KR101826927B1 - A microorganism having enhanced levansucrase productivity and a method of producing levan using the microorganism - Google Patents

Abstract

The present invention relates to a Levans sucrase secretion expression cassette comprising a nucleic acid encoding levansucrase and a nucleic acid encoding a translational fusion partner (TFP), a vector comprising the cassette, Or a vector, and a method for producing Levanschraze and Levan using the transformed microorganism.
The transforming microorganism of the present invention can secrete recombinant levanshucrase with good efficiency by using a protein secretion fusion factor and can be obtained by reacting levanshucrage produced from the microorganism with sugar or by mixing the microorganism with sugar And thus can effectively produce levan.

Description

[0001] The present invention relates to a microorganism having enhanced production ability of levansucrase and a method of producing the same using levansucrase,

The present invention relates to a Levans sucrase secretion expression cassette comprising a nucleic acid encoding Levansucrase and a nucleic acid encoding a translational fusion partner (TFP), a vector comprising the cassette, Or a vector, and a method for producing Levanschraze or Levan using the transformed microorganism.

Levan, known as a fructose polymer, is a natural substance found in a small amount in some plants and microorganisms. Unlike inulin, levan is the only water soluble fructose polymer. Therefore, levan is a diverse variety of foods and functional cosmetics, agricultural and livestock industry, It is a functional substance that is in demand in the industry.

Although Levan is a new biomaterial with diverse uses and market potential, research on levan production has been mainly focused on microbial fermentation and small-scale production and characterization using purified enzymes. Until the early 1990s, research on Levan production by direct fermentation with microorganisms such as Zymomonas mobilis or Bacillus polymyxa was dominant. However, in the case of the microbial fermentation method, the expression amount of levansucrase involved in levan biosynthesis is low, and the yield of levan is only about 30 to 50% of the theoretical yield due to inhibition of biosynthesis by the reaction products. In addition, Levan-producing microorganisms have most of the Levan hydrolysis-activating enzymes, which causes the yield of levan to decrease.

Therefore, since the Levanshukraze gene derived from Zymomonas mobilis has been reported, until recently, recombinant levanshucrase has been produced and an enzyme process using it has produced levans, replacing the direct fermentation process. Therefore, efficient recombinant expression of levansucrase is an indispensable technology for mass production of Levans industrially.

In the case of mass-expressing exogenous proteins such as levansucrase using microorganisms, the expression amount, the water availability, the expression site, and the modification of the protein may be different depending on the characteristics of the host cell and the target protein, In order to construct a production system, it is important to select the most suitable expression system for the target protein.

Although E. coli is generally used as a host system in the production of recombinant Levanshukraze, Levanschucrase, which is produced when overexpression or expression conditions do not match, is accumulated in the form of insoluble inclusion bodies, thereby lowering the overall activity It is often the case. In addition, since E. coli is not safe for human body, the use of Levas is limited by the use of the produced enzyme. Therefore, there is a need to use Generally Recognized As Safe microorganisms harmless to human body such as yeast . Saccharomyces cerevisiae is a GRAS microorganism, which is the same eukaryotic cell as a higher organism, has a protein transcription and translation system similar to that of a higher organism, and has a protein secretion system such as an endoplasmic reticulum and a Golgi apparatus, it is advantageous to secrete active protein through post-translational modification. In particular, when the recombinant fusion protein is secreted in a culture medium, the enzyme can be easily separated without complicated purification steps. Despite the various technical advantages of such a yeast system, there has been no case in which levansucrase has been efficiently produced in yeast since the productivity of the enzyme is low in the existing yeast protein secretion expression system, (PLoS One, Franken J et al., Biosynthesis of levan a bacterial extracellular polysaccharide in the yeast Saccharomyces cerevisiae, 2013, 8 (10), e77499).

Accordingly, the present inventors have made intensive efforts to develop a technique capable of secretion production of levanshucraza with high efficiency. As a result, it has been found that secretion production of recombinant levanshucrase using the GRAS yeast protein secretion fusion technology (Korean Patent No. 0975596) It was confirmed that the strain can secrete recombinant Levanshukra efficiently by producing a transformant having excellent efficiency. Furthermore, it has been confirmed that the recombinant levanshucrage produced from the strain can be applied to the production process of levan, or the levan can be efficiently produced through the fermentation process using the strain directly. Thus, the present invention has been completed.

One object of the present invention is to provide a levansucrase secretion expression cassette comprising a nucleic acid encoding levansucrase 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 transforming microorganism comprising a Levanschrage expression cassette comprising a nucleic acid encoding Levansucrase and a nucleic acid encoding a protein secretion fusion factor.

Still another object of the present invention is to provide a method for producing a microorganism which comprises (i) culturing the transformed microorganism; And (ii) recovering levansucrase from the cultured microorganism or culture supernatant thereof.

It is still another object of the present invention to provide a process for producing Levan, which comprises reacting the transforming microorganism or levansucrase prepared by the above-described method with sugar.

In order to achieve the above object, the present invention provides, as one embodiment, a nucleic acid encoding levansucrase and a nucleic acid encoding a translational fusion partner (TFP), wherein the secretion of levansucrase Expression cassettes are provided.

In the present invention, levansucrase was produced by using a protein secretion fusion factor to secrete levan from microorganisms with high efficiency. To this end, a nucleic acid and a protein secretion factor encoding levansucrase A recombinant vector containing the cassette, and a transforming microorganism were prepared and then used to produce levanshucrase useful for production of Levan.

The "levan" is commonly referred to as a polymer of fructose C ₆ H ₁₂ O ₆ . The levan is also referred to as levuloic acid, polyfructanoic acid, fluoplexose, and polyhedra. The levan is a polysaccharide having a beta- (2-> 6) bond between the fructose rings, wherein the number represents the carbon atom in the fructose ring to which it is linked, and the beta represents a stereochemical relationship. Levan is also described as a fructan in which the dominant glycoside bond between the D-fructopuranoside monomer units is beta - (2-> 6). Levan is generally produced by microorganisms, and some low-molecular-weight levans with molecular weights less than 100,000 daltons can be produced, although they are not produced as high molecular weight compounds in plants. In particular, Levan in the present invention may be one produced by Levansucrase.

The term " levansucrase "in the present invention refers to an enzyme that produces levan using sugar as a substrate. The Levanschraze may be a Levan-producing microorganism, such as Rhanella aquatilis , Zymomonas mobilis , Pseudomonas aurantiaca , Pseudomonas syringae , But are not limited to, bacteria such as Gluconobacter oxydans , Aerobacter levanicum , Bacillus amyloliquifaciens , Bacillus polymixa , Bacillus subtilis , Can be derived from Corynebacterium laevaniformans , Erwinia amylovora , or Streptococcus salivarius , and more specifically from Rhanella aquatilis , It may be from Zymomonas mobilis or Pseudomonas syringae More specifically, it can be derived from Lanella aquaticis having the amino acid sequence of SEQ ID NO: 12, from Zymomonas mobilis having the amino acid sequence of SEQ ID NO: 13, or from Bacillus subtilis derived from the amino acid sequence of SEQ ID NO: Sucrase, but is not limited thereto, and any enzyme having levan-producing ability can be included regardless of its origin or sequence.

And thus comprises the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14, or the conserved sequence of the amino acid sequence of SEQ ID NO: 12, 13 or SEQ ID NO: 14, (Specifically, 2 to 20 amino acid residues, more specifically 2 to 10 amino acid residues, and more particularly 2 to 5 amino acid residues) differ depending on the position or type of the amino acid residue in the protein. 12, SEQ ID NO: 13, or SEQ ID NO: 14, as long as it can maintain or enhance the activity of the Levanschuraza. The amino acid sequence of SEQ ID NO: 12, SEQ ID NO: , 80% or more, specifically 90% or more, more specifically 95% or more, particularly specifically 97% or more homologous to the amino acid sequence And, the substitution of the amino acid, deletion, insertion, addition or inversion or the like may include even the mutant sequence or artificial sequence variations produced in a microorganism containing the activity of levan sucrase shoe 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 fusion factor may be one disclosed in Korean Patent Laid-Open No. 10-2008-0042823, and more specifically, TFP 1 of SEQ ID NO: 15, TFP 2 of SEQ ID NO: 16, TFP 3 of SEQ ID NO: 17, TFP 4, TFP 5 of SEQ ID NO: 20, TFP 6 of SEQ ID NO: 21, TFP 7 of SEQ ID NO: 21, TFP 8 of SEQ ID NO: 22, TFP 9 of SEQ ID NO: 23, TFP 10 of SEQ ID NO: TFP 14 of SEQ ID NO: 28, TFP 14 of SEQ ID NO: 28, TFP 15 of SEQ ID NO: 29, TFP 16 of SEQ ID NO: 30, TFP 17 of SEQ ID NO: 31, TFP 18 of SEQ ID NO: TFP 19 of SEQ ID NO: 33, TFP 20 of SEQ ID NO: 34, TFP 21 of SEQ ID NO: 35, TFP 22 of SEQ ID NO: 36, TFP 23 of SEQ ID NO: 37 or TFP 24 of SEQ ID NO: 38, Any factor capable of improving expression can be included without limitation.

Thus, the nucleotide sequences of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, The amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: Lt; RTI ID = 0.0 > of the < / RTI > amino acid sequence of one or more (Specifically, 2 to 20 amino acid residues, more specifically 2 to 10 amino acid residues, and more particularly 2 to 5 amino acid residues) of the amino acid residues of the protein are substituted SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: More preferably 90% or more, more particularly 95% or more, more preferably 95% or more, more preferably 90% or more, more preferably 90% or more, More specifically, 97% or more Or substitution, deletion, insertion, addition, or inversion of the amino acid may include a naturally occurring mutant sequence or an artificial mutation sequence in the microorganism containing the TFP activity .

Further, when the recombinant levanshucrase is derived from lanella aquaticis, TFP 3 of SEQ ID NO: 17, TFP 4 of SEQ ID NO: 18, TFP 8 of SEQ ID NO: 22, or TFP 16 of SEQ ID NO: 30 TFP6 of SEQ ID NO: 20, TFP8 of SEQ ID NO: 22, TFP9 of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: TFP 11 of SEQ ID NO: 30, TFP 19 of SEQ ID NO: 33 or TFP 22 of SEQ ID NO: 36, TFP 1 of SEQ ID NO: 15, TFP 5 of SEQ ID NO: 19, TFP9 of SEQ ID NO: 20, TFP8 of SEQ ID NO: 22, TFP9 of SEQ ID NO: 23, TFP13 of SEQ ID NO: 27, TFP16 of SEQ ID NO: 30 or TFP19 of SEQ ID NO: 33 It is not.

In the Levans sucrase secretion expression cassette, the nucleic acid encoding the contained levanshucrase 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 encoding levanshucrase 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 levanshucrase can be performed by conventional DNA techniques, such as PCR and / or restriction enzyme cleavage and linkage.

The linker DNA of the present invention should have a sufficient length and be introduced into a host cell so that the nucleic acid sequence encoding levanshucrase in the cell can be recombined into a linear vector containing a nucleic acid sequence encoding TFP. May be of sufficient homology with a portion of the sequence. Specifically, the linker DNA has a length of 20 bp or more, such as 30 or 40 bp or more. More specifically, the linker DNA is at least 80% homologous to the linear vector and may have 85%, 90%, 95% or 99% homology.

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 thereto.

The affinity tag may be linked to the carboxy terminal or the amino terminal of levansucrase, and in particular, it may be connected to the carboxy terminal but is not limited thereto.

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 Levans sucrase secretion expression cassette comprising a nucleic acid encoding levanshucrase 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 levanshucrase, in particular a "levansucrase secretion expression cassette " &Quot;, which can secrete Levanshucrase.

In one example, the levallapurans-derived Levanschraja of the present invention may be encoded by the nucleic acid sequence of SEQ ID NO: 1, and the Zymomonas mobilis-derived Levanschuraza may be encoded by the nucleic acid sequence of SEQ ID NO: 6 And the Bacillus subtilis-derived levanshukraze may be one encoded by the nucleic acid sequence of SEQ ID NO: 9, but is not limited thereto.

As examples of the fusion protein of the present invention, TFP 1 is SEQ ID NO: 39, TFP 2 is SEQ ID NO: 40, TFP 3 is SEQ ID NO: 41, TFP 4 is SEQ ID NO 42, TFP 5 is SEQ ID NO 43, TFP 6 44, TFP 7 is SEQ ID NO: 45, TFP 8 is SEQ ID NO: 46, TFP 9 is SEQ ID NO: 47, TFP 10 is SEQ ID NO: 48, TFP 11 is SEQ ID NO 49, TFP 12 is SEQ ID NO 50, TFP 16 is SEQ ID NO: 54, TFP 17 is SEQ ID NO: 55, TFP 18 is SEQ ID NO: 56, TFP 19 is SEQ ID NO: 57, TFP 20 is SEQ ID NO: 51, TFP 14 is SEQ ID NO: 52, TFP 15 is SEQ ID NO: 58, TFP 21 is SEQ ID NO: 59, TFP 22 is SEQ ID NO: 60, TFP 23 is SEQ ID NO: 61, or TFP 24 is SEQ ID NO: 62.

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 levanshucrase 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 one specific example of the present invention, YGaST16-lsrA-6H containing TFP16 and levan's aquaticis-derived Levanshukrage gene was used as an expression vector for producing high secretion of levan's aquaticus-derived levanshucrase (Fig. 16).

In another specific embodiment of the present invention, an expression vector for producing high secretion of levanshucrazase derived from Zymomonas mobilis is YGaST8-levU-TFP8, which contains TFP8 and Zymomonas mobilis- 6H was prepared (Fig. 17).

In another specific embodiment of the present invention, an expression vector for producing high secretion of levanshucrase derived from Bacillus subtilis is YGaST8-BsSacB-1 comprising TFP8 and a Levanshukrage gene derived from Bacillus subtilis, 6H was prepared (Fig. 18).

The present invention provides, in yet another aspect, a transgenic microorganism comprising a Levans sucrase secretion expression cassette comprising a nucleic acid encoding Levansucrase and a nucleic acid encoding a protein secretion fusion factor do.

Levanshucrase, a protein secretion fusion factor, a nucleic acid, and a levansucrase secretion expression cassette have been described above.

The term "transformed" of the present invention means that DNA is introduced into a host cell so that the DNA can be replicated as an extrachromosomal factor or by chromosomal integration.

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 high introduction efficiency and expression efficiency of the Levanshukrage gene of the present invention, For example, it can include bacteria, fungi, and yeast. May be the transformed microorganism The specifically yeasts, more specifically, the yeast is Candida (Candida), di berry Oh, my process (Debaryomyces), Hanse Cronulla (Hansenula), Cluj Vero My process (Kluyveromyces), Pichia (Pichia) , A skiing survey can be one belonging to the genus Schizosaccharomyces , Yarrowia , Saccharomyces , Schwanniomyces or Arxula , and more specifically, Candida utilis For example, Candida utilis , Candida boidinii , Candida albicans , Kluyveromyces lactis , Pichia pastoris , Pichia stipitis , , Skiing investigation, Schizosaccharomyces pombe , Saccharomyces cerevisiae , Haensenula polyphora ( Ha but are not limited to, nsenula polymorpha , Yarrowia lipolytica , Schwanniomyces occidentalis , or Arxula adeninivorans .

Specific embodiment of the present invention, the La Nella aqua subtilis (Rhanella aquatilis), Eisai thigh eggplant Mobilis (Zymomonas mobilis), or Bacillus subtilis (Bacillus subtilis) derived Levan shoe sucrase (levansucrase) to secretory expressing the gene (Fig. 2 to Fig. 26). The recombinant levanshucrase production was confirmed by using the respective expression vectors prepared from the yeast secretion fusion factors and the yeast transformed with the respective vectors.

In one embodiment of the present invention, the GAL10 promoter used for the expression of Levanschuracia 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. Invertase, which is a product of the SUC2 gene, is an enzyme that decomposes sugar into glucose and fructose. Therefore, when levansucrase is expressed using a strain in which an invertase is present, some of the sugar is not converted to levan, To reduce the production capacity of Levan. For the above-mentioned reasons, a specific example of the present invention expresses Levanschraja using a strain in which GAL80 gene and SUC2 gene are simultaneously inactivated.

As a specific example for constructing the transformed 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 by 40 bases, the GAL7 transcription terminator end And 50 bases are the same, when introduced into a yeast cell together with a linearized vector, crossing occurs in the cell, and a Levanshukrage gene and a 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).

In addition, the transformed yeast is subjected to oil-bed culturing to refine levanshucrase and produce levan using the purified enzyme (FIG. 27, FIG. 28, and Tables 4 to 9), or the transformed It was confirmed that yeast could be fermented to produce levan (Table 10).

Specifically, analysis of Levan production ability of secreted Levanshucraze showed that the turbidity typically exhibited during Levan production was increased, and in HPLC analysis, the product of E. coli-derived Levanschuraja Was produced by using Levan, which has the same molecular weight as levan. In addition, when the transformed recombinant yeast was cultured in a medium containing sucrose, it was confirmed that levans were produced from the sugar by the Levanschraja secreted out of the cells during the culturing.

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

The transforming microorganism and levanshucrase are as described above.

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

In a specific embodiment of the present invention, fed-batch culture is carried out using the above-mentioned transforming microorganism to obtain levanshucrase.

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) (30% yeast extract, 30% glucose) according to the growth rate of the cells at 30 ° C for 48 hours. The cells were cultured in 200 ml of YEPD liquid medium, Supply.

According to another aspect of the present invention, there is provided a process for producing levan, which comprises reacting the transforming microorganism or levanshucrase produced by the above-described method with sugar.

The method of producing the transformed microorganism, levanshukraze, and Levan are as described above.

The reaction may be carried out at a pH of from 4.5 to pH 7.5, and the reaction may be carried out at 1 to 45 ° C, but is not limited thereto.

With respect to reacting levanshucrase with the sugar, in one specific embodiment of the present invention, the prepared levanshucrase was purified using affinity chromatography (Fig. 26), and the activity of the purified levanshucrase Optimal conditions were analyzed (Table 3, FIG. 27 and FIG. 28), confirming that levan from levansucrase could be produced in good yield (Tables 4 to 9).

Meanwhile, the step of reacting the transformed microorganism with sugar may include, but not limited to, fermenting the transformed microorganism in a medium containing sugar or fermenting it in a medium containing molasses.

Compared with an enzyme process in which levanshcraze is produced and then reacted with sugar to produce levan, the transformant strain is directly fermented in a medium containing sugar to produce levan, thereby eliminating the cost of enzyme production and purification, The high purity Levan can be produced because the transformant strain can be used as a carbon source in the amount of fructose, glucose, and sugar produced by the action of the enzyme.

In a specific example of the present invention, recombinant yeasts derived from Lanella aquaticus and Bacillus subtilis were cultivated in a medium supplemented with 5% sugar for 2 days to confirm that levan was produced at a high yield ), And in another specific example, recombinant yeasts derived from Ranella aquaticus and Bacillus subtilis were cultured in a medium containing molasses for 72 hours to confirm that Levan was produced at a high yield (Table 11).

The transgenic microorganism of the present invention can secrete recombinant levanshucrase with excellent efficiency by a protein secretion fusion factor and can be obtained by reacting levanshucrage produced from the microorganism with sugar or by mixing the microorganism with sugar It is possible to effectively produce levan by fermentation in a medium containing

Brief Description of the Drawings Fig. 1 is a diagram showing the results of a method of linking three kinds of Levanshukraze genes (lsrA, levU, BsSacB) to a GAL10 promoter vector containing 24 yeast protein secretion fusion factors (TFP) and introducing them into Saccharomyces cerevisiae It is a schematic diagram showing the recombination process in vivo.
FIG. 2 is a result of SDS-PAGE analysis of Levan sucrose protein-derived supernatant derived from Ranella aquacillus secreted in Saccharomyces cerevisiae and expressed in 24 strains.
Fig. 3 is a result of western blot analysis of the Levan sucralase protein supernatant derived from Ranella aquacillus secreted in Saccharomyces cerevisiae and expressed in 24 strains.
FIG. 4 is a result of SDS-PAGE analysis of the secreted levanshucrase protein supernatant by culturing three single strains of five strains selected first among the 24 TFP strains.
FIG. 5 shows Western blot analysis of secreted levanshucrase protein supernatants obtained by culturing three strains of 5 strains selected from among 24 TFP strains.
FIG. 6 is a result of SDS-PAGE analysis of the levanshucrase protein supernatant derived from Zymomonas mobilis expressed in 24 strains of Saccharomyces cerevisiae and secreted out of the cells.
Fig. 7 is a result of western blot analysis of the levanshucrase protein supernatant derived from Zymomonas mobilis expressed in 24 strains of Saccharomyces cerevisiae and secreted out of the cells.
Fig. 8 shows the results of analysis of enzyme activity from the protein supernatant derived from Zymomonas mobilis expressed in 24 strains of Saccharomyces cerevisiae and secreted out of the cells.
FIG. 9 is a result of SDS-PAGE analysis of the secreted protein of levamus sphaericase derived from Zymomonas mobilis by culturing a single strain in three strains of eight strains selected from among the 24 TFP strains.
FIG. 10 is a result of Western blot analysis of secreted protein levels of Levamuscular protein derived from Zymomonas mobilis by culturing a single strain in three strains of eight strains selected first among the 24 TFP strains.
FIG. 11 shows the results of analysis of enzyme activities from the secreted protein of Zymomonas mobilis-derived levanshucrase by culturing a single strain of three strains in eight strains selected first among the 24 TFP strains.
12 is a result of SDS-PAGE analysis of the Levanschrage protein supernatant derived from Bacillus subtilis secreted in Saccharomyces cerevisiae and expressed in 24 strains.
FIG. 13 shows the results of activity measurement of the protein supernatant derived from Bacillus subtilis-derived levanshucrase expressed in 24 strains of Saccharomyces cerevisiae and secreted out of the cells by DNS analysis method.
FIG. 14 shows the result of SDS-PAGE analysis of the Bacillus subtilis-derived levanshucrase protein supernatant secreted out of the cells by culturing a single strain in three strains of eight strains selected from among the 24 TFP strains.
FIG. 15 shows the activity of the Bacillus subtilis-derived levanshucrase protein supernatant derived from the cell culture obtained by culturing a single strain in three strains of eight strains selected first among the 24 TFP strains by the DNS analysis method to be.
16 is a schematic diagram of YGa-ST16-lsrA-6H expression and secretion vector containing secretory fusion factor TFP 16 and lsrA gene.
17 is a schematic diagram of YGa-ST8-levU-6H expression and secretion vector containing the secretion fusion factor TFP8 and the levU gene.
18 is a schematic diagram of YGa-ST8-BsSacB-6H expression and secretion vector containing secretory fusion factor TFP8 and BsSacB gene.
Fig. 19 is a graph showing the activity of the yeast Saccharomyces cerevisiae containing the recombinant vector YGa-ST16-lsrA-6H And the growth of the cells when fermenting the strain in a 5 L fermenter.
Fig. 20 is a graph showing the effect of the recombinant vector YGa-ST16-lsrA-6H on yeast Saccharomyces cerevisiae The results of SDS-PAGE analysis of 20 μl of the culture medium and 1 μl of the cell lysate in a 5 L fermenter while fermenting the fermentation broth are shown.
FIG. 21 is a graph showing changes in the secretion amount of enzymes inside and outside the cell by measuring the enzyme activity with the medium and the cell lysate taken at each fermentation time. FIG.
22 is a graph showing the effect of the recombinant vector YGa-ST16-lsrA-6H on yeast Saccharomyces cerevisiae The Y2805GS strain was analyzed by SDS-PAGE and Western blotting in a 5 L fermentor, and 20 μl of the fermentation broth obtained by fermenting the cells by growth time.
FIG. 23 is a graph showing the effect of the recombinant vector YGa-ST8-levU-6H on yeast Saccharomyces cerevisiae The Y2805GS strain was analyzed by SDS-PAGE and Western blotting in a 5 L fermentor, and 20 μl of the fermentation broth obtained by fermenting the cells by growth time.
24 is a graph showing the effect of the recombinant vector YGa-ST8-BsSacB-6H on yeast Saccharomyces cerevisiae And the growth of the cells when the fermentation was carried out in a 5 L fermentation tank.
25 is a graph showing the effect of the recombinant vector YGa-ST8-BsSacB-6H on yeast Saccharomyces cerevisiae The Y2805GS strain was subjected to fed-batch fermentation in a 5 L fermenter and subjected to SDS-PAGE and analyzed by Western blotting
Fig. 26 shows the result of SDS-PAGE analysis of the final purified fraction of the purified two kinds of levanshchrazas.
FIG. 27 shows the results of analysis of the optimal pH of the purified two kinds of levanshchrazas.
Fig. 28 shows the results of analysis of the optimal temperatures of the two kinds of refined levanshukraze.

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 1: Preparation of Lanolin aquatiles ( Rhanella aquatilis Selection of Yeast Protein Secretion Fusion Factor (TFP) for Expression of Levansucrase Derived

(Seo et al., Journal of Biotechnology 81, 63 (2000)) as a template and SEQ ID NO: 2 as a template for the production of a secretion expression vector for lanescacclase gene (lsrA, The lsrA gene was amplified by the first PCR using the primer of SEQ ID NO: 3 (94 캜 for 5 minutes, 94 캜 for 30 seconds, 55 캜 for 30 seconds, 72 캜 for 1 minute, 25 times, 72 캜 for 7 minutes once) . The gene amplified by the first PCR was subjected to second amplification with a primer of LNK39 (SEQ ID NO: 4) and HisGT50R (SEQ ID NO: 5), and a vector containing a protein secretion fusion factor was used for the sequence necessary for in vivo recombination And a recombinant gene in which a histidine tag to be used for protein purification 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: 15) TFP 2 MTPYAVAITVALLIVTVSALQVNNSCVAFPPSNLRGKNGDGTNEQYATALLSIPWNGPPESSRDINLIELEPQVALYLLENYINHYYNTTRDNKCPNNHYLMGGQLGSSSDNRSLNEAASASAGLALDKR (SEQ ID NO: 16) TFP 3 MQFKNVALAASVAALSATASAEGYTPGEPWSTLTPTGSISCGAAEYTTTFGIAVQAITSSKAKRDVISQIGDGQVQATSAATAQATDSQAQATTTATPTSSEKMAASASAGLALDKR (SEQ ID NO: 17) TFP 4 MRFAEFLVVFATLGGGMAAPVESLAGTQRYLVQMKERFTTEKLCALDDKAASASAGLALDKR (SEQ ID NO: 18) TFP 5 MFNRFNKFQAAVALALLSRGALGAPVNTTTEDETALIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVAASASAGLALDKR (SEQ ID NO: 19) TFP 6 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVAASASAGLALDKR (SEQ ID NO: 20) TFP 7 MVFGQLYALFIFTLSCCISKTVQADSSKESSSFISFDKESNWDTISTISSTADVISSVDSAIAVFEFDNFSLLDSLMIDEEYPFFNRFFANDVSLTVHDDSPLNISQSLSPIMEQFTVDELPESASDLLYEYSLDDKSIVLFKFTSDAYDLKKLDEFIDSCLSFLEDKSGDNLTVVINSLGWAFEDEDGDDEYATEETLSHHDNNKGKEGDDLAASASAGLALDKR (SEQ ID NO: 21) TFP 8 MLQSVVFFALLTFASSVSAIYSNNTVSTTTTLAPSYSLVPQETTISYADDLAASASAGLALDKR (SEQ ID NO: 22) TFP 9 MKFSTAVTTLISSGAIVSALPHVDVHQEDAHQHKRAVAYKYVYETVVVDSDGHTVTPAASEVATAATSAIITTSVLAPTSSAAAADSSASIAVSSAALAKNEKISDAAASATASTSQGASSSSYLAASASAGLALDKR (SEQ ID NO: 23) TFP 10 MNWLFLVSLVFFCGVSTHPALAMSSNRLLKLANKSPKKIIPLKDSSFENILAPPHENAYIVALFTATAPEIGCSLCLELESEYDTIVASWFDDHPDAKSSNSDTSIFFTKVNLEDPSKTIPKAFQFFQLNNVPRLFIFKLNSPSILDHSVISISTDTGSERMKQIIQAQQQQVNDFSLHLPVGLAASASAGLALDKR (SEQ ID NO: 24) TFP 11 MKFSSVTAITLATVATVATAKKGEHDFTTTLTLSSDGSLTTTTSTHTTHKYGKFNKTSKSKTPLAASASAGLALDKR (SEQ ID NO: 25) TFP 12 MASFATKFVIACFLFFSASAHNVLLPAYGRRCFFEDLSKGDELSISFQFGDRNPQSSSQLTGDFIIYGPERHEVLKTVRELAASASAGLALDKR (SEQ ID NO: 26) TFP 13 MQYKKTLVASALAATTLAAYAPSEPWSTLTPTATYSGGVTDYASTFGIAVQPISTTSSASSAATTASSKAKRAASQIGDGQVQAATTTASVSTKSTAAAVSQIGDGQIQATTKTTAAAVSQIGDGQIQATTKTTSAKTTAAAVSQISDGQIQATTTTLAPLAASASAGLALDKR (SEQ ID NO: 27) TFP 14 MQFKNALTATAILSASALAANSTTSIPSSCSIGTSATATAQATDSQAQATTTAPLAASASAGLALDKR (SEQ ID NO: 28) TFP 15 MVSKTWICGFISIITVVQALSCEKHDVLKKYQVGKFSSLTSTERDTPPSTTIEKWWINVCEEHNVEPPEECKKNDMLCGLTDVILPGKDAITTQIIDFDKNIGFNVEETESALTLTLNGATWGANSFDAKLEFQCNDNMKQDELAASASAGLALDKR (SEQ ID NO: 29) TFP 16 MKLSALLALSASTAVLAAPAVHHSDNHHHNDKRAVVTVTQYVNADGAVVIPAATTATSAAADGKVESVAAATTTLSSTAAAATTLAASASAGLALDKR (SEQ ID NO: 30) TFP 17 MKLSTVLLSAGLASTTLAQFSNSTSASSTDVTSSSSISTSSGSVTITSSEAPESDNGTSTAAPTETSTEAPTTAIPTNGTSTEAPTTAIPTNGTSTEAPTDTTTEAPTTALPTNGTSTEAPTDTTTEAPTTGLPTNGTTSAFPPTTSLPPSNTTTTPPYNPSTDYTTDYTVVTEYTTYCPERAASASAGLALDKR (SEQ ID NO: 31) TFP 18 MRFSTTLATAATALFFTASQVSAIGELAFNLGVKNNDGTCKSTSDYETELQALKSYTSTVKVYAASDCNTLQNLGPAAEAEGFTIFVGVWPLAASASAGLALDKR (SEQ ID NO: 32) TFP 19 MRLSNLIASASLLSAATLAAPANHEHKDKRAVVTTTVQKQTTIIVNGAASTPVAALEENAVVNSAPAAATSTTSSAASVATAASSSENNSQVSAAASPASSSAATSTQSSLAASASAGLALDKR (SEQ ID NO: 33) TFP 20 MQFSTVASIAAVAAVASAAANVTTATVSQESTTLVTITSCEDHVCSETVSPALVSTATVTVDDVITQYTTWCPLTTEAPKNGTSTAAPVTSTEAPKNTTSAAPTHSVTSYTGAAAKALPAAGALLAASASAGLALDKR (SEQ ID NO: 34) TFP 21 MKFSSALVLSAVAATALAESITTTITATKNGHVYTKTVTQDATFVWGGEDSYASSTSAAESSAAETSAAETSAAATTSAAATTSAAETSSAAETSSADEGSGSSITTTITATKNGHVYTKTVTQDATFVWTGEGSSNTWSPSSTSTSSEAATSSASTTATTLLAASASAGLALDKR (SEQ ID NO: 35) TFP 22 MKFQVVLSLSACACSAVVASPIENLFKYRAVKASHSKNINSTLPAWNGSNSSNVTYANGTNSTTNTTTAESSQLQIIVTGGQVPITNSSLTHTNYTRLFNSSSALNITELYNVARVVNETIQDNLAASASAGLALDKR (SEQ ID NO: 36) TFP 23 MRAITLLSSVVSLALLSKEVLATPPACLLACVAQVGKSSSTCDSLNQVTCYCEHENSAVKKCLDSICPNNDADAAYSAFKSSCSEQNASLGDSSGSASSSVLAASASAGLALDKR (SEQ ID NO: 37) TFP 24 MKLSTVLLSAGLASTTLAQFSNSTSASSTDVTSSSSSTSSSTVTTSSEAPESDNGTSTAAPTETSTEAPTTAIPTNGTSTEAPTTAIPTNGTSTEAPTDTTTEAPTTALPTNGTSTEAPTDTTTEAPTTGLPTNGTTSAFPPTTSLPPSNTTTTLAASASAGLALDKR (SEQ ID NO: 38)

The nucleotide sequence of the protein secretion fusion factor Protein secretion factor Nucleotide sequence TFP 1 ATGTTCAATCGTTTTAACAAATTCCAAGCTGCTGTCGCTTTGGCCCTACTCTCTCGCGGCGCTCTCGGTGACTCTTACACCAATAGCACCTCCTCCGCAGACTTGAGTTCTATCACTTCCGTCTCGTCAGCTAGTGCAAGTGCCACCGCTTCCGACTCACTTTCTTCCAGTGACGGTACCGTTTATTTGCCATCCACAACAATTAGCGGTGATCTCACAGTTACTGGTAAAGTAATTGCAACCGAGGCCGTGGAAGTCGCTGCCGGTGGTAAGTTGACTTTACTTGACGGTGAAAAATACGTCTTCTCATCTGAGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 39) TFP 2 ATGACGCCCTATGCAGTAGCAATTACCGTGGCCTTACTAATTGTAACAGTGAGCGCACTCCAGGTCAACAATTCATGTGTCGCTTTTCCGCCATCAAATCTCAGGGGCAAGAATGGAGACGGTACTAATGAACAGTATGCAACTGCACTACTTTCTATTCCCTGGAATGGGCCTCCTGAGTCATCGAGGGATATTAATCTTATCGAACTCGAACCGCAAGTTGCACTCTATTTGCTCGAAAATTATATTAACCATTACTACAACACCACAAGAGACAATAAGTGCCCTAATAACCACTACCTAATGGGAGGGCAGTTGGGTAGCTCATCGGATAATAGGAGTTTGAACGAGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 40) TFP 3 ATGCAATTCAAAAACGTCGCCCTAGCTGCCTCCGTTGCTGCTCTATCCGCCACTGCTTCTGCTGAAGGTTACACTCCAGGTGAACCATGGTCCACCTTAACCCCAACCGGCTCCATCTCTTGTGGTGCAGCCGAATACACTACCACCTTTGGTATTGCTGTTCAAGCTATTACCTCTTCAAAAGCTAAGAGAGACGTTATCTCTCAAATTGGTGACGGTCAAGTCCAAGCCACTTCTGCTGCTACTGCTCAAGCCACCGATAGTCAAGCCCAAGCTACTACTACCGCTACCCCAACCAGCTCCGAAAAGATGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 41) TFP 4 ATGAGATTTGCAGAATTCTTGGTGGTATTTGCCGTTAGGCGGGGGGATGGCTGCACCGGTTGAGTCTCTGGCCGGGACCCAACGGTATCTGGTGCAAATGAAGGAGCGGTTCACCACAGAGAAGCTGTGTGCTTTGGACGACAAGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 42) TFP 5 ATGTTCAATCGTTTTAACAAATTCCAAGCTGCTGTCGCTTTGGCCCTACTCTCTCGCGGCGCTCTCGGTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACTAATTCCGGCTGAAGCTGTCATCGGTTACTTAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 43) TFP 6 ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTTAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 44) TFP 7 ATGGTGTTCGGTCAGCTGTATGCCCTTTTCATCTTCACGTTATCATGTTGTATTTCCAAAACTGTGCAAGCAGATTCATCCAAGGAAAGCTCTTCCTTTATTTCGTTCGACAAAGAGAGTAACTGGGATACCATCAGCACTATATCTTCAACGGCAGATGTTATATCATCCGTTGACAGTGCTATCGCTGTTTTTGAATTTGACAATTTCTCATTATTGGACAGCTTGATGATTGACGAAGAATACCCATTCTTCAATAGATTCTTTGCCAATGATGTCAGTTTAACTGTTCATGACGATTCGCCTTTGAACATCTCTCAATCATTATCTCCCATTATGGAACAATTTACTGTGGATGAATTACCTGAAAGTGCCTCTGACTTACTATATGAATACTCCTTAGATGATAAAAGCATCGTTTTGTTCAAGTTTACCTCGGATGCCTACGATTTGAAAAAATTAGATGAATTTATTGATTCTTGCTTATCGTTTTTGGAAGATAAATCTGGCGACAATTTGACTGTGGTTATTAACTCTCTTGGTTGGGCTTTTGAAGATGAAGATGGTGACGATGAATATGCAACAGAAGAGACTTTGAGCCATCATGATAACAACAAGGGTAAAGAAGGCGACGATCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 45) TFP 8 ATGCTTCAATCCGTTGTCTTTTTCGCTCTTTTAACCTTCGCAAGTTCTGTGTCAGCGATTTATTCAAACAATACTGTTTCTACAACTACCACTTTAGCGCCCAGCTACTCCTTGGTGCCCCAAGAGACTACCATATCGTACGCCGACGACCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 46) TFP 9 ATGAAATTCTCAACTGCCGTTACTACGTTGATTAGTTCTGGTGCCATCGTGTCTGCTTTACCACACGTGGATGTTCACCAAGAAGATGCCCACCAACATAAGAGGGCCGTTGCGTACAAATACGTTTACGAAACTGTTGTTGTCGATTCTGATGGCCACACTGTAACTCCTGCTGCTTCAGAAGTCGCTACTGCTGCTACCTCTGCTATCATTACAACATCTGTGTTGGCTCCAACCTCCTCCGCAGCCGCTGCGGATAGCTCCGCTTCCATTGCTGTTTCATCTGCTGCCTTAGCCAAGAATGAGAAAATCTCTGATGCCGCTGCATCTGCCACTGCCTCAACATCTCAAGGGGCATCCTCCTCATCCTACCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 47) TFP 10 ATGAATTGGCTGTTTTTGGTCTCGCTGGTTTTCTTCTGCGGCGTGTCAACCCATCCTGCCCTGGCAATGTCCAGCAACAGACTACTAAAGCTGGCTAATAAATCTCCCAAGAAAATTATACCTCTGAAGGACTCAAGTTTTGAAAACATCTTGGCACCACCTCACGAAAATGCCTATATAGTTGCTCTGTTTACTGCCACAGCGCCCGAAATTGGCTGTTCTCTGTGTCTCGAGCTAGAATCCGAATACGACACCATAGTGGCCTCCTGGTTTGATGATCATCCGGATGCAAAATCGTCCAATTCCGATACATCTATTTTCTTCACAAAGGTCAATTTGGAGGACCCTTCTAAGACCATTCCTAAAGCGTTCCAGTTTTTCCAACTAAACAATGTTCCTAGATTGTTCATCTTCAAACTAAACTCTCCCTCTATTCTGGACCACAGCGTGATCAGTATTTCCACTGATACTGGCTCAGAAAGAATGAAGCAAATCATACAAGCCATTAAGCAGTTCTCGCAAGTAAACGACTTCTCTTTACACTTACCTGTGGGTCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 48) TFP 11 ATGAAGTTCTCTTCTGTTACTGCTATTACTCTAGCCACCGTTGCCACCGTTGCCACTGCTAAGAAGGGTGAACATGATTTCACTACCACTTTAACTTTGTCATCGGACGGTAGTTTAACTACTACCACCTCTACTCATACCACTCACAAGTATGGTAAGTTCAACAAGACTTCCAAGTCCAAGACCCCCCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 49) TFP 12 ATGGCCTCATTTGCTACTAAGTTTGTCATTGCTTGCTTCCTGTTCTTCTCGGCGTCCGCCCATAATGTCCTTCTTCCAGCTTATGGCCGTAGATGCTTCTTCGAAGACTTGAGTAAGGGTGACGAGCTCTCCATTTCGTTCCAGTTCGGTGATAGAAACCCTCAATCCAGTAGCCAGCTGACTGGTGACTTTATCATCTACGGGCCGGAAAGACATGAAGTTTTGAAAACGGTTAGGGAACTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 50) TFP 13 ATGCAATACAAAAAGACTTTGGTTGCCTCTGCTTTGGCCGCTACTACATTGGCCGCCTATGCTCCATCTGAGCCTTGGTCCACTTTGACTCCAACAGCCACTTACAGCGGTGGTGTTACCGACTACGCTTCCACCTTCGGTATTGCCGTTCAACCAATCTCCACTACATCCAGCGCATCATCTGCAGCCACCACAGCCTCATCTAAGGCCAAGAGAGCTGCTTCCCAAATTGGTGATGGTCAAGTCCAAGCTGCTACCACTACTGCTTCTGTCTCTACCAAGAGTACCGCTGCCGCCGTTTCTCAGATCGGTGATGGTCAAATCCAAGCTACTACCAAGACTACCGCTGCTGCTGTCTCTCAAATTGGTGATGGTCAAATTCAAGCTACCACCAAGACTACCTCTGCTAAGACTACCGCCGCTGCCGTTTCTCAAATCAGTGATGGTCAAATCCAAGCTACCACCACTACTTTAGCCCCTCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 51) TFP 14 ATGCAATTCAAGAACGCTTTGACTGCTACTGCTATTCTAAGTGCCTCCGCTCTAGCTGCTAACTCAACTACTTCTATTCCATCTTCATGTAGTATTGGTACTTCTGCCACTGCTACTGCTCAAGCCACCGATAGTCAAGCCCAAGCTACTACTACCGCACCCCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 52) TFP 15 ATGGTATCGAAGACTTGGATATGTGGCTTCATCAGTATAATTACAGTGGTACAGGCCTTGTCCTGCGAGAAGCATGATGTATTGAAAAAGTATCAGGTGGGAAAATTTAGCTCACTAACTTCTACGGAAAGGGATACTCCGCCAAGCACAACTATTGAAAAGTGGTGGATAAACGTTTGCGAAGAGCATAACGTAGAACCTCCTGAAGAATGTAAAAAAAATGACATGCTATGTGGTTTAACAGATGTCATCTTGCCCGGTAAGGATGCTATCACCACTCAAATTATAGATTTTGACAAAAACATTGGCTTCAATGTCGAGGAAACTGAGAGTGCGCTTACATTGACACTAAACGGCGCTACGTGGGGCGCCAATTCTTTTGACGCAAAACTAGAATTTCAGTGTAATGACAATATGAAACAAGACGAACTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 53) TFP 16 ATGAAATTATCCGCTCTATTAGCTTTATCAGCCTCCACCGCCGTCTTGGCCGCTCCAGCTGTCCACCATAGTGACAACCACCACCACAACGACAAGCGTGCCGTTGTCACCGTTACTCAGTACGTCAACGCAGACGGCGCTGTTGTTATTCCAGCTGCCACCACCGCTACCTCGGCGGCTGCTGATGGAAAGGTCGAGTCTGTTGCTGCTGCCACCACTACTTTGTCCTCGACTGCCGCCGCCGCTACAACCCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 54) TFP 17 ATGAAATTATCAACTGTCCTATTATCTGCCGGTTTAGCCTCGACTACTTTGGCCCAATTTTCCAACAGTACATCTGCTTCTTCCACCGATGTCACTTCCTCCTCTTCCATCTCCACTTCCTCTGGCTCAGTAACTATCACATCTTCTGAAGCTCCAGAATCCGACAACGGTACCAGCACAGCTGCACCAACTGAAACCTCAACAGAGGCTCCAACCACTGCTATCCCAACTAACGGTACCTCTACTGAAGCTCCAACCACTGCTATCCCAACTAACGGTACCTCTACTGAAGCTCCAACTGATACTACTACTGAAGCTCCAACCACCGCTCTTCCAACTAACGGTACTTCTACTGAAGCTCCAACTGATACTACTACTGAAGCTCCAACCACCGGTCTTCCAACCAACGGTACCACTTCAGCTTTCCCACCAACTACATCTTTGCCACCAAGCAACACTACCACCACTCCTCCTTACAACCCATCTACTGACTACACCACTGACTACACTGTAGTCACTGAATATACTACTTACTGTCCGGAACGGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 55) TFP 18 ATGCGTTTCTCTACTACACTCGCTACTGCAGCTACTGCGCTATTTTTCACAGCCTCCCAAGTTTCAGCTATTGGTGAACTAGCCTTTAACTTGGGTGTCAAGAACAACGATGGTACTTGTAAGTCCACTTCCGACTATGAAACCGAATTACAAGCTTTGAAGAGCTACACTTCCACCGTCAAAGTTTACGCTGCCTCAGATTGTAACACTTTGCAAAACTTAGGTCCTGCTGCTGAAGCTGAGGGATTTACTATCTTTGTCGGTGTTTGGCCACTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 56) TFP 19 ATGCGTCTCTCTAACCTAATTGCTTCTGCCTCTCTTTTATCTGCTGCTACTCTTGCTGCTCCCGCTAACCACGAACACAAGGACAAGCGTGCTGTGGTCACTACCACTGTTCAAAAACAAACCACTATCATTGTTAATGGTGCCGCTTCAACTCCAGTTGCTGCTTTGGAAGAAAATGCTGTTGTCAACTCCGCTCCAGCTGCCGCTACCAGTACAACATCGTCTGCTGCTTCTGTAGCTACCGCTGCTTCCTCTTCTGAGAACAACTCACAAGTTTCTGCTGCCGCATCTCCAGCCTCCAGCTCTGCTGCTACATCTACTCAATCTTCTCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 57) TFP 20 ATGCAATTTTCTACTGTCGCTTCTATCGCCGCTGTCGCCGCTGTCGCTTCTGCCGCTGCTAACGTTACCACTGCTACTGTCAGCCAAGAATCTACCACTTTGGTCACCATCACTTCTTGTGAAGACCACGTCTGTTCTGAAACTGTCTCCCCAGCTTTGGTTTCCACCGCTACCGTCACCGTCGATGACGTTATCACTCAATACACCACCTGGTGCCCATTGACCACTGAAGCCCCAAAGAACGGTACTTCTACTGCTGCTCCAGTTACCTCTACTGAAGCTCCAAAGAACACCACCTCTGCTGCTCCAACTCACTCTGTCACCTCTTACACTGGTGCTGCTGCTAAGGCTTTGCCAGCTGCTGGTGCTTTGCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 58) TFP 21 ATGAAATTCTCTTCCGCTTTGGTTCTATCTGCTGTTGCCGCTACTGCTCTTGCTGAGAGTATCACCACCACCATCACTGCCACCAAGAACGGTCATGTCTACACTAAGACTGTCACCCAAGATGCTACTTTTGTTTGGGGTGGTGAAGACTCTTACGCCAGCAGCACTTCTGCCGCTGAATCTTCTGCCGCCGAAACTTCTGCCGCCGAAACCTCTGCTGCCGCTACCACTTCTGCTGCCGCTACCACTTCTGCTGCTGAGACTTCTTCTGCTGCTGAGACTTCTTCTGCTGATGAAGGTTCTGGTTCTAGTATCACTACCACTATCACTGCCACCAAGAACGGTCACGTCTACACTAAGACTGTCACCCAAGATGCTACTTTTGTCTGGACTGGTGAAGGCAGCAGCAACACCTGGTCTCCAAGTAGTACCTCTACCAGCTCAGAAGCTGCTACCTCTTCTGCTTCAACCACTGCAACCACCCTGCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 59) TFP 22 ATGAAGTTCCAAGTTGTTTTATCTGCCCTTTTGGCATGTTCATCTGCCGTCGTCGCAAGCCCAATCGAAAACCTATTCAAATACAGGGCAGTTAAGGCATCTCACAGTAAGAATATCAACTCCACTTTGCCGGCCTGGAATGGGTCTAACTCTAGCAATGTTACCTACGCTAATGGAACAAACAGTACTACCAATACTACTACTGCCGAAAGCAGTCAATTACAAATCATTGTAACAGGTGGTCAAGTACCAATCACCAACAGTTCTTTGACCCACACAAACTACACCAGATTATTCAACAGTTCTTCTGCTTTGAACATTACCGAATTGTACAATGTTGCCCGTGTTGTTAACGAAACGATCCAAGATAACCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 60) TFP 23 ATGCGTGCCATCACTTTATTATCTTCAGTCGTTTCTTTGGCATTGTTGTCGAAGGAAGTCTTAGCAACACCTCCAGCTTGTTTATTGGCCTGTGTTGCGCAAGTCGGCAAATCCTCTTCCACATGTGACTCTTTGAATCAAGTCACCTGTTACTGTGAACACGAAAACTCCGCCGTCAAGAAATGTCTAGACTCCATCTGCCCAAACAATGACGCTGATGCTGCTTATTCTGCTTTCAAGAGTTCTTGTTCCGAACAAAATGCTTCATTGGGCGATTCCAGCGGCAGTGCCTCCTCATCCGTTCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 61) TFP 24 ATGAAATTATCAACTGTCCTATTATCTGCCGGTTTGGCCTCGACTACTTTGGCCCAATTTTCCAACAGTACATCTGCTTCTTCCACCGATGTCACTTCCTCCTCTTCCATCTCCACTTCCTCTGGCTCAGTAACTATCACATCTTCTGAAGCTCCAGAATCCGACAACGGTACCAGCACAGCTGCACCAACTGAAACCTCAACAGAGGCTCCAACCACTGCTATCCCAACTAACGGTACCTCTACTGAAGCTCCAACCACTGCTATCCCAACTAACGGTACCTCTACTGAAGCTCCAACTGATACTACTACTGAAGCTCCAACCACCGCTCTTCCAACTAACGGTACTTCTACTGAAGCTCCAACTGATACTACTACTGAAGCTCCAACCACCGGTCTTCCAACCAACGGTACCACTTCAGCTTTCCCACCAACTACATCTTTGCCACCAAGCAACACTACCACCACTCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 62)

Twenty-four transformants were cultured in YPDG medium (1% yeast extract, 2% peptone, 1% glucose, 1% galactose) for 40 hours and then centrifuged to remove the cells. 0.6 ml of the culture supernatant was suspended in 0.4 ml of acetone SDS-PAGE analysis was performed (FIG. 2), and secretion of lsrA was confirmed by Western blotting using an anti-His antibody (FIG. 3).

As a result, Levanshukraze is known to be a protein that is difficult to secrete in yeast. However, when a protein secretion fusion factor is used, most of 24 protein secretion fusion factors can secrete lsrA as shown in Fig. Respectively. Among these, TFPs of four species (TFP 3, TFP 4, TFP 8, TFP 16) which are relatively excellent in the expression of levansucrase were selected.

Since the strains selected by the above method were obtained by analyzing a mixture of transformants rather than a single colony, the strain was cultured from a single colony for accurate analysis and the protein synthesis was analyzed. To obtain a single strain from each strain, three single colonies were selected for each TFP and cultured in the same manner. The obtained culture medium was subjected to SDS-PAGE analysis in the same manner (Fig. 4), and the anti-His antibody To perform Western blotting (FIG. 5). In order to compare the secretion of levansucrase from each TFP, the secretion signal of yeast was compared with the secretion expression of levanshucrase using the MF secretion signal most widely used as a secretion signal in yeast.

As a result of the confirmation, it was confirmed that the selected TFP secreted Levansucrase more effectively than the MF secretion signal, and it was confirmed that TFP 16 could secrete levansucrase most efficiently.

Based on the above results, YGaST16-lsrA-6H containing TFP16 and levan's aquaticis-derived levanshuclaca gene was prepared as an expression vector which secreted and produced Raban's aquaticus-derived levanshucrase in high yield (Fig. 16). The strain transformed with the above-mentioned expression vector was deposited on June 1, 2015 in the Korean Collection for Type Culture (KCTC), an international depository institution under the Budapest Treaty, and deposited with Accession No. KCTC18393P .

Example 2. Zymomonas mobilis ( Zymomonas mobilis Selection of Yeast Protein Secretion Fusion Factor (TFP) for Expression of Levansucrase Derived

Zymomonas mobilis is widely used as a strain producing Levan, and Levanschraze derived from the strain is known to be highly active at low temperatures because it can be recombinantly produced in Escherichia coli.

(Song, et al., Biochim Biophys Acta. 1173, 320 (1993)) as a template and SEQ ID NO: 7 and SEQ ID NO: 6 as a template to produce a secretion expression vector for levum shukla gene (levU, SEQ ID NO: 6) derived from Zymomonas mobilis. The levU gene was amplified by the first PCR using primer No. 8 (94 ° C for 5 minutes; 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute, 25 cycles; 72 ° C for 7 minutes) . The gene amplified by the first PCR was secondly amplified with a primer of LNK39 (SEQ ID NO: 4) and HisGT50R (SEQ ID NO: 5), and the sequence containing the protein secretion factor was introduced into the vector for in vivo recombination And a histidine tag-introduced recombinant gene was obtained at the carboxy terminus.

Then, the levU gene amplified using the same method as in Example 1 was transformed with a linear vector containing 24 protein secretion fusion factors (Korean Patent No. 0975596) Y2805GS. SDS-PAGE analysis was performed (FIG. 6) and Western blot was performed using anti-His antibody (FIG. 7) to confirm secretion of levU. As shown in FIG. 7, it was confirmed that most of 24 protein secretion fusion factors can secrete levu.

Next, in order to compare the activity of secreted enzymes, the amount of glucose produced when 0.2 ml of 4% sugar solution was reacted with 5 L of the culture medium of each strain was measured by DNS (FIG. 8). The enzyme activity and the Western blot results corresponded to each other. Eight species (TFP 3, TFP 6, TFP 8, TFP 9, TFP 11, TFP 16, TFP 19, and TFP 22) exhibiting relatively high levansucrase secretion- Of TFP were selected.

Then, 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 obtained culture medium was subjected to SDS-PAGE analysis (FIG. 9) Western blot was performed using the His antibody (Fig. 10), and the enzyme activities were compared (Fig. 11). As a result, it was confirmed that TFP 8 can secrete levansucrase most efficiently.

Based on the above results, YGaST8-levU-6H containing TFP8 and levanshuclaze gene derived from Zymomonas mobilis was produced as an expression vector which secretion-produced Levanshukraze from Zymomonas mobilis finally (Fig. 17), and it was confirmed that Levanschracia was successfully secreted in the strain transformed with the expression vector

Example 3. Bacillus subtilis ( Bacillus subtilis Selection of Yeast Protein Secretion Fusion Factor (TFP) for Expression of Levansucrase Derived

(BsSacB, SEQ ID NO: 9) secretion expression vector of Bacillus subtilis derived from Gram-positive bacteria, Bacillus subtilis genomic DNA was used as a template and primers of SEQ ID NO: 10 and SEQ ID NO: 11 were used to prepare 1 The BsSacB gene was amplified by PCR (94 ° C for 5 minutes; 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute, 25 cycles, 72 ° C for 7 minutes). The gene amplified by the first PCR was secondly amplified again with primers LNK39 (SEQ ID NO: 4) and HisGT50R (SEQ ID NO: 5), and the sequence containing the protein secretion factor and the sequence necessary for in vivo recombination And a recombinant gene in which a histidine tag was introduced at the carboxy terminal was obtained.

Then, the BsSacB gene amplified using the same method as in Example 1 was introduced into yeast strain Y2805GS along with a linear vector containing 24 protein secretion fusion factors, and secretion of the protein through SDS-PAGE (Fig. 12).

Next, in order to compare enzyme activities, the amount of glucose produced when 0.2 ml of 4% sugar solution was reacted with 5 L of the culture medium of each strain was measured by DNS (FIG. 13). Among these, TFP of eight species (TFP 1, TFP 5, TFP 6, TFP 8, TFP 9, TFP 13, TFP 16, and TFP 19) which are relatively excellent in the efficacy of levansucrase secretion were selected. 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 obtained culture medium was subjected to SDS-PAGE analysis in the same manner (Fig. 14) (Fig. 15).

As a result, it was confirmed that TFP 8 can secrete levansucrase most efficiently.

Based on the above results, YGaST8-BsSacB-6H (see FIG. 18 (a)) containing TFP8 and a Levanshukrage gene derived from Bacillus subtilis was used as an expression vector for high yield production of levanshucrase derived from Bacillus subtilis ), And the strain transformed with the expression vector was deposited on June 1, 2015 in the Korean Collection for Type Culture (KCTC), an international depository institution under the Budapest Treaty, and deposited under accession number KCTC18392P .

Example 4 Fermentation Production of Levanshucrazas by Fed-Batch Fermentation of Recombinant Yeast

Example 4-1. Fermentation production of levanshucrazas derived from Ranella aquaticis

The recombinant yeast Y2805GS containing YGaST16-lsrA-6H as described above was cultivated in a 5 L fermenter to mass-produce the levan's aquaculture-derived levanshucrase.

Specifically, the cells were firstly cultured in 50 ml of SD-Ura medium (0.67% amino acid-deficient yeast substrate, 0.77% uracil deficient nutritional supplement, 2% glucose) before entering the present culture, and then 200 ml of YEPD liquid medium (2% glucose, 4% yeast extract, 1% peptone) and then 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 while gradually adding 20 g / L per hour from 2 g / L according to the growth of the cells. The degree of cell growth during culturing was determined by taking the samples in time and measuring the absorbance at a wavelength of 600 nm (Fig. 19). The culture supernatant (20 쨉 l), which had not been concentrated, was analyzed by SDS-PAGE (Fig. 20). To confirm the presence of Levanschracase in the cells without being secreted into the culture supernatant, the cells in 1 ml of the culture were centrifuged, and 500 μl of a lysis buffer (20 mM sodium phosphate (pH 7.4), 100 mM NaCl , 2 mM EDTA, 5% SDS), 0.5 mm glass beads were added thereto, stirred for 10 minutes, centrifuged, and the supernatant recovered was subjected to SDS-PAGE analysis (Fig. 22). And the change of enzyme activity with respect to the secretion amount of the enzyme inside and outside of the cell growth time was confirmed (FIG. 21).

As a result, some levanshucracases were confirmed in the cells, but most of the levanshcracases were secreted outside the cells, and the enzyme activity of the cell culture medium was confirmed to be about twice as high as the intracellular activity.

As can be seen from the SDS-PAGE results (FIG. 22), most of the proteins contained in the fermentation broth are recombinant Levanschraclase. Therefore, it is considered that a high purity enzyme concentrate can be produced by simple purification or simple concentration.

Example 4-2. Zai Momonas Mobilis Fermentation production of Levanschraze derived

In order to mass-produce levanshcrazine derived from Zymomonas mobilis The above recombinant yeast Y2805GS containing YGaST8-levU-6H was fed-fed using a 5 L fermenter.

Specifically, the cells were firstly cultured in 50 ml of SD-Ura medium (0.67% amino acid-deficient yeast substrate, 0.77% uracil deficient nutritional supplement, 2% glucose) before entering the present culture, and then 200 ml of YEPD liquid medium (2% glucose, 4% yeast extract, 1% peptone) and then 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 while gradually adding 20 g / L per hour from 2 g / L according to the growth of the cells. Levanschracase secreted in the medium was confirmed by performing 10 μl of the non-concentrated culture supernatant by SDS-PAGE and Western blot analysis using an anti-His antibody (FIG. 23).

As a result, the amount of secreted protein did not reach Levanschlajera derived from Ranella aquaticus, but the protein occupying most of the secreted protein was identified as Levanschrajera derived from Zymomonas mobilis

Example 4-3. Fermentation production of levanshucrazas derived from Bacillus subtilis

To mass-produce the Bacillus subtilis-derived Levanschlaja, recombinant yeast Y2805GS containing YGaST8-BsSacB-6H was fed-fed using a 5 L fermenter.

Specifically, the cells were firstly cultured in 50 ml of SD-Ura medium (0.67% amino acid-deficient yeast substrate, 0.77% uracil deficient nutritional supplement, 2% glucose) before entering the present culture, and then 200 ml of YEPD liquid medium (2% glucose, 4% yeast extract, 1% peptone) and then 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 while gradually adding 20 g / L of 2 g / L per hour depending on the growth of the cells. The degree of cell growth during culturing was determined by measuring the absorbance at a wavelength of 600 nm by taking a sample with time, and 20 μl of the culture supernatant, which had not been concentrated, was analyzed by Western blot analysis using an anti-His antibody Respectively. Although the cell growth of the recombinant yeast proceeded smoothly to a level of 150 or more at an absorbance of 600 nm (Fig. 24), the amount of the protein thus expressed was lower than that of the expression of Levanschraja derived from Gram- (Fig. 25).

Example 5. Purification of levansucrase using affinity chromatography method

The Levanschraclase expressed in the present invention is 6X histidine tagged at the carboxyl end, and purification can be performed by an affinity chromatography method using Ni-NTA resin.

In order to purify the levanshucrase secreted by the fermentation medium, the fermentation medium was concentrated 10 times with 30,000 MWCO Quick-stand (GE healthcare) after first purification using a filter (Sartoclear, Sartorious). Then, 100 ml of the fermentation concentrate was applied to the HisPrep FF column, and the fusion protein was bound to the Ni-NTA resin. Then, 50 mM Tris-HCl (pH 7.5), 0.5 The protein was eluted with M NaCl, 0.25 M imidazole solution. The final purified fractions were desalted using a Hiprep Desalting column, followed by SDS-PAGE at 2 and analyzed for enzyme activity.

As can be seen from the results of SDS-PAGE, a small protein band was observed in all levanshucrachea fractions (FIG. 26), and it was confirmed that most of the proteins were present in an intact size.

Example 6. Analysis of the optimal conditions for the activity of levanshukraze of purified two species (lsrA, levU)

Example 6-1. Determination of optimal pH

In order to confirm the optimal pH of the purified levanshakraze (lsrA, levU), 50 mM of buffer at various pH was used (Table 3) and 1 g / L of purified enzyme solution Were reacted at 37 占 폚 for 30 minutes. After 30 minutes, the reaction was heated at 65 ° C for 15 minutes in order to stop the enzyme reaction. Then, HPLC (1100 series) analysis by Agilent was carried out to confirm the amounts of glucose, fructose and levan produced from the sugar. The analysis was carried out using a Shodex KS-802 (8 x 300 mm) column at flow rates of 0.5 ml / min at 50 < 0 > C. The solvent was HPLC solution and confirmed by Refractive Index Detector. HPLC results were calculated by quantitative calculation according to standard curve.

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

A comparison of the levans production of recombinase at various pH ranges confirmed that only significant lamb production was found in lsrA among the two enzymes (Figure 27). The best conditions for the synthesis of Levan in a wide range of pH were confirmed using pH 5.0 acetate buffer and pH 6.0 maleate buffer.

Example 6-2. Determine the Optimum Temperature

To determine the optimum temperature of levanshucrazide in the purified two species (lsrA, levU), 1 g / L of purified enzyme solution in 1% sugar substrate solution and 50 mM malate buffer was added to a solution of 4 , 20, 30, 37, 50 and 60 ° C) for 30 minutes. After 30 minutes, the reaction was heated at 65 ° C for 15 minutes in order to stop the enzyme reaction, and HPLC analysis was performed with Agilent's 1100 series to check the amount of glucose, fructose, and levan produced from the sugar. The analysis was carried out using a Shodex KS-802 (8 x 300 mm) column at flow rates of 0.5 ml / min at 50 < 0 > C. The solvent was HPLC solution and confirmed by Refractive Index Detector. HPLC results were calculated by quantitative calculation according to standard curve.

The amount of levan produced in a wide range of temperatures was calculated by comparing with the standard curve, and it was confirmed that only significant amount of Levan was produced in lsrA among the two enzymes (Fig. 28). Specifically, the optimum temperature for the digestion of lsrA was 50 ° C, but the optimum temperature for the production of levan was 30-37 ° C.

From the above results, it was found that lsrA had superior levan synthesizing activity to other enzymes at all temperatures, and it was confirmed that lsrA had optimal activity at 30 to 37 ° C.

Example 6-3. Comparison of Levan production ability of Levanschraze of two purified (lsrA, levU)

Enzyme activities were compared with the Levans conversion of the two kinds of Levanschracca purified first by Hisprep column.

Specifically, 1 ml of 5% sucrose (50 mM sodium phosphate buffer (pH 6.0)) was treated with 1 의 of enzyme and reacted at 4 캜 (Table 4) and 20 캜 (Table 5) for 24 hours and shodex KS-802 Column chromatography was used to compare the changes in sugar content.

As a result, as shown in Table 4 and Table 5, it was confirmed that Levanschraze expressed in yeast forms Levans of two sizes.

Comparison of levan production (4 ° C) Levanshkraje Polymer Levan Low-molecular Levan Sugar glucose fruit sugar Lunella Aquaticus
(lsrA) 10.5 4.2 13 18.4 6.3 Zymomonas mobilis (levU) - 1.5 32.9 9.2 7.7

(Unit: g / L)

Comparison of levan production (20 ° C) Levanshkraje Polymer Levan Low-molecular Levan Sugar glucose fruit sugar Lunella Aquaticus
(lsrA) 13.7 5.8 3.9 22.4 10.3 Zymomonas mobilis (levU) 1.1 1.3 20.7 13.8 15.8

(Unit: g / L)

Levansucrase is known to have an activity of levan-producing enzyme and a sugar-degrading enzyme. Due to this activity, a certain amount of fructose is formed during the reaction. Since fructose produced from sugar degradation is not used as a substrate for levan synthesis, enzymes that degrade sugar at low efficiency with high efficiency of levan sum performance are desirable to improve Levan productivity. The amount of glucose accumulated in the sugar was proportional to the amount of the produced levan because glucose produced in the sugar accumulates without being converted into levan. Lanella Aquatiles The resulting Levans sucrase was found to be the most potent at both temperatures and was more active at 20 ° C than at 4 ° C. It was also confirmed that the sugar resolution was lowest at the two temperatures.

Example 6-4. Comparison of Levan production of yeast recombinant Levanshucraze and E. coli recombinant levanshucrazas

In order to compare the activity of a Levanshukraze enzyme derived from Ranella aquacillus obtained from Escherichia coli expression and a Levanshukrasae fermentation concentrate derived from Ranella aquacillus produced in yeast, HPLC analysis was performed to determine the activity As a result of comparing the contents of the converted levan, the activity was shown to be equivalent (conversion rate of 21.8%) as shown in Table 6 below.

Enzyme type content(%) Reaction time (h) Levan Sugar glucose fruit sugar Existing enzymes (E. coli)
(295.33 U / ml) 21.5 9.1 49.1 11.3 38 Yeast Enzyme / Enrichment
(505.49 U / ml) 21.8 2.6 51.4 18.4 65

Therefore, it was confirmed that levanshucrazide secreted from yeast could be applied to the Levan production process only by the concentration of the fermentation broth without purification process, and this characteristic will contribute to the reduction of the production cost of the enzyme in the mass production of the enzyme.

Example 6-5. Confirmation of Levan production yield according to substrate concentration and enzyme concentration

The optimum conditions of the substrate sugar were established at the optimum buffer concentration of 10 mM sodium acetate buffer (pH 5.5) and the reaction temperature of 30 ℃. Experiments were carried out at 10%, 15%, and 20% sugar concentrations, and the enzyme concentration was increased to 30 U or 40 U with increasing sugar concentration. As shown in Table 7 below, when the concentration of the sugar was increased and the amount of the enzyme was increased, it could not induce a higher efficiency improvement. Therefore, the optimum sugar concentration was established at 10%.

Levan content (%) Sugar concentration (%) / Enzyme concentration (U) Reaction time (h) 10% / 20U 15% / 20U 15% / 30U 20% / 20U 20% / 30U 20% / 40U 20 22 19.1 20.6 16.6 17.8 18.5 40 22.5 20.6 20.9 18.6 18.3 18.4 50 23.9 20.6 20.5 18 17.9 18.5

Next, we attempted to establish optimal enzyme concentrations at 10 mM sodium acetate buffer (pH 5.5), 30 ° C reaction temperature, and 10% sugar concentration, which is the established optimal condition. Since it was not effective to increase the enzyme concentration in the previous experiment, the yield of Levan conversion at an enzyme concentration of 30 U or less was confirmed.

As a result, when the enzymes of 15, 20, and 30 U were used, there was slight difference in the conversion rate in the initial 20 hours, but no significant difference in the conversion rate of Levan after 40 hours (Table 8). Therefore, when the unit price of production is taken into consideration, 15 U is established as an optimal condition.

Levan content (%) Enzyme concentration (U) Reaction time (h) 2.5 5 10 15 20 30 20 6.4 10.9 16.1 20.9 22.6 23.9 40 9 15.7 23.6 23 27 26.2 50 9.8 17 23.3 24.4 23.9 23.8 70 13.7 19.8 24.6 24.2 23.9 23.6

Example 7: Verification of activity of Levanschraze derived from fermented Bacillus subtilis

Through the above examples, it was confirmed that Levan sucralose derived from Lanella aquaticus can express high secretion in yeast and possesses high levan production ability. However, as confirmed in Example 6-3, fructose corresponding to 50% of the produced levan concentration was generated. This sugar degrading ability is a reason for lowering the efficiency of levan biosynthesis.

Therefore, the secretory form of Levanschuracil derived from Bacillus subtilis, which is a thermophilic Gram-positive bacterium, has different properties from the non-dividing levanshucracase derived from Gram-negative bacteria as confirmed in the above Examples. In order to compare the activity with the levan's aquacillus-derived levanshucrase fermented in Example 5, 50 μl of the fermentation supernatant was reacted at 30 ° C. for 48 hours in 5 ml of a 5% sugar solution. The samples were collected every 12 hours after the reaction and incubated at 90 ° C for 10 minutes to stop the enzymatic reaction. Then, using the Shodex KS-802 column, the sugar consumed by the RID analysis at 65 ° C and 1 ml / Quantitative analysis of the produced Levan, glucose and fructose was carried out. Mobile phase HPLC grade water was used.

As a result, it was found that when the reaction was continued for 48 hours, the amount of levan synthesized by levanshucrase (BsSacB) derived from Bacillus subtilis was similar to that of lanraacquartile derived lansacurase (lsrA) It was confirmed that levan was synthesized (Table 9). It was also confirmed that the amount of glucose and fructose present in the reaction product was less than lsrA. Therefore, Levan production rate is far superior to Levan's aquaticus-derived Levanshukrabas, but levansucrase derived from Bacillus subtilis has a low ability to decompose sugar, and as a whole, the production efficiency of Levanshucrajan levan derived from bacillus subtilis is high .

sample Sugar (g / L) Glucose (g / L) Fructose (g / L) Levan (g / L) Reaction time (h) lsrA 1.63 27.3 23.7 1.0 12 BsSacB 16.7 17.0 7.1 1.2 48

Therefore, two kinds of recombinant yeast expressing lsrA having the best initial reaction rate and BsSacB having the best reaction efficiency were finally selected as recombinant yeast for producing Levan.

Example 8. Direct production of Levan by recombinant yeast fermentation

When levan is directly produced from sugar without enzyme purification using microbial fermentation, low activity of levanshucrase and many fermentation by-products are produced, so no industrial application has been reported yet. However, unlike other microorganisms, yeast strain producing levanshucrase is fermented directly in the medium containing sugar to produce no levigirage by-products, and the amount of fructose, glucose or sugar produced by the action of levanshucrazas Since yeast strain can be used as a carbon source, Levans can be produced with high purity.

Expression of lanella aquaticus and bacillus subtilis-derived levanshucrase as described above The recombinant yeast Y2805GS was used to confirm whether Levans could be produced by direct fermentation. Two types of recombinant yeast (YGaST16-lsrA-6H / Y2805GS, YGaST8-BsSacB-6H / Y2805GS) that secrete and express levansucrase were cultured in YP medium supplemented with 5% sugar for 72 hours, As a result of HPLC analysis (shodex sugar KS-802 column), recombinant yeast secreted and expressed Levan sucralase derived from Ranella aquaticus showed cell growth more favorably, resulting in synthesis of 7.0 g / On the other hand, it was confirmed that the recombinant yeast secreted and expressed Bacillus subtilis-derived levanshucrase was relatively low in cell growth but formed a levan of 10.4 g / L. (Table 10). In contrast, the reaction rate of levan synthesis was faster than that of recombinant yeast expressing Bacillus subtilis-derived levanshukerase in the early stage, while recombinant yeast secreted and expressing levanchuracil- And the rate of recombinant yeast secreted from Bacillus subtilis - derived levanshucrase was continuously increased up to 72 hours.

YGaST16-lsrA-6H / Y2805GS YGaST8-BsSacB-6H / Y2805GS Cell growth rate (OD600) 31.0 22.5 Levan (g / L) 7.0 10.4

Example  9. Using molasses Levan  Direct production

Molasses is a by-product of mucus produced in the production of sugar, and is used not only in food industry such as confectionery coloring but also as a carbon source for fermentation of microorganisms such as alcohol, acetone, butanol, and citric acid. As a result of analyzing the components of molasses, 40% sugar, 14% fructose and 10% of glucose were contained, and it was considered that it could be used as a substrate for production of Levan. Thus, using the levan-produced recombinant yeast obtained in the above- To confirm the availability of Levan directly.

To verify this, the YPM medium (1% yeast extract, 2% peptone, 10% diluted molasses stock solution (containing 4% sugar)) containing carbon source in molasses (Daemyung Chemical, Korea) The culture supernatant was analyzed by HPLC (shodex sugar KS-802 column) after culturing the recombinant yeast strains (YGaST16-lsrA-6H / Y2805GS, YGaST8-BsSacB-6H / Y2805GS) for 72 hours.

As a result, it was confirmed that levan was produced in both recombinant yeasts as in the case of using sugar as a substrate. Recombinant yeast expressing lanabula-aquaticis-derived levanshucrase produced 3.7 g / L of levan after 72 hours and recombinant yeast expressing bacillus subtilis-derived levanshucrase 72 hours after culture It was confirmed that levans of 13.8 g / L were produced. Levan conversion was 34.5% (the theoretical maximum conversion rate was 50%) considering that 4% of the sugar contained in the 10-fold diluted molasses was included, and levan was produced at a higher efficiency than the case of using the sugar directly (Table 11). Since the price of molasses sold in the market today is generally one-half of sugar, the technology to produce levans from molasses at high efficiency has the advantage of significantly reducing Levan's production costs.

YGaST16-lsrA-6H / Y2805GS YGaST8-BsSacB-6H / Y2805GS Cell growth rate (OD600) 38 36 Levan (g / L) 3.7 13.8

In conclusion, in the present invention, levanshucrase is produced from recombinant yeast (YGaST16-lsrA-6H / Y2805GS, YGaST8-BsSacB-6H / Y2805GS) which secretes and expresses Lanella aquaticus and Bacillus subtilis-derived levanshukraze And it was confirmed that Levan could be produced through the separation and purification of Levanschraze, and that the recombinant yeast could be directly fermented to produce Levan.

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 KCTC18392P 20150601 Korea Research Institute of Bioscience and Biotechnology KCTC18393P 20150601

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> A microorganism having enhanced levansucrase productivity and a          method of producing levan using the microorganism <130> KPA150364-KR <160> 62 <170> Kopatentin 2.0 <210> 1 <211> 1248 <212> DNA <213> Rhanella aquatilis-lsrA <400> 1 atgacaaatt taaattatac accgacaatc tggacacgtg ctgatgctct gaaggtcaac 60 gaaaacgacc cgaccaccac ccagcctatt gttgacgccg attttccggt tatgagtgat 120 gaagtattta tttgggatac tatgcccctg cggtcattag acggtacggt ggtttctgta 180 gacggatggt ccgttatttt cacgcttacc gctcagcgta ataacaataa ttcagagtat 240 ctcgacgcag aaggaaatta tgacatcacc agtgactgga ataatcgcca cgggcgcgca 300 agaatttgtt actggtattc ccgtacgggt aaagattgga tttttggcgg gcgtgtgatg 360 gcagaaggtg tctcccctac atcccgcgaa tgggcaggga ctcccatttt gcttaatgaa 420 gacggtgata ttgatcttta ttatacctgc gtcacgccgg gagcgactat tgcaaaagtg 480 agaggcaaag tgctgacctc agaagaaggt gtaacgctgg ccggtttcaa cgaggttaaa 540 tcattatttt cagctgacgg cgtgtactac caaactgaaa gtcaaaatcc gtactggaac 600 ttcagggatc caagcccgtt tatcgatcct catgatggca agctttacat ggtatttgaa 660 ggaaatgtgg cgggtgaacg gggttctcac gtcattggca aacaggaaat gggtaccctg 720 cccccgggcc atcgtgatgt gggcaatgcg agataccagg cgggttgtat tggtatggcc 780 gtcgccaaag atctttcagg agacgaatgg gaaatcctcc cacctctggt cacggctgtc 840 ggtgtgaatg atcaaaccga acgcccgcat tttgtcttcc aggatggaaa atattactta 900 ttcactatca gtcataaatt tacttatgca gatggtctga caggtcctga cggtgtttac 960 ggcttcctga gtgataacct taccggcccg tattctccga tgaacggctc cggtttggtg 1020 ttaggcaatc cgccttctca gcccttccag acctactcac attgcgtcat gcccaatggc 1080 ctggtgacat cctttattga taacgttccg acaagcgacg gtaattatcg tattggtggg 1140 acagaagctc ctaccgtaaa aattgtcctt aagggaaacc gttcctttgt agagcgcgtg 1200 tttgattacg ggtatatccc gccgatgaaa aacattattt taaattaa 1248 <210> 2 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> lsRA primer-1 <400> 2 ctcgccttag ataaaagaat gacaaattta aattatac 38 <210> 3 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> lsRA primer-2 <400> 3 gtgatggtga tggtgatgat ttaaaataat gtttttcatc gg 42 <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> 1272 <212> DNA <213> Zymomonas mobilis-levU <400> 6 atgttgaata aagcaggcat tgcagagccg agcttgtgga ctcgtgcgga tgctatgaaa 60 gtgcataccg atgatcccac ggcaaccatg cctaccattg attatgactt tcctgtcatg 120 actgataaat attgggtttg ggacacttgg cccttacgcg atattaacgg tcaggttgtc 180 agcttccaag gttggtcggt gatctttgct ttggtcgctg atcgcaccaa atatggttgg 240 cataatcgca atgatggcgc cagaattggt tatttctatt cacgtggtgg aagcaactgg 300 atttttggtg gtcatcttct gaaagatggt gccaatccgc gttcttggga atggtctggt 360 tgcacgatta tggcaccggg tacggccaat tctgtcgaag tattctttac gtctgtcaat 420 gatacgccgt cagaatccgt tcctgcccag tgcaagggct acatctatgc cgatgataaa 480 tcggtatggt ttgacggttt tgataaagtg accgatctgt ttcaggcaga tggcctttat 540 tatgctgatt atgcagaaaa taatttctgg gatttccgcg atccgcatgt cttcattaac 600 cccgaagatg gcaaaacata tgccttgttt gaaggtaatg ttgccatgga gcgcggtacg 660 gtcgctgttg gcgaagagga aattggccct gttccaccaa aaaccgaaac gcctgatggc 720 gctcgctatt gtgctgctgc cattggtatt gcacaggccc ttaatgaagc ccgcaccgaa 780 tggaaattgt taccgccttt ggtaaccgcc tttggtgtca atgaccagac ggagcggcct 840 catgtcgttt tccagaatgg cttgacctat ctctttacga tcagtcatca ttcgacttat 900 gccgatggtt tgtcgggtcc tgatggggtt tatggctttg tttctgaaaa cggcattttt 960 ggcccttatg aaccgctgaa tggttccggt ttggttctcg gtaacccctc ttcacagcct 1020 tatcaggctt attcccatta tgtgatgaca aatgggctgg tgacctcctt cattgatacc 1080 attccgagtt ctgacccgaa tgtctatcgt tatggtggca ccttggcacc gaccatcaaa 1140 ttggaattgg ttggccatcg cagcttcgtt accgaagtga agggttatgg ctatattccg 1200 ccacagatcg agtggttggc agaagatgaa tcttctaatt ctgcggcagc cctgtcttta 1260 ttgaataaat aa 1272 <210> 7 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> levU primer-1 <400> 7 ctcgccttag ataaaagaat gttgaataaa gcaggcat 38 <210> 8 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> levU primer-2 <400> 8 gtgatggtga tggtgatgtt tattcaataa agacaggg 38 <210> 9 <211> 1422 <212> DNA <213> Artificial Sequence <220> <223> Bacillus subtilis SacB <400> 9 atgaacatca aaaagtttgc aaaacaagca acagtattaa cctttactac cgcactgctg 60 gcaggaggcg caactcaagc gtttgcgaaa gaaacgaacc aaaagccata taaggaaaca 120 tacggcattt cccatattac acgccatgat atgctgcaaa tccctgaaca gcaaaaaaat 180 gaaaaatatc aagttcctga attcgattcg tccacaatta aaaatatctc ttctgcaaaa 240 ggcctggacg tttgggacag ctggccatta caaaacgctg acggcactgt cgcaaactat 300 cacggctacc acatcgtctt tgcattagcc ggagatccta aaaatgcgga tgacacatcg 360 atttacatgt tctatcaaaa agtcggcgaa acttctattg acagctggaa aaacgctggc 420 cgcgtcttta aagacagcga caaattcgat gcaaatgatt ctatcctaaa agaccaaaca 480 caagaatggt caggttcagc cacatttaca tctgacggaa aaatccgttt attctacact 540 gatttctccg gtaaacatta cggcaaacaa acactgacaa ctgcacaagt taacgtatca 600 gcatcagaca gctctttgaa catcaacggt gtagaggatt ataaatcaat ctttgacggt 660 gacggaaaaa cgtatcaaaa tgtacagcag ttcatcgatg aaggcaacta cagctcaggc 720 gacaaccata cgctgagaga tcctcactac gtagaagata aaggccacaa atacttagta 780 tttgaagcaa acactggaac tgaagatggc taccaaggcg aagaatcttt atttaacaaa 840 gcatactatg gcaaaagcac atcattcttc cgtcaagaaa gtcaaaaact tctgcaaagc 900 gataaaaaac gcacggctga gttagcaaac ggcgctctcg gtatgattga gctaaacgat 960 gattacacac tgaaaaaagt gatgaaaccg ctgattgcat ctaacacagt aacagatgaa 1020 attgaacgcg cgaacgtctt taaaatgaac ggcaaatggt acctgttcac tgactcccgc 1080 ggatcaaaaa tgacgattga cggcattacg tctaacgata tttacatgct tggttatgtt 1140 tctaattctt taactggccc atacaagccg ctgaacaaaa ctggccttgt gttaaaaatg 1200 gatcttgatc ctaacgatgt aacctttact tactcacact tcgctgtacc tcaagcgaaa 1260 ggaaacaatg tcgtgattac aagctatatg acaaacagag gattctacgc agacaaacaa 1320 tcaacgtttg cgccaagctt cctgctgaac atcaaaggca agaaaacatc tgttgtcaaa 1380 gacagcatcc ttgaacaagg acaattaaca gttaacaaat aa 1422 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> BsSacB primer-1 <400> 10 ctcgccttag ataaaagaaa agaaacgaac caaaag 36 <210> 11 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> BsSacB primer-2 <400> 11 ttagtgatgg tgatggtgat gtttgttaac tgttaattg 39 <210> 12 <211> 415 <212> PRT <213> Rhanella aquatilis-lsrA <400> 12 Met Thr Asn Leu Asn Tyr Thr Pro Thr Ile Trp Thr Arg Ala Asp Ala   1 5 10 15 Leu Lys Val Asn Glu Asn Asp Pro Thr Thr Thr Gln Pro Ile Val Asp              20 25 30 Ala Asp Phe Pro Val Met Ser Asp Glu Val Phe Ile Trp Asp Thr Met          35 40 45 Pro Leu Arg Ser Leu Asp Gly Thr Val Val Ser Val Asp Gly Trp Ser      50 55 60 Val Ile Phe Thr Leu Thr Ala Gln Arg Asn Asn Asn Asn Ser Glu Tyr  65 70 75 80 Leu Asp Ala Glu Gly Asn Tyr Asp Ile Thr Ser Asp Trp Asn Asn Arg                  85 90 95 His Gly Arg Ala Arg Ile Cys Tyr Trp Tyr Ser Arg Thr Gly Lys Asp             100 105 110 Trp Ile Phe Gly Gly Arg Val Met Ala Glu Gly Val Ser Pro Thr Ser         115 120 125 Arg Glu Trp Ala Gly Thr Pro Ile Leu Leu Asn Glu Asp Gly Asp Ile     130 135 140 Asp Leu Tyr Tyr Thr Cys Val Thr Pro Gly Ala Thr Ile Ala Lys Val 145 150 155 160 Arg Gly Lys Val Leu Thr Ser Glu Glu Gly Val Thr Leu Ala Gly Phe                 165 170 175 Asn Glu Val Lys Ser Leu Phe Ser Ala Asp Gly Val Tyr Tyr Gln Thr             180 185 190 Glu Ser Gln Asn Pro Tyr Trp Asn Phe Arg Asp Pro Ser Pro Phe Ile         195 200 205 Asp Pro His Asp Gly Lys Leu Tyr Met Val Phe Glu Gly Asn Val Ala     210 215 220 Gly Glu Arg Gly Ser His Val Ile Gly Lys Gln Glu Met Gly Thr Leu 225 230 235 240 Pro Pro Gly His Arg Asp Val Gly Asn Ala Arg Tyr Gln Ala Gly Cys                 245 250 255 Ile Gly Met Ala Val Ala Lys Asp Leu Ser Gly Asp Glu Trp Glu Ile             260 265 270 Leu Pro Pro Leu Val Thr Ala Val Gly Val Asn Asp Gln Thr Glu Arg         275 280 285 Pro His Phe Val Phe Gln Asp Gly Lys Tyr Tyr Leu Phe Thr Ile Ser     290 295 300 His Lys Phe Thr Tyr Ala Asp Gly Leu Thr Gly Pro Asp Gly Val Tyr 305 310 315 320 Gly Phe Leu Ser Asp Asn Leu Thr Gly Pro Tyr Ser Pro Met Asn Gly                 325 330 335 Ser Gly Leu Val Leu Gly Asn Pro Pro Ser Gln Pro Phe Gln Thr Tyr             340 345 350 Ser His Cys Val Met Pro Asn Gly Leu Val Thr Ser Phe Ile Asp Asn         355 360 365 Val Pro Thr Ser Asp Gly Asn Tyr Arg Ile Gly Gly Thr Glu Ala Pro     370 375 380 Thr Val Lys Ile Val Leu Lys Gly Asn Arg Ser Phe Val Glu Arg Val 385 390 395 400 Phe Asp Tyr Gly Tyr Ile Pro Met Lys Asn Ile Ile Leu Asn                 405 410 415 <210> 13 <211> 423 <212> PRT <213> Zymomonas mobilis-levU <400> 13 Met Leu Asn Lys Ala Gly Ile Ala Glu Pro Ser Leu Trp Thr Arg Ala   1 5 10 15 Asp Ala Met Lys Val His Thr Asp Asp Pro Thr Ala Thr Met Pro Thr              20 25 30 Ile Asp Tyr Asp Phe Pro Val Met Thr Asp Lys Tyr Trp Val Trp Asp          35 40 45 Thr Trp Pro Leu Arg Asp Ile Asn Gly Gln Val Ser Ser Phe Gln Gly      50 55 60 Trp Ser Val Ile Phe Ala Leu Val Ala Asp Arg Thr Lys Tyr Gly Trp  65 70 75 80 His Asn Arg Asn Asp Gly Ala Arg Ile Gly Tyr Phe Tyr Ser Arg Gly                  85 90 95 Gly Ser Asn Trp Ile Phe Gly Gly His Leu Leu Lys Asp Gly Ala Asn             100 105 110 Pro Arg Ser Trp Glu Trp Ser Gly Cys Thr Ile Met Ala Pro Gly Thr         115 120 125 Ala Asn Ser Val Glu Val Phe Phe Thr Ser Val Asn Asp Thr Pro Ser     130 135 140 Glu Ser Val Pro Ala Gln Cys Lys Gly Tyr Ile Tyr Ala Asp Asp Lys 145 150 155 160 Ser Val Trp Phe Asp Gly Phe Asp Lys Val Thr Asp Leu Phe Gln Ala                 165 170 175 Asp Gly Leu Tyr Tyr Ala Asp Tyr Ala Glu Asn Asn Phe Trp Asp Phe             180 185 190 Arg Asp Pro His Val Phe Ile Asn Pro Glu Asp Gly Lys Thr Tyr Ala         195 200 205 Leu Phe Glu Gly Asn Val Ala Met Glu Arg Gly Thr Val Ala Val Gly     210 215 220 Glu Glu Ile Gly Pro Val Pro Pro Lys Thr Glu Thr Pro Asp Gly 225 230 235 240 Ala Arg Tyr Cys Ala Ala Ile Gly Ile Ala Gln Ala Leu Asn Glu                 245 250 255 Ala Arg Thr Glu Trp Lys Leu Leu Pro Pro Leu Val Thr Ala Phe Gly             260 265 270 Val Asn Asp Gln Thr Glu Arg Pro His Val Val Phe Gln Asn Gly Leu         275 280 285 Thr Tyr Leu Phe Thr Ile Ser His His Ser Thr Tyr Ala Asp Gly Leu     290 295 300 Ser Gly Pro Asp Gly Val Tyr Gly Phe Val Ser Glu Asn Gly Ile Phe 305 310 315 320 Gly Pro Tyr Glu Pro Leu Asn Gly Ser Gly Leu Val Leu Gly Asn Pro                 325 330 335 Ser Ser Gln Pro Tyr Gln Ala Tyr Ser His Tyr Val Met Thr Asn Gly             340 345 350 Leu Val Thr Ser Phe Ile Asp Thr Ile Pro Ser Ser Asp Pro Asn Val         355 360 365 Tyr Arg Tyr Gly Gly Thr Leu Ala Pro Thr Ile Lys Leu Glu Leu Val     370 375 380 Gly His Arg Ser Phe Val Thr Glu Val Lys Gly Tyr Gly Tyr Ile Pro 385 390 395 400 Pro Gln Ile Glu Trp Leu Ala Glu Asp Glu Ser Ser Asn Ser Ala Ala                 405 410 415 Ala Leu Ser Leu Leu Asn Lys             420 <210> 14 <211> 473 <212> PRT <213> Artificial Sequence <220> <223> Bacillus subtilis SacB <400> 14 Met Asn Ile Lys Lys Phe Ala Lys Gln Ala Thr Val Leu Thr Phe Thr   1 5 10 15 Thr Ala Leu Lea Ala Gly Gly Ala Thr Gln Ala Phe Ala Lys Glu Thr              20 25 30 Asn Gln Lys Pro Tyr Lys Glu Thr Tyr Gly Ile Ser His Ile Thr Arg          35 40 45 His Asp Met Leu Gln Ile Pro Glu Gln Gln Lys Asn Glu Lys Tyr Gln      50 55 60 Val Pro Glu Phe Asp Ser Ser Thr Ile Lys Asn Ile Ser Ser Ala Lys  65 70 75 80 Gly Leu Asp Val Trp Asp Ser Trp Pro Leu Gln Asn Ala Asp Gly Thr                  85 90 95 Val Ala Asn Tyr His Gly Tyr His Ile Val Phe Ala Leu Ala Gly Asp             100 105 110 Pro Lys Asn Ala Asp Asp Thr Ser Ile Tyr Met Phe Tyr Gln Lys Val         115 120 125 Gly Glu Thr Ser Ile Asp Ser Trp Lys Asn Ala Gly Arg Val Phe Lys     130 135 140 Asp Ser Asp Lys Phe Asp Ala Asn Asp Ser Ile Leu Lys Asp Gln Thr 145 150 155 160 Gln Glu Trp Ser Gly Ser Ala Thr Phe Thr Ser Asp Gly Lys Ile Arg                 165 170 175 Leu Phe Tyr Thr Asp Phe Ser Gly Lys His Tyr Gly Lys Gln Thr Leu             180 185 190 Thr Thr Ala Gln Val Asn Val Ser Ala Ser Asp Ser Ser Leu Asn Ile         195 200 205 Asn Gly Val Glu Asp Tyr Lys Ser Ile Phe Asp Gly Asp Gly Lys Thr     210 215 220 Tyr Gln Asn Val Gln Gln Phe Ile Asp Glu Gly Asn Tyr Ser Ser Gly 225 230 235 240 Asp Asn His Thr Leu Arg Asp Pro His Tyr Val Glu Asp Lys Gly His                 245 250 255 Lys Tyr Leu Val Phe Glu Ala Asn Thr Gly Thr Glu Asp Gly Tyr Gln             260 265 270 Gly Glu Glu Ser Leu Phe Asn Lys Ala Tyr Tyr Gly Lys Ser Thr Ser         275 280 285 Phe Phe Arg Gln Glu Ser Gln Lys Leu Leu Gln Ser Asp Lys Lys Arg     290 295 300 Thr Ala Glu Leu Ala Asn Gly Ala Leu Gly Met Ile Glu Leu Asn Asp 305 310 315 320 Asp Tyr Thr Leu Lys Lys Val Lys Pro Leu Ile Ala Ser Asn Thr                 325 330 335 Val Thr Asp Glu Ile Glu Arg Ala Asn Val Phe Lys Met Asn Gly Lys             340 345 350 Trp Tyr Leu Phe Thr Asp Ser Arg Gly Ser Lys Met Thr Ile Asp Gly         355 360 365 Ile Thr Ser Asn Asp Ile Tyr Met Leu Gly Tyr Val Ser Asn Ser Leu     370 375 380 Thr Gly Pro Tyr Lys Pro Leu Asn Lys Thr Gly Leu Val Leu Lys Met 385 390 395 400 Asp Leu Asp Pro Asn Asp Val Thr Phe Thr Tyr Ser His Phe Ala Val                 405 410 415 Pro Gln Ala Lys Gly Asn Asn Val Val Ile Thr Ser Tyr Met Thr Asn             420 425 430 Arg Gly Phe Tyr Ala Asp Lys Gln Ser Thr Phe Ala Pro Ser Phe Leu         435 440 445 Leu Asn Ile Lys Gly Lys Lys Thr Ser Val Val Lys Asp Ser Ile Leu     450 455 460 Glu Gln Gly Gln Leu Thr Val Asn Lys 465 470 <210> 15 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> TFP 1 a.a. <400> 15 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> 16 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> TFP 2 a.a. <400> 16 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> 17 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> TFP 3 a.a. <400> 17 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> 18 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> TFP 4 a.a. <400> 18 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> 19 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> TFP 5 a.a. <400> 19 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> 20 <211> 93 <212> PRT <213> Artificial Sequence <220> <223> TFP 6 a.a. <400> 20 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> 21 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> TFP 7 a.a. <400> 21 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> 22 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> TFP 8 a.a. <400> 22 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> 23 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> TFP 9 a.a. <400> 23 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> 24 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> TFP 10 a.a. <400> 24 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> 25 <211> 77 <212> PRT <213> Artificial Sequence <220> <223> TFP 11 a.a. <400> 25 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> 26 <211> 94 <212> PRT <213> Artificial Sequence <220> <223> TFP 12 a.a. <400> 26 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> 27 <211> 174 <212> PRT <213> Artificial Sequence <220> <223> TFP 13 a.a. <400> 27 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> 28 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> TFP 14 a.a. <400> 28 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> 29 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> TFP 15 a.a. <400> 29 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> 30 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> TFP 16 a.a. <400> 30 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> 31 <211> 195 <212> PRT <213> Artificial Sequence <220> <223> TFP 17 a.a. <400> 31 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> 32 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> TFP 18 a.a. <400> 32 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> 33 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> TFP 19 a.a. <400> 33 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> 34 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> TFP 20 a.a. <400> 34 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> 35 <211> 176 <212> PRT <213> Artificial Sequence <220> <223> TFP 21 a.a. <400> 35 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> 36 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> TFP 22 a.a. <400> 36 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> 37 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> TFP 23 a.a. <400> 37 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> 38 <211> 170 <212> PRT <213> Artificial Sequence <220> <223> TFP 24 a.a. <400> 38 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> 39 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> TFP 1 nt <400> 39 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> 40 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> TFP 2 nt <400> 40 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> 41 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> TFP 3 nt <400> 41 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> 42 <211> 186 <212> DNA <213> Artificial Sequence <220> <223> TFP 4 nt <400> 42 atgagatttg cagaattctt ggtggtattt gccacgttag gcggggggat ggctgcaccg 60 gttgagtctc tggccgggac ccaacggtat ctggtgcaaa tgaaggagcg gttcaccaca 120 gagaagctgt gtgctttgga cgacaaggcc gcctcggcct ctgctggcct cgccttagat 180 aaaaga 186 <210> 43 <211> 291 <212> DNA <213> Artificial Sequence <220> <223> TFP 5 nt <400> 43 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> 44 <211> 279 <212> DNA <213> Artificial Sequence <220> <223> TFP 6 nt <400> 44 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> 45 <211> 678 <212> DNA <213> Artificial Sequence <220> <223> TFP 7 nt <400> 45 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> 46 <211> 192 <212> DNA <213> Artificial Sequence <220> <223> TFP 8 nt <400> 46 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> 47 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> TFP 9 nt <400> 47 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> 48 <211> 597 <212> DNA <213> Artificial Sequence <220> <223> TFP 10 nt <400> 48 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> 49 <211> 231 <212> DNA <213> Artificial Sequence <220> <223> TFP 11 nt <400> 49 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> 50 <211> 282 <212> DNA <213> Artificial Sequence <220> <223> TFP 12 nt <400> 50 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> 51 <211> 522 <212> DNA <213> Artificial Sequence <220> <223> TFP 13 nt <400> 51 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> 52 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> TFP 14 nt <400> 52 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> 53 <211> 471 <212> DNA <213> Artificial Sequence <220> <223> TFP 15 nt <400> 53 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> 54 <211> 294 <212> DNA <213> Artificial Sequence <220> <223> TFP 16 nt <400> 54 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> 55 <211> 585 <212> DNA <213> Artificial Sequence <220> <223> TFP 17 nt <400> 55 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> 56 <211> 315 <212> DNA <213> Artificial Sequence <220> <223> TFP 18 nt <400> 56 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> 57 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> TFP 19 nt <400> 57 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> 58 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> TFP 20 nt <400> 58 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> 59 <211> 528 <212> DNA <213> Artificial Sequence <220> <223> TFP 21 nt <400> 59 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> 60 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> TFP 22 nt <400> 60 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> 61 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> TFP 23 nt <400> 61 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> 62 <211> 510 <212> DNA <213> Artificial Sequence <220> <223> TFP 24 nt <400> 62 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 (22)

A Levans sucrase secretion expression cassette comprising a nucleic acid encoding levansucrase and a nucleic acid encoding a translational fusion partner (TFP)
The protein secretion factor may be,
TFP 2 of SEQ ID NO: 17, TFP 3 of SEQ ID NO: 18, TFP 4 of SEQ ID NO: 18, TFP 5 of SEQ ID NO: 19, TFP 6 of SEQ ID NO: 20, TFP 7 of SEQ ID NO: 21, SEQ ID NO: TFP 9 of SEQ ID NO: 24, TFP 10 of SEQ ID NO: 24, TFP 11 of SEQ ID NO: 25, TFP 12 of SEQ ID NO: 26, TFP 13 of SEQ ID NO: 27, TFP 14 of SEQ ID NO: 29, TFP 15 of SEQ ID NO: 30, TFP 17 of SEQ ID NO: 31, TFP 18 of SEQ ID NO: 32, TFP 19 of SEQ ID NO: 33, TFP 20 of SEQ ID NO: 34, TFP 21 of SEQ ID NO: 35, SEQ ID NO: The TFP 23 of SEQ ID NO: 37, and TFP 24 of SEQ ID NO: 38.
The secretory expression cassette according to claim 1, wherein the Levanschraze is derived from Rhanella aquatilis , Zymomonas mobilis , or Bacillus subtilis .
3. The method of claim 2, wherein the Levanschraze is
(i) levanshucrase derived from Lanella aquaticis having the amino acid sequence of SEQ ID NO: 12;
(ii) Levanshukrage from Zymomonas mobilis having the amino acid sequence of SEQ ID NO: 13; or
(iii) Levanschuracase derived from Bacillus subtilis having the amino acid sequence of SEQ ID NO: 14,
Secretion expression cassette.
delete 3. The method of claim 2,
Wherein the Levan sucrase derived from lanella aquaticis is linked to TFP 3 of SEQ ID NO: 17, TFP 4 of SEQ ID NO: 18, TFP 8 of SEQ ID NO: 22 or TFP 16 of SEQ ID NO: 30.
3. The method of claim 2,
Levanshukraze derived from Zymomonas mobilis comprises TFP 3 of SEQ ID NO: 17, TFP 6 of SEQ ID NO: 20, TFP 8 of SEQ ID NO: 22, TFP 9 of SEQ ID NO: 23, TFP 11 of SEQ ID NO: 30, TFP 19 of SEQ ID NO: 33, or TFP 22 of SEQ ID NO: 36.
3. The method of claim 2,
Levanshukraze derived from Bacillus subtilis contains TFP 1 of SEQ ID NO: 15, TFP 5 of SEQ ID NO: 19, TFP 6 of SEQ ID NO: 20, TFP 8 of SEQ ID NO: 22, TFP 9 of SEQ ID NO: 23, SEQ ID NO: Of TFP 13 of SEQ ID NO: 30, or TFP 19 of SEQ ID NO: 33.
The secretory expression cassette according to claim 1, wherein the nucleic acid encoding Levansucrase and the nucleic acid encoding the protein secretion factor are linked to each other by a linker.
9. The secretory expression cassette of claim 8, wherein the linker comprises a protease recognition sequence or affinity tag.
The secretory expression cassette of claim 1, wherein said secretory expression cassette further comprises an affinity tag.
11. The method of claim 10, 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.
11. A vector comprising a levansucrase secretion expression cassette of any one of claims 1 to 3 and 5 to 11.
1. A transforming microorganism comprising a Levansucrase secretion expression cassette comprising a nucleic acid encoding Levansucrase and a nucleic acid encoding a protein secretion fusion factor,
The protein secretion factor may be,
TFP 2 of SEQ ID NO: 17, TFP 3 of SEQ ID NO: 18, TFP 4 of SEQ ID NO: 18, TFP 5 of SEQ ID NO: 19, TFP 6 of SEQ ID NO: 20, TFP 7 of SEQ ID NO: 21, SEQ ID NO: TFP 9 of SEQ ID NO: 24, TFP 10 of SEQ ID NO: 24, TFP 11 of SEQ ID NO: 25, TFP 12 of SEQ ID NO: 26, TFP 13 of SEQ ID NO: 27, TFP 14 of SEQ ID NO: 29, TFP 15 of SEQ ID NO: 30, TFP 17 of SEQ ID NO: 31, TFP 18 of SEQ ID NO: 32, TFP 19 of SEQ ID NO: 33, TFP 20 of SEQ ID NO: 34, TFP 21 of SEQ ID NO: 35, SEQ ID NO: The TFP 22 of SEQ ID NO: 37, and the TFP 24 of SEQ ID NO: 38.
14. The microorganism according to claim 13, wherein the transforming microorganism is yeast.
15. The method of claim 14, 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).
15. The method of claim 14, wherein the yeast is selected from the group consisting of Candida utilis , Candida boidinii , Candida albicans , Kluyveromyces lactis , Pichia pastoris), Pichia Stevenage avoid tooth (Pichia stipitis), ski investigation Caro Mai Seth pombe (Schizosaccharomyces pombe), the Saccharomyces Roman Isis serenity busy (Saccharomyces cerevisiae), a century Cronulla poly Maurepas (Hansenula polymorpha), Jaroslaw's Li poly Utica Wherein the microorganism is selected from the group consisting of Yarrowia lipolytica , Schwanniomyces occidentalis , and Arxula adeninivorans .
(i) culturing the transformed microorganism of claim 13; And
(ii) recovering levansucrase from the cultured microorganism or culture supernatant thereof.
A method for producing levan comprising the step of reacting the transforming microorganism of claim 13 or levanshucrase prepared by the method of claim 17 with sugar.
19. The process according to claim 18, wherein the reaction is carried out at a pH of from 4.5 to pH 7.5.
19. The process according to claim 18, wherein the reaction is carried out at a temperature of from 1 占 폚 to 45 占 폚.
19. The method of claim 18,
Wherein said step comprises fermenting the transforming microorganism in a medium comprising sugar.
19. The method of claim 18,
Wherein said step comprises fermenting the transforming microorganism in a medium containing molasses.

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