweetener. However, due to the dangers of sugar, the World Health Organization (WHO) recently issued a recommendation to limit the use of sugar to the current 10%. The US government (New York City, July 2003, New Jersey, September 2004, Illinois, March 2006, Connecticut) April 2006) banned the sale of sugar-based foods and high content beverages. In addition, in Korea, a National Obesity Countermeasure Committee was formed to announce a policy to label sugar risk warnings. Therefore, the emergence of a new sweetener that can replace sugar is required. In 1879, Ira Remson of the United States and Constantine Falberg of Germany discovered saccharin, which is 500 times sweeter than sugar. Saccharin has the advantage of being excreted without being broken down in the body, but it has caused controversy that it is carcinogenic. Eventually, it was concluded that it is harmless to the human body, but it is not used much recently because of the disadvantage of using aftertaste. In 1937, at the University of Illinois in the United States, sodium cyclohexylsulfamate was found to be sweet. It was called cyclamate under the brand name and began to be used in the early 1950s, and dominated the world sweetener market in the 1960s. However, since it proved to be a carcinogenic substance, its use has been banned in Korea since the 1970s. The most widely used artificial sweetener in recent years is aspartame, discovered by James Schretter in 1965. Aspartame has a sugar content of about 180 to 200 times higher than sugar. Most diet drinks currently on the market contain aspartame, which, when ingested, produces phenylalanine during metabolism. Therefore, it is disadvantageous that phenylketonuria patients who are inherently deficient in a specific enzyme that degrades phenylalanine are not available.

Research into the development of natural sweeteners as well as such artificial sweeteners has continued, and as a result, there is a substance called stevioside in the leaves of the Stevia rabaudiana, which is classified as an herb. Indigenous peoples from the borders of Paraguay and Brazil have used this substance as a sweetener for more than 400 years. It is also added to Korean soju, which is 200 times sweeter than sugar.

Recently, interest in sweet protein extracted from tropical fruits has increased. Thaumatin is a perennial plant called Thaumatococcus , a miracle fruit in West Africa. It is a protein contained in the fruit of daniellii ) and is 2,000 to 3,000 times sweeter than sugar. Monellin is a protein derived from the fruit of a vine-like plant called Serendipiti berry, which grows in the rain forests of Africa, and is 3,000 times sweeter than sugar. However, it is not easy to cultivate and it is difficult to extract from the fruit. In addition, the thermal stability is low, the heat treatment during the food processing process loses the three-dimensional protein structure has a disadvantage that does not give a sweet taste. Currently, research is being conducted to improve thermal stability using protein engineering techniques to overcome these disadvantages.

On the other hand, brazzein is a sweet protein first extracted from the fruit of Pentadipladra brazzeana Baillon in West Africa [Ming et al., FEBS Letters , 355: 106-108, 1994]. Brazein has a sweet taste of about 500 to 2,000 or more times as compared to sucrose [Jin et al., Chem . Senses . 28: 491-498, 2003]. There are two types, major type and minor type. The major type of plant-derived brazein has 54 amino acids, including pyroglutamic acid residues at its amino terminus. On the other hand, minor types of brazein have only 53 amino acid residues without pyroglutamic acid residues at the amino terminus, and are about twice as sweet as major types of brazein [Assadi-Porter et al. ., Arch .. Biochem . Biophys. 376: 259-265, 2000. Brazein is the smallest sweet protein, has a molecular weight of about 6.5 kDa, and is a monomer composed of one subunit. It consists of a single polypeptide and consists of one α-helix and two β-sheets. Brazein has eight cysteine residues to form four disulfide bonds in the molecule, resulting in very high thermal stability. In addition, the solubility and pH stability in water are very high [Gao et al., Int . J. Biol . macromol . 24: 351-359, 1999.

US Pat. No. 6,274,707 and Asadi-Porter et al., Arch .. Biochem. Biophys. 376: 259-265, 2000 describe recombinant braze as described above by using a genetic engineering method using E. coli. It describes a method of producing a protein, which synthesizes a gene encoding brazein and inserts it into a recombinant vector containing a SNase to generate a new transformation vector, and then introduces it into E. coli to fusion with the final SNase. Disclosed are methods for expressing and purifying proteins. However, brazein expressed by fusion with SNase produces an insoluble inclusion body, refolds it, and removes SNase and methionine by using cyanobromide (CNBr). Due to the separation and purification, it is technically complicated and difficult to commercialize by mass production.

Accordingly, the present inventors patented a polynucleotide containing E. coli pelB signal sequence and brazein gene and a method for producing brazein using the same through prior studies (Domestic Patent Registration No. 809100). have. In addition, the present inventors have patented a method for preparing a variant and multiple variants of amino acids at specific positions which are expected to not affect the structure among the amino acids constituting brazein in order to find a natural sweetener having high thermal stability and excellent sweetness. The application (domestic patent application 2007-0117013, domestic patent application 2008-0019008, international patent application PCT / KR2009 / 04855) was performed.

The present inventors completed the present invention by searching for and developing quaternary variants having a higher sweetness while having thermal stability equivalent to that of conventional brazein.

Accordingly, it is an object of the present invention to provide new brazein quaternary variants and uses thereof having thermal stability equivalent to that of conventional brazein subtypes and having a sweet taste of at least 15-50 times.

In order to achieve the above object, the present invention provides a new brazein quaternary variant sweeter than the conventional natural brazein.

In order to achieve another object of the present invention, the present invention provides a polynucleotide encoding the brazein quaternary variant.

In order to achieve another object of the present invention, the present invention provides a recombinant expression vector comprising the polynucleotide, E. coli transformed with the vector and a method for producing a brazein quaternary variant using the E. coli.

In order to achieve another object of the present invention, the present invention provides a food composition for enhancing sugar content comprising a brazein quaternary variant as an active ingredient.

The brazain quaternary variant of the present invention has excellent thermal stability like the conventional braze and has a sweet taste of at least 15 times and up to 50 times higher than that of the conventional brazein. Therefore, the brazein quaternary variant of the present invention can replace other sweeteners such as sugar (sucrose) in a small amount, and can be used in various ways as a sweetener in food compositions and the like.

1 is a schematic diagram showing a process for preparing a recombinant expression vector for preparing a brazein quaternary variant according to the present invention.
Figure 2 shows the results of electrophoresis to confirm the expression of the brazein multiple quaternary variants according to the present invention [lane M: molecular weight marker, lane 1: brazein fourth variant (H30R_E35D_E40A_E52K) crude extract, lane 2: bra Jane Quaternary Variants (H30R_E35D_E40A_E52R) crude extract, Lane 3: Brazein Quaternary variants (H30R_E35D_E40A_E52A) crude extract, Lane 4: Brazain Quaternary variants (H30R_E35D_E40A_E52H) crude extract, D: R_E35E35E30E 35E Crude extracts].
Figure 3 shows the results of electrophoresis to confirm the purification of brazein multiple quaternary variants according to the present invention (lane M: molecular weight marker, lane 1: purified brazein quaternary variant (H30R_E35D_E40A_E52K), lane 2: purification Brazaine Quaternary Variants (H30R_E35D_E40A_E52R), Lane 3: Purified Brazain Quaternary Variants (H30R_E35D_E40A_E52A), Lane 4: Purified Brazain Quaternary Variants (H30R_E35D_E40A_E52H), Lane 5: Purified Brazine D5E40E40E40E40E40E )].
Figure 4 is the result of performing the thermal stability by selecting the brazein variant showing a high sweetness after measuring the sweetness of the brazein variant according to the present invention [lane 1: relative activity of the brazein minor (minor type) after heat treatment, Lane 2: Relative activity of brazein quaternary variant (H30R_E35D_E40A_E52K) after heat treatment, lane 3: Relative activity of brazein quaternary variant (H30R_E35D_E40A_E52R) after heat treatment, lane 4: Relative activity of brazein quaternary variant (H30R_E35D_E40A) after heat treatment Relative activity, lane 5: relative activity of brazein quaternary variant after heat treatment (H30R_E35D_E40A_E52H), lane 6: relative activity of brazein quaternary variant after heat treatment (H30R_E35D_E40A_E52D).

Hereinafter, the present invention will be described in detail.

The present invention is characterized in that it provides a novel brazein quaternary variant with better sugar content than conventional wild type brazein.

The brazine quaternary variant of the present invention has a residue of glutamic acid, which is the 52nd amino acid of the brazein tertiary variant (H30R_E35D_E40A) having the amino acid sequence of SEQ ID NO: 31, of SEQ ID NO: 18 substituted with a lysine residue. Brazein variants having an amino acid sequence, brazein variants having an amino acid of SEQ ID NO: 19 substituted with an arginine residue, brazein variants having an amino acid of SEQ ID NO: 20 substituted with an alanine residue, histidine A brazein variant having an amino acid of SEQ ID NO: 21 substituted with a residue may be a brazein variant having an amino acid of SEQ ID NO: 22 substituted with an aspartic acid residue.

On the other hand, the present invention provides a polynucleotide encoding the amino acid sequence of the E. coli pelB signal sequence and the brazein quaternary variant. E. coli pelB signal sequence is a type of cell membrane gap signal sequence of Escherichia coli (Rietsch et al., Proc . Natl . Acad . Sci. USA 93: 130408-13053, 1996, Raina et al., Ann . Rev. Microbiol . 51: 179-202, 1997, Sone et al., J. Biol . Chem . 272: 10349-10352, 1997), the synthesis of the brazein of the present invention can be transferred to the cell membrane gap of E. coli to induce accurate disulfide bonds, inhibit the formation of insoluble aggregates of brazein protein, and minimize unnecessary E. coli-derived protein This can facilitate the purification process. E. coli pelB signal sequence of the present invention preferably has a nucleotide sequence of SEQ ID NO: 28, and is linked to have the same frame when translated into a protein on the 5 'top of the nucleotide sequence of the brazein variant of the present invention.

The amino acid sequence of the brazein quaternary variant has an amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, preferred polynucleotides SEQ ID NO: 12 for the amino acid sequence of SEQ ID NO: 18 Nucleotide sequence of SEQ ID NO: 13 for the amino acid sequence of SEQ ID NO: 19, nucleotide sequence of SEQ ID NO: 14 for the amino acid sequence of SEQ ID NO: 20, nucleotide sequence of SEQ ID NO: 15 for the amino acid sequence of SEQ ID NO: 21, and sequence The amino acid sequence of No. 22 may have a nucleotide sequence of SEQ ID NO: 16.

In addition, the present invention provides a promoter and a recombinant expression vector for brazein quaternary variant expression comprising the polynucleotide operably linked thereto.

The term 'promoter' refers to a DNA sequence that regulates 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. Its function or expression is affected 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 promoter 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 E. coli pelB Promoter, U6 promoter, cytomegalovirus (CMV) promoter, SV40 promoter, CAG promoter (Hitoshi Niwa et al., Gene, 108: 193-199, 1991; Monahan et al., Gene Therapy 7: 24-30, 2000 ? , CaMV 35S promoter (Odell et al., Nature 313: 810-812, 1985), Rsyn7 promoter (US Patent Application Serial No. 08 / 991,601), rice actin promoter (McElroy et al ., Plant Cell 2: 163-171, 1990), ubiquitin promoters (Christensen et al., Plant Mol. Biol. 12: 619-632, 1989), ALS promoters (US Patent Application No. 08 / 409,297) and the like. In addition, U.S. Patents 5,608,149; The promoters disclosed in 5,608,144 5,604,121 5,569,597, 5,466,785, 5,399,680 5,268,463, 5,608,142, and the like can all be used. E. coli pelB promoter can be used as the most preferred promoter in the present invention.

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. .

Vectors of the invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors include signal or leader sequences for membrane targeting or secretion in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation signals, and enhancers (promoter genes) Can be. In addition, the expression vector includes a selection marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, includes the origin of replication.

Recombinant expression vector for the expression of the brazain quaternary variant of the present invention is preferably pET26B (+)-brazein (H30R_E35D_E40A_E52K), pET26B (+)-brazein (H30R_E35D_E40A_E52R), pET26B (+)-brain ( H30R_E35D_E40A_E52A), pET26B (+)-Brazin (H30R_E35D_E40A_E52H), pET26B (+)-Brazin (H30R_E35D_E40A_E52D), which is the main sequence of pET26B (+)-Brazin (H30R40A35D_A35D_A35D_E35D) , 3, 4, 5, 6, 7, 8, 9, 10, 11 can be prepared by site-directed mutagenesis method using the primers.

The present invention also provides E. coli transformed with the recombinant expression vector. The E. coli is transformed according to a conventional transformation method with the recombinant expression vector, wherein the transformation includes any method of introducing a nucleic acid into the host cell, a standard suitable for the host cell as known in the art The technique can be selected and performed. These methods include electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microprojectile bombardment, electroporation, PEG-mediated fusion (PEG) mediated fusion, microinjection, liposome-mediated methods, etc., but are not limited thereto.

In another aspect, the present invention provides a method for producing a brazine quaternary variant comprising culturing the transformed Escherichia coli, separating the membrane gap protein from the cultured Escherichia coli, and heat treatment to purify the brazein.

The transformed E. coli of the present invention can be cultured under appropriate media and conditions so that the polynucleotide encoding the brazein quaternary variant is expressed, which is the same or similar to the culture conditions of conventional E. coli. While the transformed Escherichia coli is cultured, brazein including a pelB signal sequence is expressed by an expression control sequence in an expression vector, and the expression of brazein in the present invention is IPTG (isopropyl-beta-D-thiogalactopyranoside). Even without compounds that promote the expression of conventional inducible promoters. The brazein containing the expressed pelB signal sequence is moved to the cell membrane gap of E. coli by the signal sequence, and the signal sequence is removed by the signal peptidase of E. coli to synthesize brazein.

In order to isolate brazein expressed in transformed cells from the cell membrane gap of Escherichia coli, a known method of separating proteins from the cell membrane gap of Escherichia coli (Snyder et al., J. Bacteriology) 177: 953963, 1995), for example, but not limited to, 30 mM tris-hydrochloric acid (Tri-HCl, pH 8) containing 20% sucrose after collecting cultured E. coli. ), And eluted the protein of the cell membrane gap of Escherichia coli using EDTA (pH 8) solution and MgSO ₄ .

The method of separating brazein of the present invention from the cell membrane gap protein of E. coli can be carried out through various separation and purification methods known in the art, for example, salting out (ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation The brazein of the present invention can be isolated by applying techniques such as (precipitation of protein fractions using acetone, ethanol, etc.), dialysis, gel filtration, ion exchange chromatography, reverse phase column chromatography and affinity chromatography, alone or in combination. have. Since the brazein of the present invention is heat stable, the method of separating the brazein may be preferably performed by heat treatment. Although not limited thereto, the heat treatment is preferably performed by heat-denatured proteins other than brazein by heating at 70-90 ° C. for 15-60 minutes, followed by heat-denatured protein by centrifugation at 18000 g for 30 minutes at 4 ° C. You can separate the brazein.

As described above, the enzymatic properties of brazein quaternary variants having amino acids consisting of SEQ ID NOs: 18, 19, 20, 21, and 22 having higher sweetness and high thermal stability are summarized as follows.

1) Molecular Weight: 6.5 kDa

2) high thermal stability and acid resistance

3) high water solubility

4) Brazein Quaternary Variants Sweetness Ratio of Protein Subtype of Brazein: 15-50 times or more

5) The ratio of sweetness of the 4th variant of brazein to sucrose of 1g / 100ml: about 30,000 ~ 100,000 times or more

As described above, the brazein quaternary variant according to the present invention is a subtype protein of brazein, that is, a wild type subtype of protein and the present product, which are expressed and purified in the inventor's patent registration (Domestic Patent Registration No. 809100). Compared with the brazein variants expressed and purified in the inventors' domestic patent application No. 2007-0117013 and domestic patent application No. 2008-0019008, the characteristics such as thermal stability and acid resistance and water solubility are similar to those of wild type brazein and are higher. It is characterized by having a novel amino acid sequence exhibiting a sweet taste.

In addition, brazein quaternary variants in this invention are described in US Pat. No. 6,274,707; It exhibits a higher sweetness and effect than the brazein variants known by No. 7,153,535.

For reference, reference may be made to the above-mentioned nucleotide and protein operations (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982); Sambrook et al. ., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989); Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182. Academic Press. Inc., San Diego, CA (1990). )).

In an embodiment of the present invention, a recombinant vector encoding three multiple quaternary variants in which the 52 th glutamic acid residue of the brazein subtype is mutated to a lysine residue, an arginine residue and an alanine residue is prepared based on the tertiary variant (H30R_E35D_E40A). The expression, purification and activity (sweetness) were measured in the same manner as in the above example. As a result, it was possible to obtain a high-purity Brazain multiple quaternary variant, having the same stability as the Brazein subtype protein and having a sweet taste of at least 15 to 50 times sweeter than the Brazein subtype protein Variants could be produced. Through this, it can be seen that the brazein quaternary variants prepared above can also be used as an excellent sweetener.

Accordingly, the present invention provides a food composition for enhancing sugar content comprising the brazein quaternary variant of the present invention as an active ingredient.

The food composition of the present invention includes all forms such as functional foods, nutritional supplements, health foods 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), edible vegetable oils, margarine, vegetable protein, retort It can be prepared by adding the brazein quaternary variant of the present invention to food, frozen foods, various seasonings (eg, miso, soy sauce, sauce, etc.).

In addition, in order to use the food composition containing the brazein quaternary variant of the present invention in the form of a food additive, it can be prepared in powder or concentrate form.

The preferred content of the brazein quaternary variant of the present invention in the food composition of the present invention may include about 0.01 to 10% by weight relative to the total composition weight.

Hereinafter, the present invention will be described in more detail by way of examples. It should be noted, however, that the following examples are illustrative of the invention and are not intended to limit the scope of the invention.

Example  One: Brazane  4th Variants  Of polynucleotides encoding Cloning

In order to prepare a brazain multiple variant having a higher sweetness than brazein, one specific amino acid constituting the tertiary variant of brazein (H30R_E35D_E40A) was selected and mutated to another specific amino acid.

First, through the structural analysis of brazein as a specific amino acid to be mutated, the 52nd residue having the outer chain (side chain) facing outward and polarity was selected. Based on this structural information, the sequence of SEQ ID NO: 1 from which the unnecessary "ATG" sequence was removed to synthesize the protein of the third variant (H30R_E35D_E40A) of brazein in E. coli used in the inventors' patent registration (Domestic Patent Registration No. 809100) A primer pair having a sequence complementary to each of the five primers specified in Table 1 below and a recombinant expression vector (pET26B (+)-Brazzein (H30R_E35D_E40A), Korean Patent Publication No. 2009-93470) containing Was synthesized. The primers were designed in consideration of the external residue length and electrical properties of the specific amino acid (SEQ ID NO: 17) constituting the brazein tertiary variant (H30R_E35D_E40A) protein (see Table 1). Using the primers of Table 1 and the QuikChange ^™ Site-Directed Mutagenesis Kit (Stratagene, USA), the five Brazain variants were substituted for position 52 of the Brazein tertiary variant (H30R_E35D_E40A) protein according to the manufacturer's instructions. The expression vector containing the nucleotide which carried out was obtained. At this time, the underlined portion of the primer sequence of Table 1 below shows the changed sequence for the brazein variant.

Primers used for the preparation of the Brazain 4th variant location Before variation
Amino acid residues After variation
Amino acid residues Used
primer Remarks Before variation After variation SEQ ID NO: 52 Glu (E) Lys (K) tac tgc aag tac taa
(SEQ ID NO: 23) positive positive SEQ ID NOs: 2, 3 Arg (R) tac tgc cgt tac taa
(SEQ ID NO: 24) positive SEQ ID NOs: 4, 5 Ala (E) tac tgc gct tac taa
(SEQ ID NO: 25) neutral SEQ ID NOs: 6, 7 His (H) tac tgc cac tac taa
(SEQ ID NO 26) positive SEQ ID NOs: 8, 9 Asp (D) tac tgc gac tac taa
(SEQ ID NO 27) negative SEQ ID NOs: 10, 11

Specifically, 10 ng of pET26B (+)-brazein (H30R_E35D_E40A) vector, 0.2 mL of dNTP mixture at a final concentration of 0.2 mM each, 125 ng of the primers specified in Table 1 above, 5 μl of 10 × reaction buffer, PfuTurbo DNA polymerase (2.5 U / μl, Stratagene, USA) PCR was performed with the reaction solution with a total of 50 μl containing 1 μl. The PCR reaction was denatured at 95 ° C. for 1 minute, then 95 ° C. for 30 seconds, and 55 ° C. for 1 minute. The reaction was conducted 20 times at 68 ° C. for 15 minutes and the final reaction was performed at 68 ° C. for 15 minutes. After the reaction, the amplified product was confirmed by 1.0% agarose gel electrophoresis, and the amplified product was treated with Dpn I for 1 hour at 37 ° C. Then, E. coli XL1-Blue, a supercompetent cell, was transformed. The transformed XL1-Blue was selected by incubating for 12 hours in an LB-agar plate containing 50 µg / ml kanamycin, and the selected colonies were cultured in LB-agar medium. DNA was isolated from. It was confirmed by genetic analysis that the isolated DNA is linked to the E. coli pelB signal sequence and the nucleotides encoding the respective brazein variants. It is named by the SEQ ID NO and nucleotide name indicated in Table 2 below. One letter designating each amino acid is designated according to a known amino acid code.

Polynucleotide name and sequence number encoding the Brazein quaternary variant Brazain Quaternary Variant Locations Each of the brazein quaternary variants
Nucleotide name encoding SEQ ID NO: H30R_E35D_E40A_E52K E. coli pelB + Brazein (H30R_E35D_E40A_E52K) gene SEQ ID NO: 12 H30R_E35D_E40A_E52R E. coli pelB + Brazein (H30R_E35D_E40A_E52R) gene SEQ ID NO: 13 H30R_E35D_E40A_E52A E. coli pelB + Brazein (H30R_E35D_E40A_E52A) gene SEQ ID NO: 14 H30R_E35D_E40A_E52H E. coli pelB + Brazein (H30R_E35D_E40A_E52H) gene SEQ ID NO: 15 H30R_E35D_E40A_E52D E. coli pelB + Brazein (H30R_E35D_E40A_E52D) gene SEQ ID NO: 16

The result of recombinant expression vectors (pET26B (+) for a bra Jane the fourth variant of the total 5 kinds - Brazzein (H30R_E35D_E40A_E52K), pET26B (+) - Brazzein (H30R_E35D_E40A_E52R), pET26B (+) - Brazzein (H30R_E35D_E40A_E52A), pET26B ( +)-Brazzein (H30R_E35D_E40A_E52H), pET26B (+)-Brazzein (H30R_E35D_E40A_E52D)) could be produced, again E. coli Transformation with BL21 (star) was used for mass expression (see FIG. 1).

Example  2: Brazane  4th Mutant  Expression and Purification

<2-1> Brazane  4th Mutant  Expression

E. coli BL21 (star), each of which introduced the five recombinant expression vectors for the brazein quaternary variants prepared in Example 1, was derived from a protein in a 1 L LB medium containing 30 µl / ml kanamycin. Incubated at 37 ° C. for 12 hours without the addition of phosphorous IPTG (isopropyl β-D-thoigalactopyranoside) to express the respective brazein variants in each transformed Escherichia coli [see FIG. 2].

<2-2> Brazane  4th Mutant  refine

Each Escherichia coli cultured in Example <2-1> was collected by centrifugation at 8,000 g for 10 minutes. After collection, the cells were suspended in 30 mM Tris-HCl (pH 8.0) solution containing 20% sucrose, and then 0.5 M EDTA (pH 8.0) solution was added to a final concentration of 1 mM and room temperature. The mixture was stirred slowly for 10 minutes. It was centrifuged at 10,000 g, 4 ° C. for 10 minutes and the supernatant was removed, then cold 5 mM MgSO ₄ was added and stirred slowly on ice for 10 minutes. In this process, proteins of the periplasm are released into the buffer solution. Thereafter, the supernatant was separated by centrifugation at 10,000 g for 10 minutes at 4 ° C., and heat treated at 80 ° C. for 30 minutes to purify the brazein quaternary variants present in the periplasm. Thereafter, dialysis was performed for 24 hours with distilled water, followed by freeze-drying to obtain purified brazein quaternary variants represented by the sequence numbers shown in Table 3 below, and the degree of purification was primarily confirmed through SDS-PAGE.

Brazain 4th variant name and sequence number Brazein 4th
Variant location
Name of each brazein variant
SEQ ID NO: H30R_E35D_E40A_E52K Brazzein (H30R_E35D_E40A_E52K) SEQ ID NO: 18 H30R_E35D_E40A_E52R Brazzein (H30R_E35D_E40A_E52R) SEQ ID NO: 19 H30R_E35D_E40A_E52A Brazzein (H30R_E35D_E40A_E52A) SEQ ID NO: 20 H30R_E35D_E40A_E52H Brazzein (H30R_E35D_E40A_E52H) SEQ ID NO: 21 H30R_E35D_E40A_E52D Brazzein (H30R_E35D_E40A_E52D) SEQ ID NO: 22

As a result, the brazein protein was purified to high purity, and the molecular weight thereof was about 6.5 kDa [see FIG. 3].

Example  3: Brazane  4th Mutant  Activity (sweetness) and thermal stability measurements

<3-1> Brazane  4th Mutant  Sweetness measurement

Since the recombinant brazein in the present invention is not a compound of the sugar family having a cyclic ring, sweetness cannot be measured using a sugar meter, and thus activity was measured using human taste. The sugar measurement was performed on 20 subjects who were trained to have a similar level of sucrose minimum sucrose with a solution of sucrose, and the initial sweet taste concentration using each variant. Measured. In addition, the comparative sweetness ratio of brazein was 1g / 100ml for the sweetness of the sucrose solution, and the minimum amount of stimulation for the sweetness of the brazein subtype protein was 500 µg / 100ml, and was calculated based on this. Ie 1 / 0.0005 = 2000 for the brazein subtype.

Sweetness test results for each of the brazein quaternary variants Primary variant
location SEQ ID NO: Lowest amount of irritation with an initial sweet taste
(Μ / 100 ml) Brussels Quaternary Variants vs. Sucrose (1g / 100ml)
(Brazin subtype: 2000) Brazein type comparison sweetness
Increase drainage H30R_E35D_E40A_E52K SEQ ID NO: 18 110 100,000 50 H30R_E35D_E40A_E52R SEQ ID NO: 19 230 56,000 28 H30R_E35D_E40A_E52A SEQ ID NO: 20 270 42,000 21 H30R_E35D_E40A_E52H SEQ ID NO: 21 280 38,000 19 H30R_E35D_E40A_E52D SEQ ID NO: 22 300 30,000 15

As a result, as shown in Table 4 above, the brazein (H30R_E35D_E40A_E52K) represented by SEQ ID NO: 18, the brazein (H30R_E35D_E40A_E52R) represented by SEQ ID NO: 19, and the brazein represented by SEQ ID NO: 20 (H30R_E35D_E40A_E52A), and SEQ ID NO: 21 When compared to brazein (H30R_E35D_E40A_E52H) and brazein (H30R_E35D_E40A_E52D) represented by SEQ ID NO: 22 with the protein of the brazein subtype, at least 15 to about 50 times or more (minimum to 1 g / 100 ml of sucrose) Up to about 100,000 times). In particular, brazein (H30R_E35D_E40A_E52K) showed the highest sweetness increase rate.

<3-2> Brazane  4th Mutant  Thermal stability measurement

Based on the results measured in Example <3-1>, a brazein variant exhibiting a high sweet taste, that is, a brazein (H30R_E35D_E40A_E52K) represented by SEQ ID NO: 18, a brazein represented by SEQ ID NO: 19 (H30R_E35D_E40A_E52R), SEQ ID NO: 50 mM tris-HCl (100T) -HCl (100 mM tris-HCl) with 100 mg of brazine variants such as brazein (H30R_E35D_E40A_E52A) represented by SEQ ID NO: 21, brazein (H30R_E35D_E40A_E52H) represented by SEQ ID NO: 21, and H. B. , pH 8.0) and 20 subjects in the same manner as in Example <3-1> on the basis of the sweetness before heat treatment for each of the brazein quaternary variants after heating for 4 hours at 80 ℃ The degree of sweetness change was measured, and the result is shown in relative activity, and is shown in FIG. 4.

As a result, a series of Brine variants, such as brazein (H30R_E35D_E40A_E52K) represented by SEQ ID NO: 18, brazein (H30R_E35D_E40A_E52R) represented by SEQ ID NO: 19, and brazein (H30R_E35D_E40A_E52A) represented by SEQ ID NO: 20 It was shown to maintain stability.

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Method for preparing novel mutated and 4th-mutated Brazzein          having higher sweetness <160> 28 <170> KopatentIn 1.71 <210> 1 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> E. coli pelB + Brazzein gene (H30R_E35D_E40A) <400> 1 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgct aagcgtgcta gatctggaga ttgcttttac 180 gatgccaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> H30R_E35D_E40A_E52K forward primer <400> 2 cttcaatgca tttgcgatta ctgcaagtac taa 33 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> H30R_E35D_E40A_E52K reverse primer <400> 3 ttagtacttg cagtaatcgc aaatgcattg aag 33 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> H30R_E35D_E40A_E52R forward primer <400> 4 cttcaatgca tttgcgatta ctgccgttac taa 33 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> H30R_E35D_E40A_E52R reverse primer <400> 5 ttagtaacgg cagtaatcgc aaatgcattg aag 33 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> H30R_E35D_E40A_E52A forward primer <400> 6 cttcaatgca tttgcgatta ctgcgcttac taa 33 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> H30R_E35D_E40A_E52A reverse primer <400> 7 ttagtaagcg cagtaatcgc aaatgcattg aag 33 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> H30R_E35D_E40A_E52H forward primer <400> 8 cttcaatgca tttgcgatta ctgccactac taa 33 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> H30R_E35D_E40A_E52H reverse primer <400> 9 ttagtagtgg cagtaatcgc aaatgcattg aag 33 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> H30R_E35D_E40A_E52D forward primer <400> 10 cttcaatgca tttgcgatta ctgcgactac taa 33 <210> 11 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> H30R_E35D_E40A_E52D reverse primer <400> 11 ttagtagtcg cagtaatcgc aaatgcattg aag 33 <210> 12 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> E. coli pelB + Brazzein gene (H30R_E35D_E40A_E52K) <400> 12 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgct aagcgtgcta gatctggaga ttgcttttac 180 gatgccaaga gaaatcttca atgcatttgc gattactgca agtactaa 228 <210> 13 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> E. coli pelB + Brazzein gene (H30R_E35D_E40A_E52R) <400> 13 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgct aagcgtgcta gatctggaga ttgcttttac 180 gatgccaaga gaaatcttca atgcatttgc gattactgcc gttactaa 228 <210> 14 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> E. coli pelB + Brazzein gene (H30R_E35D_E40A_E52A) <400> 14 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgct aagcgtgcta gatctggaga ttgcttttac 180 gatgccaaga gaaatcttca atgcatttgc gattactgcg cttactaa 228 <210> 15 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> E. coli pelB + Brazzein gene (H30R_E35D_E40A_E52H) <400> 15 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgct aagcgtgcta gatctggaga ttgcttttac 180 gatgccaaga gaaatcttca atgcatttgc gattactgcc actactaa 228 <210> 16 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> E. coli pelB + Brazzein gene (H30R_E35D_E40A_E52D) <400> 16 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgct aagcgtgcta gatctggaga ttgcttttac 180 gatgccaaga gaaatcttca atgcatttgc gattactgcg actactaa 228 <210> 17 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> Brazzein (H30R_E35D_E40A) <400> 17 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 Ala 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> 18 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> Brazzein (H30R_E35D_E40A_E52K) <400> 18 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 Ala 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 Lys Tyr      50 <210> 19 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> Brazzein (H30R_E35D_E40A_E52R) <400> 19 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 Ala 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 Arg Tyr      50 <210> 20 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> Brazzein (H30R_E35D_E40A_E52A) <400> 20 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 Ala 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 Ala Tyr      50 <210> 21 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> Brazzein (H30R_E35D_E40A_E52H) <400> 21 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 Ala 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 His Tyr      50 <210> 22 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> Brazzein (H30R_E35D_E40A_E52D) <400> 22 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 Ala 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 Asp Tyr      50 <210> 23 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 tactgcaagt actaa 15 <210> 24 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 tactgccgtt actaa 15 <210> 25 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 tactgcgctt actaa 15 <210> 26 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 tactgccact actaa 15 <210> 27 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 tactgcgact actaa 15 <210> 28 <211> 60 <212> DNA <213> Artificial Sequence <220> E223 coli pelB signal sequence <400> 28 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60                                                                           60

Claims (10)

A brazine quaternary variant having an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.
A polynucleotide for expression of a brazein quaternary variant encoding an E. coli pelB signal sequence and an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.
The polynucleotide of claim 2, wherein the E. coli pelB signal sequence has a nucleotide sequence of SEQ ID NO: 28. 4.
The polynucleotide of claim 2, wherein the polynucleotide has a nucleotide sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
Recombinant expression vector for the expression of brazein quaternary variants comprising a promoter and a polynucleotide of claim 2 operably linked thereto.
The recombinant expression vector of claim 5, wherein the promoter is an E. coli pelB promoter.
The recombinant expression vector of claim 5, wherein the recombinant expression vector is pET26B (+)-brazein (H30R_E35D_E40A_E52K), pET26B (+)-brazein (H30R_E35D_E40A_E52R), pET26B (+)-brazein (H30R_E35D_E40A + E-A52) Recombinant expression vector selected from the group consisting of brazein (H30R_E35D_E40A_E52H) and pET26B (+)-brazein (H30R_E35D_E40A_E52D).
E. coli transformed with the recombinant expression vector of claim 5.
Culturing E. coli of claim 8;
Separating the protein of the cell membrane gap of the cultured Escherichia coli; And
Heat treating the separated cell membrane protein
Method for producing a brazine quaternary variant comprising a.
The composition for enhancing sugar content of claim 1 comprising the brazein fourth mutant as an active ingredient.

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