KR20090093470A - Method for preparing novel mutated and multi-mutated Brazzein having higher sweetness - Google Patents

Abstract

A novel multi-mutated Brazzein having high sweetness is provided to improve sweetness by maintaining pH stability and replace or mix to more various sweetner. A method for producing a multi-mutated Brazzein comprises: a step of culturing E.coli which is transformed with mutant gene which encodes E.coli pelB signal sequence and the mutated Brazzein; a step of isolating a protein of cell membrane of E.coli; and a step of heating the protein of cell membrane.

Description

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

The present invention relates to a novel brazein multiple variant having a high sweet taste and its use, and more particularly, a brazein variant having a higher sweetness than a wild type brazein (minor type) protein, a method for preparing the same, and a same It relates to a food composition for enhancing sugar content.

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, the National Obesity Countermeasure Committee was formed to announce a policy to label sugar risk warnings, and from 2010, food advertisements above the sugar standard will be regulated. 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, as it turns out 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, patients with phenylketonuria who are inherently deficient in phenylalanine hydroxylase 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, there has been a growing interest in sweet protein extracted from tropical fruits. Thaumatin is a protein contained in the fruit of the perennial plant (Thaumatococcus daniellii), a miracle fruit in West Africa. ~ 3,000 times sweeter. Monellin is a protein derived from the fruit of a vine-like plant called Serendipiti berry, which grows in the rain forests of Africa. It is 3,000 times sweeter than sugar. But it is not easy to cultivate and it is difficult to extract from the fruit. In addition, the heat stability of the low heat stability 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 the thermal stability using protein engineering techniques to overcome these disadvantages.

Brazzein, meanwhile, is Pentadipladra, West African Pentadi Flandra. A sweet protein originally extracted from the fruit of brazzeana Baillon (Ming et al., FEBS) Letters , 355: 106-108, 1994). Brazein has a sweet taste of about 500 to 2,000 times more than sucrose (Jin et al., Chem . Senses . 28: 491-498, 2003), major type and minor There are two types of types. The major type of plant-derived brazein has 54 amino acids, including pyroglutamic acid residues at its amino terminus. On the other hand, minor type brazein has only 53 amino acid residues without pyroglutamic acid residues at the amino terminus and has about twice as sweet taste as major type 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 of the water is very high (Gao et al, Int J. Biol macromol 24:.... 351-359, 1999).

U.S. Patent (UP 6,274,707 B1) and Asadi Porter et al. (Assadi-Porter et al., Arch .. Biochem. Biophys. 376: 259-265, 2000) recombine such brazein by genetic engineering using E. coli. It describes a method for producing brazein, by synthesizing a gene encoding brazein and inserting it into a recombinant vector containing a SNase to generate a new transformation vector, which is then introduced into Escherichia coli and the final SNase and A method of expressing and purifying linked fusion proteins is disclosed. However, brazein expressed by fusion with SNase produces an 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 have applied for a polynucleotide including Escherichia coli pelB signal sequence and brazein gene and a method for producing brazein using the same through prior studies (Korean Patent Application No. 2006-97619). There is a bar.

Accordingly, the present inventors prepared variants and multiple variants of amino acids at specific positions which are expected to not affect the structure of the amino acids constituting the brazein in order to find a natural sweetener with high thermal stability and excellent sweetness, among which The present invention has been completed by searching for and developing variants and multiple variants having higher sweetness while having properties such as thermal stability and pH stability and high water solubility equivalent to those of conventional brazein.

Accordingly, an object of the present invention is a novel brazein variant and multiple variants having properties such as high thermal stability, pH stability and high water solubility, which are equivalent to those of the conventional brazein subtype, and exhibiting a sweet taste of at least 2 times to 20 times or more. And uses thereof.

In order to achieve the above object, the present invention provides a novel brazein variant whose sweet taste is superior to conventional natural brazein.

In order to achieve another object of the present invention, the present invention provides a polynucleotide encoding the brazein variants and multiple variants.

In order to achieve another object of the present invention, the present invention provides a recombinant expression vector containing the polynucleotide, E. coli transformed with the vector and a method for producing a brazein 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 variant and multiple variants as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention is characterized by providing novel brazein variants and multiple variants with superior sugar content compared to conventional natural forms.

The brazein variant of the present invention is a substitution of the histidine residue, the thirtieth amino acid glutamic acid residue, or the thirty-third amino acid glutamic acid residue of the brazein subtype, for example, A brazein variant having the amino acid sequence of SEQ ID NO: 100 in which the thirtieth amino acid histidine residue is replaced with an arginine residue, and the glutamic acid residue thirty-third amino acid replaced with an aspartic acid residue. The brazein variant having the amino acid sequence of SEQ ID NO: 109, or the 40th glutamic acid residue is an alanine residue, an aspartic acid residue, a lysine residue, and an arginine residue, respectively. Brazein variant 1 having an amino acid sequence of SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 117 There.

In addition, the brazein variant of the present invention is a multiple variant in which two or more of the above-mentioned 30th amino acid histidine residue, 35th amino acid glutamic acid residue or 40th glutamic acid residue are modified two or more times. For example, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, It may be a brazein variant having the amino acid sequence of SEQ ID NO: 153 or SEQ ID NO: 154. In addition, the brazein variant of the present invention comprises a residue between the 29th lysine residue and the 30th histidine residue of the brazein variant having the amino acid sequence of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153 and SEQ ID NO: 154. The lysine residue is inserted and may preferably have the amino acid sequence of SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, or SEQ ID NO: 158.

On the other hand, the present invention provides a polynucleotide encoding the brazein variant. The polynucleotide may be a polynucleotide encoding the amino acid sequence of SEQ ID NO: 100, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 117, and preferably for the amino acid sequence of SEQ ID NO: 100 The nucleotide sequence of SEQ ID NO: 59, the nucleotide sequence of SEQ ID NO: 68 for the amino acid sequence of SEQ ID NO: 109, the nucleotide sequence of SEQ ID NO: 72 for the amino acid sequence of SEQ ID NO: 113, and the base of SEQ ID NO: 73 for the amino acid sequence of SEQ ID NO: 114 The nucleotide sequence of SEQ ID NO: 74 for the amino acid sequence of SEQ ID NO: 115 and the nucleotide sequence of SEQ ID NO: 76 for the amino acid sequence of SEQ ID NO: 117 may be used.

In addition, the polynucleotide encoding the brazein multiple variant of the present invention is SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, or polynucleotide encoding the amino acid sequence of SEQ ID NO: 158, preferably SEQ ID NO: The nucleotide sequence of SEQ ID NO: 123 for the amino acid sequence of 142, the nucleotide sequence of SEQ ID NO: 124 for the amino acid sequence of SEQ ID NO: 143, the nucleotide sequence of SEQ ID NO: 125 for the amino acid sequence of SEQ ID NO: 144, and the amino acid sequence of SEQ ID NO: 145; For the nucleotide sequence of SEQ ID NO: 126, for the amino acid sequence of SEQ ID NO: 146, for the nucleotide sequence of SEQ ID NO: 127; The base sequence of SEQ ID NO: 128 for an aboric acid sequence, the base sequence of SEQ ID NO: 129 for an amino acid sequence of SEQ ID NO: 148, the base sequence of SEQ ID NO: 130 for an amino acid sequence of SEQ ID NO: 149, and a sequence for the amino acid sequence of SEQ ID NO: 150 The nucleotide sequence of SEQ ID NO: 131, the nucleotide sequence of SEQ ID NO: 132 for the amino acid sequence of SEQ ID NO: 151, the nucleotide sequence of SEQ ID NO: 133 for the amino acid sequence of SEQ ID NO: 152, and the nucleotide sequence of SEQ ID NO: 134 for the amino acid sequence of SEQ ID NO: 153; For the amino acid sequence of SEQ ID NO: 154, the nucleotide sequence of SEQ ID NO: 135 for the amino acid sequence of SEQ ID NO: 154, the nucleotide sequence of SEQ ID NO: 138 for the amino acid sequence of SEQ ID NO: 155, the nucleotide sequence of SEQ ID NO: 139 for the amino acid sequence of SEQ ID NO: 156: For the amino acid sequence, the nucleotide sequence of SEQ ID NO: 140, and the amino acid of SEQ ID NO: 158 You can have the nucleotide sequence of SEQ ID NO: 141 for columns.

In addition, the present invention provides a promoter and a recombinant expression vector for expression of brazein variants and multiple variants 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 promoter (Christensen et al ., Plant Mol . Biol . 12: 619-632, 1989) and ALS promoters (US Patent Application No. 08 / 409,297). 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. Preferably, the promoter may use the E. coli pelB promoter.

E. coli, pelB signal sequence is a kind of membrane gap signal sequence of E. 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), when the brazein of the present invention is synthesized, it moves to the cell membrane gap of E. coli to induce accurate disulfide bonds, Inhibiting the formation of insoluble aggregates of brazein protein, it is possible to facilitate the purification process by minimizing unnecessary E. coli-derived protein. E. coli pelB signal sequence of the present invention preferably has a nucleotide sequence of SEQ ID NO: 137, 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.

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.

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 sequences 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) and can be prepared in various ways depending on the purpose. Can be. The expression vector also includes a selection marker for selecting a host cell containing the vector and, in the case of a replicable expression vector, a replication origin.

Recombinant expression vector for the expression of the brazein variant of the present invention is preferably pET26B (+)-Brazzein (H30R), pET26B (+)-Brazzein (E35D), pET26B (+)-Brazzein (E40A), pET26B ( +)-Brazzein (E40D), pET26B (+)-Brazzein (E40K) or pET26B (+)-Brazzein (E40R), each with pET26B (+)-Brazzein (Met-) as a template SEQ ID NO: 19 for pET26B (+)-Brazzein (H30R), SEQ ID NO: 28 for pET26B (+)-Brazzein (E35D), SEQ ID NO: 32 for pET26B (+)-Brazzein (E40A), pET26B ( +)-Brazzein (E40D) for SEQ ID NO: 33, pET26B (+)-Brazzein (E40K) for SEQ ID NO: 34, and for pET26B (+)-Brazzein (E40R) using SEQ ID NO: 36 It can be prepared by site-directed mutagenesis.

In addition, the recombinant expression vector for the expression of the brazein secondary variant of the brazein multiple variant of the present invention is preferably pET26B (+)-Brazzein (H30R_E35D), pET26B (+)-Brazzein (H30R_E40A), pET26B ( +)-Brazzein (H30R_E40D), pET26B (+)-Brazzein (H30R_E40K) or pET26B (+)-Brazzein (H30R_E40R), each with pET26B (+)-Brazzein (H30R) as a template, SEQ ID NO: 28 for pET26B (+)-Brazzein (H30R_E35D), SEQ ID NO: 32 for pET26B (+)-Brazin (H30R_E40A), SEQ ID NO: 33 for pET26B (+)-Brazzein (H30R_E40D) +)-Brazzein (H30R_E40K) can be prepared by site-directed mutagenesis using SEQ ID NO: 34 and pET26B (+)-Brazzein (H30R_E40R) using SEQ ID NO: 36 have.

In addition, the recombinant expression vector for the expression of another brazein of the braze multiple variant of the present invention is preferably pET26B (+)-Brazzein (E35D_E40A), pET26B (+)-Brazzein (E35D_E40D), pET26B (+)-Brazzein (E35D_E40K) or pET26B (+)-Brazzein (E35D_E40R), respectively, with pET26B (+)-Brazzein (E35D) as the template, pET26B (+)-Brazzein (E35D_E40A) ), SEQ ID NO: 32, pET26B (+)-Brazzein (E35D_E40D), SEQ ID NO: 33, pET26B (+)-Brazzein (E35D_E40K), SEQ ID NO: 34, and pET26B (+)-Brazzein (E35D_E40R) Cases can be prepared by site-directed mutagenesis using SEQ ID NO: 36.

In addition, the recombinant expression vector for the expression of the brazein third variant of the brazein multiple variant of the present invention is preferably pET26B (+)-Brazzein (H30R_E35D_E40A), pET26B (+)-Brazzein (H30R_E35D_E40D), pET26B ( +)-Brazzein (H30R_E35D_E40K) or pET26B (+)-Brazzein (H30R_E35D_E40R), each of which is pET26B (+)-Brazzein (H30R_E35D) as a template, and H_R_E_A_E_A_E_A_E_A_E_A_E_A_E_A_E_E_B_E_35_E40K) SEQ ID NO: 32, pET26B (+)-Brazin (H30R_E35D_E40D), SEQ ID NO: 33, pET26B (+)-Brazzzein (H30R_E35D_E40K), SEQ ID NO: 34, pET26B (+)-Brazzein (H30R_E35D_E40R) Using SEQ ID NO: 36 can be prepared by site-directed mutagenesis.

In addition, the recombinant expression vector for the expression of the brazein fourth variant of the brazein multiple variants of the present invention is preferably pET26B (+)-Brazzein (29ins30 Lys_H30R_E35D_E40A), pET26B (+)-Brazzein (29ins30 Lys_H30R_E35D_E40D) pET26B (+)-Brazzein (29ins30 Lys_H30R_E35D_E40K) or pET26B (+)-Brazzein (29ins30 Lys_H30R_E35D_E40R), respectively, using as primers of SEQ ID NO: 136, which is pET26B (+)-Brazzein (29R30E26_35) +)-Brazzein (H30R_E35D_E40A), pET26B (+)-Brazzein (29ins30 Lys_H30R_E35D_E40D), pET26B (+)-Brazzein (H30R_E35D_E40D), and pET26B (+)-BrazKin_H_R_B_E_R_E_D_E_D_E_D_E_D_E40D) ) -Brazzein (H30R_E35D_E40K) is used for pET26B (+)-Brazzein (29ins30 Lys_H30R_E35D_E40R) as pET26B (+)-Brazzein (H30R_E35D_E40R) as a template (site-directed mutagenesis mutagenesis) It can manufacture by.

The present invention also provides E. coli comprising 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 a host cell, and is suitable for the host cell as known in the art. Standard techniques 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.

The present invention provides a method for producing a variant of brazein comprising culturing the transformed Escherichia coli, separating the cell membrane gap protein from the cultured Escherichia coli, and heat treatment to purify the brazein.

E. coli transformed to include the polynucleotide of the present invention may be cultured under appropriate media and conditions so that the polynucleotide encoding the brazein variant is expressed, which is the same as 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.

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 can be used (Snyder et al., J. Bacteriology , 177: 953963, 1995). For example, but not limited to, the cultured E. coli was collected, suspended in 30 mM Tris-HCl (Pri-HCl, pH 8) solution containing 20% sucrose, and EDTA (pH). 8) A solution and MgSO ₄ can be used to elute the protein of the cell membrane gap of E. coli.

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 for separating the brazein may be preferably performed by heat treatment. Although not limited thereto, the heat treatment is preferably heat-denatured protein other than braze by heating at 70-90 ° C. for 15-60 minutes, and then heat-denatured protein and brazein by centrifugation at 18000 g at 4 ° C. for 30 minutes. Can be separated.

As described above, brazein variants and sequences having amino acids consisting of SEQ ID NO: 100, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 117 with higher sweetness and higher thermostability in the present invention SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154 , Brazein multiple variant enzymatic properties having an amino acid consisting of SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157 or SEQ ID NO: 158 is as follows.

1) Molecular Weight: 6.5kDa

2) high thermal stability and acid resistance

3) high water solubility

4) The protein-based brazein variant sweetness ratio of the brazein subtype: 2 to 3.3 times or more

5) The ratio of sweetness of the brazine variant to sucrose of 1g / 100ml: about 4,000 to about 6,600 times or more

6) Brazein's subtype of protein-based brazein multiple variants sweetness ratio: 4-20 times or more

7) 1 g / 100ml sucrose to braze multiple variant sweetness ratio: about 8,000 ~ about 40,000 times or more

As described above, the brazein variant according to the present invention and the brazein multiple variant prepared based on the same are proteins of the subtype of brazein that is expressed and purified in the present patent application (Korean Patent Application No. 2006-97619), that is, a natural type. Compared with the Brazein subtype protein of Korea and Korean Patent Application No. 10-2007-0117013 expressed and purified brazein variants, the characteristics of thermal stability, acid resistance and water solubility are similar to those of natural type It is characterized by having a novel amino acid sequence that exhibits a higher sweetness.

In addition, the brazein variants in this invention exhibit higher sweetness and effect than the brazein variants known by the US patents (US 6,274,707B1; US 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 one embodiment of the present invention by the site-directed mutagenesis method, the fifth amino acid lysine residue of the brazein subtype, the 28th amino acid aspartic acid residue, the 30th amino acid histidine residue, 35th A recombinant vector encoding 40 brazein variants in which the amino acid glutamic acid residue, the 40th glutamic acid residue, or the 42nd amino acid arginine residue was substituted with another amino acid was prepared.

In another embodiment of the present invention, using each of the recombinant vectors prepared above, each of the brazein variants was expressed and purified. As a result, a high purity brazein variant was obtained.

In another embodiment of the present invention, the braze variant and braze subtype protein activity (sweetness) and thermal stability prepared above were measured. As a result, the histidine residue, the 30th amino acid, of the brazein subtype protein is an arginine residue, the glutamic acid residue, the 35th amino acid, is an aspartic acid residue, the 40th glutamic acid residue, an alanine residue, an aspartic acid residue, a lysine residue, and an arginine residue. The mutant was selected for the production of multiple varieties of braze for higher sweetness with higher activity (sweetness) and comparable thermal stability than brazein subtype proteins, and can be used as an excellent sweetener by itself. I could see that.

In another embodiment of the present invention, the brazein variant of the above prepared by combining appropriately the 9th secondary variant, 4 kinds of tertiary variant and the 29th lysine residue of the brazein subtype based on the tertiary variant And a recombinant vector encoding a total of 17 Brazain multiple variants of four quaternary variants in which the lysine residue was inserted between the 30th histidine residue and the expression, purification and activity in the same manner as in the above example. (Sweetness) was measured. As a result, it was possible to obtain a high-purity brazein variant, it was possible to produce a brazein variant having the same stability as the brazein subtype protein and showing a sweet taste of at least 4 to up to 20 times as compared to the brazein subtype protein. Through this, it can be seen that the brazein multiple variants prepared using the selected brazein variants can also be used as an excellent sweetener.

Accordingly, the present invention provides a food composition for enhancing sugar content comprising the brazein 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 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 variant of the present invention in the form of a food additive, it may be prepared and used in the form of powder or concentrate.

The preferred content of the brazein 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 weight of the food.

As described above, the brazein variant of the present invention has properties such as thermal stability and acid resistance and water solubility superior to the conventional braze, and has a sweet taste of at least two to up to 3.3 times or more than the conventional brazein Multiple variants have the same stability and at least four to up to twenty times the sweetness of the brazein subtype proteins as well as the brazein variants. Therefore, the brazein 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 expressing a brazein variant according to the present invention.

Figure 2 is a schematic diagram showing a process for producing a brazein variant and a brazein multiple variant according to the present invention.

Figure 3 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 brazein minor type after heat treatment

Lane 2: Relative Activity of Brazein Variant (H30K) After Heat Treatment

Lane 3: Relative Activity of Brazein Variant (H30R) After Heat Treatment

Lane 4: Relative Activity of Brazein Variant (E35D) After Heat Treatment

Lane 5: relative activity of brazein variant (E40A) after heat treatment

Lane 6: Relative Activity of Brazein Variant (E40D) After Heat Treatment

Lane 7: Relative Activity of Brazein Variant (E40K) After Heat Treatment

Lane 8: Relative Activity of Brazein Variant (E40H) After Heat Treatment

Lane 9: Relative Activity of Brazein Variant (E40R) After Heat Treatment

Figure 4 shows the results of electrophoresis to identify the brazein multiple variants according to the present invention.

Lane M: molecular weight marker

Lane 1: purified brazein secondary variant (H30R_E35D)

Lane 2: purified brazein secondary variant (H30R_E40A)

Lane 3: purified brazein secondary variant (H30R_E40D)

Lane 4: purified brazein secondary variant (H30R_E40K)

Lane 5: purified brazein secondary variant (H30R_E40R)

Lane 6: purified brazein secondary variant (E35D_E40A)

Lane 7: purified brazein secondary variant (E35D_E40D)

Lane 8: purified brazein secondary variant (E35D_E40K)

Lane 9: purified brazein secondary variant (E35D_E40R)

Lane 10: purified brazein tertiary variant (H30R_E35D_E40A)

Lane 11: purified brazein tertiary variant (H30R_E35D_E40D)

Lane 12: purified brazein tertiary variant (H30R_E35D_E40K)

Lane 13: purified brazein tertiary variant (H30R_E35D_E40R)

Lane 14: purified brazein quaternary variant (29ins30 Lys_H30R_E35D_E40A)

Lane 15: purified brazein quaternary variant (29ins30 Lys_H30R_E35D_E40D)

Lane 16: purified brazein quaternary variant (29ins30 Lys_H30R_E35D_E40K)

Lane 17: purified brazein quaternary variant (29ins30 Lys_H30R_E35D_E40R)

Figure 5 shows the results of performing reversed phase chromatography to compare the degree of purification and structural differences of the brazein variants according to the present invention.

A: refined brazein minor type

B: purified brazain quaternary variant (29ins30 Lys_H30R_E35D_E40A)

C: purified brazein quaternary variant (29ins30 Lys_H30R_E35D_E40D)

D: purified brazein quaternary variant (29ins30 Lys_H30R_E35D_E40K)

E: purified brazein quaternary variant (29ins30 Lys_H30R_E35D_E40R)

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1>

Brazane  Primary Variants  Of polynucleotides encoding Cloning

In order to prepare a brazain multiple variant having a higher sweetness than brazein, one specific amino acid constituting a minor type of brazein was selected and mutated to another specific amino acid.

First, the residues of which the outer chain was facing out and polar residues were selected through structural analysis of brazein as a specific amino acid to be mutated. On the basis of this structural information, the sequence of SEQ ID NO: 1 from which the unnecessary "ATG" sequence was removed to synthesize the brazein subtype protein in Escherichia coli used in the inventor's patent application (Korean Patent Application No. 2006-97619) Using a recombinant expression vector (pET26B (+)-Brazzein (Met-), see Example 6) as a template to synthesize the primers 40 and the primers having a sequence complementary to each primer shown in Table 1 below. The primers were designed in consideration of the external residue length and electrical properties of the specific amino acids (see Table 1) constituting the brazein subtype protein. Using the primers of Table 1 and the QuikChange ^™ Site-Directed Mutagenesis Kit (Stratagene, USA), the nucleotides encoding 40 brazein variants that substituted specific positions of the brazein subtype protein according to the manufacturer's instructions were identified. An expression vector 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 production of the Brazain primary variants location Mutant Total Amino Acid Residues Amino acid residues after mutation Primer used Remarks Before variation After variation SEQ ID NO: 5 Lys (K) Ala (A) tgc aaa gct gtt tac positive neutral SEQ ID NO: 2 Asp (D) tgc aaa gac gtt tac negative SEQ ID NO: 3 Glu (E) tgc aaa gaa gtt tac negative SEQ ID NO: 4 His (H) tgc aaa cac gtt tac positive SEQ ID NO: 5 Arg (R) tgc aaa cgt gtt tac positive SEQ ID NO: 6 28 Asp (D) Ala (A) aag ctt gct aag cat negative neutral SEQ ID NO: 7 His (H) aag ctt cac aag cat positive SEQ ID NO: 8 Lys (K) aag ctt aaa aag cat positive SEQ ID NO: 9 Arg (R) aag ctt cgt aag cat positive SEQ ID NO: 10 Glu (E) aag ctt gaa aag cat negative SEQ ID NO: 11 29 Lys (K) Ala (A) ctt gat gct cat gct positive neutral SEQ ID NO: 12 Arg (R) ctt gat cgt cat gct positive SEQ ID NO: 13 His (H) ctt gat cgc cat gct positive SEQ ID NO: 14 Asp (D) ctt gat gac cat gct negative SEQ ID NO: 15 Glu (E) ctt gat gaa cat gct negative SEQ ID NO: 16 30 His (H) Ala (A) gat aag gct gct cga positive neutral SEQ ID NO: 17 Lys (K) gat aag aaa gct cga positive SEQ ID NO: 18 Arg (R) gat aag cgt gct cga positive SEQ ID NO: 19 Asp (D) gat aag gac gct cga negative SEQ ID NO: 20 Glu (E) gat aag gaa gct cga negative SEQ ID NO: 21 32 Arg (R) Ala (A) cat gct gct tct gga positive neutral SEQ ID NO: 22 Lys (K) cat gct aaa tct gga positive SEQ ID NO: 23 His (H) cat gct cac tct gga positive SEQ ID NO: 24 Asp (D) cat gct gac tct gga negative SEQ ID NO: 25 Glu (E) cat gct gaa tct gga negative SEQ ID NO: 26 35 Glu (E) Ala (A) tct gga gct tgc ttt negative neutral SEQ ID NO: 27 Asp (D) tct gga gac tgc ttt negative SEQ ID NO: 28 Lys (K) tct gga aaa tgc ttt positive SEQ ID NO: 29 His (H) tct gga cac tgc ttt positive SEQ ID NO: 30 Arg (R) tct gga cgt tgc ttt positive SEQ ID NO: 31 40 Glu (E) Ala (A) tac gat gct aag aga negative neutral SEQ ID NO: 32 Asp (D) tac gat gac aag aga negative SEQ ID NO: 33 Lys (K) tac gat aaa aag aga positive SEQ ID NO: 34 His (H) tac gat cac aag aga positive SEQ ID NO: 35 Arg (R) tac gat cgt aag aga positive SEQ ID NO: 36 42 Arg (R) Ala (A) gaa aag gct aat ctt positive neutral SEQ ID NO: 37 Lys (K) gaa aag aaa aat ctt positive SEQ ID NO: 38 His (H) gaa aag cac aat ctt positive SEQ ID NO: 39 Asp (D) gaa aag gac aat ctt negative SEQ ID NO: 40 Glu (E) gaa aag gaa aat ctt negative SEQ ID NO: 41

Specifically, 10 ng of the pET26B (+)-Brazzein (Met-) vector, a mixture of dNTPs each having a final concentration of 0.2 mM, 125 ng of each 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 performed 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. Immediately afterwards, 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 through genetic analysis that the nucleotides encoding the pelB signal sequence and each of the brazein variants were linked to the separated DNA. It is named by the SEQ ID NO and nucleotide name indicated in Table 2 below. At this time, the method of referring to the variants in Table 2 and below, for example, in the case of K5D, the lysine residue at position 5 of the minor type protein of brazein is replaced with an aspartic aicd residue. It is shown. One letter designating each amino acid is designated according to a known amino acid code.

Polynucleotide name and sequence number encoding the Brazain primary variant Brazain Primary Variant Locations Nucleotide Names Encoding Each Brazein Primary Variant SEQ ID NO: K5A E. coli pelB + Brazein (K5A) gene SEQ ID NO: 42 K5D E. coli pelB + Brazein (K5D) gene SEQ ID NO: 43 K5E E. coli pelB + Brazein (K5E) gene SEQ ID NO: 44 K5H E. coli pelB + Brazein (K5H) gene SEQ ID NO: 45 K5R E. coli pelB + Brazein (K5R) gene SEQ ID NO: 46 D28A E. coli pelB + Brazein (D28A) gene SEQ ID NO: 47 D28H E. coli pelB + Brazein (D28H) gene SEQ ID NO: 48 D28K E. coli pelB + Brazein (D28K) gene SEQ ID NO: 49 D28R E. coli pelB + Brazein (D28R) gene SEQ ID NO: 50 D28E E. coli pelB + Brazein (D28E) gene SEQ ID NO: 51 K29A E. coli pelB + Brazein (K29A) gene SEQ ID NO: 52 K29R E. coli pelB + Brazein (K29R) gene SEQ ID NO: 53 K29H E. coli pelB + Brazein (K29H) gene SEQ ID NO: 54 K29D E. coli pelB + Brazein (K29D) gene SEQ ID NO: 55 K29E E. coli pelB + Brazein (K29E) gene SEQ ID NO: 56 H30A E. coli pelB + Brazein (H30A) gene SEQ ID NO: 57 H30K E. coli pelB + Brazein (H30K) gene SEQ ID NO: 58 H30R E. coli pelB + Brazein (H30R) gene SEQ ID NO: 59 H30D E. coli pelB + Brazein (H30D) gene SEQ ID NO: 60 H30E E. coli pelB + Brazein (H30E) gene SEQ ID NO: 61 R32A E. coli pelB + Brazein (R32A) gene SEQ ID NO: 62 R32K E. coli pelB + Brazein (R32K) gene SEQ ID NO: 63 R32H E. coli pelB + Brazein (R32H) gene SEQ ID NO: 64 R32D E. coli pelB + Brazein (R32D) gene SEQ ID NO: 65 R32E E. coli pelB + Brazein (R32E) gene SEQ ID NO: 66 E35A E. coli pelB + Brazein (E35A) gene SEQ ID NO: 67 E35D E. coli pelB + Brazein (E35D) gene SEQ ID NO: 68 E35K E. coli pelB + Brazein (E35K) gene SEQ ID NO: 69 E35H E. coli pelB + Brazein (E35H) gene SEQ ID NO: 70 E35R E. coli pelB + Brazein (E35R) gene SEQ ID NO: 71 E40A E. coli pelB + Brazein (E40A) gene SEQ ID NO: 72 E40D E. coli pelB + Brazein (E40D) gene SEQ ID NO: 73 E40K E. coli pelB + Brazein (E40K) gene SEQ ID NO: 74 E40H E. coli pelB + Brazein (E40H) gene SEQ ID NO: 75 E40R E. coli pelB + Brazein (E40R) gene SEQ ID NO: 76 R42A E. coli pelB + Brazein (R42A) gene SEQ ID NO: 77 R42K E. coli pelB + Brazein (R42K) gene SEQ ID NO: 78 R42H E. coli pelB + Brazein (R42H) gene SEQ ID NO: 79 R42D E. coli pelB + Brazein (R42D) gene SEQ ID NO: 80 R42E E. coli pelB + Brazein (R42E) gene SEQ ID NO: 81

As a result of the experiment, it was possible to prepare an expression vector for all species of Brazein. Transformation with BL21 (star) was used for mass expression (see FIG. 1).

< Example  2>

Brazane  Primary Variant  Expression and Purification

<2-1> Brazane  Primary Variant  Expression

Each of E. coli BL21 (star), in which 32 expression vectors for the brazein primary variants prepared in <Example 1> were introduced, was used as a protein inducing agent in 1 L of LB medium containing 30 μl / ml of kanamycin. Incubated at 37 ° C. for 12 hours without the addition of phosphorous IPTG (isopropyl β-D-thoigalactopyranoside) to express each brazein variant in each transformed Escherichia coli.

<2-2> Brazane Variant  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 0.5 M EDTA (pH 8.0) solution was added to a final concentration of 1 mM. Stir slowly at room temperature for 10 minutes. It was centrifuged at 10,000 g, 4 ° C. for 10 minutes, 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 and 4 ° C. for 10 minutes, and heat-treated at 80 ° C. for 30 minutes to purify the brazein variant present in the periplasm. Thereafter, after dialysis with distilled water for 24 hours, lyophilization was carried out to obtain a purified brazein primary variant represented by the sequence number of Table 3 below, and the degree of purification was primarily confirmed through SDS-PAGE.

BRAZINE primary variant name and sequence number  Brazain Primary Variant Locations Name of each brazein variant SEQ ID NO: - Brazzein (minor type) SEQ ID NO: 82 K5A Brazzein (K5A) SEQ ID NO: 83 K5D Brazzein (K5D) SEQ ID NO: 84 K5E Brazzein (K5E) SEQ ID NO: 85 K5H Brazzein (K5H) SEQ ID NO: 86 K5R Brazzein (K5R) SEQ ID NO: 87 D28A Brazzein (D28A) SEQ ID NO: 88 D28H Brazzein (D28H) SEQ ID NO: 89 D28K Brazzein (D28K) SEQ ID NO: 90 D28R Brazzein (D28R) SEQ ID NO: 91 D28E Brazzein (D28E) SEQ ID NO: 92 K29A Brazzein (K29A) SEQ ID NO: 93 K29R Brazzein (K29R) SEQ ID NO: 94 K29H Brazzein (K29H) SEQ ID NO: 95 K29D Brazzein (K29D) SEQ ID NO: 96 K29E Brazzein (K29E) SEQ ID NO: 97 H30A Brazzein (H30A) SEQ ID NO: 98 H30K Brazzein (H30K) SEQ ID NO: 99 H30R Brazzein (H30R) SEQ ID NO: 100 H30D Brazzein (H30D) SEQ ID NO: 101 H30E Brazzein (H30E) SEQ ID NO: 102 R32A Brazzein (R32A) SEQ ID NO: 103 R32K Brazzein (R32K) SEQ ID NO: 104 R32H Brazzein (R32H) SEQ ID NO: 105 R32D Brazzein (R32D) SEQ ID NO: 106 R32E Brazzein (R32E) SEQ ID NO: 107 E35A Brazzein (E35A) SEQ ID NO: 108 E35D Brazzein (E35D) SEQ ID NO: 109 E35K Brazzein (E35K) SEQ ID NO: 110 E35H Brazzein (E35H) SEQ ID NO: 111 E35R Brazzein (E35R) SEQ ID NO: 112 E40A Brazzein (E40A) SEQ ID NO: 113 E40D Brazzein (E40D) SEQ ID NO: 114 E40K Brazzein (E40K) SEQ ID NO: 115 E40H Brazzein (E40H) SEQ ID NO: 116 E40R Brazzein (E40R) SEQ ID NO: 117 R42A Brazzein (R42A) SEQ ID NO: 118 R42K Brazzein (R42K) SEQ ID NO: 119 R42H Brazzein (R42H) SEQ ID NO: 120 R42D Brazzein (R42D) SEQ ID NO: 121 R42E Brazzein (R42E) SEQ ID NO: 122

As a result of the experiment, the purity of brazein protein was purified to a high molecular weight of about 6.5kDa.

<2-3> Brazane  Primary Variant  Confirm Purity and Change of Structure

Refined Reverse phase chromatography on Varina's High Performance Liquid Chromatography to analyze the structural differences after confirming the degree of purification of the subtypes of brazein and each of the brazein variants purified from Example 2-2. Analysis was performed using a reverse-phase chromatography column, Vydac 214TP54 (USA) column. Solvent conditions consist of solvent A with 0.05% triflouroacetic acid in water and solvent B with 0.05% triflouroacetic acid in acetonitrile at a flow rate of 1 ml per minute. For 30 minutes, solvent B was eluted in a linear gradient from 10% to 50%. The eluted solution observed the change of absorbance at 210 nm.

As a result, it was confirmed that most of the brazein variants were eluted after a retention time of 15 minutes, which indicates that there is almost no structural difference between the expressed brazein variants.

< Example  3>

Brazane  Primary Variant  Active (sweet) and Thermal stability  Measure

<3-1> Brazane  Primary Variant  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 glucose measurement was conducted on 20 subjects who were trained to have a similar level of sucrose minimum sucrose with a solution of sucrose in solution, and to determine 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 first varieties of brazein  Primary variant location SEQ ID NO: Lowest irritant amount (㎍ / 100㎖) feels the initial sweetness Comparison of sweetness ratio of sucrose (1g / 100ml) to the first variant of brazein (Brazin subtype: 2000) Increased sweetness compared to braze boolean type K5A SEQ ID NO: 83 6,000 167 0.08 K5D SEQ ID NO: 84 6,000 167 0.08 K5E SEQ ID NO: 85 6,000 167 0.08 K5H SEQ ID NO: 86 10,000 100 0.05 K5R SEQ ID NO: 87 10,000 100 0.05 D28A SEQ ID NO: 88 10,000 100 0.05 D28H SEQ ID NO: 89 6,000 167 0.08 D28K SEQ ID NO: 90 6,000 167 0.08 D28R SEQ ID NO: 91 6,000 167 0.08 D28E SEQ ID NO: 92 2,000 500 0.25 K29A SEQ ID NO: 93 10,000 100 0.05 K29R SEQ ID NO: 94 10,000 100 0.05 K29H SEQ ID NO: 95 10,000 100 0.05 K29D SEQ ID NO: 96 10,000 100 0.05 K29E SEQ ID NO: 97 10,000 100 0.05 H30A SEQ ID NO: 98 6,000 167 0.08 H30K SEQ ID NO: 99 250 4,000 2 H30R SEQ ID NO: 100 150 6,600 3.3 H30D SEQ ID NO: 101 3,000 334 0.16 H30E SEQ ID NO: 102 3,000 334 0.16 R32A SEQ ID NO: 103 6,000 167 0.08 R32K SEQ ID NO: 104 3,000 334 0.16 R32H SEQ ID NO: 105 3,000 334 0.16 R32D SEQ ID NO: 106 10,000 100 0.05 R32E SEQ ID NO: 107 10,000 100 0.05 E35A SEQ ID NO: 108 10,000 100 0.05 E35D SEQ ID NO: 109 150 6,600 3.3 E35K SEQ ID NO: 110 6,000 167 0.08 E35H SEQ ID NO: 111 6,000 167 0.08 E35R SEQ ID NO: 112 6,000 167 0.08 E40A SEQ ID NO: 113 150 6,600 3.3 E40D SEQ ID NO: 114 150 6,600 3.3 E40K SEQ ID NO: 115 150 6,600 3.3 E40H SEQ ID NO: 116 250 4,000 2 E40R SEQ ID NO: 117 250 4,000 2 R42A SEQ ID NO: 118 10,000 100 0.05 R42K SEQ ID NO: 119 3,000 334 0.16 R42H SEQ ID NO: 120 3,000 334 0.16 R42D SEQ ID NO: 121 10,000 100 0.05 R42E SEQ ID NO: 122 10,000 100 0.05

As a result, as shown in Table 4 above, brazein represented by SEQ ID NO: 99 (H30K), brazein represented by SEQ ID NO: 100 (H30R), brazein represented by SEQ ID NO: 109 (E35D), and SEQ ID NO: 113. Brazein (E40A) represented by SEQ ID NO: 113, brazein (E40A) represented by SEQ ID NO: 113, brazein (E40D) represented by SEQ ID NO: 114, brazein (E40K) represented by SEQ ID NO: 115, represented by SEQ ID NO: 116 When compared to the variant (E40H) and the variant represented by SEQ ID NO: 117 (E40R) with the protein of the brazein subtype, at least 2 times up to about 3.3 times (at least about 4,000 times up to 1 g / 100ml sucrose) About 6,600 times). In particular, brazein (E40D) showed the highest sweetness increase rate.

<3-2> Thermal Stability Measurement of Brazain Primary Variants

Based on the results measured in Example <3-1> brazein variant showing a high sweet taste, that is, brazein (H30K) represented by SEQ ID NO: 99, brazein (H30R) represented by SEQ ID NO: 100, SEQ ID NO: Brazein (E35D) represented by 109, brazein (E40A) represented by SEQ ID NO: 113, brazein (E40A) represented by SEQ ID NO: 113, brazein (E40D) represented by SEQ ID NO: 114, and SEQ ID NO: 115. 100 mg of a brazein variant, such as the indicated brazein (E40K), the variant represented by SEQ ID NO: 116 (E40H) and the variant represented by SEQ ID NO: 117 (E40R), was prepared in 50 mM Tris-HCl (Ph 8.0) solution. After melting for 4 hours at 80 ° C., the degree of sweetness change was measured in 20 test subjects in the same manner as in <Example 3-1> based on the sweetness before heat treatment for each of the primary varieties of brazein. And show the result as relative active Control are shown in Fig.

As a result, as shown in Figure 3, brazein (H30R) represented by SEQ ID NO: 100, brazein (E35D) represented by SEQ ID NO: 109, brazein (E40A) represented by SEQ ID NO: 113, SEQ ID NO: 113 Brazein variants such as brazein (E40A), brazein (E40D) represented by SEQ ID NO: 114, brazein (E40K) represented by SEQ ID NO: 115, and variant (E40R) represented by SEQ ID NO: 117 The thermal stability of was maintained as it is.

<Example 4>

Cloning of Polynucleotides Encoding Brazein Multiple Variants

Based on the results of <Example 3-2> and compared to the brazein subtype protein, compared to brazein primary variants (H30R, E35D, E40A, E40D, E40R, E40K) having a high sweet taste Was used to create a brazein secondary variant with a higher sweetness.

Specifically, the template (expression vector containing the polynucleotide encoding the brazyin primary variant) and the primers used for the primary variant preparation are used in Tables 5 to 7 below to prepare the brazein secondary variants. By using the site-directed mutagenesis method of Example 1, a total of nine polynucleotides encoding the brazein secondary variant were prepared. At this time, the nomenclature in Tables 5 to 7 below, for example, means that H30R_E35D means that the histidine residue at position 30 of the minor type protein of braze is 35 times as an arginine residue. Glutamic acid residues at the position is substituted with aspartic aicd residues at the same time, 29ins30 Lys_ means that the lysine residues are inserted at positions 29 and 30. In addition, the underlined portion of the primer sequence refers to the sequence changed for the brazein variant.

Templates and Primers Used for the Fabrication of Brazein Secondary Variants Template for writing Brazein secondary variants (SEQ ID NO) Primers used to create secondary variants Created Brazain Secondary Variants Primer sequence SEQ ID NO: E. coli pelB + Brazein (H30R) gene (SEQ ID NO: 59) tct gga gac tgc ttt SEQ ID NO: 28 H30R_E35D tac gat gct aag aga SEQ ID NO: 32 H30R_E40A tac gat gac aag aga SEQ ID NO: 33 H30R_E40D tac gat aaa aag aga SEQ ID NO: 34 H30R_E40K tac gat cgt aag aga SEQ ID NO: 36 H30R_E40R E. coli pelB + Brazein (E35D) gene (SEQ ID NO: 68) tac gat gct aag aga SEQ ID NO: 32 E35D_E40A tac gat gac aag aga SEQ ID NO: 33 E35D_E40D tac gat aaa aag aga SEQ ID NO: 34 E35D_E40K tac gat cgt aag aga SEQ ID NO: 36 E35D_E40R

Template and Primer Used for the Preparation of Brazein Tertiary Variants Template for writing Brazein tertiary variants (SEQ ID NO) Primers used to create tertiary variants Created Brazain Tertiary Variants Primer sequence SEQ ID NO: E. coli pelB + Brazein (H30R_E35D) gene (SEQ ID NO: 123) tac gat gct aag aga SEQ ID NO: 32 H30R_E35D_E40A tac gat gac aag aga SEQ ID NO: 33 H30R_E35D_E40D tac gat aaa aag aga SEQ ID NO: 34 H30R_E35D_E40K tac gat cgt aag aga SEQ ID NO: 36 H30R_E35D_E40R

Templates and Primers Used for the Fabrication of Brazain Quaternary Variants Template (SEQ ID NO) for writing Brazein 4th variant Primers Used for Quaternary Variants Created Brazain Quaternary Variants Primer sequence SEQ ID NO: E. coli pelB + Brazein (H30R_E35D_E40A) gene (SEQ ID NO: 132) gataag aaa catgct SEQ ID NO: 136 29ins30 Lys_H30R_E35D_E40A E. coli pelB + Brazein (H30R_E35D_E40D) gene (SEQ ID NO: 133) gataag aaa catgct SEQ ID NO: 136 29ins30 Lys_H30R_E35D_E40D E. coli pelB + Brazein (H30R_E35D_E40K) gene (SEQ ID NO: 134) gataag aaa catgct SEQ ID NO: 136 29ins30 Lys_H30R_E35D_E40K E. coli pelB + Brazein (H30R_E35D_E40R) gene (SEQ ID NO: 135) gataag aaa catgct SEQ ID NO: 136 29ins30 Lys_H30R_E35D_E40R

In addition, in order to prepare a brazein tertiary variant having a higher sweet taste, the template (expression vector containing polynucleotide encoding the brazein secondary variant) and primers used for preparing the primary variant are shown in Table 5 above. Using four sites, a total of four polynucleotides encoding brazein tertiary variants were prepared by the site-directed mutagenesis method.

In addition, the results of the sweetness test of the primary variant of brazein of <Example 3> assumes that the lysine residues at position 29 and the histidine residues at position 30 of the brazein subtype protein are important positions for showing sweetness. A brazein quaternary variant was prepared by inserting a lysine residue between the lysine residue at position 29 and the arginine residue at position 30 of the tertiary variant. To this end, the template (expression vector containing a polynucleotide encoding a brazein tertiary variant) described in Table 5, a primer encoding a lysine residue represented by SEQ ID NO: 136 and a primer having a sequence complementary thereto Was synthesized and a total of four polynucleotides encoding the brazein quaternary variants were prepared by the site-directed mutagenesis method of <Example 1>. This was named by the SEQ ID NO and nucleotide name specified in Table 8 below.

As a result, expression vectors for a total of 17 Brazain multiple variants were produced. It was transformed into BL21 (star) and used for mass expression.

Polynucleotide name and sequence number encoding the Brazein multiple variant Brazein multiple variant positions Nucleotide Names Encoding Each Brazein Multiple Variant SEQ ID NO: H30R_E35D E. coli pelB + Brazein (H30R_E35D) gene SEQ ID NO: 123 H30R_E40A E. coli pelB + Brazein (H30R_E40A) gene SEQ ID NO: 124 H30R_E40D E. coli pelB + Brazein (H30R_E40D) gene SEQ ID NO: 125 H30R_E40K E. coli pelB + Brazein (H30R_E40K) gene SEQ ID NO: 126 H30R_E40R E. coli pelB + Brazein (H30R_E40R) gene SEQ ID NO: 127 E35D_E40A E. coli pelB + Brazein (E35D_E40A) gene SEQ ID NO: 128 E35D_E40D E. coli pelB + Brazein (E35D_E40D) gene SEQ ID NO: 129 E35D_E40K E. coli pelB + Brazein (E35D_E40K) gene SEQ ID NO: 130 E35D_E40R E. coli pelB + Brazein (E35D_E40R) gene SEQ ID NO: 131 H30R_E35D_E40A E. coli pelB + Brazein (H30R_E35D_E40A) gene SEQ ID NO: 132 H30R_E35D_E40D E. coli pelB + Brazein (H30R_E35D_E40D) gene SEQ ID NO: 133 H30R_E35D_E40K E. coli pelB + Brazein (H30R_E35D_E40K) gene SEQ ID NO: 134 H30R_E35D_E40R E. coli pelB + Brazein (H30R_E35D_E40R) gene SEQ ID NO: 135 29ins30 Lys_H30R_E35D_E40A E. coli pelB + Brazein (29ins30 Lys_H30R_E35D_E40A) gene SEQ ID NO: 138 29ins30 Lys_H30R_E35D_E40D E. coli pelB + Brazein (29ins30 Lys_H30R_E35D_E40D) gene SEQ ID NO: 139 29ins30 Lys_H30R_E35D_E40K E. coli pelB + Brazein (29ins30 Lys_H30R_E35D_E40K) gene SEQ ID NO: 140 29ins30 Lys_H30R_E35D_E40R E. coli pelB + Brazein (29ins30 Lys_H30R_E35D_E40R) gene SEQ ID NO: 141

< Example  5>

Brazane  multiple Variants  Expression Purification and Characterization

The same as in <Example 2-1> and <Example 2-2> using the respective E. coli BL21 (star) introduced with 17 expression vectors for the brazein multiple variant prepared in <Example 4> By expression and purification it was possible to obtain a purified brazein variant represented by SEQ ID NOS of Table 9 to Table 11, the degree of purification was confirmed primarily through SDS-PAGE.

Brazein secondary variant name and sequence number Brazein Secondary Variant Position Brazain Secondary Variants SEQ ID NO: H30R_E35D Brazzein (H30R_E35D) SEQ ID NO: 142 H30R_E40A Brazzein (H30R_E40A) SEQ ID NO: 143 H30R_E40D Brazzein (H30R_E40D) SEQ ID NO: 144 H30R_E40K Brazzein (H30R_E40K) SEQ ID NO: 145 H30R_E40R Brazzein (H30R_E40R) SEQ ID NO: 146 E35D_E40A Brazzein (E35D_E40A) SEQ ID NO: 147 E35D_E40D Brazzein (E35D_E40D) SEQ ID NO: 148 E35D_E40K Brazzein (E35D_E40K) SEQ ID NO: 149 E35D_E40R Brazzein (E35D_E40R) SEQ ID NO: 150

Brazein 3rd variant name and sequence number Brazein tertiary variant positions Bra Jane Tertiary Variant SEQ ID NO: H30R_E35D_E40A Brazzein (H30R_E35D_E40A) SEQ ID NO: 151 H30R_E35D_E40D Brazzein (H30R_E35D_E40D) SEQ ID NO: 152 H30R_E35D_E40K Brazzein (H30R_E35D_E40K) SEQ ID NO: 153 H30R_E35D_E40R Brazzein (H30R_E35D_E40R) SEQ ID NO: 154

Brazain 4th variant name and sequence number Brazain Quaternary Variant Locations Brazain 4th variant name SEQ ID NO: 29ins30 Lys_ H30R_E35D_E40A Brazzein (29ins30 Lys_H30R_E35D_E40A) SEQ ID NO: 155 29ins30 Lys_ H30R_E35D_E40D Brazzein (29ins30 Lys_H30R_E35D_E40D) SEQ ID NO: 156 29ins30 Lys_ H30R_E35D_E40K Brazzein (29ins30 Lys_H30R_E35D_E40K) SEQ ID NO: 157 29ins30 Lys_ H30R_E35D_E40R Brazzein (29ins30 Lys_H30R_E35D_E40R) SEQ ID NO: 158

As a result, as shown in Figure 4, the brazein protein was purified to high purity, the molecular weight was about 6.5kDa.

In addition, the structural differences of the Brazain multiple variants were analyzed using Varina's High Performance Liquid Chromatography in the same manner as in <Example 2-3>, and as a result, most of the Brazain quaternary variants were excluded. It was confirmed that the Brazein multiple variant of was eluted after a retention time of about 15 minutes the same as the Brazein variant. However, it was confirmed that the Brazein 4th variant was eluted after a delay time of about 20 minutes. These results were thought to result in structural differences with the native brazein protein as the lysine residue was inserted between the lysine residue at position 29 and the arginine residue at position 30 of the brazein tertiary variant (see FIG. 5).

In addition, the activity (sweetness) measurement for the brazein multiple variants in the same manner as in <Example 3-1> was performed and the results are shown in Table 12 below.

Sweetness test results for Brazain multiple variants Multiple variant types Lowest irritant amount to feel the initial sweetness (㎍ / ㎖) Comparison of sweetness ratio of brazein multiple variants to sucrose (1g / 100ml) Increased sweetness compared to braze boolean type Variant Location (SEQ ID NO) Secondary variants H30R_E35D (SEQ ID NO: 142) 1,250 8,000 4 H30R_E40A (SEQ ID NO: 143) 1,250 8,000 4 H30R_E40D (SEQ ID NO: 144) 1,250 8,000 4 H30R_E40K (SEQ ID NO: 145) 1,000 10,000 5 H30R_E40R (SEQ ID NO: 146) 1,000 10,000 5 E35D_E40A (SEQ ID NO: 147) 1,000 10,000 5 E35D_E40D (SEQ ID NO: 148) 1,000 10,000 5 E35D_E40K (SEQ ID NO: 149) 1,250 8,000 4 E35D_E40R (SEQ ID NO: 150) 850 12,000 6 Tertiary variant H30R_E35D_E40A (SEQ ID NO: 151) 650 15,000 7.5 H30R_E35D_E40D (SEQ ID NO: 152) 500 20,000 10 H30R_E35D_E40K (SEQ ID NO: 153) 500 20,000 10 H30R_E35D_E40R (SEQ ID NO: 154) 450 22,000 11 Quaternary variant 29ins30 Lys_ H30R_E35D_E40A (SEQ ID NO: 155) 400 25,000 12.5 29ins30 Lys_ H30R_E35D_E40D (SEQ ID NO: 156) 350 28,000 14 29ins30 Lys_ H30R_E35D_E40K (SEQ ID NO: 157) 350 28,000 14 29ins30 Lys_ H30R_E35D_E40R (SEQ ID NO: 158) 250 40,000 20

As shown in Table 12 above, all of the brazein multiple variants are at least 4 times up to about 20 times higher (at least about 8,000 times up to about 40,000 times higher than 1g / 100ml sucrose) when compared to the proteins of the brazein subtype. It was shown to indicate sweetness.

In addition, as a result of measuring the thermal stability using the brazein multiple variants in the same manner as in <Example 3-2>, all of the brazein multiple variants showed the same thermal stability as the brazein subtype protein. High Performance Liquid Chromatography analysis of the 4th variant of the Brazain multiple variant confirmed that the thermal stability was maintained the same despite the structural characteristics different from that of the Brazein subtype protein.

By combining these results, we analyze the structure and amino acids of the protein of the braze minor type, and select the specific amino acid having the external chain toward the outside of the protein and having the electrical polarity. Forty brazein primary variants were prepared. Among them, 6 kinds of brazein (H30R, E35D, E40A, E40D, E40R, E40K), which show the same heat stability and high sweetness of at least 2 times and up to 3.3 times as compared to the protein of brazein type 1 Primary variants were selected. Selected brazein primary variants were used to prepare brazein multiple variants that exhibited the same thermal stability and higher sweetness as compared to subtype proteins. Most of the Brazain multiple variants, except for the Brazain quaternary variant, in which the lysine residue was inserted between the lysine residue at position 29 and the arginine residue at position 30 of the brazein tertiary variant, form the same structure as the protein of the brazein subtype. It was. Structural differences of the brazein quaternary variants are believed to be influenced by the inserted lysine residues. However, all of the multiple varieties of Brazain, including the Brazain 4th variant, showed the same thermostability as that of the Brazein subtype protein, and it was confirmed that sweetness increased from 4 times to 40 times.

< Example  6>

Recombinant Expression Vector pET26B (+)- Brazzein ( Met -) Made

<6-1> Brazein  New Artificial Gene Synthesis Encoding

Pentadipladra Brazena Byron by using a sequence (Genbank registered number P56552) the first sequence (codon (E. coli codon usage) that is based on the amino acid sequence except for pyroglutamic acid), and there are many in the interior of the E. coli in the fruit extract Bra agent brazzeana Baillon) The sequence of SEQ ID NO: 159 was determined as follows. At this time, the portion shown in bold indicates a portion modified in the sequence of Genbank Accession No. P56552 based on codons present in E. coli: GA T AA G TGCAA G AA G GTTTACGAAAA T TACCC A GTTTC T AA G TGCCA A CT T GCTAA T CA A TGCAA T TACGA T TGCAA G CT T G CT AA G CA T GCT A G A TC T GG A GAATGCTT T TACGA T GAAAA GA G A AA T CT T CA A TGCAT TT G C GA T TACTGCG AA TACTAA .

Based on the sequence information of SEQ ID NO: 159, the polynucleotide of the brazein gene was artificially synthesized by Takara Korea Biomedical.

<6-2> primer  making

In order to link the synthesized polynucleotide with the pelB signal sequence of pET26B (+) (Novagen, USA), it is included in the multi cloning site (MCS) of pET26B (+). Restriction enzyme site The primers were synthesized to include Nco I and Xho I and were called SEQ ID NO: 160 (forward primer, CATG CCATGG ATAAGTGCAAGAAGGTTTAC) and SEQ ID NO: 161 (reverse primer, CCG CTCGAG TTAGTATTCGCAGTAATCG). At this time, Nco I and Xho I sites are underlined, respectively.

<6-3> PCR On by Brazane  Gene amplification

The brazein gene synthesized in <Example 6-1> was used as a template, and the brazein gene was amplified using two primers synthesized in <Example 6-2>. The PCR reaction was performed with 1.5 μl of the template gene (synthesized brazein gene, SEQ ID NO: 159), 2 μl of the forward primer (SEQ ID NO: 160), 1 μl of the reverse primer (SEQ ID NO: 161), 25 mM MgCl ₂ 3 PCR was performed using a final volume of 50 μl, including the reaction solution, 4 μl of 2.5 mM dNTP, 5 μl of 10 × Ex-taq buffer, 1 μl of Ex-taq polymerase (Takara, Japan), and 3 μl of H ₂ O as a reaction solution. . The PCR reaction was denatured at 94 ° C. for 2 minutes, and then reacted 30 times at 98 ° C. for 30 seconds, 58 ° C. for 2 minutes, and 74 ° C. for 3 minutes, followed by a final reaction at 74 ° C. for 10 minutes. After the reaction was completed, the azerose gel amplified by 2.0% agarose gel electrophoresis was identified, the azerose gel was recovered from the agarose gel and extracted using a QIAquick Gel extraction kit (Qiagen, USA). And purified. The extracted brazein gene was inserted into the pGEM-T Easy vector (Promega, USA) (called pGEM-T Easy-Brazzein), and then the pGEM-T Easy vector into which the braze gene was inserted was transformed into E. coli JM109. . This was cultured on plate L-Broth medium containing 50 μg / ml Ampicillin to select transformed Escherichia coli, which was incubated in liquid L-broth medium again, and then from therein, brazein PGEM-T Easy vectors with genes were obtained in large quantities.

<6-4> Recombinant Expression Vector pET26B (+)- Brazzein ( Met -) Made

The pGEM-T Easy-Brazzein vector cloned in <Example 6-3> was restriction enzyme Nco. I and Xho I (using 10 × K buffer and 0.1% BSA) were digested at 37 ° C. for 6 hours. Expression vector pET26B (+) vector with T7 promoter was also cut under the same conditions. The portion of the brazein gene and the cleaved pET26B (+) vector in the pGEM-T Easy-Brazzein vector were purified using the QIAquick Gel extraction kit (Qiagen, USA). The mixture was reacted with T4 DNA ligase (Takara, Japan) for 12 hours at 16 ° C., and then transformed into JM109 supercompetent cells (see FIG. 2). The recombinant expression vector produced as a result of ligation was named pET26B (+)-Brazzein.

Meanwhile, the recombinant brazein of the present invention is transferred to the periplasm of Escherichia coli as a translation process progresses after transcription, and then fused with recombinant braze by a signal peptidase in E. coli. The pelB signal sequence is removed. However, Met, which is translated by ATG inside the restriction enzyme Nco I in the primer, is not removed by signal peptidase.

Therefore, in order to express the same type of brazein extracted from natural products, the restriction enzyme ( Nco I) following the pelB signal sequence in the pET26B (+)-Brazzein vector through site-directed mutagenesis using PCR. ) “ATG” inside was removed (see FIG. 2). This will be described in detail as follows.

Primers of SEQ ID NO: 162 (CAGCCGGCGATGGCCGACAAATGCAAAAAA) and SEQ ID NO: 163 (TTTTTTGCATTTGTCGGCCATCGCCGGCTG) were synthesized with a length of about 15 bp, which is the same as the base sequence of brazein on both sides of the base to be removed in the brazein gene. They are capable of binding complementary to each single strand of brazein, except for ATG, which is each removed. The pET26B (+)-Brazzein vector was used as a template, and described above according to the manufacturer's instructions using a QuikChange ^™ Site-Directed Mutagenesis Kit (Stratagene, USA) using primers of SEQ ID NO: 162 and SEQ ID NO: 163. As described above, pET26B (+)-Brazzein (Met-) was obtained from which ATG was removed from the pET26B (+)-Brazzein vector. That is, 10 ng of the pET26B (+)-Brazzein vector, a mixture of dNTPs having a final concentration of 0.2 mM, respectively, 125 ng of SEQ ID NO: 162 and a primer of SEQ ID NO: 163, 5 μl of 10 × reaction buffer, Pfu - Turbo DNA polymerase (2.5 U / Μl, Stratagene, USA) PCR was performed with the reaction solution with a total of 50 μl containing 1 μl. PCR reaction was pre-denatured at 95 ℃ for 2 minutes, 98 ℃ 30 seconds, 55 ℃ 1 minutes. The reaction was performed 15 times at 68 ° C. for 15 minutes and the final reaction was performed at 68 ° C. for 10 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 immediately transformed into E. coli XL1-Blue, a supercompetent cell. Transformed Escherichia coli XL1-Blue was selected by incubating for 12 hours in an LB-agar plate containing 50 µg / ml kanamycin, and the selected colony was cultured in LB-agar medium to separate DNA from E. coli. The vector identified as a variant from which ATG was removed by sequencing the isolated DNA was transformed into E. coli BL21 (DE3) -Star and used for mass expression. The recombinant expression vector generated by site-specific mutation was named pET26B (+)-Brazzein (Met-).

The brazein variant of the present invention has properties such as heat stability and acid resistance and water solubility that are superior to the conventional brazein, and has a sweet taste of at least 2 to up to 3.3 times or more than the brazein, and in the case of the brazein multiple variants Like the Jane variants, they have the same stability and at least four to up to twenty times the sweetness of the proteins of the Brazein subtype. Therefore, the brazein 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.

<110> G O ELEMENT CO., LTD. <120> Method for preparing novel mutated and multi-mutated Brazzein having higher sweetness <130> NP08-0019 <160> 163 <170> KopatentIn 1.71 <210> 1 <211> 228 <212> DNA <213> E. coli pelB + Brazzein gene (Met-) <400> 1 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgcta gatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_1 <400> 2 tgcaaggctg tttac 15 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_2 <400> 3 tgcaaggacg tttac 15 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Primer_3 <400> 4 tgcaaggaag tttac 15 <210> 5 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Primer_4 <400> 5 tgcaagcacg tttac 15 <210> 6 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_5 <400> 6 tgcaagcgtg tttac 15 <210> 7 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_6 <400> 7 aagcttgcta agcat 15 <210> 8 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_7 <400> 8 aagcttcaca agcat 15 <210> 9 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_8 <400> 9 aagcttaaaa agcat 15 <210> 10 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_9 <400> 10 aagcttcgta agcat 15 <210> 11 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_10 <400> 11 aagcttgaaa agcat 15 <210> 12 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_11 <400> 12 cttgatgctc atgct 15 <210> 13 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_12 <400> 13 cttgatcgtc atgct 15 <210> 14 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_13 <400> 14 cttgatcgcc atgct 15 <210> 15 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_14 <400> 15 cttgatgacc atgct 15 <210> 16 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_15 <400> 16 cttgatgaac atgct 15 <210> 17 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_16 <400> 17 gataaggctg ctcga 15 <210> 18 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_17 <400> 18 gataagaaag ctcga 15 <210> 19 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_18 <400> 19 gataagcgtg ctcga 15 <210> 20 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_19 <400> 20 gataaggacg ctcga 15 <210> 21 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_20 <400> 21 gataaggaag ctcga 15 <210> 22 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_21 <400> 22 catgctgctt ctgga 15 <210> 23 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_22 <400> 23 catgctaaat ctgga 15 <210> 24 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_23 <400> 24 catgctcgct ctgga 15 <210> 25 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_24 <400> 25 catgctgact ctgga 15 <210> 26 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_25 <400> 26 catgctgaat ctgga 15 <210> 27 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_26 <400> 27 tctggagctt gcttt 15 <210> 28 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_27 <400> 28 tctggagact gcttt 15 <210> 29 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_28 <400> 29 tctggaaaat gcttt 15 <210> 30 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_29 <400> 30 tctggacgct gcttt 15 <210> 31 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_30 <400> 31 tctggacgtt gcttt 15 <210> 32 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_31 <400> 32 tacgatgcta agaga 15 <210> 33 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_32 <400> 33 tacgatgaca agaga 15 <210> 34 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_33 <400> 34 tacgataaaa agaga 15 <210> 35 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_34 <400> 35 tacgatcgca agaga 15 <210> 36 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_35 <400> 36 tacgatcgta agaga 15 <210> 37 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_36 <400> 37 gaaaaggcta atctt 15 <210> 38 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_37 <400> 38 gaaaaggaca atctt 15 <210> 39 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_38 <400> 39 gaaaagcgca atctt 15 <210> 40 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_39 <400> 40 gaaaaggaca atctt 15 <210> 41 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_40 <400> 41 gaaaaggaaa atctt 15 <210> 42 <211> 228 <212> DNA <213> E. coli pelB + Brazein (K5A) gene <400> 42 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaaagc tgtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga ctgcttttac 180 gatcgtaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 43 <211> 228 <212> DNA <213> E. coli pelB + Brazein (K5D) gene <400> 43 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaaaga cgtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga ctgcttttac 180 gatcgtaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 44 <211> 228 <212> DNA <213> E. coli pelB + Brazein (K5E) gene <400> 44 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaaaga agtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga ctgcttttac 180 gatcgtaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 45 <211> 228 <212> DNA <213> E. coli pelB + Brazein (K5H) gene <400> 45 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaaaca cgtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga ctgcttttac 180 gatcgtaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 46 <211> 228 <212> DNA <213> E. coli pelB + Brazein (K5R) gene <400> 46 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaaacg tgtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga ctgcttttac 180 gatcgtaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 47 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> E. coli pelB + Brazein (D28A) gene <400> 47 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgct aagcatgcta gatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 48 <211> 228 <212> DNA <213> E. coli pelB + Brazein (D28H) gene <400> 48 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaaacg tgtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgct aagcgtgcta gatctggaga ctgcttttac 180 gatcgtaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 49 <211> 228 <212> DNA <213> E. coli pelB + Brazein (D28K) gene <400> 49 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttaaa aagcatgcta gatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 50 <211> 228 <212> DNA <213> E. coli pelB + Brazein (D28R) gene <400> 50 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttcgt aagcatgcta gatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 51 <211> 228 <212> DNA <213> E. coli pelB + Brazein (D28E) gene <400> 51 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgaa aagcatgcta gatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 52 <211> 228 <212> DNA <213> E. coli pelB + Brazein (K29A) gene <400> 52 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat gctcatgcta gatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 53 <211> 228 <212> DNA <213> E. coli pelB + Brazein (K29R) gene <400> 53 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat cgtcatgcta gatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 54 <211> 228 <212> DNA <213> E. coli pelB + Brazein (K29H) gene <400> 54 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat cgccatgcta gatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 55 <211> 228 <212> DNA <213> E. coli pelB + Brazein (K29D) gene <400> 55 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat gaccatgcta gatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 56 <211> 228 <212> DNA <213> E. coli pelB + Brazein (K29E) gene <400> 56 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat gaacatgcta gatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 57 <211> 228 <212> DNA <213> E. coli pelB + Brazein (H30A) gene <400> 57 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aaggctgcta gatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 58 <211> 228 <212> DNA <213> E. coli pelB + Brazein (H30K) gene <400> 58 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 59 <211> 228 <212> DNA <213> E. coli pelB + Brazein (H30R) gene <400> 59 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 60 <211> 228 <212> DNA <213> E. coli pelB + Brazein (H30D) gene <400> 60 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aaggacgcta gatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 61 <211> 228 <212> DNA <213> E. coli pelB + Brazein (H30E) gene <400> 61 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aaggaagcta gatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 62 <211> 228 <212> DNA <213> E. coli pelB + Brazein (R32A) gene <400> 62 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgctg cttctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 63 <211> 228 <212> DNA <213> E. coli pelB + Brazein (R32K) gene <400> 63 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgcta aatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 64 <211> 228 <212> DNA <213> E. coli pelB + Brazein (R32H) gene <400> 64 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgctc actctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 65 <211> 228 <212> DNA <213> E. coli pelB + Brazein (R32D) gene <400> 65 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgctg actctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 66 <211> 228 <212> DNA <213> E. coli pelB + Brazein (R32E) gene <400> 66 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgctg aatctggaga atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 67 <211> 228 <212> DNA <213> E. coli pelB + Brazein (E35A) gene <400> 67 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgctg aatctggagc ttgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 68 <211> 229 <212> DNA <213> E. coli pelB + Brazein (E35D) gene <400> 68 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga ctgcttttac 180 gatgaataag agaaatcttc aatgcatttg cgattactgc gaatactaa 229 <210> 69 <211> 228 <212> DNA <213> E. coli pelB + Brazein (E35K) gene <400> 69 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttaaa aagcatgctg aatctggaaa atgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 70 <211> 228 <212> DNA <213> E. coli pelB + Brazein (E35H) gene <400> 70 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttcac aagcatgctc actctggaca ctgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 71 <211> 228 <212> DNA <213> E. coli pelB + Brazein (E35R) gene <400> 71 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttcgt aagcatgctc actctggaca ctgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 72 <211> 228 <212> DNA <213> E. coli pelB + Brazein (E40A) gene <400> 72 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgctc actctggaca ctgcttttac 180 gatgctaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 73 <211> 228 <212> DNA <213> E. coli pelB + Brazein (E40D) gene <400> 73 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgctc actctggaca ctgcttttac 180 gatgacaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 74 <211> 228 <212> DNA <213> E. coli pelB + Brazein (E40K) gene <400> 74 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgctc actctggaca ctgcttttac 180 gataaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 75 <211> 228 <212> DNA <213> E. coli pelB + Brazein (E40H) gene <400> 75 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgctc actctggaca ctgcttttac 180 gatcacaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 76 <211> 228 <212> DNA <213> E. coli pelB + Brazein (E40R) gene <400> 76 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgctc actctggaca ctgcttttac 180 gatcgtaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 77 <211> 228 <212> DNA <213> E. coli pelB + Brazein (R42A) gene <400> 77 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgcta gatctggaga atgcttttac 180 gatgaaaagg ctaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 78 <211> 228 <212> DNA <213> E. coli pelB + Brazein (R42K) gene <400> 78 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgcta gatctggaga atgcttttac 180 gatgaaaaga aaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 79 <211> 228 <212> DNA <213> E. coli pelB + Brazein (R42H) gene <400> 79 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgcta gatctggaga atgcttttac 180 gatgaaaagc acaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 80 <211> 228 <212> DNA <213> E. coli pelB + Brazein (R42D) gene <400> 80 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgcta gatctggaga atgcttttac 180 gatgaaaagg acaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 81 <211> 228 <212> DNA <213> E. coli pelB + Brazein (R42E) gene <400> 81 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcatgcta gatctggaga atgcttttac 180 gatgaaaagg aaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 82 <211> 53 <212> PRT <213> Brazzein (minor type) <400> 82 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 Arg 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> 83 <211> 53 <212> PRT <213> Brazzein (K5A) <400> 83 Asp Lys Cys Lys Ala 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 Arg 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> 84 <211> 53 <212> PRT <213> Brazzein (K5D) <400> 84 Asp Lys Cys Lys Asp 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 Arg 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> 85 <211> 53 <212> PRT <213> Brazzein (K5E) <400> 85 Asp Lys Cys Lys Glu 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 Arg 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> 86 <211> 53 <212> PRT <213> Brazzein (K5H) <400> 86 Asp Lys Cys Lys His 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 Arg 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> 87 <211> 53 <212> PRT <213> Brazzein (K5R) <400> 87 Asp Lys Cys Lys Arg 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 Arg 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> 88 <211> 53 <212> PRT <213> Brazzein (D28A) <400> 88 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 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> 89 <211> 53 <212> PRT <213> Brazzein (D28H) <400> 89 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 His 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> 90 <211> 53 <212> PRT <213> Brazzein (D28K) <400> 90 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 Lys 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> 91 <211> 53 <212> PRT <213> Brazzein (D28R) <400> 91 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 Arg 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> 92 <211> 53 <212> PRT <213> Brazzein (D28E) <400> 92 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 Glu 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> 93 <211> 53 <212> PRT <213> Brazzein (K29A) <400> 93 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 Arg Ala 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> 94 <211> 53 <212> PRT <213> Brazzein (K29R) <400> 94 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 Arg Arg 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> 95 <211> 53 <212> PRT <213> Brazzein (K29H) <400> 95 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 Arg His 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> 96 <211> 53 <212> PRT <213> Brazzein (K29D) <400> 96 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 Arg Asp 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> 97 <211> 53 <212> PRT <213> Brazzein (K29E) <400> 97 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 Arg Glu 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> 98 <211> 53 <212> PRT <213> Brazzein (H30A) <400> 98 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 Arg Lys Ala 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> 99 <211> 53 <212> PRT <213> Brazzein (H30K) <400> 99 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 Arg Lys Lys 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> 100 <211> 53 <212> PRT <213> Brazzein (H30R) <400> 100 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 Arg Lys Arg 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> 101 <211> 53 <212> PRT <213> Brazzein (H30D) <400> 101 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 Arg Lys Asp 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> 102 <211> 53 <212> PRT <213> Brazzein (H30E) <400> 102 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 Arg Lys Glu 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> 103 <211> 53 <212> PRT <213> Brazzein (R32A) <400> 103 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 Arg Lys His Ala Ala 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> 104 <211> 53 <212> PRT <213> Brazzein (R32K) <400> 104 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 Arg Lys His Ala Lys 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> 105 <211> 53 <212> PRT <213> Brazzein (R32H) <400> 105 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 Arg Lys His Ala His 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> 106 <211> 53 <212> PRT <213> Brazzein (R32D) <400> 106 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 Arg Lys His Ala Asp 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> 107 <211> 53 <212> PRT <213> Brazzein (R32E) <400> 107 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 Arg Lys His Ala Glu 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> 108 <211> 53 <212> PRT <213> Brazzein (E35A) <400> 108 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 Arg Lys His Ala Arg 20 25 30 Ser Gly Ala Cys Phe Tyr Asp Glu Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 109 <211> 53 <212> PRT <213> Brazzein (E35D) <400> 109 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 Arg Lys His Ala Arg 20 25 30 Ser Gly Asp Cys Phe Tyr Asp Glu Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 110 <211> 53 <212> PRT <213> Brazzein (E35K) <400> 110 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 Arg Lys His Ala Arg 20 25 30 Ser Gly Lys Cys Phe Tyr Asp Glu Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 111 <211> 53 <212> PRT <213> Brazzein (E35H) <400> 111 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 Arg Lys His Ala Arg 20 25 30 Ser Gly His Cys Phe Tyr Asp Glu Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 112 <211> 53 <212> PRT <213> Brazzein (E35R) <400> 112 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 Arg Lys His Ala Arg 20 25 30 Ser Gly Arg Cys Phe Tyr Asp Glu Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 113 <211> 53 <212> PRT <213> Brazzein (E40A) <400> 113 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 Arg Lys His Ala Arg 20 25 30 Ser Gly Glu Cys Phe Tyr Asp Ala Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 114 <211> 53 <212> PRT <213> Brazzein (E40D) <400> 114 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 Arg Lys His Ala Arg 20 25 30 Ser Gly Glu Cys Phe Tyr Asp Asp Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 115 <211> 53 <212> PRT <213> Brazzein (E40K) <400> 115 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 Arg Lys His Ala Arg 20 25 30 Ser Gly Glu Cys Phe Tyr Asp Lys Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 116 <211> 53 <212> PRT <213> Brazzein (E40H) <400> 116 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 Arg Lys His Ala Arg 20 25 30 Ser Gly Glu Cys Phe Tyr Asp His Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 117 <211> 53 <212> PRT <213> Brazzein (E40R) <400> 117 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 Arg Lys His Ala Arg 20 25 30 Ser Gly Glu Cys Phe Tyr Asp Arg Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 118 <211> 53 <212> PRT <213> Brazzein (R42A) <400> 118 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 Arg Lys His Ala Arg 20 25 30 Ser Gly Glu Cys Phe Tyr Asp Glu Lys Ala Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 119 <211> 53 <212> PRT <213> Brazzein (R42K) <400> 119 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 Arg Lys His Ala Arg 20 25 30 Ser Gly Glu Cys Phe Tyr Asp Glu Lys Lys Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 120 <211> 53 <212> PRT <213> Brazzein (R42H) <400> 120 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 Arg Lys His Ala Arg 20 25 30 Ser Gly Glu Cys Phe Tyr Asp Glu Lys His Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 121 <211> 53 <212> PRT <213> Brazzein (R42D) <400> 121 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 Arg Lys His Ala Arg 20 25 30 Ser Gly Glu Cys Phe Tyr Asp Glu Lys Asp Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 122 <211> 53 <212> PRT <213> Brazzein (R42E) <400> 122 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 Arg Lys His Ala Arg 20 25 30 Ser Gly Glu Cys Phe Tyr Asp Glu Lys Glu Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 123 <211> 228 <212> DNA <213> E. coli pelB + Brazein (H30R_E35D) gene <400> 123 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga ctgcttttac 180 gatgaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 124 <211> 228 <212> DNA <213> E. coli pelB + Brazein (H30R_E40A) gene <400> 124 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga atgcttttac 180 gatgctaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 125 <211> 228 <212> DNA <213> E. coli pelB + Brazein (H30R_E40D) gene <400> 125 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga atgcttttac 180 gatgacaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 126 <211> 228 <212> DNA <213> E. coli pelB + Brazein (H30R_E40K) gene <400> 126 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga atgcttttac 180 gataaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 127 <211> 228 <212> DNA <213> E. coli pelB + Brazein (H30R_E40R) gene <400> 127 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga atgcttttac 180 gatcgtaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 128 <211> 228 <212> DNA <213> E. coli pelB + Brazein (E35D_E40A) gene <400> 128 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttcgt aagcatgctc actctggaca ctgcttttac 180 gatgctaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 129 <211> 228 <212> DNA <213> E. coli pelB + Brazein (E35D_E40D) gene <400> 129 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttcgt aagcatgctc actctggaca ctgcttttac 180 gatgacaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 130 <211> 228 <212> DNA <213> E. coli pelB + Brazein (E35D_E40K) gene <400> 130 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttcgt aagcatgctc actctggaca ctgcttttac 180 gataaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 131 <211> 228 <212> DNA <213> E. coli pelB + Brazein (E35D_E40R) gene <400> 131 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttcgt aagcatgctc actctggaca ctgcttttac 180 gatcgtaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 132 <211> 228 <212> DNA <213> E. coli pelB + Brazein (H30R_E35D_E40A) gene <400> 132 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga ctgcttttac 180 gatgctaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 133 <211> 228 <212> DNA <213> E. coli pelB + Brazein (H30R_E35D_E40D) gene <133> 133 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga ctgcttttac 180 gatgacaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 134 <211> 228 <212> DNA <213> E. coli pelB + Brazein (H30R_E35D_E40K) gene <400> 134 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga ctgcttttac 180 gataaaaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 135 <211> 228 <212> DNA <213> E. coli pelB + Brazein (H30R_E35D_E40R) gene <400> 135 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagcgtgcta gatctggaga ctgcttttac 180 gatcgtaaga gaaatcttca atgcatttgc gattactgcg aatactaa 228 <210> 136 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer_41 <400> 136 gataagaaac gtgct 15 <210> 137 <211> 66 <212> DNA 213 pelB signal sequence <400> 137 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggcc 66 <210> 138 <211> 231 <212> DNA <213> E. coli pelB + Brazein (29ins30 Lys_H30R_E35D_E40A) gene <400> 138 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagaaacgtg ctagatctgg agactgcttt 180 tacgatgcta agagaaatct tcaatgcatt tgcgattact gcgaatacta a 231 <139> <211> 231 <212> DNA <213> E. coli pelB + Brazein (29ins30 Lys_H30R_E35D_E40D) gene <400> 139 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagaaacgtg ctagatctgg agactgcttt 180 tacgatgcta agagaaatct tcaatgcatt tgcgattact gcgaatacta a 231 <210> 140 <211> 231 <212> DNA <213> E. coli pelB + Brazein (29ins30 Lys_H30R_E35D_E40K) gene <400> 140 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagaaacgtg ctagatctgg agactgcttt 180 tacgataaaa agagaaatct tcaatgcatt tgcgattact gcgaatacta a 231 <210> 141 <211> 231 <212> DNA <213> E. coli pelB + Brazein (29ins30 Lys_H30R_E35D_E40R) gene <400> 141 atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccgata agtgcaagaa ggtttacgaa aattacccag tttctaagtg ccaacttgct 120 aatcaatgca attacgattg caagcttgat aagaaacgtg ctagatctgg agactgcttt 180 tacgatcgta agagaaatct tcaatgcatt tgcgattact gcgaatacta a 231 <210> 142 <211> 53 <212> PRT <213> Brazzein (H30R_E35D) <400> 142 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 Arg Lys Arg Ala Lys 20 25 30 Ser Gly Asp Cys Phe Tyr Asp Glu Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 143 <211> 53 <212> PRT <213> Brazzein (H30R_E40A) <400> 143 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 Arg Lys Arg Ala Lys 20 25 30 Ser Gly Glu Cys Phe Tyr Asp Ala Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 144 <211> 53 <212> PRT <213> Brazzein (H30R_E40D) <400> 144 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 Arg Lys Arg Ala Lys 20 25 30 Ser Gly Glu Cys Phe Tyr Asp Asp Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 145 <211> 53 <212> PRT <213> Brazzein (H30R_E40K) <400> 145 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 Arg Lys Arg Ala Lys 20 25 30 Ser Gly Glu Cys Phe Tyr Asp Lys Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 146 <211> 53 <212> PRT <213> Brazzein (H30R_E40R) <400> 146 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 Arg Lys Arg Ala Lys 20 25 30 Ser Gly Glu Cys Phe Tyr Asp Arg Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 147 <211> 53 <212> PRT <213> Brazzein (E35D_E40A) <400> 147 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 Arg Lys His Ala Lys 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> 148 <211> 53 <212> PRT <213> Brazzein (E35D_E40D) <400> 148 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 Arg Lys His Ala Lys 20 25 30 Ser Gly Asp Cys Phe Tyr Asp Asp Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 149 <211> 53 <212> PRT <213> Brazzein (E35D_E40K) <400> 149 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 Arg Lys His Ala Lys 20 25 30 Ser Gly Asp Cys Phe Tyr Asp Lys Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 150 <211> 53 <212> PRT <213> Brazzein (E35D_E40R) <400> 150 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 Arg Lys His Ala Lys 20 25 30 Ser Gly Asp Cys Phe Tyr Asp Arg Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 151 <211> 53 <212> PRT <213> Brazzein (H30R_E35D_E40A) <400> 151 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 Arg Lys Arg Ala Lys 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> 152 <211> 53 <212> PRT <213> Brazzein (H30R_E35D_E40D) <400> 152 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 Arg Lys Arg Ala Lys 20 25 30 Ser Gly Asp Cys Phe Tyr Asp Asp Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 153 <211> 53 <212> PRT <213> Brazzein (H30R_E35D_E40K) <400> 153 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 Arg Lys Arg Ala Lys 20 25 30 Ser Gly Asp Cys Phe Tyr Asp Lys Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 154 <211> 53 <212> PRT <213> Brazzein (H30R_E35D_E40R) <400> 154 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 Arg Lys Arg Ala Lys 20 25 30 Ser Gly Asp Cys Phe Tyr Asp Arg Lys Arg Asn Leu Gln Cys Ile Cys 35 40 45 Asp Tyr Cys Glu Tyr 50 <210> 155 <211> 54 <212> PRT <213> Brazzein (29ins30 Lys_H30R_E35D_E40A) <400> 155 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 Arg Lys Lys Arg Ala 20 25 30 Lys 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> 156 <211> 54 <212> PRT <213> Brazzein (29ins30 Lys_H30R_E35D_E40D) <400> 156 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 Arg Lys Lys Arg Ala 20 25 30 Lys Ser Gly Asp Cys Phe Tyr Asp Asp Lys Arg Asn Leu Gln Cys Ile 35 40 45 Cys Asp Tyr Cys Glu Tyr 50 <210> 157 <211> 54 <212> PRT <213> Brazzein (29ins30 Lys_H30R_E35D_E40K) <400> 157 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 Arg Lys Lys Arg Ala 20 25 30 Lys Ser Gly Asp Cys Phe Tyr Asp Lys Lys Arg Asn Leu Gln Cys Ile 35 40 45 Cys Asp Tyr Cys Glu Tyr 50 <210> 158 <211> 54 <212> PRT <213> Brazzein (29ins30 Lys_H30R_E35D_E40R) <400> 158 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 Arg Lys Lys Arg Ala 20 25 30 Lys Ser Gly Asp Cys Phe Tyr Asp Arg Lys Arg Asn Leu Gln Cys Ile 35 40 45 Cys Asp Tyr Cys Glu Tyr 50 <210> 159 <211> 162 <212> DNA <213> Artificial Sequence <220> <223> Brazzein <400> 159 gataagtgca agaaggttta cgaaaattac ccagtttcta agtgccaact tgctaatcaa 60 tgcaattacg attgcaagct tgctaagcat gctagatctg gagaatgctt ttacgatgaa 120 aagagaaatc ttcaatgcat ttgcgattac tgcgaatact aa 162 <210> 160 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for brazzein cloning <400> 160 catgccatgg ataagtgcaa gaaggtttac 30 <210> 161 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for brazzein cloning <400> 161 ccgctcgagt tagtattcgc agtaatcg 28 <210> 162 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> ATG deletion primer 1 <400> 162 cagccggcga tggccgacaa atgcaaaaaa 30 <210> 163 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> ATG deletion primer 2 <400> 163 ttttttgcat ttgtcggcca tcgccggctg 30

Claims (9)

(a) Escherichia coli pelB signal sequence and SEQ ID NO: 100, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158 Culturing E. coli transformed with a brazein variant gene encoding a brazein variant having an amino acid sequence selected from the group consisting of: (b) separating the protein of the cell membrane gap of the cultured Escherichia coli; And (C) a method for producing a brazein variant comprising the step of heat-treating the separated cell membrane protein. SEQ ID NO: 100, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158 Brazein variants. A polynucleotide encoding the brazein variant of claim 2. The method of claim 3, wherein the polynucleotide is SEQ ID NO: 59, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: Polynucleotide having a base sequence selected from the group consisting of 141. A recombinant expression vector for expression of a brazein variant, comprising a promoter and a polynucleotide of claim 3 operably linked thereto. The recombinant expression vector according to claim 5, wherein the recombinant expression vector is pET26B (+)-Brazzein (H30R), pET26B (+)-Brazzein (E35D), pET26B (+)-Brazzein (E35D), pET26B (+)-Brazzein (E40D), pET26B (+)-Brazzein (E40K), pET26B (+)-Brazzein (E40R), pET26B (+)-Brazzein (H30R_E35D), pET26B (+)-Brazzein (H30R_E40A), pET26B (+)-Brazzein (H30R_ pET26B (+)-Brazzein (H30R_E40K) or pET26B (+)-Brazzein (H30R_E40R), pET26B (+)-Brazzein (H30R_E35D_E40A), pET26B (+)-Brazzein (H30R_E35D_E40B (D) E40D) pET26B (+)-Brazzein (H30R_E35D_E40R), pET26B (+)-Brazzein (29ins30 Lys_H30R_E35D_E40A), pET26B (+)-Brazzein (29ins30 Lys_H30R_E35D_E40D), pET26Bzein_H30R (zH30) 29ins30 Lys_H30R_E35D_E40R) recombinant expression vector, characterized in that selected from the group consisting of. E. coli comprising the recombinant expression vector of claim 5 or 6. (a) culturing E. coli of claim 7; (b) separating the protein of the cell membrane gap of the cultured Escherichia coli; And (C) a method for producing a brazein variant comprising the step of heat-treating the separated cell membrane protein. A composition for enhancing sugar content of claim 2 comprising the brazein mutant as an active ingredient.