KR101779890B1 - A microorganism having enhanced levan fructotransferase productivity and a method of producing difructose anhydride IV using the microorganism - Google Patents

Abstract

The present invention relates to a Levan fructosyltransferase secretion expression cassette comprising a nucleic acid encoding levan fructotransferase and a nucleic acid encoding a translational fusion partner (TFP), a vector containing the cassette, A cassette or a vector, and a process for producing a levansucrose transferase and a difructose anhydride IV using the recombinant microorganism.
The recombinant microorganism of the present invention can secrete recombinant levan fructose transferase with excellent efficiency by using a protein secretion fusion factor and can be obtained by reacting the levan fructose transferase produced from the microorganism with levan, Can be fermented in a medium containing Levan to effectively produce diproprathiane hydride IV.

Description

[0001] The present invention relates to a microorganism having enhanced levulinotransferase activity and a method for producing the same,

The present invention relates to a Levan fructosyltransferase secretion expression cassette comprising a nucleic acid encoding levan fructotransferase and a nucleic acid encoding a translational fusion partner (TFP), a vector containing the cassette, A cassette or a vector, and a process for producing a levansucrose transferase or difructose anhydride IV using the recombinant microorganism.

Difructose anhydride (DFA) is a disaccharide produced from fructose polymers. It is the smallest cyclic disaccharide in which two molecules of fructose are bound to each other by a reducing end and a non-reducing hydroxyl group. (J Ind Microbiol Biotechnol 2014, 41: 893-906), which has an excellent absorption-promoting function of mineral ions (calcium and iron) and has an effect of preventing and improving anemia and osteoporosis.

Among them, DFA-IV is produced by Levan fructosyl transferase from Levan, another fructose polymer. The microorganisms found to produce DFA were found mainly in Arthrobacter . However, when Levan fructose transferase expressed from microorganisms of the genus Arthrobacter is used for the production of DFA-IV, the expression level of levan fructose transferase is low, There was a disadvantage in increasing. In order to overcome this disadvantage, a method of producing a recombinant levan fructosyltransferase using E. coli prepared by cloning Levan fructosyltransferase gene derived from a microorganism of the genus Astrobacter has been reported in various groups, and DFA-IV production (Letters in Applied Microbiology 2005, 40: 228-234), the conversion was low.

A key technology for the efficient production of DFA-IV from Levan is the recombinant mass production technology of levan fructose transferase. In the case of mass expression of exogenous proteins such as levan fructosyltransferase using microorganisms, depending on the characteristics of the host cell and the target protein, the expression level, water solubility, expression site, and modification of the protein are different from each other, An efficient production system can be constructed only by selecting an appropriate expression system. Generally, Escherichia coli is used as a host system. However, when Escherichia coli produces recombinant levan fructose-transferase, it is mostly expressed as an active form, but when it is over-expressed or inadequate in expression conditions, it accumulates as insoluble inclusion bodies, Is often lowered. In addition, since E. coli is not safe for human body, the use of DFA produced by using the produced enzyme is restricted, and therefore, there is a need to use a microorganism called GRAS (Generally Recognized As Safe) such as yeast.

Yeast Saccharomyces cerevisiae cerevisiae ) is a GRAS microorganism, which is the same eukaryotic cell as a higher organism, similar in transcription and translation system to genes derived from higher organisms, and is similar to the intron removal system through splicing and secretion similar to the Golgi apparatus of higher animals It has an institution. Therefore, it is possible to secrete an active protein through post-translational modification, and when the recombinant protein is secreted by a culture medium, the pure enzyme can be easily separated without complicated purification steps . Nevertheless, there is no report on the production of levansuclease transferase in yeast, since the conventional secretion expression system was not able to secure competitiveness due to low enzyme productivity.

Accordingly, the inventors of the present invention have found that, in order to develop a system for mass production of levan fructosyltransferase from microorganisms, recombinant levan fructose transfection enzyme can be effectively used as a microorganism cell by using a protein secretion fusion technology (Korean Patent No. 10-0975596) The protein secretion factors that can secrete out were selected. Through the above process, it was confirmed that a transformant was introduced into a microorganism by linking the selected protein secretion factor gene and a Levan fructosyltransferase gene-linked expression vector to produce a recombinant lepane fructosyltransferase. Further, it was confirmed that DFA-IV was efficiently produced from Levan by the production of levan-fransonase from Levan using the produced levan fructose-transferase, and it was confirmed that the recombinant microorganism secreted and expressed Levan- The present inventors completed the present invention by confirming that DFA-IV can be directly produced from Levan.

One object of the present invention is to provide a Levan fructosylase secretion expression cassette comprising a nucleic acid encoding levan fructotransferase and a nucleic acid encoding a translational fusion partner (TFP) .

Yet another object of the present invention is to provide a vector comprising said cassette.

It is another object of the present invention to provide a recombinant microorganism containing a Levan fructose transferase secretion expression cassette comprising a nucleic acid encoding levan fructotransferase and a nucleic acid encoding a protein secretion fusion factor.

Yet another object of the present invention is to provide a method for producing a recombinant microorganism which comprises (i) culturing the recombinant microorganism; And (ii) recovering levan fructosyltransferase from the cultured microorganism or culture supernatant thereof. The present invention also provides a method for producing Levan fructotransferase.

It is still another object of the present invention to provide a process for preparing difreuctose anhydride IV comprising reacting the recombinant microorganism or the levan fructose transferase produced by the production method with levan .

In order to achieve the above object, the present invention provides, as an embodiment, a nucleic acid encoding a levan fructotransferase and a nucleic acid encoding a translational fusion partner (TFP) Secretion expression cassette.

In order to produce difructose anhydride IV with high efficiency, a recombinant fibrin glycosyltransferase was secreted from a microorganism using a protein secretion fusion factor. For this purpose, a nucleic acid encoding a levan fructose transferase A recombinant vector containing the cassette, and a recombinant microorganism having the Levan fructosyltransferase secretion expression cassette comprising the nucleic acid encoding the protein secretion fusion factor were prepared and then used to obtain Levan fructosyl transferase Enzymes.

The above-mentioned "difructose anhydride (DFA)" is produced during the process of producing fructose syrup by treatment of inulin with sulfuric acid, and the reducing end of the two fructose is formed at the end of the fructose Cyclic divalent sugar bound to a non-reducing hydroxyl group. Up to now, five classes of DFA I to V have been known. Of these, the structure of diproxthanhydride IV is represented by the following formula (1).

[Chemical Formula 1]

Diprutosteride hydride is a non-digestible, non-toxic, non-degradable disaccharide that can be used as a low calorie sweetener in the body of an animal and can be used as a bifidus microflora growth factor to inhibit tooth decay. It can also be used as a mineral absorption promoting factor in vivo. However, it is not limited thereto.

The term " levan fructotransferase " in the present invention refers to an enzyme that produces diplactose anhydride IV (DFA-IV) using levan as a substrate. In the present invention, it is also called lftA. The levan fructose transferase is specifically an Arthrobacter ureafaciens , more specifically, a levan fumaryltransferase having the amino acid sequence of SEQ ID NO: 8, but is not limited thereto, and any enzyme having the ability to produce diproprathionidolide IV may be used regardless of its origin or sequence Can all be included.

Thus, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 8, or the conserved sequence of the amino acid sequence of SEQ ID NO: 8, and has one or more amino acid residues at one or more positions Amino acid sequence may be substituted, deleted, inserted, added or inverted, specifically 2 to 20, more particularly 2 to 10, more particularly 2 to 5 amino acids) As long as it can maintain or enhance the activity of the levan fructosyltransferase, it is preferably 80% or more, more preferably 90% or more, more particularly 95% or more, particularly 97 % Or more of the amino acid sequence, and the substitution, deletion, insertion, addition, or inversion of the amino acid may include, for example, In the micro-organisms containing an active can include even the mutant sequence or artificial sequence variation occurring naturally.

The term " homology " of the present invention is intended to indicate a degree similar to an amino acid sequence of a wild-type protein or a nucleotide sequence encoding the amino acid sequence, and the amino acid sequence or nucleotide sequence of the present invention is preferably identical to the above- Lt; / RTI > This homology can be determined by comparing the two sequences visually, but can be determined using a bioinformatic algorithm that aligns the sequences to be compared and analyzes the degree of homology. The homology between the two amino acid sequences can be expressed as a percentage. Useful automated algorithms are available in the GAP, BESTFIT, FASTA and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, Wis. USA). The automated array algorithms in this module include Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman sequence alignment algorithms. Algorithm and homology determination for other useful arrays is automated in software including FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL W.

In the present invention, the term " translational fusion partner (TFP) " refers to a factor useful for secretion of a target protein. The protein secretion factor may be one disclosed in Korean Patent Laid-Open No. 10-2008-0042823, and more specifically, TFP 1 of SEQ ID NO: 9, TFP 2 of SEQ ID NO: 10, TFP 3 of SEQ ID NO: 11, TFP 4, TFP 5 of SEQ ID NO: 13, TFP 6 of SEQ ID NO: 15, TFP 7 of SEQ ID NO: 15, TFP 8 of SEQ ID NO: 16, TFP 9 of SEQ ID NO: 17, TFP 10 of SEQ ID NO: 18, TFP 11 of SEQ ID NO: 19 TFP 14 of SEQ ID NO: 22, TFP 14 of SEQ ID NO: 22, TFP 15 of SEQ ID NO: 23, TFP 16 of SEQ ID NO: 24, TFP 17 of SEQ ID NO: 25, TFP 18 of SEQ ID NO: 26, TFP 19 of SEQ ID NO: 27, TFP 20 of SEQ ID NO: 28, TFP 21 of SEQ ID NO: 29, TFP 22 of SEQ ID NO: 30, TFP 23 of SEQ ID NO: 31 or TFP 24 of SEQ ID NO: 32, Any factor capable of improving expression can be included without limitation.

Thus, the TFP may comprise an amino acid sequence of any one of SEQ ID NOS: 9 to 32. Alternatively, the TFP may contain one or more amino acid residues at one or more positions (including amino acid residues in the amino acid sequence of SEQ ID NOS: 9 to 32) Specifically, 2 to 20 amino acid residues, more specifically 2 to 10 amino acid residues, more specifically 2 to 5 amino acid residues, more specifically 2 to 5 amino acid residues) As long as it can improve the secretory expression of the target protein, 80% or more, specifically 90% or more, more particularly 95% or more, of the amino acid sequence of SEQ ID NOS: 9 to 32 , Specifically, an amino acid sequence having a homology of 97% or more, and substitution, deletion, insertion, addition, or inversion of the amino acid may include It can be included in a microorganism containing a mutant sequence of the TFP activated even or artificial mutant sequence produced naturally.

Further, when the recombinant levan fructose transferase is derived from Arthrobacter ureafaciens , it is preferable that the Levan fructosyl transferase is selected from the group consisting of TFP7 of SEQ ID NO: 15, TFP8 of SEQ ID NO: 16, TFP9 of SEQ ID NO: 17, TFP 13 of SEQ ID NO: 21 or TFP 19 of SEQ ID NO: 27 may be linked, but are not limited thereto.

In the Levan fructosyltransferase expression cassette, the nucleic acid encoding the contained levan fructose transferase and the nucleic acid encoding the protein secretion fusion factor may be linked to each other by a linker.

The linker may comprise a protease recognition sequence or an affinity tag.

In addition, the nucleic acid sequence coding for the levan fructose transferase used in the present invention may include linker DNA used for recombination in cells with the linear vector of the present invention. Addition of such a linker sequence to a nucleic acid sequence encoding a levan fructosyltransferase can be carried out by conventional DNA techniques such as PCR and / or restriction enzyme cleavage and linkage.

The linker DNA of the present invention should be of sufficient length and introduced into a host cell so that the nucleic acid sequence encoding the Levan fucosyltransferase in the cell can be recombined into a linear vector containing the nucleic acid sequence encoding TFP, May be of sufficient homology with a portion of the sequence. Specifically, the linker DNA may have a length of 20 bp or more, such as 30 or 40 bp or more, but is not limited thereto. More specifically, the linker DNA is at least 80% homologous to the linear vector and may have 85%, 90%, 95% or 99% homology, but is not limited thereto.

As an example, the linker DNA can encode a protease recognition sequence to cause cleavage at the junction between TFP and the target protein. For example, the linker DNA comprises a yeast kex2p protease recognition sequence (amino acid sequence comprising Lys-Arg), a mammal purine recognition sequence (amino acid sequence comprising Arg-XX-Arg), a Factor-Xa recognition sequence (Ile-Glu-Gly -Ag), an enterokinase-recognition sequence (amino acid sequence containing Asp-Asp-Lys), a subtilisin recognition sequence (amino acid sequence comprising Ala-His-Tyr) (Amino acid sequence containing Glu-Asn-Leu-Tyr-Phe-Gln-Gly), ubiquitin hydrolase recognition sequence (amino acid sequence containing Arg-Gly-Gly) or thrombin recognition sequence (Arg- Pro-Arg). ≪ / RTI >

The term " affinity tag " in the present invention can be used for various purposes as a peptide or nucleic acid sequence that can be introduced into a recombinant fusion protein or a nucleic acid encoding the same, for example, to enhance purification efficiency of a target protein. The affinity tag may be GST, MBP, NusA, thioredoxin, ubiquitin, FLAG, BAP, HIS, STREP, CBP, CBD or S-tag, But is not limited to.

The affinity tag may be linked to the carboxy terminus or the amino terminus of the levan fructosyl transferase, in particular, but not exclusively, to the carboxy terminus.

The term " nucleic acid " in the present invention has a meaning inclusive of DNA (gDNA and cDNA) and RNA molecules, and the nucleotide, which is a basic constituent unit in the nucleic acid molecule, includes not only natural nucleotides, It also includes analogues. The present invention provides a Levan fructitetransferase expression cassette comprising a nucleic acid encoding levan fructose transferase and a nucleic acid encoding a protein secretion fusion factor.

The term " expression cassette " of the present invention has a nucleic acid element capable of affecting the expression of a structural gene in a cell. Naturally occurring or synthesized nucleic acid constructs generally include an expression cassette that contains a transcribed nucleic acid and a promoter. For the purpose of the present invention, the expression cassette may comprise a nucleic acid encoding a protein secretion fusion factor and a nucleic acid encoding a levan fructose transferase, and in particular, a " Levan fructose transferase secretion expression cassette " "And can secrete the levan fructose transgene by expressing it.

For example, the Archaeobacter Ureapassin-derived levan fructose transferase of the present invention may be, but is not limited to, be encoded by the nucleic acid sequence of SEQ ID NO: 1.

As examples of the fusion protein of the present invention, TFP 1 is SEQ ID NO: 33, TFP 2 is SEQ ID NO: 34, TFP 3 is SEQ ID NO: 35, TFP 4 is SEQ ID NO: 36, TFP 5 is SEQ ID NO: SEQ ID NO: 38, TFP 7, SEQ ID NO: 39, TFP 8, SEQ ID NO: 40, TFP 9, SEQ ID NO: 41, TFP 10, SEQ ID NO: SEQ ID NO: 45, TFP 14 is SEQ ID NO: 46, TFP 15 is SEQ ID NO: 47, TFP 16 is SEQ ID NO: 48, TFP 17 is SEQ ID NO 49, TFP 18 is SEQ ID NO 50, TFP 19 is SEQ ID NO 51, 52, TFP 21 is SEQ ID NO: 53, TFP 22 is SEQ ID NO: 54, TFP 23 is SEQ ID NO: 55, or TFP 24 is SEQ ID NO: 56.

The present invention provides, as yet another aspect, a vector comprising said cassette.

The cassette is as described above.

The term " vector " in the present invention means a DNA product containing a nucleotide sequence encoding a desired protein operably linked to a suitable regulatory sequence so as to be able to express the desired protein in a suitable host, , The target protein refers to a recombinant fusion protein comprising a levan fructose transferase and a protein secretion fusion factor.

These vectors include expression-regulating fragments having various functions for suppressing, amplifying, or inducing expression of a target gene, genes encoding a marker for selection of a transformant, a gene resistant to antibiotics, A suitable customized fusion factor, and the like. In particular, it may be preferable to use the protein secretion fusion factor as a customized fusion factor suitable for the above-mentioned hairless protein.

In a specific example of the present invention, YGaST13-lftA-6H containing TFP13 and Levantub fucoxan-transferase gene derived from Astrobepture urea pathces as an expression vector producing high secretion of Levan fructosyltransferase derived from Agrobacterium ureapathianae (Fig. 7).

The present invention provides, in yet another aspect, a recombinant microorganism containing a Levan fructose transferase secretion expression cassette, comprising a nucleic acid encoding levan fructotransferase and a nucleic acid encoding a protein secretion fusion factor do.

Levan fructosyltransferase, protein secretion fusion factor, nucleic acid, and levansucose transferase secretion expression cassette have been described above.

The recombinant microorganism may be a microorganism transformed with the nucleic acid.

The term " transformed " of the present invention means that a nucleic acid is introduced into a host cell so that the nucleic acid becomes replicable as an extrachromosomal element or by chromosome integration completion.

Any transformation method of the present invention can be used, and can be easily carried out according to a conventional method in the art.

The host cell used for transformation according to the present invention may be any host cell well known in the art. However, it is possible to use a host having a high efficiency of introduction and expression efficiency of the levan fructose transferase gene of the present invention, For example, it can include bacteria, fungi, and yeast. Specifically, the recombinant microorganism may be yeast. More specifically, the yeast may be selected from the group consisting of Candida , Debaryomyces , Hansenula , Kluyveromyces , Pichia , The skiing can be one belonging to the genus Schizosaccharomyces , Yarrowia , Saccharomyces , Schwanniomyces or Arxula , more particularly Candida utilis Candida utilis , Candida boidinii), Candida albicans (Candida albicans), Cluj Vero Mai Seth lactis (Kluyveromyces lactis , Pichia pastoris , Pichia stipitis ), skiing investigation Schizosaccharomyces pombe ), Saccharomyces cerevisiae , Hansenula ( Hansenula polymorpha , Yarrowia lipolytica , Schwanniomyces occidentalis , or Arxula adeninivorans , but are not limited thereto.

In a specific embodiment of the present invention, an optimal yeast secretion fusion factor capable of secretion and expression of Levan fructotransferase gene derived from Arthrobacter ureafaciens is selected, and each expression The production of recombinant levan fructose transferase was confirmed using the vector and the yeast transformed with each vector (FIGS. 2 to 10 and Table 3).

In the present invention, for example, the GAL10 promoter used for the expression of levan fructose transferase is a promoter that is induced to be transcribed by galactose, but a strain in which the GAL80 gene is inactivated can maintain high gene expression regardless of the presence or absence of galactose. For this reason, in the present invention, Levan fugetransferase was expressed using a strain in which the GAL80 gene was inactivated.

As a specific example for constructing the recombinant microorganism, the PCR product amplified with LNK39 (SEQ ID NO: 4) and HisGT50R primer (SEQ ID NO: 5) was linked with the linker end of the vector and 40 base, the GAL7 transcription terminator end Since the 50 bases are the same, when introduced into yeast cells together with the linearized vector, crossing occurs in the cells, and the levan fugetransferase gene and the protein secretion factor vector can be connected in a certain direction. Further, the PCR product and the vector into which the vector was introduced were subjected to SD-Ura medium (0.67% amino acid-deficient yeast substrate, 0.77% uracil-deficient nutritional supplement, 2% glucose) as a selective medium without uracil When selected, the transformant into which each expression vector is introduced can be prepared directly without vector production using E. coli (FIG. 1).

Also, the transfected yeast may be fed in an oil-in-water culture to refrain the levan fructose transferase, and then the purified enzyme may be used to produce diprolactone anhydride IV (FIGS. 11, 12, and 5) It was confirmed that the transformed yeast could be fermented to produce diprolactone anhydride IV (FIG. 13).

Specifically, an analysis of the production ability of dipeptide anhydride IV of secreted recombinant fibrin glycosyltransferase showed that the fermentation broth of 5-fold concentrated recombinant yeast was 128% higher than that of E. coli-produced fibrin glycosyltransferase Respectively. Also, when the transformed recombinant yeast was cultured in a culture medium containing levan, it was confirmed that levulinic anhydride IV was produced from levan by the levan fructose transferase secreted out of the cells during the culturing process.

In another aspect, the present invention provides a method for producing a recombinant microorganism, comprising: (i) culturing the recombinant microorganism; And recovering the levan fumaryl transferase from the cultured microorganism or culture supernatant thereof.

The recombinant microorganism and levansucose transferase are as described above.

The medium for culturing the recombinant microorganism and the culture conditions may be appropriately selected depending on the host cell. Specifically, the cells were cultured in a YEPD medium (4% yeast extract, 2% glucose, 1% peptone) containing glucose as a carbon source, and supplemented with feed medium (30% glucose, 15% yeast extract) But is not limited thereto.

In one specific embodiment of the present invention, the recombinant microorganism was used for fed-batch culture to obtain levan fructose transferase.

The term " fed-batch cultivation " of the present invention is a method of culturing having high final concentration and productivity of a product, and is very often used for culturing high-concentration cells. After initially filling the culture medium with a new culture medium, A precursor of the product, etc., but the reaction product may remain in the fermenter.

For example, but not by way of limitation, the preferred method of fed-batch cultivation is a one-step seed culture in 50 ml of minimal liquid medium (yeast nitrogen source substrate lacking 0.67% amino acid, 0.5% casamino acid, 2% glucose) Then, the cells were cultured in 200 ml of YEPD liquid medium, and the cells were inoculated into the culture medium. The cells were cultured at 30 ° C. for 50 hours, and then fed with a culture medium (15% yeast extract, 30% glucose) It may be to supply.

According to another aspect of the present invention, there is provided a process for producing diprolactone anhydride IV, which comprises reacting the recombinant microorganism or the levan fructose transferase produced by the production method with Levan.

The recombinant microorganism, the method for producing levan fructose transferase, and the difluntosanhydride IV are as described above.

The reaction may be carried out at a pH of from 3.5 to 9.5, and the reaction may be conducted at a temperature of from 1 ° C to 70 ° C, but is not limited thereto.

With respect to reacting levan fructose transferase with Levan, in one specific embodiment of the present invention, the prepared levan fumetate transferase was purified using affinity chromatography (FIG. 10), and the activity of the purified levan fructose transferase Optimal conditions were analyzed (Table 4, Figs. 11 and 12), and it was confirmed that diphenylmethane dihydroidide IV can be produced in good yield from levan fructose transferase (Table 5).

Meanwhile, the step of reacting the recombinant microorganism with Levan may include, but not limited to, fermentation of the recombinant microorganism in a medium containing Levan.

Compared to an enzyme process in which levansucrose transition enzyme is produced and then reacted with levan to produce diprolactone anhydride IV, the transformant strain is directly fermented in a medium containing Levan to produce diproprathone anhydride IV, Production of high purity diproprathone anhydride IV can be possible because the conversion product can be used as a carbon source for fructose and glucose produced by the action of levan fructose transferase without the cost of enzyme production and purification.

In a specific embodiment of the present invention, a recombinant yeast strain secreted and produced from Avian influenza-derived levan fructose-transferase is cultivated in a medium supplemented with 4% levan to produce diproprathone anhydride IV in a high yield (Fig. 13).

The recombinant microorganism of the present invention can secrete and express the recombinant levan fructosyltransferase by a protein secretion fusion factor with excellent efficiency and can be obtained by reacting the levan fructose transactivation enzyme produced from the microorganism with Levan or by reacting the microorganism with levan And can be fermented in a medium to effectively produce diproprathiane hydride IV.

Figure 1 shows that the lftA gene and 24 protein secretion fusion factors (TFP) were ligated to the GAL 10 promoter to produce Saccharomyces cerevisiae cerevisiae strain of the present invention is a schematic view showing intracellular homologous recombination process.
Fig. 2 shows the result of SDS-PAGE analysis of the protein supernatant expressed in 24 strains of Saccharomyces cerevisiae and secreted out of the cells.
FIG. 3 is a result of Western blot analysis of protein supernatant expressed in 24 strains of Saccharomyces cerevisiae and secreted out of the cells using an anti-HIS antibody.
FIG. 4 shows the results of SDS-PAGE analysis of secreted protein supernatants obtained by culturing a single strain in three strains of the five strains selected first among the 24 TFP strains.
FIG. 5 shows the result of Western blotting using the anti-HIS antibody to determine the secreted protein supernatant by culturing three strains of 5 strains selected from the 24 TFP strains.
Fig. 6 shows SDS-PAGE analysis results for comparison of the expression using yeast secretion signal MFa and the results of selection of levansucrose transferase using selected TFP.
FIG. 7 is a diagram illustrating a YGaST13-lftA-6H expression vector containing the protein secretion factor TFP 13 and the lftA gene.
FIG. 8 shows the result of confirming the growth of cells by fermentation of the strain in a 5 L fermenter in yeast Saccharomyces cerevisiae containing the recombinant vector YGaSTP13-lftA-6H.
FIG. 9 is a result of SDS-PAGE analysis of 10 μl of a strain obtained by fermenting a yeast Saccharomyces cerevisiae strain containing YGaSTP13-lftA-6H as a recombinant vector in a 5 L fermenter by time course.
FIG. 10 shows the results of (A) chromatographic analysis of protein purified using Ni-NTA adsorption column and (B) analysis of each fraction by SDS-PAGE.
Fig. 11 shows the results of confirming the optimum pH of the purified levan fructose transferase
Figure 12 shows the result of confirming the optimal temperature of the purified levan fructose transferase
13 is a graph showing the growth of the strain and the amount of DFA IV produced by culturing the yeast Saccharomyces cerevisiae containing YGaST13-lftA-6H in a medium containing Levan.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified in various forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example  One. Levan Fructose-transferase  Yeast protein secretion for secretion production Fusion factor (TFP) selection

Arthrobacter < RTI ID = 0.0 > ureafaciens ) was used as a template to amplify the gene (lftA (SEQ ID NO: 1)) corresponding to the mature protein region except for the signal peptide using pUDFA81 (Lett Appl Microbiol. 2005, , SEQ ID NO: 1) was subjected to first PCR (95 DEG C for 5 minutes, 95 to 30 seconds, 55 to 30 seconds, 72 for 1 minute, 25 cycles, 72 DEG C for 7 minutes, and 1 time using primers of SEQ ID NO: ). The gene amplified by the first PCR is amplified again by the second LNK39 (SEQ ID NO: 4) and HisGT50R (SEQ ID NO: 5) primers, vectors and recombinant cells (in vivo recombination was introduced and a gene in which a histidine tag was introduced at the carboxy terminal was obtained.

The amplified gene was transformed with a linear vector containing 24 protein secretion fusion factors (Korean Patent No. 0975596) that helped express the protein secretion, and a yeast strain Y2805GS (Mat a pep4 :: HIS3 prb1 can1 his3-200 ura3- 52, gal80, suc2) to produce a transformant through intracellular recombination (Fig. 1). The amino acid sequences (Table 1) and the nucleotide sequences (Table 2) of the protein secretion fusion factors used in the present invention are as follows.

Amino acid sequence of protein secretion fusion factor Protein secretion factor Amino acid sequence TFP 1 MFNRFNKFQAAVALALLSRGALGDSYTNSTSSADLSSITSVSSASASATASDSLSSSDGTVYLPSTTISGDLTVTGKVIATEAVEVAAGGKLTLLDGEKYVFSSEAASASAGLALDKR (SEQ ID NO: 9) TFP 2 MTPYAVAITVALLIVTVSALQVNNSCVAFPPSNLRGKNGDGTNEQYATALLSIPWNGPPESSRDINLIELEPQVALYLLENYINHYYNTTRDNKCPNNHYLMGGQLGSSSDNRSLNEAASASAGLALDKR (SEQ ID NO: 10) TFP 3 MQFKNVALAASVAALSATASAEGYTPGEPWSTLTPTGSISCGAAEYTTTFGIAVQAITSSKAKRDVISQIGDGQVQATSAATAQATDSQAQATTTATPTSSEKMAASASAGLALDKR (SEQ ID NO: 11) TFP 4 MRFAEFLVVFATLGGGMAAPVESLAGTQRYLVQMKERFTTEKLCALDDKAASASAGLALDKR (SEQ ID NO: 12) TFP 5 MFNRFNKFQAAVALALLSRGALGAPVNTTTEDETALIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVAASASAGLALDKR (SEQ ID NO: 13) TFP 6 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVAASASAGLALDKR (SEQ ID NO: 14) TFP 7 MVFGQLYALFIFTLSCCISKTVQADSSKESSSFISFDKESNWDTISTISSTADVISSVDSAIAVFEFDNFSLLDSLMIDEEYPFFNRFFANDVSLTVHDDSPLNISQSLSPIMEQFTVDELPESASDLLYEYSLDDKSIVLFKFTSDAYDLKKLDEFIDSCLSFLEDKSGDNLTVVINSLGWAFEDEDGDDEYATEETLSHHDNNKGKEGDDLAASASAGLALDKR (SEQ ID NO: 15) TFP 8 MLQSVVFFALLTFASSVSAIYSNNTVSTTTTLAPSYSLVPQETTISYADDLAASASAGLALDKR (SEQ ID NO: 16) TFP 9 MKFSTAVTTLISSGAIVSALPHVDVHQEDAHQHKRAVAYKYVYETVVVDSDGHTVTPAASEVATAATSAIITTSVLAPTSSAAAADSSASIAVSSAALAKNEKISDAAASATASTSQGASSSSYLAASASAGLALDKR (SEQ ID NO: 17) TFP 10 MNWLFLVSLVFFCGVSTHPALAMSSNRLLKLANKSPKKIIPLKDSSFENILAPPHENAYIVALFTATAPEIGCSLCLELESEYDTIVASWFDDHPDAKSSNSDTSIFFTKVNLEDPSKTIPKAFQFFQLNNVPRLFIFKLNSPSILDHSVISISTDTGSERMKQIIQAIKQFSQVNDFSLHLPVGLAASASAGLALDKR (SEQ ID NO: 18) TFP 11 ≪ / RTI & TFP 12 MASFATKFVIACFLFFSASAHNVLLPAYGRRCFFEDLSKGDELSISFQFGDRNPQSSSQLTGDFIIYGPERHEVLKTVRELAASASAGLALDKR (SEQ ID NO: 20) TFP 13 MQYKKTLVASALAATTLAAYAPSEPWSTLTPTATYSGGVTDYASTFGIAVQPISTTSSASSAATTASSKAKRAASQIGDGQVQAATTTASVSTKSTAAAVSQIGDGQIQATTKTTAAAVSQIGDGQIQATTKTTSAKTTAAAVSQISDGQIQATTTTLAPLAASASAGLALDKR (SEQ ID NO: 21) TFP 14 MQFKNALTATAILSASALAANSTTSIPSSCSIGTSATATAQATDSQAQATTTAPLAASASAGLALDKR (SEQ ID NO: 22) TFP 15 MVSKTWICGFISIITVVQALSCEKHDVLKKYQVGKFSSLTSTERDTPPSTTIEKWWINVCEEHNVEPPEECKKNDMLCGLTDVILPGKDAITTQIIDFDKNIGFNVEETESALTLTLNGATWGANSFDAKLEFQCNDNMKQDELAASASAGLALDKR (SEQ ID NO: 23) TFP 16 MKLSALLALSASTAVLAAPAVHHSDNHHHNDKRAVVTVTQYVNADGAVVIPAATTATSAAADGKVESVAAATTTLSSTAAAATTLAASASAGLALDKR (SEQ ID NO: 24) TFP 17 MKLSTVLLSAGLASTTLAQFSNSTSASSTDVTSSSSISTSSGSVTITSSEAPESDNGTSTAAPTETSTEAPTTAIPTNGTSTEAPTTAIPTNGTSTEAPTDTTTEAPTTALPTNGTSTEAPTDTTTEAPTTGLPTNGTTSAFPPTTSLPPSNTTTTPPYNPSTDYTTDYTVVTEYTTYCPERAASASAGLALDKR (SEQ ID NO: 25) TFP 18 MRFSTTLATAATALFFTASQVSAIGELAFNLGVKNNDGTCKSTSDYETELQALKSYTSTVKVYAASDCNTLQNLGPAAEAEGFTIFVGVWPLAASASAGLALDKR (SEQ ID NO: 26) TFP 19 MRLSNLIASASLLSAATLAAPANHEHKDKRAVVTTTVQKQTTIIVNGAASTPVAALEENAVVNSAPAAATSTTSSAASVATAASSSENNSQVSAAASPASSSAATSTQSSLAASASAGLALDKR (SEQ ID NO: 27) TFP 20 ≪ RTI ID = TFP 21 MKFSSALVLSAVAATALAESITTTITATKNGHVYTKTVTQDATFVWGGEDSYASSTSAAESSAAETSAAETSAAATTSAAATTSAAETSSAAETSSADEGSGSSITTTITATKNGHVYTKTVTQDATFVWTGEGSSNTWSPSSTSTSSEAATSSASTTATTLLAASASAGLALDKR (SEQ ID NO: 29) TFP 22 MKFQVVLSLSACSAVVASPIENLFKYRAVKASHSKNINSTLPAWNGSNSSNVTYANGTNSTTNTTTAESSQLQIIVTGGQVPITNSSLTHTNYTRLFNSSSALNITELYNVARVVNETIQDNLAASASAGLALDKR (SEQ ID NO: 30) TFP 23 ≪ RTI ID = TFP 24 MKLSTVLLSAGLASTTLAQFSNSTSASSTDVTSSSSISTSSGSVTITSSEAPESDNGTSTAAPTETSTEAPTTAIPTNGTSTEAPTTAIPTNGTSTEAPTDTTTEAPTTALPTNGTSTEAPTDTTTEAPTTGLPTNGTTSAFPPTTSLPPSNTTTTLAASASAGLALDKR (SEQ ID NO: 32)

The nucleotide sequence of the protein secretion fusion factor Protein secretion factor Nucleotide sequence TFP 1 ATGTTCAATCGTTTTAACAAATTCCAAGCTGCTGTCGCTTTGGCCCTACTCTCTCGCGGCGCTCTCGGTGACTCTTACACCAATAGCACCTCCTCCGCAGACTTGAGTTCTATCACTTCCGTCTCGTCAGCTAGTGCAAGTGCCACCGCTTCCGACTCACTTTCTTCCAGTGACGGTACCGTTTATTTGCCATCCACAACAATTAGCGGTGATCTCACAGTTACTGGTAAAGTAATTGCAACCGAGGCCGTGGAAGTCGCTGCCGGTGGTAAGTTGACTTTACTTGACGGTGAAAAATACGTCTTCTCATCTGAGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 33) TFP 2 ATGACGCCCTATGCAGTAGCAATTACCGTGGCCTTACTAATTGTAACAGTGAGCGCACTCCAGGTCAACAATTCATGTGTCGCTTTTCCGCCATCAAATCTCAGGGGCAAGAATGGAGACGGTACTAATGAACAGTATGCAACTGCACTACTTTCTATTCCCTGGAATGGGCCTCCTGAGTCATCGAGGGATATTAATCTTATCGAACTCGAACCGCAAGTTGCACTCTATTTGCTCGAAAATTATATTAACCATTACTACAACACCACAAGAGACAATAAGTGCCCTAATAACCACTACCTAATGGGAGGGCAGTTGGGTAGCTCATCGGATAATAGGAGTTTGAACGAGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 34) TFP 3 ATGCAATTCAAAAACGTCGCCCTAGCTGCCTCCGTTGCTGCTCTATCCGCCACTGCTTCTGCTGAAGGTTACACTCCAGGTGAACCATGGTCCACCTTAACCCCAACCGGCTCCATCTCTTGTGGTGCAGCCGAATACACTACCACCTTTGGTATTGCTGTTCAAGCTATTACCTCTTCAAAAGCTAAGAGAGACGTTATCTCTCAAATTGGTGACGGTCAAGTCCAAGCCACTTCTGCTGCTACTGCTCAAGCCACCGATAGTCAAGCCCAAGCTACTACTACCGCTACCCCAACCAGCTCCGAAAAGATGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 35) TFP 4 ATGAGATTTGCAGAATTCTTGGTGGTATTTGCCGTTAGGCGGGGGGATGGCTGCACCGGTTGAGTCTCTGGCCGGGACCCAACGGTATCTGGTGCAAATGAAGGAGCGGTTCACCACAGAGAAGCTGTGTGCTTTGGACGACAAGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 36) TFP 5 ATGTTCAATCGTTTTAACAAATTCCAAGCTGCTGTCGCTTTGGCCCTACTCTCTCGCGGCGCTCTCGGTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACTAATTCCGGCTGAAGCTGTCATCGGTTACTTAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 37) TFP 6 ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTTAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 38) TFP 7 ATGGTGTTCGGTCAGCTGTATGCCCTTTTCATCTTCACGTTATCATGTTGTATTTCCAAAACTGTGCAAGCAGATTCATCCAAGGAAAGCTCTTCCTTTATTTCGTTCGACAAAGAGAGTAACTGGGATACCATCAGCACTATATCTTCAACGGCAGATGTTATATCATCCGTTGACAGTGCTATCGCTGTTTTTGAATTTGACAATTTCTCATTATTGGACAGCTTGATGATTGACGAAGAATACCCATTCTTCAATAGATTCTTTGCCAATGATGTCAGTTTAACTGTTCATGACGATTCGCCTTTGAACATCTCTCAATCATTATCTCCCATTATGGAACAATTTACTGTGGATGAATTACCTGAAAGTGCCTCTGACTTACTATATGAATACTCCTTAGATGATAAAAGCATCGTTTTGTTCAAGTTTACCTCGGATGCCTACGATTTGAAAAAATTAGATGAATTTATTGATTCTTGCTTATCGTTTTTGGAAGATAAATCTGGCGACAATTTGACTGTGGTTATTAACTCTCTTGGTTGGGCTTTTGAAGATGAAGATGGTGACGATGAATATGCAACAGAAGAGACTTTGAGCCATCATGATAACAACAAGGGTAAAGAAGGCGACGATCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 39) TFP 8 ATGCTTCAATCCGTTGTCTTTTTCGCTCTTTTAACCTTCGCAAGTTCTGTGTCAGCGATTTATTCAAACAATACTGTTTCTACAACTACCACTTTAGCGCCCAGCTACTCCTTGGTGCCCCAAGAGACTACCATATCGTACGCCGACGACCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 40) TFP 9 ATGAAATTCTCAACTGCCGTTACTACGTTGATTAGTTCTGGTGCCATCGTGTCTGCTTTACCACACGTGGATGTTCACCAAGAAGATGCCCACCAACATAAGAGGGCCGTTGCGTACAAATACGTTTACGAAACTGTTGTTGTCGATTCTGATGGCCACACTGTAACTCCTGCTGCTTCAGAAGTCGCTACTGCTGCTACCTCTGCTATCATTACAACATCTGTGTTGGCTCCAACCTCCTCCGCAGCCGCTGCGGATAGCTCCGCTTCCATTGCTGTTTCATCTGCTGCCTTAGCCAAGAATGAGAAAATCTCTGATGCCGCTGCATCTGCCACTGCCTCAACATCTCAAGGGGCATCCTCCTCATCCTACCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 41) TFP 10 ATGAATTGGCTGTTTTTGGTCTCGCTGGTTTTCTTCTGCGGCGTGTCAACCCATCCTGCCCTGGCAATGTCCAGCAACAGACTACTAAAGCTGGCTAATAAATCTCCCAAGAAAATTATACCTCTGAAGGACTCAAGTTTTGAAAACATCTTGGCACCACCTCACGAAAATGCCTATATAGTTGCTCTGTTTACTGCCACAGCGCCCGAAATTGGCTGTTCTCTGTGTCTCGAGCTAGAATCCGAATACGACACCATAGTGGCCTCCTGGTTTGATGATCATCCGGATGCAAAATCGTCCAATTCCGATACATCTATTTTCTTCACAAAGGTCAATTTGGAGGACCCTTCTAAGACCATTCCTAAAGCGTTCCAGTTTTTCCAACTAAACAATGTTCCTAGATTGTTCATCTTCAAACTAAACTCTCCCTCTATTCTGGACCACAGCGTGATCAGTATTTCCACTGATACTGGCTCAGAAAGAATGAAGCAAATCATACAAGCCATTAAGCAGTTCTCGCAAGTAAACGACTTCTCTTTACACTTACCTGTGGGTCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 42) TFP 11 ATGAAGTTCTCTTCTGTTACTGCTATTACTCTAGCCACCGTTGCCACCGTTGCCACTGCTAAGAAGGGTGAACATGATTTCACTACCACTTTAACTTTGTCATCGGACGGTAGTTTAACTACTACCACCTCTACTCATACCACTCACAAGTATGGTAAGTTCAACAAGACTTCCAAGTCCAAGACCCCCCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 43) TFP 12 ATGGCCTCATTTGCTACTAAGTTTGTCATTGCTTGCTTCCTGTTCTTCTCGGCGTCCGCCCATAATGTCCTTCTTCCAGCTTATGGCCGTAGATGCTTCTTCGAAGACTTGAGTAAGGGTGACGAGCTCTCCATTTCGTTCCAGTTCGGTGATAGAAACCCTCAATCCAGTAGCCAGCTGACTGGTGACTTTATCATCTACGGGCCGGAAAGACATGAAGTTTTGAAAACGGTTAGGGAACTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 44) TFP 13 ATGCAATACAAAAAGACTTTGGTTGCCTCTGCTTTGGCCGCTACTACATTGGCCGCCTATGCTCCATCTGAGCCTTGGTCCACTTTGACTCCAACAGCCACTTACAGCGGTGGTGTTACCGACTACGCTTCCACCTTCGGTATTGCCGTTCAACCAATCTCCACTACATCCAGCGCATCATCTGCAGCCACCACAGCCTCATCTAAGGCCAAGAGAGCTGCTTCCCAAATTGGTGATGGTCAAGTCCAAGCTGCTACCACTACTGCTTCTGTCTCTACCAAGAGTACCGCTGCCGCCGTTTCTCAGATCGGTGATGGTCAAATCCAAGCTACTACCAAGACTACCGCTGCTGCTGTCTCTCAAATTGGTGATGGTCAAATTCAAGCTACCACCAAGACTACCTCTGCTAAGACTACCGCCGCTGCCGTTTCTCAAATCAGTGATGGTCAAATCCAAGCTACCACCACTACTTTAGCCCCTCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 45) TFP 14 ATGCAATTCAAGAACGCTTTGACTGCTACTGCTATTCTAAGTGCCTCCGCTCTAGCTGCTAACTCAACTACTTCTATTCCATCTTCATGTAGTATTGGTACTTCTGCCACTGCTACTGCTCAAGCCACCGATAGTCAAGCCCAAGCTACTACTACCGCACCCCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 46) TFP 15 ATGGTATCGAAGACTTGGATATGTGGCTTCATCAGTATAATTACAGTGGTACAGGCCTTGTCCTGCGAGAAGCATGATGTATTGAAAAAGTATCAGGTGGGAAAATTTAGCTCACTAACTTCTACGGAAAGGGATACTCCGCCAAGCACAACTATTGAAAAGTGGTGGATAAACGTTTGCGAAGAGCATAACGTAGAACCTCCTGAAGAATGTAAAAAAAATGACATGCTATGTGGTTTAACAGATGTCATCTTGCCCGGTAAGGATGCTATCACCACTCAAATTATAGATTTTGACAAAAACATTGGCTTCAATGTCGAGGAAACTGAGAGTGCGCTTACATTGACACTAAACGGCGCTACGTGGGGCGCCAATTCTTTTGACGCAAAACTAGAATTTCAGTGTAATGACAATATGAAACAAGACGAACTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 47) TFP 16 ATGAAATTATCCGCTCTATTAGCTTTATCAGCCTCCACCGCCGTCTTGGCCGCTCCAGCTGTCCACCATAGTGACAACCACCACCACAACGACAAGCGTGCCGTTGTCACCGTTACTCAGTACGTCAACGCAGACGGCGCTGTTGTTATTCCAGCTGCCACCACCGCTACCTCGGCGGCTGCTGATGGAAAGGTCGAGTCTGTTGCTGCTGCCACCACTACTTTGTCCTCGACTGCCGCCGCCGCTACAACCCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 48) TFP 17 ATGAAATTATCAACTGTCCTATTATCTGCCGGTTTAGCCTCGACTACTTTGGCCCAATTTTCCAACAGTACATCTGCTTCTTCCACCGATGTCACTTCCTCCTCTTCCATCTCCACTTCCTCTGGCTCAGTAACTATCACATCTTCTGAAGCTCCAGAATCCGACAACGGTACCAGCACAGCTGCACCAACTGAAACCTCAACAGAGGCTCCAACCACTGCTATCCCAACTAACGGTACCTCTACTGAAGCTCCAACCACTGCTATCCCAACTAACGGTACCTCTACTGAAGCTCCAACTGATACTACTACTGAAGCTCCAACCACCGCTCTTCCAACTAACGGTACTTCTACTGAAGCTCCAACTGATACTACTACTGAAGCTCCAACCACCGGTCTTCCAACCAACGGTACCACTTCAGCTTTCCCACCAACTACATCTTTGCCACCAAGCAACACTACCACCACTCCTCCTTACAACCCATCTACTGACTACACCACTGACTACACTGTAGTCACTGAATATACTACTTACTGTCCGGAACGGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 49) TFP 18 ATGCGTTTCTCTACTACACTCGCTACTGCAGCTACTGCGCTATTTTTCACAGCCTCCCAAGTTTCAGCTATTGGTGAACTAGCCTTTAACTTGGGTGTCAAGAACAACGATGGTACTTGTAAGTCCACTTCCGACTATGAAACCGAATTACAAGCTTTGAAGAGCTACACTTCCACCGTCAAAGTTTACGCTGCCTCAGATTGTAACACTTTGCAAAACTTAGGTCCTGCTGCTGAAGCTGAGGGATTTACTATCTTTGTCGGTGTTTGGCCACTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 50) TFP 19 ATGCGTCTCTCTAACCTAATTGCTTCTGCCTCTCTTTTATCTGCTGCTACTCTTGCTGCTCCCGCTAACCACGAACACAAGGACAAGCGTGCTGTGGTCACTACCACTGTTCAAAAACAAACCACTATCATTGTTAATGGTGCCGCTTCAACTCCAGTTGCTGCTTTGGAAGAAAATGCTGTTGTCAACTCCGCTCCAGCTGCCGCTACCAGTACAACATCGTCTGCTGCTTCTGTAGCTACCGCTGCTTCCTCTTCTGAGAACAACTCACAAGTTTCTGCTGCCGCATCTCCAGCCTCCAGCTCTGCTGCTACATCTACTCAATCTTCTCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 51) TFP 20 ATGCAATTTTCTACTGTCGCTTCTATCGCCGCTGTCGCCGCTGTCGCTTCTGCCGCTGCTAACGTTACCACTGCTACTGTCAGCCAAGAATCTACCACTTTGGTCACCATCACTTCTTGTGAAGACCACGTCTGTTCTGAAACTGTCTCCCCAGCTTTGGTTTCCACCGCTACCGTCACCGTCGATGACGTTATCACTCAATACACCACCTGGTGCCCATTGACCACTGAAGCCCCAAAGAACGGTACTTCTACTGCTGCTCCAGTTACCTCTACTGAAGCTCCAAAGAACACCACCTCTGCTGCTCCAACTCACTCTGTCACCTCTTACACTGGTGCTGCTGCTAAGGCTTTGCCAGCTGCTGGTGCTTTGCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 52) TFP 21 ATGAAATTCTCTTCCGCTTTGGTTCTATCTGCTGTTGCCGCTACTGCTCTTGCTGAGAGTATCACCACCACCATCACTGCCACCAAGAACGGTCATGTCTACACTAAGACTGTCACCCAAGATGCTACTTTTGTTTGGGGTGGTGAAGACTCTTACGCCAGCAGCACTTCTGCCGCTGAATCTTCTGCCGCCGAAACTTCTGCCGCCGAAACCTCTGCTGCCGCTACCACTTCTGCTGCCGCTACCACTTCTGCTGCTGAGACTTCTTCTGCTGCTGAGACTTCTTCTGCTGATGAAGGTTCTGGTTCTAGTATCACTACCACTATCACTGCCACCAAGAACGGTCACGTCTACACTAAGACTGTCACCCAAGATGCTACTTTTGTCTGGACTGGTGAAGGCAGCAGCAACACCTGGTCTCCAAGTAGTACCTCTACCAGCTCAGAAGCTGCTACCTCTTCTGCTTCAACCACTGCAACCACCCTGCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 53) TFP 22 ATGAAGTTCCAAGTTGTTTTATCTGCCCTTTTGGCATGTTCATCTGCCGTCGTCGCAAGCCCAATCGAAAACCTATTCAAATACAGGGCAGTTAAGGCATCTCACAGTAAGAATATCAACTCCACTTTGCCGGCCTGGAATGGGTCTAACTCTAGCAATGTTACCTACGCTAATGGAACAAACAGTACTACCAATACTACTACTGCCGAAAGCAGTCAATTACAAATCATTGTAACAGGTGGTCAAGTACCAATCACCAACAGTTCTTTGACCCACACAAACTACACCAGATTATTCAACAGTTCTTCTGCTTTGAACATTACCGAATTGTACAATGTTGCCCGTGTTGTTAACGAAACGATCCAAGATAACCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 54) TFP 23 ATGCGTGCCATCACTTTATTATCTTCAGTCGTTTCTTTGGCATTGTTGTCGAAGGAAGTCTTAGCAACACCTCCAGCTTGTTTATTGGCCTGTGTTGCGCAAGTCGGCAAATCCTCTTCCACATGTGACTCTTTGAATCAAGTCACCTGTTACTGTGAACACGAAAACTCCGCCGTCAAGAAATGTCTAGACTCCATCTGCCCAAACAATGACGCTGATGCTGCTTATTCTGCTTTCAAGAGTTCTTGTTCCGAACAAAATGCTTCATTGGGCGATTCCAGCGGCAGTGCCTCCTCATCCGTTCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 55) TFP 24 ATGAAATTATCAACTGTCCTATTATCTGCCGGTTTGGCCTCGACTACTTTGGCCCAATTTTCCAACAGTACATCTGCTTCTTCCACCGATGTCACTTCCTCCTCTTCCATCTCCACTTCCTCTGGCTCAGTAACTATCACATCTTCTGAAGCTCCAGAATCCGACAACGGTACCAGCACAGCTGCACCAACTGAAACCTCAACAGAGGCTCCAACCACTGCTATCCCAACTAACGGTACCTCTACTGAAGCTCCAACCACTGCTATCCCAACTAACGGTACCTCTACTGAAGCTCCAACTGATACTACTACTGAAGCTCCAACCACCGCTCTTCCAACTAACGGTACTTCTACTGAAGCTCCAACTGATACTACTACTGAAGCTCCAACCACCGGTCTTCCAACCAACGGTACCACTTCAGCTTTCCCACCAACTACATCTTTGCCACCAAGCAACACTACCACCACTCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 56)

Twenty-four transformants were cultured in YPD medium (1% yeast extract, 2% peptone, 2% glucose) for 40 hours, centrifuged to remove the cells, and 0.4 ml of acetone was added to the culture supernatant (0.6 ml) , Followed by SDS-PAGE analysis (FIG. 2), and Western blotting using an anti-HIS antibody confirmed the expression of lftA (FIG. 3).

Levan fructose transferase is known to be a protein difficult to secrete in yeast. However, when the protein secretion factor technique is used, as shown in FIG. 3, most of the 24 protein secretion fusion factors can secrete lftA . Among these, TFP of five species (TFP 7, TFP 8, TFP 9, TFP 13, and TFP 19) which are relatively excellent in the ability to express levansuclease transferase were selected.

Since the strains selected by the above method were obtained by analyzing the transformants rather than the single colonies, the strains were cultured from a single colony to analyze the protein synthesis for accurate analysis. In order to obtain a single strain from each strain, three single colonies were selected for each TFP and cultured in the same manner. The culture medium was subjected to SDS-PAGE analysis (FIG. 4) in the same manner and Western blot (Fig. 5). In addition, in order to compare the secretion ability of levansuclease transferase of each TFP, the MFα secretion signal which is most widely used as a secretion signal in yeast was used to compare with the secretory expression of levansuclease transferase (FIG. 6).

As a result, it was confirmed that all of the selected TFPs secreted Levan-fructose-transferase more efficiently than MFa secretion signal, and it was confirmed that TFP 13 can secrete Levan-fructose-transferase most efficiently.

Based on the above results, YGaST13-lftA-6H was produced as an expression vector which was finally produced by high-yield production of Levan fructosyltransferase derived from Agrobacterium tumefaciens (Fig. 7), and the strain transformed with the expression vector under the Budapest Treaty Was deposited on June 30, 2015 and granted accession number KCTC18396P to the international depositary, the Korean Collection for Type Culture (KCTC).

Example  2. Levan Fructose-transferase  Enzyme activity confirmation

The Levansolytic activity of the Levan fructose transferase-producing strains obtained in Example 1 was compared using the DNS method. For the DNS analysis method, the amount of enzyme producing 1 μM of reducing sugar per minute at 37 ° C was defined as one unit. Add 5 μl of the test tube culture to 100 μl of 4% levan in 50 mM sodium phosphate buffer (pH 6.0), add 95 μl of the buffer solution, and adjust the total volume to 200 μl And then reacted at 37 ° C for several hours (2 to 24 hours). Then, 700 μl of DNS was added, boiled for 5 minutes, and the absorbance at 550 nm was measured to calculate the enzyme activity. Glucose was used to determine the standard curve and the amount of enzyme.

As a result, as in the above SDS-PAGE analysis (FIG. 6), the strain transformed with the vector containing each of the above-mentioned five TFPs exhibited higher activity than that expressed using the MFa secretion signal, In particular, the strain transformed with the YGaST13-lftA-6H vector showed a 1.8-fold higher activity than that expressed using the MFa secretion signal (Table 3).

TFP OD ₅₅₀ U / ml / min Mfα 0.423 1.60 ST7 0.457 1.71 ST8 0.535 1.97 ST9 0.647 2.33 ST13 0.796 2.82 ST19 0.637 2.30

The expression vector YGaST13-lftA-6H (FIG. 7), which expresses the levan fructosyltransferase at high secretion level, was transfected with the GAL10 promoter which is strongly induced by galactose expression and the lftA gene was transcribed by TFP 13 (SEQ ID NO: 6) It is a structure in which protein synthesis and secretion of fructose transferase are induced. Six histidine amino acids were added to the carboxy terminus for the enzyme purification in the amino acid sequence (SEQ ID NO: 7) of the levan fructose transferase linked to TFP 13. YEG-HIR525 vector (Sohn et al., Process Biochem. 1995, 653) were used.

Example  3. Recombinant yeast Oil price formula  Through fermentation Levan Fructose transferase  massive production

Recombinant yeast (YGaST13-lftA-6H / Y2805GS) containing the above-mentioned YGaST13-lftA-6H vector was cultivated in a 5 L fermenter to mass-produce levan fructose transferase. The cells were cultured in a 50 ml YNB medium (yeast substrate lacking 0.67% amino acid, 0.5% casamino ester, 2% glucose) medium, and then resuspended in 200 ml YEPD liquid medium (2% glucose, 4% yeast extract , 1% peptone), and inoculated into 1.8 L of the culture medium (2% glucose, 4% yeast extract, 1% peptone) and fermented at 30 ° C. When the glucose was completely consumed during the culture, the feed medium (30% glucose, 15% yeast extract) was fermented for 50 hours with the addition of 2 g / L to 20 g / L per hour gradually as the cells were grown. The degree of cell growth during the culture was measured by taking samples over time and measuring the absorbance at a wavelength of 600 nm (FIG. 8). The lftA secreted in the medium was confirmed by SDS-PAGE analysis of 10 μl of the culture supernatant not concentrated ).

The fermented protein was centrifuged at 3500 rpm for 20 minutes for purification, and then passed through a 0.1 micron filter membrane to completely remove the cells. Then, the culture was concentrated by repeatedly adding 20 mM Tris buffer (pH 7.5) and passing ultrafiltration (cut-off molecular weight: 30 kDa) membrane (FIG. 9).

Example  4. Levan using affinity chromatography method Fructose-transferase  refine

The levan fructosyltransferase produced in the present invention has 6X histidine tagging at the carboxy terminus and can be purified by an affinity chromatography method using Ni-NTA resin.

The fusion protein was bound to the Ni-NTA resin by applying 150 ml of the fermentation medium concentrate obtained in Example 3 to the HisPrep FF column, and then the supernatant was digested with 50 mM Tris-HCl (pH 7.4) using AKTA FPLC (Fast Protein Liquid Chromatography, GE healthcare) (pH 7.5), 0.5 M NaCl, and 0.25 M imidazole. The final purified fractions were desalted using a Hiprep Desalting column to obtain a final purified product (FIG. 10).

Example  5. Refined Levan Fructose transferase  Optimal condition verification using

To confirm the optimal pH of the levan fructose transferase purified in Example 4, 100 μl of 1% Levan solution was mixed with 100 μl of 50 mM buffer (Table 4) in various pH ranges, and then 1 μg / Enzyme was added and enzyme reaction was performed at a temperature of 37 ° C for 1 hour. After the reaction, 300 μl of DNS solution was added to measure the reducing sugar liberated from Levan, boiled for 5 minutes, and the absorbance at 550 nm was measured to compare the relative activities.

pH 3 4 5 6 7 8 9 Buffer glycine-
HCl Acetate Acetate Maleate Tris-
maleate Tris-
maleate Tris-
HCl

In order to determine the optimal temperature of the enzyme, 100 μl of 1% Levan solution was mixed with 100 μl of pH 5.0 buffer, which was an optimal pH condition. Then, the mixture was incubated at various temperatures (4, 20, 30, 37, 50, And 1 μg / μl of the purified enzyme was added and reacted for 1 hour. Then, the relative activities were compared by the DNS method in the same manner as above.

As a result, the optimum pH of the recombinant levan fructose transferase was pH 5.0 (FIG. 11) and the optimum temperature was 50 ° C. (FIG. 12).

Example  6. HPLC Using DFA -VI Productivity Analysis

As shown in Example 3, when the culture solution of the recombinant yeast (YGaST13-lftA-6H / Y2805GS) secreted and expressed by the levan fructose transferase was concentrated, most of the proteins were recombinant levan fructose transferase, The culture was applied to DFA-IV production using an enzyme solution 5-fold concentrated without further purification.

0.1 ml of the enzyme solution was added to 0.9 ml of a 4% Levan solution (pH 5.0), and the product was subjected to enzymatic reaction at 45 ° C for 30 minutes. The product was analyzed with a YMC-pack ODS-AQ (250 × 10 mm) Respectively.

As a result of comparing the levan fructose transferase produced from Escherichia coli with the levan fructose transferase produced from yeast, the fermentation broth of 5-fold concentrated recombinant yeast was 128% higher than that of E. coli-produced levan fructose transferase 5).

Levan Fructose transferase Activity (U / ml) Relative activity (%) YGaST13-lftA-6H / Y2805GS culture medium 6783 48 YGaST13-lftA-6H / Y2805GS culture concentrate 18088 128 Recombinant E. coli-derived levan fructose transferase 14183 100

Therefore, the levan fructose transferase produced from the yeast can produce DFA-VI from levan, and can produce DFA-VI at a higher efficiency than the E. coli-derived levan fructose transferase even if the culture solution is concentrated without further purification Respectively.

Example  7. Levan  Through direct fermentation of recombinant yeast in the medium containing DFA -IV Production

The existing DFA-IV production process involves extracting and partially purifying the enzyme produced in the cell and then converting the Levan substrate to DFA-IV, but this method requires the extraction and purification cost of the enzyme. However, if the yeast strain secreted by the levan fructose transferase can be directly cultured in a medium containing Levan to produce DFA-IV, the production cost can be reduced. Also, since the glucose and fructose produced in the DFA-IV production process from levan are used as the carbon source of the yeast, the purity of the produced DFA-IV can be increased.

Thus, the present inventors confirmed the possibility of producing DFA-IV through direct fermentation of the recombinant yeast (YGaST13-lftA-6H / Y2805) prepared above. Specifically, yeast (YGaST13-lftA-6H / Y2805GS) expressing levan fructose transferase was cultured in YP medium (1% yeast extract, 2% peptone, 4% Levan) containing 4% The absorbance of the culture was measured at 540 nm, and the amount of DFA-IV produced by the YMC-pack ODS-AQ (250 X 10 mm) column was quantitatively analyzed using water as a supernatant after centrifugation (FIG. 13).

As a result, about 20 g / L of DFA IV was produced through fermentation of recombinant yeast strains in a medium containing 4% Levan, which corresponds to a conversion rate of about 50%, indicating that DFA-IV production efficiency is higher than enzyme reaction Respectively.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the spirit and scope of the present invention as defined by the appended claims.

Korea Research Institute of Bioscience and Biotechnology KCTC18396P 20150630

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> A microorganism having enhanced levan fructotransferase          productivity and a method of producing difructose anhydride IV          using the microorganism <130> KPA150510-KR <160> 56 <170> Kopatentin 2.0 <210> 1 <211> 1467 <212> DNA <213> Artificial Sequence <220> <223> lftA <400> 1 tccgctccgg gctcgctccg tgccgtctac cacatgacgc cccccagcgg ctggctctgc 60 gacccccaac gcccggtcac cacccacggc gcctaccagc tgtactacct gcactccgac 120 cagaacaacg gccccggcgg ctgggaccac gcgagcacga ccgacggcgt cgccttcacg 180 caccacggca ccgtgatgcc gctgcggccc gacttccccg tgtggtccgg gtcggcggtc 240 gtcggcaccg cgaacacggc agggttcggc gccggcgcgg tcgtcgcgct cgcgacccag 300 ccgaccgacg gcgtccgcaa gtaccaggag cagtacctct actggtcgac cgacggcggg 360 ttcacgttca ccgccctgcc cgaccccgtc atcgtcaaca ccgacggtcg cgccgccacc 420 acgcccgccg agatcgagaa cgccgagtgg ttccgcgacc ccaagatcca ctgggacacc 480 gcccgcggag aatgggtctg cgtcatcgga cgactgcggt acgccgcgtt ctacacctcg 540 ccgaacctgc gcgactggac acttcgccgc aacttcgact acccgaacca cgccctcggc 600 ggcatcgagt gccccgacct gttcgagatc accgcagacg acgggacacg ccactgggtg 660 ctcgccgcca gcatggacgc ctacggcatc ggcctcccca tgacgtacgc ctactggaca 720 ggcacctggg acggcgagca gttccacgcc gacgacctca ccccgcaatg gctcgactgg 780 ggctgggact ggtacgcggc cgtcacctgg ccatcgatcg acgcgcccga gaccaagcgc 840 ctcgccatcg cgtggatgaa caactggaag tacgccgcac gcgacgtccc caccgacgca 900 tccgacggct acaacgggca gaactcgatc gtccgcgagc tgcggctcgc ccgacagcct 960 ggcggctggt acaccctcct gagcaccccc gtggcagcgc tgacgaacta cgtcaccgcc 1020 accaccacac tccccgaccg gaccgtcgac ggcagcgccg tcctgccatg gaacggacgc 1080 gcatacgaga tcgagctcga catcgcctgg gacaccgcga cgaacgtcgg catctcggtg 1140 ggccgctccc ccgacggaac ccggcacacg aacatcggca agtacggagc agacctgtac 1200 gtcgaccgag gaccctccga cctcgccggg tactcgctcg ccccctactc gcgagccgcc 1260 gcccccatcg accccggcgc ccgatccgtg cacctgcgca tcctcgtcga cacccagagc 1320 gtcgaggtct tcgtcaacgc cggccacacc gtgctctccc agcaggtcca cttcgccgag 1380 ggcgacacgg gaatctcgct ctacaccgac ggcggccccg cacacttcac cggcatcgtc 1440 gtccgcgaga ttggccaggc gatctag 1467 <210> 2 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> lftA primer 1 <400> 2 ctcgccttag ataaagatc cgctccgggc tcgctccg 38 <210> 3 <211> 38 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > lftA primer 2 <400> 3 gtgatggtga tggtgatgga tcgcctggcc aatctcgc 38 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> LNK39 primer <400> 4 ggccgcctcg gcctctgctg gcctcgcctt agataaaaga 40 <210> 5 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> HisGT50R primer <400> 5 gtcattatta aatatatata tatatatatt gtcactccgt tcaagtcgac ttagtgatgg 60 tgatggtgat g 71 <210> 6 <211> 522 <212> DNA <213> Artificial Sequence <220> <223> TFP13 <400> 6 atgcaataca aaaagacttt ggttgcctct gctttggccg ctactacatt ggccgcctat 60 gctccatctg agccttggtc cactttgact ccaacagcca cttacagcgg tggtgttacc 120 gactacgctt ccaccttcgg tattgccgtt caaccaatct ccactacatc cagcgcatca 180 tctgcagcca ccacagcctc atctaaggcc aagagagctg cttcccaaat tggtgatggt 240 caagtccaag ctgctaccac tactgcttct gtctctacca agagtaccgc tgccgccgtt 300 tctcagatcg gtgatggtca aatccaagct actaccaaga ctaccgctgc tgctgtctct 360 caaattggtg atggtcaaat tcaagctacc accaagacta cctctgctaa gactaccgcc 420 gctgccgttt ctcaaatcag tgatggtcaa atccaagcta ccaccactac tttagcccct 480 ctggccgcct cggcctctgc tggcctcgcc ttagataaaa ga 522 <210> 7 <211> 668 <212> PRT <213> Artificial Sequence <220> <223> TFP13-lftA amino acid <400> 7 Met Gln Tyr Lys Lys Thr Leu Val Ala Ser Ala Leu Ala Ala Thr Thr   1 5 10 15 Leu Ala Ala Tyr Ala Pro Ser Glu Pro Trp Ser Thr Leu Thr Pro Thr              20 25 30 Ala Thr Tyr Ser Gly Gly Val Thr Asp Tyr Ala Ser Thr Phe Gly Ile          35 40 45 Ala Val Gln Pro Ile Ser Thr Thr Ser Ser Ala Ser Ser Ala Ala Thr      50 55 60 Thr Ala Ser Ser Lys Ala Lys Arg Ala Ser Ser Gln Ile Gly Asp Gly  65 70 75 80 Gln Val Gln Ala Ala Thr Thr Thr Ala Ser Val Ser Thr Lys Ser Thr                  85 90 95 Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Ile Gln Ala Thr Thr             100 105 110 Lys Thr Thr Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Ile Gln         115 120 125 Ala Thr Thr Lys Thr Thr Ser Ala Lys Thr Thr Ala Ala Ala Val Ser     130 135 140 Gln Ile Ser Asp Gly Gln Ile Gln Ala Thr Thr Thr Thr Leu Ala Pro 145 150 155 160 Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg Ser Ala                 165 170 175 Pro Gly Ser Leu Arg Ala Val Tyr His Met Thr Pro Pro Ser Gly Trp             180 185 190 Leu Cys Asp Pro Gln Arg Pro Val Thr Thr His Gly Ala Tyr Gln Leu         195 200 205 Tyr Tyr Leu His Ser Asp Gln Asn Asn Gly Pro Gly Gly Trp Asp His     210 215 220 Ala Ser Thr Thr Asp Gly Val Ala Phe Thr His His Gly Thr Val Met 225 230 235 240 Pro Leu Arg Pro Asp Phe Pro Val Trp Ser Gly Ser Ala Val Val Gly                 245 250 255 Thr Ala Asn Thr Ala Gly Ply Gly Ala Gly Ala Val Val Ala Leu Ala             260 265 270 Thr Gln Pro Thr Asp Gly Val Arg Lys Tyr Gln Glu Gln Tyr Leu Tyr         275 280 285 Trp Ser Thr Asp Gly Gly Phe Thr Phe Thr Ala Leu Pro Asp Pro Val     290 295 300 Ile Val Asn Thr Asp Gly Arg Ala Ala Thr Thr Pro Ala Glu Ile Glu 305 310 315 320 Asn Ala Glu Trp Phe Arg Asp Pro Lys Ile His Trp Asp Thr Ala Arg                 325 330 335 Gly Glu Trp Val Cys Val Ile Gly Arg Leu Arg Tyr Ala Ala Phe Tyr             340 345 350 Thr Ser Pro Asn Leu Arg Asp Trp Thr Leu Arg Arg Asn Phe Asp Tyr         355 360 365 Pro Asn His Ala Leu Gly Gly Ile Glu Cys Pro Asp Leu Phe Glu Ile     370 375 380 Thr Ala Asp Asp Gly Thr Arg His Trp Val Leu Ala Ala Ser Met Asp 385 390 395 400 Ala Tyr Gly Ile Gly Leu Pro Met Thr Tyr Ala Tyr Trp Thr Gly Thr                 405 410 415 Trp Asp Gly Glu Gln Phe His Ala Asp Asp Leu Thr Pro Gln Trp Leu             420 425 430 Asp Trp Gly Trp Asp Trp Tyr Ala Ala Val Thr Trp Pro Ser Ile Asp         435 440 445 Ala Pro Glu Thr Lys Arg Leu Ala Ile Ala Trp Met Asn Asn Trp Lys     450 455 460 Tyr Ala Ala Arg Asp Val Pro Thr Asp Ala Ser Asp Gly Tyr Asn Gly 465 470 475 480 Gln Asn Ser Ile Val Arg Glu Leu Arg Leu Ala Arg Gln Pro Gly Gly                 485 490 495 Trp Tyr Thr Leu Leu Thr Asn Tyr Val             500 505 510 Thr Ala Thr Thr Thr Leu Pro Asp Arg Thr Val Asp Gly Ser Ala Val         515 520 525 Leu Pro Trp Asn Gly Arg Ala Tyr Glu Ile Glu Leu Asp Ile Ala Trp     530 535 540 Asp Thr Ala Thr Asn Val Gly Ile Ser Val Gly Arg Ser Pro Asp Gly 545 550 555 560 Thr Arg His Thr Asn Ile Gly Lys Tyr Gly Ala Asp Leu Tyr Val Asp                 565 570 575 Arg Gly Pro Ser Asp Leu Ala Gly Tyr Ser Leu Ala Pro Tyr Ser Arg             580 585 590 Ala Ala Ala Pro Ile Asp Pro Gly Ala Arg Ser Val His Leu Arg Ile         595 600 605 Leu Val Asp Thr Gln Ser Val Glu Val Phe Val Asn Ala Gly His Thr     610 615 620 Val Leu Ser Gln Gln Val His Phe Ala Glu Gly Asp Thr Gly Ile Ser 625 630 635 640 Leu Tyr Thr Asp Gly Gly Pro Ala His Phe Thr Gly Ile Val Val Arg                 645 650 655 Glu Ile Gly Gln Ala Ile His His His His His             660 665 <210> 8 <211> 477 <212> PRT <213> Artificial Sequence <220> <223> lftA amino acid <400> 8 Met Thr Pro Pro Ser Gly Trp Leu Cys Asp Pro Gln Arg Pro Val Thr   1 5 10 15 Thr His Gly Ala Tyr Gln Leu Tyr Tyr Leu His Ser Asp Gln Asn Asn              20 25 30 Gly Pro Gly Gly Trp Asp His Ala Ser Thr Thr Asp Gly Val Ala Phe          35 40 45 Thr His His Gly Thr Val Met Pro Leu Arg Pro Asp Phe Pro Val Trp      50 55 60 Ser Gly Ser Ala Val Val Gly Thr Ala Asn Thr Ala Gly Phe Gly Ala  65 70 75 80 Gly Ala Val Val Ala Leu Ala Thr Gln Pro Thr Asp Gly Val Arg Lys                  85 90 95 Tyr Gln Glu Gln Tyr Leu Tyr Trp Ser Thr Asp Gly Gly Phe Thr Phe             100 105 110 Thr Ala Leu Pro Asp Pro Val Ile Val Asn Thr Asp Gly Arg Ala Ala         115 120 125 Thr Thr Pro Ala Glu Ile Glu Asn Ala Glu Trp Phe Arg Asp Pro Lys     130 135 140 Ile His Trp Asp Thr Ala Arg Gly Glu Trp Val Cys Val Ile Gly Arg 145 150 155 160 Leu Arg Tyr Ala Phe Tyr Thr Ser Pro Asn Leu Arg Asp Trp Thr                 165 170 175 Leu Arg Arg Asn Phe Asp Tyr Pro Asn His Ala Leu Gly Gly Ile Glu             180 185 190 Cys Pro Asp Leu Phe Glu Ile Thr Ala Asp Asp Gly Thr Arg His Trp         195 200 205 Val Leu Ala Ala Ser Met Asp Ala Tyr Gly Ile Gly Leu Pro Met Thr     210 215 220 Tyr Ala Tyr Trp Thr Gly Thr Trp Asp Gly Glu Gln Phe His Ala Asp 225 230 235 240 Asp Leu Thr Pro Gln Trp Leu Asp Trp Gly Trp Asp Trp Tyr Ala Ala                 245 250 255 Val Thr Trp Pro Ser Ile Asp Ala Pro Glu Thr Lys Arg Leu Ala Ile             260 265 270 Ala Trp Met Asn Asn Trp Lys Tyr Ala Ala Arg Asp Val Pro Thr Asp         275 280 285 Ala Ser Asp Gly Tyr Asn Gly Gln Asn Ser Ile Val Arg Glu Leu Arg     290 295 300 Leu Ala Arg Gln Pro Gly Gly Trp Tyr Thr Leu Leu Ser Thr Pro Val 305 310 315 320 Ala Ala Leu Thr Asn Tyr Val Thr Ala Thr Thr Thr Leu Pro Asp Arg                 325 330 335 Thr Val Asp Gly Ser Ala Val Leu Pro Trp Asn Gly Arg Ala Tyr Glu             340 345 350 Ile Glu Leu Asp Ile Ala Trp Asp Thr Ala Thr Asn Val Gly Ile Ser         355 360 365 Val Gly Arg Ser Pro Asp Gly Thr Arg His Thr Asn Ile Gly Lys Tyr     370 375 380 Gly Ala Asp Leu Tyr Val Asp Arg Gly Pro Ser Asp Leu Ala Gly Tyr 385 390 395 400 Ser Leu Ala Pro Tyr Ser Arg Ala Ala Ala Pro Ile Asp Pro Gly Ala                 405 410 415 Arg Ser Val His Leu Arg Ile Leu Val Asp Thr Gln Ser Val Glu Val             420 425 430 Phe Val Asn Ala Gly His Thr Val Leu Ser Gln Gln Val His Phe Ala         435 440 445 Glu Gly Asp Thr Gly Ile Ser Leu Tyr Thr Asp Gly Gly Pro Ala His     450 455 460 Phe Thr Gly Ile Val Val Arg Glu Ile Gly Gln Ala Ile 465 470 475 <210> 9 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> TFP 1 <400> 9 Met Phe Asn Arg Phe Asn Lys Phe Gln Ala Ala Val Ala Leu Ala Leu   1 5 10 15 Leu Ser Arg Gly Ala Leu Gly Asp Ser Tyr Thr Asn Ser Thr Ser Ser              20 25 30 Ala Asp Leu Ser Ser Ile Thr Ser Val Ser Ser Ala Ser Ala Ser Ala          35 40 45 Thr Ala Ser Asp Ser Ser Ser Ser Ser Asp Gly Thr Val Tyr Leu Pro      50 55 60 Ser Thr Thr Ile Ser Gly Asp Leu Thr Val Thr Gly Lys Val Ile Ala  65 70 75 80 Thr Glu Ala Val Glu Val Ala Ala Gly Gly Lys Leu Thr Leu Leu Asp                  85 90 95 Gly Glu Lys Tyr Val Phe Ser Ser Glu Ala Ala Ser Ala Ser Ala Gly             100 105 110 Leu Ala Leu Asp Lys Arg         115 <210> 10 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> TFP 2 <400> 10 Met Thr Pro Tyr Ala Val Ala Ile Thr Val Ala Leu Leu Ile Val Thr   1 5 10 15 Val Ser Ala Leu Gln Val Asn Asn Ser Cys Val Ala Phe Pro Ser Ser              20 25 30 Asn Leu Arg Gly Lys Asn Gly Asp Gly Thr Asn Glu Gln Tyr Ala Thr          35 40 45 Ala Leu Leu Ser Ile Pro Trp Asn Gly Pro Pro Glu Ser Ser Arg Asp      50 55 60 Ile Asn Leu Ile Glu Leu Glu Pro Gln Val Ala Leu Tyr Leu Leu Glu  65 70 75 80 Asn Tyr Ile Asn His Tyr Tyr Asn Thr Thr Arg Asp Asn Lys Cys Pro                  85 90 95 Asn Asn His Tyr Leu Met Gly Gly Gln Leu Gly Ser Ser Ser Asp Asn             100 105 110 Arg Ser Leu Asn Glu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp         115 120 125 Lys Arg     130 <210> 11 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> TFP 3 <400> 11 Met Gln Phe Lys Asn Val Ala Leu Ala Ala Ser Val Ala Ala Leu Ser   1 5 10 15 Ala Thr Ala Ser Ala Glu Gly Tyr Thr Pro Gly Glu Pro Trp Ser Thr              20 25 30 Leu Thr Pro Thr Gly Ser Ile Ser Cys Gly Ala Ala Glu Tyr Thr Thr          35 40 45 Thr Phe Gly Ile Ala Val Gln Ala Ile Thr Ser Ser Lys Ala Lys Arg      50 55 60 Asp Val Ile Ser Gln Ile Gly Asp Gly Gln Val Gln Ala Thr Ser Ala  65 70 75 80 Ala Thr Ala Gln Ala Thr Asp Ser Gln Ala Gln Ala Thr Thr Thr Ala                  85 90 95 Thr Pro Thr Ser Ser Glu Lys Met Ala Ala Ser Ala Ser Ala Gly Leu             100 105 110 Ala Leu Asp Lys Arg         115 <210> 12 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> TFP 4 <400> 12 Met Arg Phe Ala Glu Phe Leu Val Val Phe Ala Thr Leu Gly Gly Gly   1 5 10 15 Met Ala Ala Pro Val Glu Ser Leu Ala Gly Thr Gln Arg Tyr Leu Val              20 25 30 Gln Met Lys Glu Arg Phe Thr Thr Glu Lys Leu Cys Ala Leu Asp Asp          35 40 45 Lys Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg      50 55 60 <210> 13 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> TFP 5 <400> 13 Met Phe Asn Arg Phe Asn Lys Phe Gln Ala Ala Val Ala Leu Ala Leu   1 5 10 15 Leu Ser Arg Gly Ala Leu Gly Ala Pro Val Asn Thr Thr Thr Glu Asp              20 25 30 Glu Thr Ala Leu Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu          35 40 45 Glu Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn      50 55 60 Asn Gly Leu Leu Phe Ile Asn Thr Ile Ala Ser Ile Ala Ala Lys  65 70 75 80 Glu Glu Gly Val Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys                  85 90 95 Arg     <210> 14 <211> 93 <212> PRT <213> Artificial Sequence <220> <223> TFP 6 <400> 14 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser   1 5 10 15 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln              20 25 30 Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe          35 40 45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu      50 55 60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val  65 70 75 80 Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg                  85 90 <210> 15 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> TFP 7 <400> 15 Met Val Phe Gly Gln Leu Tyr Ala Leu Phe Ile Phe Thr Leu Ser Cys   1 5 10 15 Cys Ile Ser Lys Thr Val Gln Ala Asp Ser Ser Lys Glu Ser Ser Ser              20 25 30 Phe Ile Ser Phe Asp Lys Glu Ser Asn Trp Asp Thr Ile Ser Thr Ile          35 40 45 Ser Ser Thr Ser Ala Val Val Ser Ser Val Asp Ser Ala Ile Ala Val      50 55 60 Phe Glu Phe Asp Asn Phe Ser Leu Leu Asp Ser Leu Met Ile Asp Glu  65 70 75 80 Glu Tyr Pro Phe Phe Asn Arg Phe Phe Ala Asn Asp Val Ser Leu Thr                  85 90 95 Val His Asp Asp Ser Pro Leu Asn Ile Ser Gln Ser Leu Ser Pro Ile             100 105 110 Met Glu Gln Phe Thr Val Asp Glu Leu Pro Glu Ser Ala Ser Asp Leu         115 120 125 Leu Tyr Glu Tyr Ser Leu Asp Asp Lys Ser Ile Val Leu Phe Lys Phe     130 135 140 Thr Ser Asp Ala Tyr Asp Leu Lys Lys Leu Asp Glu Phe Ile Asp Ser 145 150 155 160 Cys Leu Ser Phe Leu Glu Asp Lys Ser Gly Asp Asn Leu Thr Val Val                 165 170 175 Ile Asn Ser Leu Gly Trp Ala Phe Glu Asp Glu Asp Gly Asp Asp Glu             180 185 190 Tyr Ala Thr Glu Glu Thr Leu Ser His His Asp Asn Asn Lys Gly Lys         195 200 205 Glu Gly Asp Asp Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp     210 215 220 Lys Arg 225 <210> 16 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> TFP 8 <400> 16 Met Leu Gln Ser Val Val Phe Phe Ala Leu Leu Thr Phe Ala Ser Ser   1 5 10 15 Val Ser Ala Ile Tyr Ser Asn Asn Thr Val Ser Thr Thr Thr Thr Leu              20 25 30 Ala Pro Ser Tyr Ser Leu Val Pro Gln Glu Thr Thr Ile Ser Tyr Ala          35 40 45 Asp Asp Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg      50 55 60 <210> 17 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> TFP 9 <400> 17 Met Lys Phe Ser Thr Ala Val Thr Thr Leu Ile Ser Ser Gly Ala Ile   1 5 10 15 Val Ser Ala Leu Pro His Val Asp Val His Gln Glu Asp Ala His Gln              20 25 30 His Lys Arg Ala Val Ala Tyr Lys Tyr Val Tyr Glu Thr Val Val Val          35 40 45 Asp Ser Asp Gly His Thr Val Thr Pro Ala Ala Ser Glu Val Ala Thr      50 55 60 Ala Ala Thr Ser Ala Ile Ile Thr Thr Ser  65 70 75 80 Ser Ala Ala Ala Asp Ser Ser Ala Ser Ile Ala Val Ser Ser Ala                  85 90 95 Ala Leu Ala Lys Asn Glu Lys Ile Ser Asp Ala Ala Ala Ser Ala Thr             100 105 110 Ala Ser Thr Ser Gln Gly Ala Ser Ser Ser Ser Tyr Leu Ala Ala Ser         115 120 125 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg     130 135 <210> 18 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> TFP 10 <400> 18 Met Asn Trp Leu Phe Leu Val Ser Leu Val Phe Phe Cys Gly Val Ser   1 5 10 15 Thr His Pro Ala Leu Ala Met Ser Ser Asn Arg Leu Leu Lys Leu Ala              20 25 30 Asn Lys Ser Pro Lys Lys Ile Ile Pro Leu Lys Asp Ser Ser Phe Glu          35 40 45 Asn Ile Leu Ala Pro Pro His Glu Asn Ala Tyr Ile Val Ala Leu Phe      50 55 60 Thr Ala Thr Ala Pro Glu Ile Gly Cys Ser Leu Cys Leu Glu Leu Glu  65 70 75 80 Ser Glu Tyr Asp Thr Ile Val Ala Ser Trp Phe Asp Asp His Pro Asp                  85 90 95 Ala Lys Ser Ser Asn Ser Asp Thr Ser Ile Phe Phe Thr Lys Val Asn             100 105 110 Leu Glu Asp Pro Ser Lys Thr Ile Pro Lys Ala Phe Gln Phe Phe Gln         115 120 125 Leu Asn Asn Val Pro Arg Leu Phe Ile Phe Lys Leu Asn Ser Ser Ser     130 135 140 Ile Leu Asp His Ser Val Ile Ser Ile Ser Thr Asp Thr Gly Ser Glu 145 150 155 160 Arg Met Lys Gln Ile Ile Gln Ala Ile Lys Gln Phe Ser Gln Val Asn                 165 170 175 Asp Phe Ser Leu His Leu Pro Val Gly Leu Ala Ala Ser Ala Ser Ala             180 185 190 Gly Leu Ala Leu Asp Lys Arg         195 <210> 19 <211> 77 <212> PRT <213> Artificial Sequence <220> <223> TFP 11 <400> 19 Met Lys Phe Ser Ser Val Thr Ala Ile Thr Leu Ala Thr Val Ala Thr   1 5 10 15 Val Ala Thr Ala Lys Lys Gly Glu His Asp Phe Thr Thr Thr Leu Thr              20 25 30 Leu Ser Ser Asp Gly Ser Leu Thr Thr Thr Thr Ser Thr His Thr Thr          35 40 45 His Lys Tyr Gly Lys Phe Asn Lys Thr Ser Lys Ser Lys Thr Pro Leu      50 55 60 Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg  65 70 75 <210> 20 <211> 94 <212> PRT <213> Artificial Sequence <220> <223> TFP 12 <400> 20 Met Ala Ser Phe Ala Thr Lys Phe Val Ile Ala Cys Phe Leu Phe Phe   1 5 10 15 Ser Ala Ser Ala His Asn Val Leu Leu Pro Ala Tyr Gly Arg Arg Cys              20 25 30 Phe Phe Glu Asp Leu Ser Lys Gly Asp Glu Leu Ser Ile Ser Phe Gln          35 40 45 Phe Gly Asp Arg Asn Pro Gln Ser Ser Ser Gln Leu Thr Gly Asp Phe      50 55 60 Ile Ile Tyr Gly Pro Glu Arg His Glu Val Leu Lys Thr Val Arg Glu  65 70 75 80 Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg                  85 90 <210> 21 <211> 174 <212> PRT <213> Artificial Sequence <220> <223> TFP 13 <400> 21 Met Gln Tyr Lys Lys Thr Leu Val Ala Ser Ala Leu Ala Ala Thr Thr   1 5 10 15 Leu Ala Ala Tyr Ala Pro Ser Glu Pro Trp Ser Thr Leu Thr Pro Thr              20 25 30 Ala Thr Tyr Ser Gly Gly Val Thr Asp Tyr Ala Ser Thr Phe Gly Ile          35 40 45 Ala Val Gln Pro Ile Ser Thr Thr Ser Ser Ala Ser Ser Ala Ala Thr      50 55 60 Thr Ala Ser Ser Lys Ala Lys Arg Ala Ser Ser Gln Ile Gly Asp Gly  65 70 75 80 Gln Val Gln Ala Ala Thr Thr Thr Ala Ser Val Ser Thr Lys Ser Thr                  85 90 95 Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Ile Gln Ala Thr Thr             100 105 110 Lys Thr Thr Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Ile Gln         115 120 125 Ala Thr Thr Lys Thr Thr Ser Ala Lys Thr Thr Ala Ala Ala Val Ser     130 135 140 Gln Ile Ser Asp Gly Gln Ile Gln Ala Thr Thr Thr Thr Leu Ala Pro 145 150 155 160 Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg                 165 170 <210> 22 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> TFP 14 <400> 22 Met Gln Phe Lys Asn Ala Leu Thr Ala Thr Ala Ile Leu Ser Ala Ser   1 5 10 15 Ala Leu Ala Ala Asn Ser Thr Thr Ser Ile Pro Ser Ser Cys Ser Ile              20 25 30 Gly Thr Ser Ala Thr Ala Thr Ala Gln Ala Thr Asp Ser Gln Ala Gln          35 40 45 Ala Thr Thr Ala Pro Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala      50 55 60 Leu Asp Lys Arg  65 <210> 23 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> TFP 15 <400> 23 Met Val Ser Lys Thr Trp Ile Cys Gly Phe Ile Ser Ile Ile Thr Val   1 5 10 15 Val Gln Ala Leu Ser Cys Glu Lys His Asp Val Leu Lys Lys Tyr Gln              20 25 30 Val Gly Lys Phe Ser Ser Leu Thr Ser Thr Glu Arg Asp Thr Pro Pro          35 40 45 Ser Thr Thr Ile Glu Lys Trp Trp Ile Asn Val Cys Glu Glu His Asn      50 55 60 Val Glu Pro Pro Glu Glu Cys Lys Lys Asn Asp Met Leu Cys Gly Leu  65 70 75 80 Thr Asp Val Ile Leu Pro Gly Lys Asp Ala Ile Thr Thr Gln Ile Ile                  85 90 95 Asp Phe Asp Lys Asn Ile Gly Phe Asn Val Glu Glu Thr Glu Ser Ala             100 105 110 Leu Thr Leu Thr Leu Asn Gly Ala Thr Trp Gly Ala Asn Ser Phe Asp         115 120 125 Ala Lys Leu Glu Phe Gln Cys Asn Asp Asn Met Lys Gln Asp Glu Leu     130 135 140 Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg 145 150 155 <210> 24 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> TFP 16 <400> 24 Met Lys Leu Ser Ala Leu Le Ala Leu Ser Ala Ser Thr Ala Val Leu   1 5 10 15 Ala Ala Pro Ala Val His His Ser Asp Asn His His His Asn Asp Lys              20 25 30 Arg Ala Val Val Thr Val Thr Gln Tyr Val Asn Ala Asp Gly Ala Val          35 40 45 Val Ile Pro Ala Ala Thr Ala Thr Ser Ala Ala Ala Asp Gly Lys      50 55 60 Val Glu Ser Val Ala Ala Thr Thr Thr Leu Ser Ser Thr Ala Ala  65 70 75 80 Ala Ala Thr Thr Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp                  85 90 95 Lys Arg         <210> 25 <211> 195 <212> PRT <213> Artificial Sequence <220> <223> TFP 17 <400> 25 Met Lys Leu Ser Thr Val Leu Leu Ser Ala Gly Leu Ala Ser Thr Thr   1 5 10 15 Leu Ala Gln Phe Ser Asn Ser Thr Ser Ala Ser Ser Thr Asp Val Thr              20 25 30 Ser Ser Ser Ser Ser Thr Ser Ser Ser Ser Val Thr Ile Thr Ser          35 40 45 Ser Glu Ala Pro Glu Ser Asp Asn Gly Thr Ser Thr Ala Ala Pro Thr      50 55 60 Glu Thr Ser Thr Glu Ala Pro Thr Thr Ala Ile Pro Thr Asn Gly Thr  65 70 75 80 Ser Thr Glu Ala Pro Thr Thr Ala Ile Pro Thr Asn Gly Thr Ser Thr                  85 90 95 Glu Ala Pro Thr Asp Thr Thr Thr Glu Ala Pro Thr Thr Ala Leu Pro             100 105 110 Thr Asn Gly Thr Ser Thr Glu Ala Pro Thr Asp Thr Thr Thr Glu Ala         115 120 125 Pro Thr Thr Gly Leu Pro Thr Asn Gly Thr Thr Ser Ala Phe Pro Pro     130 135 140 Thr Thr Ser Leu Pro Pro Ser Asn Thr Thr Thr Thr Pro Pro Tyr Asn 145 150 155 160 Pro Ser Thr Asp Tyr Thr Thr Asp Tyr Thr Val Val Thr Glu Tyr Thr                 165 170 175 Thr Tyr Cys Pro Glu Arg Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu             180 185 190 Asp Lys Arg         195 <210> 26 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> TFP 18 <400> 26 Met Arg Phe Ser Thr Thr Leu Ala Thr Ala Ala Thr Ala Leu Phe Phe   1 5 10 15 Thr Ala Ser Gln Val Ser Ala Ile Gly Glu Leu Ala Phe Asn Leu Gly              20 25 30 Val Lys Asn Asn Asp Gly Thr Cys Lys Ser Thr Ser Asp Tyr Glu Thr          35 40 45 Glu Leu Gln Ala Leu Lys Ser Tyr Thr Ser Thr Val Lys Val Tyr Ala      50 55 60 Ala Ser Asp Cys Asn Thr Leu Gln Asn Leu Gly Pro Ala Ala Glu Ala  65 70 75 80 Glu Gly Phe Thr Ile Phe Val Gly Val Trp Pro Leu Ala Ala Ser Ala                  85 90 95 Ser Ala Gly Leu Ala Leu Asp Lys Arg             100 105 <210> 27 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> TFP 19 <400> 27 Met Arg Leu Ser Asn Leu Ile Ala Ser Ala Ser Leu Leu Ser Ala Ala   1 5 10 15 Thr Leu Ala Ala Pro Ala Asn His Glu His Lys Asp Lys Arg Ala Val              20 25 30 Val Thr Thr Thr Val Gln Lys Gln Thr Thr Ile Ile Val Asn Gly Ala          35 40 45 Ala Ser Thr Pro Val Ala Ala Leu Glu Glu Asn Ala Val Val Asn Ser      50 55 60 Ala Pro Ala Ala Ala Thr Ser Thr Ser Ser Ala Ala Ser Val Ala  65 70 75 80 Thr Ala Ala Ser Ser Glu Asn Asn Ser Gln Val Ser Ala Ala Ala                  85 90 95 Ser Pro Ala Ser Ser Ala Ala Thr Ser Thr Gln Ser Ser Leu Ala             100 105 110 Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg         115 120 <210> 28 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> TFP 20 <400> 28 Met Gln Phe Ser Thr Val Ala Ser Ile Ala Ala Val Ala Ala Val Ala   1 5 10 15 Ser Ala Ala Asn Val Thr Thr Ala Thr Val Ser Gln Glu Ser Thr              20 25 30 Thr Leu Val Thr Ile Thr Ser Cys Glu Asp His Val Cys Ser Glu Thr          35 40 45 Val Ser Pro Ala Leu Val Ser Thr Ala Thr Val Thr Val Asp Asp Val      50 55 60 Ile Thr Gln Tyr Thr Thr Trp Cys Pro Leu Thr Thr Glu Ala Pro Lys  65 70 75 80 Asn Gly Thr Ser Thr Ala Ala Pro Val Thr Ser Thr Glu Ala Pro Lys                  85 90 95 Asn Thr Thr Ser Ala Ala Pro Thr His Ser Val Thr Ser Tyr Thr Gly             100 105 110 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Leu Ala Ala Ser         115 120 125 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg     130 135 <210> 29 <211> 176 <212> PRT <213> Artificial Sequence <220> <223> TFP 21 <400> 29 Met Lys Phe Ser Ser Ala Leu Val Seru Ser Ala Val Ala Ala Thr Ala   1 5 10 15 Leu Ala Glu Ser Ile Thr Thr Thr Ile Thr Ala Thr Lys Asn Gly His              20 25 30 Val Tyr Thr Lys Thr Val Thr Gln Asp Ala Thr Phe Val Trp Gly Gly          35 40 45 Glu Asp Ser Tyr Ala Ser Ser Thr Ser Ala Ala Glu Ser Ser Ala Ala      50 55 60 Glu Thr Ser Ala Ala Glu Thr Ser Ala Ala Ala Thr Thr Ser Ala Ala  65 70 75 80 Ala Thr Thr Ser Ala Glu Thr Ser Ser Ala Ala Glu Thr Ser Ser                  85 90 95 Ala Asp Glu Gly Ser Gly Ser Ser Ile Thr Thr Thr Ile Thr Ala Thr             100 105 110 Lys Asn Gly His Val Tyr Thr Lys Thr Val Thr Gln Asp Ala Thr Phe         115 120 125 Val Trp Thr Gly Glu Gly Ser Ser Asn Thr Trp Ser Pro Ser Ser Thr     130 135 140 Ser Thr Ser Ala Thr Ser Ser Ala Thr 145 150 155 160 Thr Leu Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg                 165 170 175 <210> 30 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> TFP 22 <400> 30 Met Lys Phe Gln Val Val Leu Ser Ala Leu Leu Ala Cys Ser Ser Ala   1 5 10 15 Val Val Ala Ser Pro Ile Glu Asn Leu Phe Lys Tyr Arg Ala Val Lys              20 25 30 Ala Ser His Ser Lys Asn Ile Asn Ser Thr Leu Pro Ala Trp Asn Gly          35 40 45 Ser Asn Ser Ser Asn Val Thr Tyr Ala Asn Gly Thr Asn Ser Thr Thr      50 55 60 Asn Thr Thr Thr Ala Glu Ser Ser Gln Leu Gln Ile Ile Val Thr Gly  65 70 75 80 Gly Gln Val Pro Ile Thr Asn Ser Ser Leu Thr His Thr Asn Tyr Thr                  85 90 95 Arg Leu Phe Asn Ser Ser Ser Ala Leu Asn Ile Thr Glu Leu Tyr Asn             100 105 110 Val Ala Arg Val Val Asn Glu Thr Ile Gln Asp Asn Leu Ala Ala Ser         115 120 125 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg     130 135 <210> 31 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> TFP 23 <400> 31 Met Arg Ala Ile Thr Leu Leu Ser Ser Val Val Ser Leu Ala Leu Leu   1 5 10 15 Ser Lys Glu Val Leu Ala Thr Pro Pro Ala Cys Leu Leu Ala Cys Val              20 25 30 Ala Gln Val Gly Lys Ser Ser Ser Thr Cys Asp Ser Leu Asn Gln Val          35 40 45 Thr Cys Tyr Cys Glu His Glu Asn Ser Ala Val Lys Lys Cys Leu Asp      50 55 60 Ser Ile Cys Pro Asn Asn Asp Ala Asp Ala Ala Tyr Ser Ala Phe Lys  65 70 75 80 Ser Ser Cys Ser Glu Gln Asn Ala Ser Leu Gly Asp Ser Ser Gly Ser                  85 90 95 Ala Ser Ser Ala Ser Ala Ser Ala Gly             100 105 110 Asp Lys Arg         115 <210> 32 <211> 170 <212> PRT <213> Artificial Sequence <220> <223> TFP 24 <400> 32 Met Lys Leu Ser Thr Val Leu Leu Ser Ala Gly Leu Ala Ser Thr Thr   1 5 10 15 Leu Ala Gln Phe Ser Asn Ser Thr Ser Ala Ser Ser Thr Asp Val Thr              20 25 30 Ser Ser Ser Ser Ser Thr Ser Ser Ser Ser Val Thr Ile Thr Ser          35 40 45 Ser Glu Ala Pro Glu Ser Asp Asn Gly Thr Ser Thr Ala Ala Pro Thr      50 55 60 Glu Thr Ser Thr Glu Ala Pro Thr Thr Ala Ile Pro Thr Asn Gly Thr  65 70 75 80 Ser Thr Glu Ala Pro Thr Thr Ala Ile Pro Thr Asn Gly Thr Ser Thr                  85 90 95 Glu Ala Pro Thr Asp Thr Thr Thr Glu Ala Pro Thr Thr Ala Leu Pro             100 105 110 Thr Asn Gly Thr Ser Thr Glu Ala Pro Thr Asp Thr Thr Thr Glu Ala         115 120 125 Pro Thr Thr Gly Leu Pro Thr Asn Gly Thr Thr Ser Ala Phe Pro Pro     130 135 140 Thr Thr Ser Leu Pro Ser Ser Asn Thr Thr Thr Thr Leu Ala Ala Ser 145 150 155 160 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg                 165 170 <210> 33 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> TFP 1 <400> 33 atgttcaatc gttttaacaa attccaagct gctgtcgctt tggccctact ctctcgcggc 60 gctctcggtg actcttacac caatagcacc tcctccgcag acttgagttc tatcacttcc 120 gtctcgtcag ctagtgcaag tgccaccgct tccgactcac tttcttccag tgacggtacc 180 gtttatttgc catccacaac aattagcggt gatctcacag ttactggtaa agtaattgca 240 accgaggccg tggaagtcgc tgccggtggt aagttgactt tacttgacgg tgaaaaatac 300 gtcttctcat ctgaggccgc ctcggcctct gctggcctcg ccttagataa aaga 354 <210> 34 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> TFP 2 <400> 34 atgacgccct atgcagtagc aattaccgtg gccttactaa ttgtaacagt gagcgcactc 60 caggtcaaca attcatgtgt cgcttttccg ccatcaaatc tcaggggcaa gaatggagac 120 ggtactaatg aacagtatgc aactgcacta ctttctattc cctggaatgg gcctcctgag 180 tcatcgaggg atattaatct tatcgaactc gaaccgcaag ttgcactcta tttgctcgaa 240 aattatatta accattacta caacaccaca agagacaata agtgccctaa taaccactac 300 ctaatgggag ggcagttggg tagctcatcg gataatagga gtttgaacga ggccgcctcg 360 gcctctgctg gcctcgcctt agataaaaga 390 <210> 35 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> TFP 3 <400> 35 atgcaattca aaaacgtcgc cctagctgcc tccgttgctg ctctatccgc cactgcttct 60 gctgaaggtt acactccagg tgaaccatgg tccaccttaa ccccaaccgg ctccatctct 120 tgtggtgcag ccgaatacac taccaccttt ggtattgctg ttcaagctat tacctcttca 180 aaagctaaga gagacgttat ctctcaaatt ggtgacggtc aagtccaagc cacttctgct 240 gctactgctc aagccaccga tagtcaagcc caagctacta ctaccgctac cccaaccagc 300 tccgaaaaga tggccgcctc ggcctctgct ggcctcgcct tagataaaag a 351 <210> 36 <211> 186 <212> DNA <213> Artificial Sequence <220> <223> TFP 4 <400> 36 atgagatttg cagaattctt ggtggtattt gccacgttag gcggggggat ggctgcaccg 60 gttgagtctc tggccgggac ccaacggtat ctggtgcaaa tgaaggagcg gttcaccaca 120 gagaagctgt gtgctttgga cgacaaggcc gcctcggcct ctgctggcct cgccttagat 180 aaaaga 186 <210> 37 <211> 291 <212> DNA <213> Artificial Sequence <220> <223> TFP 5 <400> 37 atgttcaatc gttttaacaa attccaagct gctgtcgctt tggccctact ctctcgcggc 60 gctctcggtg ctccagtcaa cactacaaca gaagatgaaa cggcactaat tccggctgaa 120 gctgtcatcg gttacttaga tttagaaggg gatttcgatg ttgctgtttt gccattttcc 180 aacagcacaa ataacgggtt attgtttata aatactacta ttgccagcat tgctgctaaa 240 gaagaagggg tggccgcctc ggcctctgct ggcctcgcct tagataaaag a 291 <210> 38 <211> 279 <212> DNA <213> Artificial Sequence <220> <223> TFP 6 <400> 38 atgagatttc cttcaatttt tactgcagtt ttattcgcag catcctccgc attagctgct 60 ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120 tacttagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180 aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggtg 240 gccgcctcgg cctctgctgg cctcgcctta gataaaaga 279 <210> 39 <211> 678 <212> DNA <213> Artificial Sequence <220> <223> TFP 7 <400> 39 atggtgttcg gtcagctgta tgcccttttc atcttcacgt tatcatgttg tatttccaaa 60 actgtgcaag cagattcatc caaggaaagc tcttccttta tttcgttcga caaagagagt 120 aactgggata ccatcagcac tatatcttca acggcagatg ttatatcatc cgttgacagt 180 gctatcgctg tttttgaatt tgacaatttc tcattattgg acagcttgat gattgacgaa 240 gaatacccat tcttcaatag attctttgcc aatgatgtca gtttaactgt tcatgacgat 300 tcgcctttga acatctctca atcattatct cccattatgg aacaatttac tgtggatgaa 360 ttacctgaaa gtgcctctga cttactatat gaatactcct tagatgataa aagcatcgtt 420 ttgttcaagt ttacctcgga tgcctacgat ttgaaaaaat tagatgaatt tattgattct 480 tgcttatcgt ttttggaaga taaatctggc gacaatttga ctgtggttat taactctctt 540 ggttgggctt ttgaagatga agatggtgac gatgaatatg caacagaaga gactttgagc 600 catcatgata acaacaaggg taaagaaggc gacgatctgg ccgcctcggc ctctgctggc 660 ctcgccttag ataaaaga 678 <210> 40 <211> 192 <212> DNA <213> Artificial Sequence <220> <223> TFP 8 <400> 40 atgcttcaat ccgttgtctt tttcgctctt ttaaccttcg caagttctgt gtcagcgatt 60 tattcaaaca atactgtttc tacaactacc actttagcgc ccagctactc cttggtgccc 120 caagagacta ccatatcgta cgccgacgac ctggccgcct cggcctctgc tggcctcgcc 180 ttagataaaa ga 192 <210> 41 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> TFP 9 <400> 41 atgaaattct caactgccgt tactacgttg attagttctg gtgccatcgt gtctgcttta 60 ccacacgtgg atgttcacca agaagatgcc caccaacata agagggccgt tgcgtacaaa 120 tacgtttacg aaactgttgt tgtcgattct gatggccaca ctgtaactcc tgctgcttca 180 gaagtcgcta ctgctgctac ctctgctatc attacaacat ctgtgttggc tccaacctcc 240 tccgcagccg ctgcggatag ctccgcttcc attgctgttt catctgctgc cttagccaag 300 aatgagaaaa tctctgatgc cgctgcatct gccactgcct caacatctca aggggcatcc 360 tcctcatcct acctggccgc ctcggcctct gctggcctcg ccttagataa aaga 414 <210> 42 <211> 597 <212> DNA <213> Artificial Sequence <220> <223> TFP 10 <400> 42 atgaattggc tgtttttggt ctcgctggtt ttcttctgcg gcgtgtcaac ccatcctgcc 60 ctggcaatgt ccagcaacag actactaaag ctggctaata aatctcccaa gaaaattata 120 cctctgaagg actcaagttt tgaaaacatc ttggcaccac ctcacgaaaa tgcctatata 180 gttgctctgt ttactgccac agcgcccgaa attggctgtt ctctgtgtct cgagctagaa 240 tccgaatacg acaccatagt ggcctcctgg tttgatgatc atccggatgc aaaatcgtcc 300 aattccgata catctatttt cttcacaaag gtcaatttgg aggacccttc taagaccatt 360 cctaaagcgt tccagttttt ccaactaaac aatgttccta gattgttcat cttcaaacta 420 aactctccct ctattctgga ccacagcgtg atcagtattt ccactgatac tggctcagaa 480 agaatgaagc aaatcataca agccattaag cagttctcgc aagtaaacga cttctcttta 540 cacttacctg tgggtctggc cgcctcggcc tctgctggcc tcgccttaga taaaaga 597 <210> 43 <211> 231 <212> DNA <213> Artificial Sequence <220> <223> TFP 11 <400> 43 atgaagttct cttctgttac tgctattact ctagccaccg ttgccaccgt tgccactgct 60 aagaagggtg aacatgattt cactaccact ttaactttgt catcggacgg tagtttaact 120 actaccacct ctactcatac cactcacaag tatggtaagt tcaacaagac ttccaagtcc 180 aagacccccc tggccgcctc ggcctctgct ggcctcgcct tagataaaag a 231 <210> 44 <211> 282 <212> DNA <213> Artificial Sequence <220> <223> TFP 12 <400> 44 atggcctcat ttgctactaa gtttgtcatt gcttgcttcc tgttcttctc ggcgtccgcc 60 cataatgtcc ttcttccagc ttatggccgt agatgcttct tcgaagactt gagtaagggt 120 gacgagctct ccatttcgtt ccagttcggt gatagaaacc ctcaatccag tagccagctg 180 actggtgact ttatcatcta cgggccggaa agacatgaag ttttgaaaac ggttagggaa 240 ctggccgcct cggcctctgc tggcctcgcc ttagataaaa ga 282 <210> 45 <211> 522 <212> DNA <213> Artificial Sequence <220> <223> TFP 13 <400> 45 atgcaataca aaaagacttt ggttgcctct gctttggccg ctactacatt ggccgcctat 60 gctccatctg agccttggtc cactttgact ccaacagcca cttacagcgg tggtgttacc 120 gactacgctt ccaccttcgg tattgccgtt caaccaatct ccactacatc cagcgcatca 180 tctgcagcca ccacagcctc atctaaggcc aagagagctg cttcccaaat tggtgatggt 240 caagtccaag ctgctaccac tactgcttct gtctctacca agagtaccgc tgccgccgtt 300 tctcagatcg gtgatggtca aatccaagct actaccaaga ctaccgctgc tgctgtctct 360 caaattggtg atggtcaaat tcaagctacc accaagacta cctctgctaa gactaccgcc 420 gctgccgttt ctcaaatcag tgatggtcaa atccaagcta ccaccactac tttagcccct 480 ctggccgcct cggcctctgc tggcctcgcc ttagataaaa ga 522 <210> 46 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> TFP 14 <400> 46 atgcaattca agaacgcttt gactgctact gctattctaa gtgcctccgc tctagctgct 60 aactcaacta cttctattcc atcttcatgt agtattggta cttctgccac tgctactgct 120 caagccaccg atagtcaagc ccaagctact actaccgcac ccctggccgc ctcggcctct 180 gctggcctcg ccttagataa aaga 204 <210> 47 <211> 471 <212> DNA <213> Artificial Sequence <220> <223> TFP 15 <400> 47 atggatatcga agacttggat atgtggcttc atcagtataa ttacagtggt acaggccttg 60 tcctgcgaga agcatgatgt attgaaaaag tatcaggtgg gaaaatttag ctcactaact 120 tctacggaaa gggatactcc gccaagcaca actattgaaa agtggtggat aaacgtttgc 180 gaagagcata acgtagaacc tcctgaagaa tgtaaaaaaa atgacatgct atgtggttta 240 acagatgtca tcttgcccgg taaggatgct atcaccactc aaattataga ttttgacaaa 300 aacattggct tcaatgtcga ggaaactgag agtgcgctta cattgacact aaacggcgct 360 acgtggggcg ccaattcttt tgacgcaaaa ctagaatttc agtgtaatga caatatgaaa 420 caagacgaac tggccgcctc ggcctctgct ggcctcgcct tagataaaag a 471 <210> 48 <211> 294 <212> DNA <213> Artificial Sequence <220> <223> TFP 16 <400> 48 atgaaattat ccgctctatt agctttatca gcctccaccg ccgtcttggc cgctccagct 60 gtccaccata gtgacaacca ccaccacaac gacaagcgtg ccgttgtcac cgttactcag 120 tacgtcaacg cagacggcgc tgttgttatt ccagctgcca ccaccgctac ctcggcggct 180 gctgatggaa aggtcgagtc tgttgctgct gccaccacta ctttgtcctc gactgccgcc 240 gccgctacaa ccctggccgc ctcggcctct gctggcctcg ccttagataa aaga 294 <210> 49 <211> 585 <212> DNA <213> Artificial Sequence <220> <223> TFP 17 <400> 49 atgaaattat caactgtcct attatctgcc ggtttagcct cgactacttt ggcccaattt 60 tccaacagta catctgcttc ttccaccgat gtcacttcct cctcttccat ctccacttcc 120 tctggctcag taactatcac atcttctgaa gctccagaat ccgacaacgg taccagcaca 180 gctgcaccaa ctgaaacctc aacagaggct ccaaccactg ctatcccaac taacggtacc 240 tctactgaag ctccaaccac tgctatccca actaacggta cctctactga agctccaact 300 gatactacta ctgaagctcc aaccaccgct cttccaacta acggtacttc tactgaagct 360 ccaactgata ctactactga agctccaacc accggtcttc caaccaacgg taccacttca 420 gctttcccac caactacatc tttgccacca agcaacacta ccaccactcc tccttacaac 480 ccatctactg actacaccac tgactacact gtagtcactg aatatactac ttactgtccg 540 gaacgggccg cctcggcctc tgctggcctc gccttagata aaaga 585 <210> 50 <211> 315 <212> DNA <213> Artificial Sequence <220> <223> TFP 18 <400> 50 atgcgtttct ctactacact cgctactgca gctactgcgc tatttttcac agcctcccaa 60 gtttcagcta ttggtgaact agcctttaac ttgggtgtca agaacaacga tggtacttgt 120 aagtccactt ccgactatga aaccgaatta caagctttga agagctacac ttccaccgtc 180 aaagtttacg ctgcctcaga ttgtaacact ttgcaaaact taggtcctgc tgctgaagct 240 gagggattta ctatctttgt cggtgtttgg ccactggccg cctcggcctc tgctggcctc 300 gccttagata aaaga 315 <210> 51 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> TFP 19 <400> 51 atgcgtctct ctaacctaat tgcttctgcc tctcttttat ctgctgctac tcttgctgct 60 cccgctaacc acgaacacaa ggacaagcgt gctgtggtca ctaccactgt tcaaaaacaa 120 accactatca ttgttaatgg tgccgcttca actccagttg ctgctttgga agaaaatgct 180 gttgtcaact ccgctccagc tgccgctacc agtacaacat cgtctgctgc ttctgtagct 240 accgctgctt cctcttctga gaacaactca caagtttctg ctgccgcatc tccagcctcc 300 agctctgctg ctacatctac tcaatcttct ctggccgcct cggcctctgc tggcctcgcc 360 ttagataaaa ga 372 <210> 52 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> TFP 20 <400> 52 atgcaatttt ctactgtcgc ttctatcgcc gctgtcgccg ctgtcgcttc tgccgctgct 60 aacgttacca ctgctactgt cagccaagaa tctaccactt tggtcaccat cacttcttgt 120 gaagaccacg tctgttctga aactgtctcc ccagctttgg tttccaccgc taccgtcacc 180 gtcgatgacg ttatcactca atacaccacc tggtgcccat tgaccactga agccccaaag 240 aacggtactt ctactgctgc tccagttacc tctactgaag ctccaaagaa caccacctct 300 gctgctccaa ctcactctgt cacctcttac actggtgctg ctgctaaggc tttgccagct 360 gctggtgctt tgctggccgc ctcggcctct gctggcctcg ccttagataa aaga 414 <210> 53 <211> 528 <212> DNA <213> Artificial Sequence <220> <223> TFP 21 <400> 53 atgaaattct cttccgcttt ggttctatct gctgttgccg ctactgctct tgctgagagt 60 atcaccacca ccatcactgc caccaagaac ggtcatgtct acactaagac tgtcacccaa 120 gatgctactt ttgtttgggg tggtgaagac tcttacgcca gcagcacttc tgccgctgaa 180 tcttctgccg ccgaaacttc tgccgccgaa acctctgctg ccgctaccac ttctgctgcc 240 gctaccactt ctgctgctga gacttcttct gctgctgaga cttcttctgc tgatgaaggt 300 tctggttcta gtatcactac cactatcact gccaccaaga acggtcacgt ctacactaag 360 actgtcaccc aagatgctac ttttgtctgg actggtgaag gcagcagcaa cacctggtct 420 ccaagtagta cctctaccag ctcagaagct gctacctctt ctgcttcaac cactgcaacc 480 accctgctgg ccgcctcggc ctctgctggc ctcgccttag ataaaaga 528 <210> 54 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> TFP 22 <400> 54 atgaagttcc aagttgtttt atctgccctt ttggcatgtt catctgccgt cgtcgcaagc 60 ccaatcgaaa acctattcaa atacagggca gttaaggcat ctcacagtaa gaatatcaac 120 tccactttgc cggcctggaa tgggtctaac tctagcaatg ttacctacgc taatggaaca 180 aacagtacta ccaatactac tactgccgaa agcagtcaat tacaaatcat tgtaacaggt 240 ggtcaagtac caatcaccaa cagttctttg acccacacaa actacaccag attattcaac 300 agttcttctg ctttgaacat taccgaattg tacaatgttg cccgtgttgt taacgaaacg 360 atccaagata acctggccgc ctcggcctct gctggcctcg ccttagataa aaga 414 <210> 55 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> TFP 23 <400> 55 atgcgtgcca tcactttatt atcttcagtc gtttctttgg cattgttgtc gaaggaagtc 60 ttagcaacac ctccagcttg tttattggcc tgtgttgcgc aagtcggcaa atcctcttcc 120 acatgtgact ctttgaatca agtcacctgt tactgtgaac acgaaaactc cgccgtcaag 180 aaatgtctag actccatctg cccaaacaat gacgctgatg ctgcttattc tgctttcaag 240 agttcttgtt ccgaacaaaa tgcttcattg ggcgattcca gcggcagtgc ctcctcatcc 300 gttctggccg cctcggcctc tgctggcctc gccttagata aaaga 345 <210> 56 <211> 510 <212> DNA <213> Artificial Sequence <220> <223> TFP 24 <400> 56 atgaaattat caactgtcct attatctgcc ggtttggcct cgactacttt ggcccaattt 60 tccaacagta catctgcttc ttccaccgat gtcacttcct cctcttccat ctccacttcc 120 tctggctcag taactatcac atcttctgaa gctccagaat ccgacaacgg taccagcaca 180 gctgcaccaa ctgaaacctc aacagaggct ccaaccactg ctatcccaac taacggtacc 240 tctactgaag ctccaaccac tgctatccca actaacggta cctctactga agctccaact 300 gatactacta ctgaagctcc aaccaccgct cttccaacta acggtacttc tactgaagct 360 ccaactgata ctactactga agctccaacc accggtcttc caaccaacgg taccacttca 420 gctttcccac caactacatc tttgccacca agcaacacta ccaccactct ggccgcctcg 480 gcctctgctg gcctcgcctt agataaaaga 510

Claims (19)

A levan fructose transferase secretion expression cassette comprising a nucleic acid encoding levan fructotransferase and a nucleic acid encoding a translational fusion partner (TFP) that secretes the protein extracellularly,
Wherein said protein secretion fusion factor is TFP9 of SEQ ID NO: 17, TFP13 of SEQ ID NO: 21 or TFP19 of SEQ ID NO: 27.
The secretory expression cassette according to claim 1, wherein the levan fructose transferase is derived from Arthrobacter ureafaciens .
The secretory expression cassette of claim 1, wherein the levan fructose transferase comprises the amino acid sequence of SEQ ID NO: 8.
delete delete The secretory expression cassette according to claim 1, wherein the nucleic acid encoding the levan fructosyltransferase and the nucleic acid encoding the protein secretion fusion factor are linked to each other by a linker.
The secretory expression cassette of claim 6, wherein the linker comprises a protease recognition sequence or affinity tag.
The secretory expression cassette of claim 1, wherein the levan fructose transferase further comprises an affinity tag.
9. The method of claim 8, wherein the affinity tag is selected from the group consisting of GST, MBP, NusA, thioredoxin, ubiquitin, FLAG, BAP, HIS, STREP, CBP, CBD, , Secreted expression cassette.
9. A vector comprising a levan fructose transferase secretion expression cassette of any one of claims 1 to 3 and 6 to 9.
Which contains a Levan fructosyltransferase expression cassette, comprising a nucleic acid encoding levan fructotransferase, and a nucleic acid encoding a protein secretion fusion partner (TFP) that secretes the protein ex vivo. As recombinant microorganisms,
Wherein said protein secretion fusion factor is TFP9 of SEQ ID NO: 17, TFP13 of SEQ ID NO: 21 or TFP19 of SEQ ID NO: 27.
12. The microorganism according to claim 11, wherein the recombinant microorganism is yeast.
13. The method of claim 12, wherein the yeast is selected from the group consisting of Candida , Debaryomyces , Hansenula , Kluyveromyces , Pichia , Schizosaccharomyces , , Jaroslaw's (Yarrowia), Saccharomyces Roman Isis (Saccharomyces), shoe Oh My wanni access (Schwanniomyces) and Sula are in, microorganisms are selected from the group consisting in (Arxula).
13. The method according to claim 12, wherein the yeast is Candida utilis , Candida boidinii), Candida albicans (Candida albicans), Cluj Vero Mai Seth lactis (Kluyveromyces lactis , Pichia pastoris , Pichia stipitis , skiing, Schizosaccharomyces pombe , Saccharomyces cerevisiae , Hansenula ( Hansenula &lt; RTI ID = 0.0 & gt ; polymorpha , Yarrowia lipolytica , Schwanniomyces occidentalis , and Arxula &lt; RTI ID = 0.0 &gt; adeninivorans ). &lt; / RTI &gt;
(i) culturing the recombinant microorganism of claim 11; And
(ii) recovering levan fructose transfection enzyme from the cultured microorganism or culture supernatant thereof.
A process for preparing difructose anhydride IV comprising reacting the recombinant microorganism of claim 11 or the levan fructose transferase produced by the method of claim 15 with Levan.
17. The process according to claim 16, wherein the reaction is carried out at a pH of from 3.5 to 9.5.
17. The process according to claim 16, wherein the reaction is carried out at a temperature of from 1 占 폚 to 70 占 폚.
17. The method of claim 16,
Wherein the step of reacting with Levan comprises fermenting the recombinant microorganism in a medium comprising Levan.

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