KR101837130B1 - Novel lipase from yeast Candida butyri SH-14 and the Use thereof - Google Patents

Abstract

The present invention relates to a Candida butyri strain having lipase activity, a nucleic acid encoding said lipase, a nucleic acid encoding said lipase, and a nucleic acid encoding a translational fusion partner (TFP) A secretory expression cassette, a vector containing the secretory expression cassette, a transformant microorganism including the lipase or secretory expression cassette, a method for producing the lipase, and a method for producing biodiesel using the lipase.
The transformant microorganism including the lipase of the present invention can be used to mass produce lipase, and the lipase can be widely used for the economical production of biodiesel.

Description

Novel lipase from yeast Candida butyri SH-14 and the Use thereof < RTI ID = 0.0 >

The present invention relates to a Candida butyri strain having lipase activity, a nucleic acid encoding said lipase, a nucleic acid encoding said lipase, and a nucleic acid encoding a translational fusion partner (TFP) A secretory expression cassette, a vector containing the secretory expression cassette, a transformant microorganism including the lipase or secretory expression cassette, a method for producing the lipase, and a method for producing biodiesel using the lipase.

Lipase (Lipase, triacylglycerol acyl hydrolases, E.C. 3.1.1.3) is an enzyme that degrades and converts lipids and is highly industrially useful. Lipase was first found in pancreas in 1834 and is an enzyme that does not require a cofactor as serine hydrolases that break down fat at the interface with fat (triacylglycerol) in aqueous solution (Rajendran et al. , 2008). This reaction is also capable of esterification and transesterification in organic solvents with minimal aqueous solution. Lipase studies have mainly focused on the expansion of substrate specificity, regioselectivity, chemoselectivity, and chiral selectivity.

On the other hand, due to the lipolytic ability of lipase and the reaction characteristics in organic solvents, it is utilized as a biocatalyst in various industries such as detergent industry, oil processing industry, biofuels, food and dairy industry, agricultural chemical industry, paper industry, (Grazia et al., Int. J. Nol. Sci 2015, 16: 20774). Lipase is an essential enzyme found in a wide range of organisms, from bacteria, yeast, fungi, plants to higher organisms, but the most studied of bacterial and yeast-derived lipases. In particular, yeast Candida rugosa and Candida antarctica , Derived lipase has been the most studied and industrially utilized. In particular, C. antarctica lipase B (CalB) is marketed by Novozymes, a global enzyme company, for the use of Novozym435 immobilized enzyme for a variety of industrial applications. In addition, a variety of lipolytic yeasts were obtained from environmental samples containing a maintenance ingredient, and research on lipase was carried out, and various yeast of Candida genus, Galactomyces geotricum , ( Arxula adeninivorans ), Yarowiari Polytica ( Yarrowia lipolytica , Trichosporon , Rhodotorula genus, Aureobasidium pullulans , and Cryptococcus , but industrially available enzymes are very limited. Lipase, which is commercially available worldwide, is an enzyme derived mainly from yeast and fungi, and it requires additional new enzymes and industrial mass production techniques for various industrial applications (Gupta et al., Progress in lipid Research 2015, 57: 40).

Under these circumstances, the present inventors have made intensive efforts to develop new lipase and technology for mass production thereof, and as a result, a novel Candida bacterium strain having excellent lipase activity was identified and a novel lipase was isolated therefrom. Also, it has been confirmed that the lipase itself can be produced in a microorganism, or secreted and expressed using a protein secretion fusion factor, and mass production of lipase, thereby producing biodiesel. Thus, the present invention has been completed.

One object of the present invention is a lipase (lipase) which is active, the accession number of Candida KCTC18455P beauty Li (Candida butyri ) strain.

Another object of the present invention is to provide a separate lipase which can be derived from said strain.

It is another object of the present invention to provide a lipase secretion expression cassette comprising a nucleic acid encoding said lipase and a nucleic acid encoding a translational fusion partner (TFP).

It is another object of the present invention to provide a vector comprising the lipase secretion expression cassette.

It is another object of the present invention to provide a transgenic microorganism comprising the lipase or the secretory expression cassette.

It is still another object of the present invention to provide a method for culturing a transformed microorganism, And recovering the lipase from the cultured microorganism or culture supernatant thereof.

It is another object of the present invention to provide a process for producing biodiesel comprising the step of reacting the lipase with a solvent and an alcohol.

One aspect of the present invention for achieving the above object is a lipase (lipase) which is active, the accession number of Candida KCTC18455P beauty Li (Candida butyri ).

This will be described in detail as follows. On the other hand, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. Further, the scope of the present invention is not limited by the detailed description described below.

In the present invention, in order to isolate a new strain exhibiting lipase activity, palm oil was recovered and lipase activity of the yeast strain isolated from the remaining EFB (empty fruit bunch) sample was confirmed (FIG. 2). As a result, the best lipase activity was found in Candida butyri ) was identified and named Candida beautiferi SH-14. Furthermore, the above-mentioned strain was deposited with the Korea Collection for Type Culture (KCTC), an international depository organization under the Budapest Treaty, on Mar. 04, 2014, and received the accession number KCTC18455P.

In the present invention, the term " lipase " refers to an enzyme that degrades fat, and specifically refers to an enzyme that acts on triglycerides and degrades fatty acids and glycerin through triacylglycerides and diacylglycerides and monoacylglycerides do. Therefore, the lipase activity means an activity to decompose fat.

In the present invention, the " strain having lipase activity " may include all strains having biologically active lipid-degrading activity, for example, Candida bacterium strain SH-14 of Accession No. KCTC18455P. The strain may produce one or more lipases and may have lipase activity.

Another aspect of the present invention is a lipase having an amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 92. The SEQ ID NO: 5, SEQ ID NO: lipase has the amino acid sequence of 8 and SEQ ID NO: 10 may be used interchangeably with each LIP2, LIP5 and LIP7 protein, or CBLIP2, CBLIP5 and CBLIP7 protein in the present invention, also LIP2, LIP5 and LIP7 Gene, or a protein encoded by the CBLIP2 , CBLIP5, and CBLIP7 genes. The lipase having the amino acid sequence of SEQ ID NO: 92 may be a lipase having the amino acid sequence of SEQ ID NO: 8, wherein the CTG codon is replaced with a TCT codon, whereby the leucine at that position is substituted with serine, The kind of the nucleotide codon that encodes it is not limited.

The lipase may also have at least 80%, 90%, 95%, 96%, 97%, 98% or 99% homology or sequence identity with the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: Lt; / RTI > polypeptide. For example, if the amino acid sequence has this homology or sequence identity and exhibits the corresponding effect of lipase comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 92, , Modified, substituted or added amino acid sequences are included within the scope of the present invention.

Also, a protein having an amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 92, a protein consisting essentially of the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: The expression of a protein consisting of an amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 92 is intended to include addition of a nonsense sequence or an unnatural sequence before or after the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 92, as long as it does not exclude a mutation or silent mutation that may occur in the above- The amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 92 of the present invention A protein consisting essentially of the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 92 or a protein consisting essentially of the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: Which is a category of proteins.

Further, although not limited thereto, the genes coding for the lipase, namely the LIP2 , LIP5 and LIP7 genes, and the polynucleotides encoding the lipase of SEQ ID NO: 92 are SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: Or at least 80%, 90%, 95%, 96%, 97%, 98% or more of the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 0.0 > 99% < / RTI > homology or sequence identity. A polynucleotide capable of being translated into a protein comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 92 or a protein having homology or sequence identity thereto by codon degeneracy It is obvious that it can be included.

In the above, the term "homology" means the degree to which a given amino acid sequence matches a nucleotide sequence and can be expressed as a percentage. In the present specification, its homologous sequence having the same or similar activity as a given amino acid sequence or nucleotide sequence is indicated as "% homology ". For example, standard software for calculating parameters such as score, identity and similarity, specifically BLAST 2.0, or by sequential hybridization experiments under defined stringent conditions, And the appropriate hybridization conditions to be defined are within the skill of the art and can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). The term " stringent conditions " as used herein refers to conditions that allow specific hybridization between polynucleotides. For example, these conditions are specifically described in the literature (e.g., J. Sambrook et al., Same as above).

In addition, the lipase may be derived from the Candida albicile strain SH-14 of the present invention, but is not limited thereto and may be included in the scope of the present invention as long as it exhibits the same or similar activity while having the same homology as described above .

In a specific embodiment of the present invention, the entire gene of the Candida albicile strain SH-14 having the novel lipase activity was analyzed, and the selected genes were expressed in yeast strains to obtain novel lipases CBLIP2, CBLIP5 and CBLIP7 (Fig. 5 to Fig. 7).

Another embodiment of the present invention is a lipase secretion expression cassette comprising a nucleic acid encoding said lipase and a nucleic acid encoding a translational fusion partner (TFP).

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: 38, TFP 2 of SEQ ID NO: 39, TFP 3 of SEQ ID NO: 40, TFP 4, TFP 5 of SEQ ID NO: 42, TFP 7 of SEQ ID NO: 44, TFP 8 of SEQ ID NO: 45, TFP 9 of SEQ ID NO: 46, TFP 10 of SEQ ID NO: 47, TFP 11 of SEQ ID NO: TFP 14 of SEQ ID NO: 51, TFP 14 of SEQ ID NO: 51, TFP 15 of SEQ ID NO: 52, TFP 16 of SEQ ID NO: 53, TFP 17 of SEQ ID NO: 54, TFP 18 of SEQ ID NO: 55, The TFP 19 of SEQ ID NO: 56, TFP 20 of SEQ ID NO: 57, TFP 21 of SEQ ID NO: 58, TFP 22 of SEQ ID NO: 59, TFP 23 of SEQ ID NO: 60 or TFP 24 of SEQ ID NO: Any factor capable of improving expression can be included without limitation.

Thus, the nucleotide sequence of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: SEQ ID NO: 52, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ ID NO: 61, SEQ ID NO: Lt; RTI ID = 0.0 > and / or < / RTI > 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: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 40 as long as it can improve the secretory expression of the target protein. SEQ ID NO: 44, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, 80% or more, specifically 90% or more, more particularly 95% or more, more preferably 95% or more, more preferably 90% or more, more preferably 95% or more, More specifically, 97% or more The substitution, deletion, insertion, addition, or inversion of the amino acid may include a naturally occurring mutant or artificial mutation sequence in the microorganism containing the activity of the TFP. have.

Further, when the lipase is a lipase having the amino acid sequence of SEQ ID NO: 5, TFP 11 of SEQ ID NO: 48, TFP 13 of SEQ ID NO: 50, TFP 19 of SEQ ID NO: 56, or TFP 23 of SEQ ID NO: In the case of a lipase having the amino acid sequence of SEQ ID NO: 8, TFP8 of SEQ ID NO: 45, TFP 9 of SEQ ID NO: 46, TFP 13 of SEQ ID NO: 50 or TFP 19 of SEQ ID NO: 56. Also, in the case of lipase having the amino acid sequence of SEQ ID NO: 10, TFP 4 of SEQ ID NO: 41, TFP 8 of SEQ ID NO: 45, TFP 13 of SEQ ID NO: 50, TFP 20 of SEQ ID NO: 57, TFP 21 of SEQ ID NO: 58, SEQ ID NO: 60, TFP 22 of SEQ ID NO: 60 or TFP 24 of SEQ ID NO: 61, Lipase having the amino acid sequence of SEQ ID NO: 92, TFP 8 of SEQ ID NO: 45, TFP 9, TFP 13 of SEQ ID NO: 50, or TFP 19 of SEQ ID NO: 56 may be linked, but the present invention is not limited thereto.

In the lipase secretion expression cassette, the nucleic acid encoding the contained lipase 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 the lipase used in the present invention may include the linker DNA used for recombination in the cell with the linear vector of the present invention. Addition of such a linker sequence to a nucleic acid sequence encoding a lipase 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 be of sufficient length and introduced into a host cell such that the nucleic acid sequence encoding the lipase in the cell is part of the nucleic acid sequence of the linear vector so that it can be recombined into a linear vector containing the nucleic acid sequence encoding TFP It may have sufficient homology. 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 the lipase, 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 the meaning that it encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides which are basic constituent units in nucleic acid molecules include not only natural nucleotides but also analogs and an analogue. The present invention provides a lipase secretion expression cassette comprising a nucleic acid encoding a lipase and a nucleic acid encoding a protein secretion fusion factor.

The term " expression cassette " of the present invention has a nucleic acid element capable of affecting the expression of a structural gene in a cell. Naturally occurring or synthesized nucleic acid constructs generally include an expression cassette that contains a transcribed nucleic acid and a promoter. For the purpose of the present invention, the expression cassette may comprise a nucleic acid encoding a protein secretion fusion factor and a nucleic acid encoding a lipase, and in particular a " lipase secretion expression cassette " which induces the secretion of a lipase, .

Another embodiment of the present invention is a vector comprising said cassette.

The cassette is as described above.

The term " vector " in the present invention means a DNA product containing a nucleotide sequence encoding a desired protein operably linked to a suitable regulatory sequence so as to be able to express the desired protein in a suitable host, , The target protein refers to a recombinant fusion protein comprising a lipase 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, but the present invention is not limited thereto.

In a specific embodiment of the present invention, a YGaST19- CBLIP2 vector containing TFP 19 and CBLIP2 gene was prepared as an expression vector for producing high secretion of lipase, and transformed into a yeast strain and cultured at about 230 KU / L / min, it was confirmed that lipase could be produced at a high concentration (Fig. 11).

Another aspect of the present invention is a transforming microorganism comprising a lipase secretion expression cassette comprising or consisting of a nucleic acid encoding a lipase and a nucleic acid encoding a protein secretion factor.

The lipase, the protein secretion fusion factor, the nucleic acid, and the lipase secretion expression cassette are as described above.

In a specific embodiment of the present invention, when the CBLIP2, CBLIP5 and CBLIP7 genes are expressed in association with a protein secretion fusion factor as well as when expressed using self-secretion signals or MFa (mating factor alpha) secretion signals of the genes (Table 7). Therefore, the lipase of the present invention may be secreted by using an MFa secretion signal or a protein secretion fusion factor, and secreted by a self-secretory signal even in a microorganism transformed with lipase itself. Therefore, it is obvious that the transformed microorganism containing the lipase is also included in the scope of the present invention.

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 a high efficiency of introduction and expression of the lipase gene of the present invention. For example, , Fungi, 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) Schizosaccharomyces , Yarrowia , Saccharomyces , Schwanniomyces , or Arxula , and more specifically, Candida bacterium , which belongs to the genus Schizosaccharomyces , Yarrowia , Saccharomyces , Schwanniomyces or Arxula , Candida butyri , Candida utilis), Candida seems dini (Candida boidinii), Candida albicans (Candida albicans , Kluyveromyces lactis , Pichia < RTI ID = 0.0 > pastoris , Pichia stipitis ), skiing investigation Schizosaccharomyces pombe ), Saccharomyces ( Saccharomyces cerevisiae , Hansenula polymorpha , Yarrowia lipolytica , Schwanniomyces < RTI ID = 0.0 > Occidentalis or Arxula < RTI ID = 0.0 > adeninivorans ), but are not limited thereto.

For example, in the present invention, the GAL10 promoter used for expression of lipase is a promoter that is transcribed by galactose, but a strain in which GAL80 gene is inactivated can maintain high gene expression regardless of the presence or absence of galactose, which is an expensive inducer. For the above-mentioned reasons, a specific example of the present invention expresses lipase using a strain in which the GAL80 gene is inactivated.

Another aspect of the present invention is a method for producing a transformed microorganism, comprising: (i) culturing the transformed microorganism; And (ii) recovering the lipase from the cultured microorganism or culture supernatant thereof.

The transforming microorganism and the lipase 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 a YPD liquid medium (2% glucose, 1% yeast extract, 2% peptone) containing glucose as a carbon source, and the culture medium (30% glucose, 15% yeast extract) For 48 hours, but not limited thereto.

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

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) After culturing in 200 ml of YPD liquid medium, the cells were inoculated into the culture medium and cultured at 30 ° C for 48 hours. Then, the medium (15% yeast extract, 30% glucose) was added according to the cell growth rate Supply.

Another embodiment of the present invention is a method for producing biodiesel comprising the step of reacting the lipase with a solvent and an alcohol.

The lipase is not limited thereto, but it can be used in a free form or immobilized form, and more specifically, it can be used in a fixed form. Immobilization of the lipase can be performed by various methods commonly used in the art, and various applications are possible without limitation. For example, physical methods such as an adsorption method and an inclusion method, and chemical methods such as a covalent bonding method and a crosslinking method can be used.

In one specific embodiment of the present invention, recombinant mass-produced CBLIP2 was immobilized using Lewatit VP OC 1600 (Bayer) as the immobilized adsorption carrier (FIG. 19).

More specifically, soybean oil, rapeseed oil, palm oil, etc. may be used, although the oil is not limited thereto. Specifically, natural oil, processed oil and lung oil may be used.

More specifically, alcohols having 2 to 4 carbon atoms can be used. More specifically, alcohols having 2 to 8 carbon atoms can be used, and more specifically, ethanol, methanol, 1-propanol, iso-propanol, 1-butanol, 2-butanol, iso-butanol or ter-butanol. The alcohol may be supplied in various feeding methods known in the art, and may be supplied in several steps or continuously. In this case, a continuous feeding method may be used.

The transformant microorganism including the lipase of the present invention can be used to mass produce lipase, and the lipase can be widely used for the economical production of biodiesel.

Figure 1 is a photograph of the yeast strains isolated pure from the composted EFB sample.
Fig. 2 is a photograph showing the lipase activity of the isolated yeasts through a transparent ring in a tributyrin medium.
Fig. 3 shows the result of a total genome analysis of Candida buttery SH-14 strain.
Fig. 4 shows a process of cloning a recombinant gene derived from Candida buttery SH-14 strain into a vector containing secretory fusion factor in Saccharomyces cerevisiae via in vivo recombination.
FIG. 5 is an electrophoresis image showing the result of SDS-PAGE analysis of CBLIP2 protein derived from Candida buttery SH-14 strain which was expressed in 24 strains of Saccharomyces cerevisiae and secreted out of the cell. The results of comparing CBLIP2 protein expressed in four strains (ST11, 13, 19, 23) before and after endo-H treatment.
6 is an electrophoresis image showing the result of SDS-PAGE analysis of CBLIP5 protein derived from Candida buttery SH-14 strain which was expressed in 24 strains of Saccharomyces cerevisiae and secreted out of the cell. Before and after Endo-H treatment.
FIG. 7 is an electrophoresis image showing the result of SDS-PAGE analysis of CBLIP7 protein derived from Candida buttery SH-14 strain which was expressed in 24 strains of Saccharomyces cerevisiae and secreted out of the cell. Before and after Endo-H treatment.
8 is a graph showing the activity of CBLIP2 lipase derived from Candida buttery SH-14 strain secreted from Saccharomyces cerevisiae in 24 strains and secreted out of the cell by substrate.
9 is a graph showing the activity of CBLIP5 lipase derived from Candida albicile SH-14 strain expressed in 24 strains of Saccharomyces cerevisiae and secreted out of the cells by substrate.
10 is a graph showing the activity of CBLIP7 lipase derived from Candida buttery SH-14 strain expressed in 24 strains of Saccharomyces cerevisiae and secreted out of the cell by substrate.
Fig. 11 is a graph showing the results of SDS-PAGE analysis of the medium obtained by fermenting the fermented product in Saccharomyces cerevisiae transformed with a recombinant vector containing the lipase gene derived from Candida buttery SH-14 strain in a 5 L fermenter, (Top), and graphs showing cell cell growth and activity of lipase at each time (bottom).
FIG. 12 shows electrophoresis (b) and SDS-PAGE analysis of the protein of the purified fraction (a) obtained through ion exchange chromatography for purification of recombinant lipase, and the lipase activity of each fraction This is the picture (c).
13 is a graph showing the effect of the purified lipase on the temperature.
14 is a graph showing the influence and stability of the purified lipase on the pH.
15 is a graph showing the effect of the purified lipase on the metal ion (a), the inhibitor, the surfactant (b), and the solvent (c).
Figure 16 shows the substrate specificity according to the number of carbons of the purified lipase.
Fig. 17 is a sequence analysis result showing the CTG codon of CBLIP5 lipase derived from Candida butterybird strain.
FIG. 18 is a graph (a) showing electrophoresis (a) and a graph (b) showing the activity of lipase, showing the results of SDS-PAGE analysis of protein expression before and after substitution of the CTG codon of CBLIP5 lipase derived from Candida buttery.
Figure 19 shows the residual activity over time while immobilizing CBLIP2 lipase on the adsorbent carrier.
20 shows the conversion of fatty acid methyl ester to water content.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these embodiments are for illustrative purposes only, and the scope of the present application is not limited to these embodiments.

Example 1 . New < RTI ID = 0.0 > Yeast caution  Isolation and identification

In order to isolate new yeast strains exhibiting lipase activity, we resuscitated for 50 days in Korea Biotechnology Research Institute using an empty fruit bunch (EFB) sample from which the palm oil was recovered from Malaysia and then harvested 10 g of the inoculated EFB sample Were placed in a 50 ml cornical tube with 40 ml of sterile distilled water and mixed for one hour using a vortex. 1 ml of the supernatant was diluted by the series dilution method and the sample was plated on YPDPS (10 g yeast extract, 10 g of peptone, 20 g of glucose, 50 g of antibiotic (penicillin / streptomycin 50 mg) And cultured for 36 hours. The medium used for the isolation of yeast strains was a medium containing a bacterial antibiotic to inhibit the bacteria. Thereafter, a variety of yeast strains of different phenotype producing opaque and smooth yeast-type colonies instead of mold spores were obtained (Fig. 1). To confirm the lipase activity of the isolated microorganism colonies, the strain was added to the medium of YPT (10 g of yeast extract per liter, 20 g of peptone, 10 g of trivitilin) and cultured at 30 ° C for one week to obtain a strain having the largest transparency (Fig. 2). The 14th strain with the best lipase activity was designated as SH-14 and deposited on March 4, 2016 in the Korea Collection for Type Culture (KCTC), an international depository organization under the Budapest Treaty And received grant number KCTC18455P.

For strain analysis of the SH-14 strain, the strain was shake-cultured at 30 DEG C and 180 rpm in a 3 mL YPDPS liquid medium for 48 hours. Then, only the cells were recovered by centrifugation, and genomic DNA was extracted using YeaStar ™ Genomic DNA Kit (Zymo Research, Orange, Calif.). In order to analyze the nucleotide sequence of the ribosomal ITS (internal transcribed spacer) region used as a molecular marker most useful for the evolutionary pathway of eukaryotic microorganisms in the extracted DNA, primers ITS1 (5'-TCCGTAGGTG AACCTGCGG-3 ' 1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3 ', SEQ ID NO: 2). After the PCR reaction, 2 μl of the reaction solution was analyzed by agarose gel electrophoresis. As a result, a fragment of about 580 bp was amplified and PCR clean-up and gel extraction kit (Macherey-Nagel, Duren, Germany) was used to purify the PCR product. The base sequence of the purified PCR product was then analyzed (Genotex, SEQ ID NO: 3). The nucleotide sequences thus identified were compared with known nucleotide sequences using the BLAST (Basic Local Alignment Search Tool) program provided by National Center for Biotechnology Information (NCBI). As a result, it showed 99.0% homology with Candida butyri .

[Sequence of the ITS region of Candida buttery SH-14 - SEQ ID NO: 3]

agaaaaaatattcttgccgcgcttactgcgcggcgagaataacaccttacacacagtggattttttttaaaagaactattgcgtttggcttggctaacgctgggccagaggattcagtaaacttcaatttttaattgaattgttattttaatatttttgtcaatttgtttgattaaattcaaaataatcttcaaaactttcaacaacggatctcttggttctcgcatcgatgaagaacgcagcgaaatgcgataagtaatatgaattgcagattttcgtgaatcatcgaatctttgaacgcacattgcgccttatggtattccataaggcatgcctgtttgagcgtcatttctctctcaaacctttgggtttggtattgagtgatactcttagtcggactaggcgtttgcttgaaaagtattggcaagagtgtactggatagtactaactggttattcaatgtattaggtttatccaactcgttgaagtgctggtggtaaatttctagtaacggctcggccttacaacaacaaacaagtttgacctcnaatcaggtaagaatacccgctgaacttaagcatatc

Example 2 . New yeast candy Beauty Lee SH -14 whole genome analysis and exploration of lipase gene

New yeast Candida butyri ) SH-14 was found to be a useful yeast strain for producing lipase through the above Example 1. However, since the information about the genome was insignificant, the whole genome was analyzed. The whole genome was analyzed by obtaining nucleotide sequence with Illumina 1.9, de novo assembly of the obtained nucleotide sequence, and annotation of the gene using Yeast Genome Annotation Pipeline. As a result of the analysis, Candida butyri SH-14 has a total genome size of 10,901,820 bp, consisting of a circular chromosome with an average G + C content of 34% and a total contig of 101, (Fig. 3).

Among the nucleotide sequences obtained through whole gene analysis, Candida butyri In order to obtain the sequence of the lipase of SH-14, a protein sequence predicted to be eight lipases among the annotated proteins was found (Table 1, SEQ ID NOS: 4 to 11), and based on the above eight protein sequences, (Table 2, SEQ ID NOS: 12 to 19). ≪ tb >< TABLE >

The amino acid sequence of the predicted lipase gene Explanation Amino acid sequence SEQ ID NO: LIP1 트리아클리칼 로 lipase precursor mklstflsasiifwratetastplsprsgdnvpkdyqklkdyanlvsfayckelipsklgsndsncpilrcqeeeyknievlnlfdfndfgsigsgytgidhqnkriilafrgtstnrdwlaninvpfkkynplsnlqlkadiecegckvhkgfyefiekhcvalikqvaqlkqkypdyqlvvlghslggvfallsgmefllmdfqplvityaspkvgnkamvrfidqlfetskvaqqsqndydfnhgyirvvhnydmvpmlpprelgyyhggveyhieksnlphnpdsvhnrgqnhfssnkdegpvekleriatslvlvftskehtqyfipitgckd 4 LIP2 LIP-domain-
containing protein micsffrlltivtlviaapttlvpptedpfytapkgfesaelgtvlayrntpapirsiyfevniknswqllvrssdsfgnpsvvvttvfepfnadpsklvsyqvaqdsayldcspsysfmnggglstinnqietvliqtaldqgyyvvspdyeglksaftggiqaghgtldsirgalsssnitgvkkdadtilwgysggslasgwaaalqptyapelasnllgvalggwvtnitatitsvsgtifsglgamgmaglsneytdlygylktampadkyeeftkaysicaaealieynfddwfegedryftdgfkvlneeptysiirnntlgliagqmpeipvfvyhgtldqivpydqaervydiwcdagiksfefatdltaghitelvqgsgaafgwikgmfegtkkpvsgcrktprisnllypgtvrsvtdvvgalldnilgfdigpngenlivennsviskanstrse 5 LIP3 alpha / beta-hydrolase mamlmllfflfeiacgniwvypgshqksgeeertpvpidievyrnmftyahlidisycissttrleepfncdlncekrfpnvtlvyqwyfddsvcgyiattysnifnyeseegkghkktiivslrgtrsifdsytdikvdmvnyynygsniqecgtdckvhrgfykyyintllkiegilrnelqtdddyellivghslggavglllglyyldegfdkitlvtmgqplvgnkqfaefvdnvmgsrlpiehntfnrkffrvihkddivatipsnnrildsysqfdnqiylnclasdtmpsleqvldcfdgdnpqcisgdienyllshnylqihttyfrsmglcgiri 6 LIP4 LIP-domain-
containing protein mmittrilvcfalialtfaapltvlkpsedefyaapdgfedeklgtilkwrktpyqiksiyfpvniknswqllvrsedaignpvavtaslfepyngntsrlvsyqvaedsasfdcapsysfvgggyhtvvakaemiliqgaldqgyyvvapdyegpnavftagvtsgmstinvlravlgegrrnetridpdaevvlwgysggtipsgwaxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxaeivdgtlfsglvpaalnglsaqyeemdeaidkflkpnklekfrsgrnecaidvvfsfayqdtfsgfdtyskdgwtilddpdvrdilaentlgvnvtkkfkpeiplfvyhgildeivpfkdaqrvydvwceegydsfefavsnstghileviegsaaglkwisdrfdgvqptkgchrqtrltnlfypglftgvtdilsalmrnifgspiglydeqiekrsdmsedekllkrlfeftdseifkrivqhsypemakkldl 7 LIP5 LIP-domain-
containing protein miltnllvyfsliacticapmtilkpseddfytapdgfedeklgtilkwrktpyqiksiyfpvniknswqllvrsedaignpvavtaslfepyngntsrlvsyqvaedsasfdcapsysfvgggyhtvvakaemiliqgaldqgyyvvapdyegpnaaftagitsgmstinvlravlsdqrsnetridsdaevvlwgysggtipsgwaasmapwyakdindnlkgaamggwvtnitataeivegslfeglvaaainglaqqyeeinealdvylvpdkletfrsaknecvidvvfsfayqsafsgsdpytldgwgvledpkvkkivnqntlglnvtekfkpeiplfvyhgildeivpfkdaqrvydvwckeginsfefavsnstghllevlegsgaalkwisdrfdgvaptkgchrqtrlsnifypglftgisdilsalvrnvmgspiglydeqvekrsdvtedekllkrlfaytdeeifrrildnsypdmaqnlgy 8 LIP6 LIP-domain-
containing protein mlittkilvyfaliiltsaapmtilkpsedefyaapdgfedeklgtilkwrktpyqiksiyfpvniknswellvrsedaigdpvavtatifepynanssrlvsyqvaedspsfncspsyafvgggyptvvtkaemiliqsalnegyyvvvpdyegpnavfgvgvtsamstinvlravlsdqrrnetridsdaevvlwgysggtipsswaasmapwyakdinqhligaalggwvsnltalmeivegslfeglvpniinglsqqfeeindafddylypskrekfrstkkqcvfdaclsfafqstflgdhpyvkgkwsvleeakvknilenntlglkvtkkfkpeipifafhgikdeivpfkdsqrlydvwceeginsfefavskssghllevvegsgaalawisdrfrgipavkgcerkerltnlfypglfssvsnilmalirnimgspiglydeqakkrsgsteeeiimkrlleytdeeiyrriiethsdmf 9 LIP7 LIP-domain-
containing protein mfkqlfllfsllifaltlptglvppsqdsfysaptgfesaelgtilkfrpspapirsvyfkvkiksswqllvrssdsfgnpsvivttvfepfnadpsklisyqvaqdsasndcspsyafmdgggfetvtsqaemlliqtaldqgyyvvspdyeglkavytggiqsghgtldsltaaltsknitginsdaksilwgysggalaagwaaalqptyapdlkqsllgaalggfvtnvtatvtavnggpfsglvgmgiaglsneypelkdylkeqmypdkyeqfqeiyglctaegalkfaftdyfngpnkyvngigvlanepaasilknntlglvpsqvpdiplfvyhgvidslvpyketervyntwcdegiesfelaadltaghltevlqgsgaafgwitkrfdgkapisgcrktkrltnllypgtvgsvtnlisalftnvlggdigpngesmkpgnseldhffhin 10 LIP8 hypothetical protein CANTEDRAFT_108669 mhmhidepvhkylspmhkhpfpmtfipflvaffmtyectvshsyqflvythkptfekmtqnlnlrnhhkqmtknnerwspirwvlglalvttvvlyihggnifkesedyisnqvmeaadlqgdsfrlkhifqhgagseyyrvhrrlditeeylsrtgldevameadttdvssnsledvyaqndwplafqghnpwnmkmpvrssnhkakirrlterhtpgfldsyldyaievkgnpqklnminlqwdeeedlilpdvhdkeslvmlalmssnayvkfpkddnekkksdwrdvgepwepdenntdiefgwdgdgvrghvfvnevnntvvialkgtsgagipgagedettnndklndnllflcccarvsylwttvcdcyekaytcnqdclekeltrkdryyqaaleiyrnvssiypsnqykiwltghslggslaslvgrtyglpvvafeapgellatqrlhlpqppgypkymehiyhigntadpifmgvcngvsstcnaagyametachtgklcvydvvtdrgwsvnvlnhrihtvvdeiilkynttaecveqppcrdcfnwryvthdddeddepklpnpliphishhstatksqgpshtaydsissssssgssspsssklpekqkclkrtwygwcrewgpadgegdqf 11

Example  3. Protein fusion factor  ( TFP ) To confirm expression of lipase and its activity

Eight lipase genes derived from Candida bauterii SH-14 strain obtained through the entire gene search were obtained from Saccharomyces cerevisiae S. cerevisiae . First, primers STFPL1-F to 8-F (Table 3, SEQ ID Nos. 20 to 27) and STFPL1-R to 8-R (Table 3, SEQ ID NOS: 28 to 35) were used to obtain a lipase gene without self- (SEQ ID NO: 36) and GT50R (Table 3, SEQ ID NO: 37) in order to obtain the respective genes by primary PCR and to introduce sequences complementary to the yeast vector ends. The primers LNK39 Secondary polymerase chain reaction was performed to prepare a vector and a gene capable of in vivo recombination.

primer order SEQ ID NO: STFPL1-F 5'-ctcgccttagataaaagagccagtaccccattgagtc-3 ' 20 STFPL2-F 5'-ctcgccttagataaaagacctaccactttagttcct-3 ' 21 STFPL3-F 5'-ctcgccttagataaaagaaatatttgggtttatcct-3 ' 22 STFPL4-F 5'-ctcgccttagataaaagagcaccattgacagtttta-3 ' 23 STFPL5-F 5'-ctcgccttagataaaagagctccaatgacgatcttg-3 ' 24 STFPL6-F 5'-ctcgccttagataaaagagctccaatgacaatcttgaa-3 ' 25 STFPL7-F 5'-ctcgccttagataaaagacttccaactggtttggttcc-3 ' 26 STFPL8-F 5'-ctcgccttagataaaagaatgcatatgcatatcgat-3 ' 27 STFPL1-R 5'-cactccgttcaagtcgacttaatctttgcaaccagtg-3 ' 28 STFPL2-R 5'-cactccgttcaagtcgacttattcacttctagtaga-3 ' 29 STFPL3-R 5'-cactccgttcaagtcgacttagattcttattccaca-3 ' 30 STFPL4-R 5'-cactccgttcaagtcgacttataagtctaattttttgg-3 ' 31 STFPL5-R 5'-cactccgttcaagtcgacttaatagcccaaattttgag-3 ' 32 STFPL6-R 5'-cactccgttcaagtcgacttaaaacatatctatgtg-3 ' 33 STFPL7-R 5'-cactccgttcaagtcgacttaattgatatggaagaagt-3 ' 34 STFPL8-R 5'-cactccgttcaagtcgacttaaaattgatcaccttctc-3 ' 35 LNK39 5'-ggccgcctcggcctctgctggcctcgccttagataaaaga-3 ' 36 GT50R Gt; 37

Next, the amplified gene was ligated with a linearized vector containing 24 kinds of protein secretion fusion factors (TFP, Korean Patent No. 10-0975596) that helped express the protein secretion, and yeast strain Y2805 (Mat alpha pep4 :: HIS3 prb1 can1 his3-200 ura3-52) to produce a transformant through intracellular recombination. The amino acid sequence (Table 4) and the nucleotide sequence (Table 5) of the protein secretion fusion factor used in the present invention are as follows.

Amino acid sequence of protein secretion fusion factor Protein secretion factor Amino acid sequence SEQ ID NO: TFP 1 MFNRFNKFQAAVALALLSRGALGDSYTNSTSSADLSSITSVSSASASATASDSLSSSDGTVYLPSTTISGDLTVTGKVIATEAVEVAAGGKLTLLDGEKYVFSSEAASASAGLALDKR SEQ ID NO: 38 TFP 2 MTPYAVAITVALLIVTVSALQVNNSCVAFPPSNLRGKNGDGTNEQYATALLSIPWNGPPESSRDINLIELEPQVALYLLENYINHYYNTTRDNKCPNNHYLMGGQLGSSSDNRSLNEAASASAGLALDKR SEQ ID NO: 39 TFP 3 MQFKNVALAASVAALSATASAEGYTPGEPWSTLTPTGSISCGAAEYTTTFGIAVQAITSSKAKRDVISQIGDGQVQATSAATAQATDSQAQATTTATPTSSEKMAASASAGLALDKR SEQ ID NO: 40 TFP 4 MRFAEFLVVFATLGGGMAAPVESLAGTQRYLVQMKERFTTEKLCALDDKAASASAGLALDKR SEQ ID NO: 41 TFP 5 MFNRFNKFQAAVALALLSRGALGAPVNTTEDETALIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVAASASAGLALDKR SEQ ID NO: 42 TFP 6 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVAASASAGLALDKR SEQ ID NO: 43 TFP 7 MVFGQLYALFIFTLSCCISKTVQADSSKESSSFISFDKESNWDTISTISSTADVISSVDSAIAVFEFDNFSLLDSLMIDEEYPFFNRFFANDVSLTVHDDSPLNISQSLSPIMEQFTVDELPESASDLLYEYSLDDKSIVLFKFTSDAYDLKKLDEFIDSCLSFLEDKSGDNLTVVINSLGWAFEDEDGDDEYATEETLSHHDNNKGKEGDDLAASASAGLALDKR SEQ ID NO: 44 TFP 8 MLQSVVFFALLTFASSVSAIYSNNTVSTTTTLAPSYSLVPQETTISYADDLAASASAGLALDKR SEQ ID NO: 45 TFP 9 MKFSTAVTTLISSGAIVSALPHVDVHQEDAHQHKRAVAYKYVYETVVVDSDGHTVTPAASEVATAATSAIITTSVLAPTSSAAAADSSASIAVSSAALAKNEKISDAAASATASTSQGASSSSYLAASASAGLALDKR SEQ ID NO: 46 TFP 10 MNWLFLVSLVFFCGVSTHPALAMSSNRLLKLANKSPKKIIPLKDSSFENILAPPHENAYIVALFTATAPEIGCSLCLELESEYDTIVASWFDDHPDAKSSNSDTSIFFTKVNLEDPSKTIPKAFQFFQLNNVPRLFIFKLNSPSILDHSVISISTDTGSERMKQIIQAIKQFSQVNDFSLHLPVGLAASASAGLALDKR SEQ ID NO: 47 TFP 11 MKFSSVTAITLATVATVATAKKGEHDFTTTLTLSSDGSLTTTTSTHTTHKYGKFNKTSKSKTPLAASASAGLALDKR SEQ ID NO: 48 TFP 12 MASFATKFVIACFLFFSASAHNVLLPAYGRRCFFEDLSKGDELSISFQFGDRNPQSSSQLTGDFIIYGPERHEVLKTVRELAASASAGLALDKR SEQ ID NO: 49 TFP 13 MQYKKTLVASALAATTLAAYAPSEPWSTLTPTATYSGGVTDYASTFGIAVQPISTTSSASSAATTASSKAKRAASQIGDGQVQAATTTASVSTKSTAAAVSQIGDGQIQATTKTTAAAVSQIGDGQIQATTKTTSAKTTAAAVSQISDGQIQATTTTLAPLAASASAGLALDKR SEQ ID NO: 50 TFP 14 MQFKNALTATAILSASALAANSTTSIPSSCSIGTSATATAQATDSQAQATTTAPLAASASAGLALDKR SEQ ID NO: 51 TFP 15 ≪ SEQ ID NO: 52 TFP 16 MKLSALLALSASTAVLAAPAVHHSDNHHHNDKRAVVTVTQYVNADGAVVIPAATTATSAAADGKVESVAAATTTLSSTAAAATTLAASASAGLALDKR SEQ ID NO: 53 TFP 17 MKLSTVLLSAGLASTTLAQFSNSTSASSTDVTSSSSSTSSASVTITSSEAPESDNGTSTAAPTETSTEAPTTAIPTNGTSTEAPTTAIPTNGTSTEAPTDTTTEAPTTALPTNGTSTEAPTDTTTEAPTTGLPTNGTTSAFPPTTSLPPSNTTTTPPYNPSTDYTTDYTVVTEYTTYCPERAASASAGLALDKR SEQ ID NO: 54 TFP 18 MRFSTTLATAATALFFTASQVSAIGELAFNLGVKNNDGTCKSTSDYETELQALKSYTSTVKVYAASDCNTLQNLGPAAEAEGFTIFVGVWPLAASASAGLALDKR SEQ ID NO: 55 TFP 19 MRLSNLIASASLLSAATLAAPANHEHKDKRAVVTTTVQKQTTIIVNGAASTPVAALEENAVVNSAPAAATSTTSSAASVATAASSSENNSQVSAAASPASSSAATSTQSSLAASASAGLALDKR SEQ ID NO: 56 TFP 20 MQFSTVASIAAVAAVASAAANVTTATVSQESTTLVTITSCEDHVCSETVSPALVSTATVTVDDVITQYTTWCPLTTEAPKNGTSTAAPVTSTEAPKNTTSAAPTHSVTSYTGAAAKALPAAGALLAASASAGLALDKR SEQ ID NO: 57 TFP 21 MKFSSALVLSAVAATALAESITTTITATKNGHVYTKTVTQDATFVWGGEDSYASSTSAAESSAAETSAAETSAAATTSAAATTSAAETSSAAETSSADEGSGSSITTTITATKNGHVYTKTVTQDATFVWTGEGSSNTWSPSSTSTSSEAATSSASTTATTLLAASASAGLALDKR SEQ ID NO: 58 TFP 22 MKFQVVLSALLACSSAVVASPIENLFKYRAVKASHSKNINSTLPAWNGSNSSNVTYANGTNSTTNTTTAESSQLQIIVTGGQVPITNSSLTHTNYTRLFNSSSALNITELYNVARVVNETIQDNLAASASAGLALDKR SEQ ID NO: 59 TFP 23 MRAITLLSSVVSLALLSKEVLATPPACLLACVAQVGKSSSTCDSLNQVTCYCEHENSAVKKCLDSICPNNDADAAYSAFKSSCSEQNASLGDSSGSASSSVLAASASAGLALDKR SEQ ID NO: 60 TFP 24 MKLSTVLLSAGLASTTLAQFSNSTSASSTDVTSSSSISTSSGSVTITSSEAPESDNGTSTAAPTETSTEAPTTAIPTNGTSTEAPTTAIPTNGTSTEAPTDTTTEAPTTALPTNGTSTEAPTDTTTEAPTTGLPTNGTTSAFPPTTSLPPSNTTTTLAASASAGLALDKR SEQ ID NO: 61

The nucleotide sequence of the protein secretion fusion factor Protein secretion factor Nucleotide sequence TFP 1 ATGTTCAATCGTTTTAACAAATTCCAAGCTGCTGTCGCTTTGGCCCTACTCTCTCGCGGCGCTCTCGGTGACTCTTACACCAATAGCACCTCCTCCGCAGACTTGAGTTCTATCACTTCCGTCTCGTCAGCTAGTGCAAGTGCCACCGCTTCCGACTCACTTTCTTCCAGTGACGGTACCGTTTATTTGCCATCCACAACAATTAGCGGTGATCTCACAGTTACTGGTAAAGTAATTGCAACCGAGGCCGTGGAAGTCGCTGCCGGTGGTAAGTTGACTTTACTTGACGGTGAAAAATACGTCTTCTCATCTGAGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 62) TFP 2 ATGACGCCCTATGCAGTAGCAATTACCGTGGCCTTACTAATTGTAACAGTGAGCGCACTCCAGGTCAACAATTCATGTGTCGCTTTTCCGCCATCAAATCTCAGGGGCAAGAATGGAGACGGTACTAATGAACAGTATGCAACTGCACTACTTTCTATTCCCTGGAATGGGCCTCCTGAGTCATCGAGGGATATTAATCTTATCGAACTCGAACCGCAAGTTGCACTCTATTTGCTCGAAAATTATATTAACCATTACTACAACACCACAAGAGACAATAAGTGCCCTAATAACCACTACCTAATGGGAGGGCAGTTGGGTAGCTCATCGGATAATAGGAGTTTGAACGAGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 63) TFP 3 ATGCAATTCAAAAACGTCGCCCTAGCTGCCTCCGTTGCTGCTCTATCCGCCACTGCTTCTGCTGAAGGTTACACTCCAGGTGAACCATGGTCCACCTTAACCCCAACCGGCTCCATCTCTTGTGGTGCAGCCGAATACACTACCACCTTTGGTATTGCTGTTCAAGCTATTACCTCTTCAAAAGCTAAGAGAGACGTTATCTCTCAAATTGGTGACGGTCAAGTCCAAGCCACTTCTGCTGCTACTGCTCAAGCCACCGATAGTCAAGCCCAAGCTACTACTACCGCTACCCCAACCAGCTCCGAAAAGATGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 64) TFP 4 ATGAGATTTGCAGAATTCTTGGTGGTATTTGCCGTTAGGCGGGGGGATGGCTGCACCGGTTGAGTCTCTGGCCGGGACCCAACGGTATCTGGTGCAAATGAAGGAGCGGTTCACCACAGAGAAGCTGTGTGCTTTGGACGACAAGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 65) TFP 5 ATGTTCAATCGTTTTAACAAATTCCAAGCTGCTGTCGCTTTGGCCCTACTCTCTCGCGGCGCTCTCGGTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACTAATTCCGGCTGAAGCTGTCATCGGTTACTTAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 66) TFP 6 ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTTAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 67) TFP 7 ATGGTGTTCGGTCAGCTGTATGCCCTTTTCATCTTCACGTTATCATGTTGTATTTCCAAAACTGTGCAAGCAGATTCATCCAAGGAAAGCTCTTCCTTTATTTCGTTCGACAAAGAGAGTAACTGGGATACCATCAGCACTATATCTTCAACGGCAGATGTTATATCATCCGTTGACAGTGCTATCGCTGTTTTTGAATTTGACAATTTCTCATTATTGGACAGCTTGATGATTGACGAAGAATACCCATTCTTCAATAGATTCTTTGCCAATGATGTCAGTTTAACTGTTCATGACGATTCGCCTTTGAACATCTCTCAATCATTATCTCCCATTATGGAACAATTTACTGTGGATGAATTACCTGAAAGTGCCTCTGACTTACTATATGAATACTCCTTAGATGATAAAAGCATCGTTTTGTTCAAGTTTACCTCGGATGCCTACGATTTGAAAAAATTAGATGAATTTATTGATTCTTGCTTATCGTTTTTGGAAGATAAATCTGGCGACAATTTGACTGTGGTTATTAACTCTCTTGGTTGGGCTTTTGAAGATGAAGATGGTGACGATGAATATGCAACAGAAGAGACTTTGAGCCATCATGATAACAACAAGGGTAAAGAAGGCGACGATCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 68) TFP 8 ATGCTTCAATCCGTTGTCTTTTTCGCTCTTTTAACCTTCGCAAGTTCTGTGTCAGCGATTTATTCAAACAATACTGTTTCTACAACTACCACTTTAGCGCCCAGCTACTCCTTGGTGCCCCAAGAGACTACCATATCGTACGCCGACGACCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 69) TFP 9 ATGAAATTCTCAACTGCCGTTACTACGTTGATTAGTTCTGGTGCCATCGTGTCTGCTTTACCACACGTGGATGTTCACCAAGAAGATGCCCACCAACATAAGAGGGCCGTTGCGTACAAATACGTTTACGAAACTGTTGTTGTCGATTCTGATGGCCACACTGTAACTCCTGCTGCTTCAGAAGTCGCTACTGCTGCTACCTCTGCTATCATTACAACATCTGTGTTGGCTCCAACCTCCTCCGCAGCCGCTGCGGATAGCTCCGCTTCCATTGCTGTTTCATCTGCTGCCTTAGCCAAGAATGAGAAAATCTCTGATGCCGCTGCATCTGCCACTGCCTCAACATCTCAAGGGGCATCCTCCTCATCCTACCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 70) TFP 10 ATGAATTGGCTGTTTTTGGTCTCGCTGGTTTTCTTCTGCGGCGTGTCAACCCATCCTGCCCTGGCAATGTCCAGCAACAGACTACTAAAGCTGGCTAATAAATCTCCCAAGAAAATTATACCTCTGAAGGACTCAAGTTTTGAAAACATCTTGGCACCACCTCACGAAAATGCCTATATAGTTGCTCTGTTTACTGCCACAGCGCCCGAAATTGGCTGTTCTCTGTGTCTCGAGCTAGAATCCGAATACGACACCATAGTGGCCTCCTGGTTTGATGATCATCCGGATGCAAAATCGTCCAATTCCGATACATCTATTTTCTTCACAAAGGTCAATTTGGAGGACCCTTCTAAGACCATTCCTAAAGCGTTCCAGTTTTTCCAACTAAACAATGTTCCTAGATTGTTCATCTTCAAACTAAACTCTCCCTCTATTCTGGACCACAGCGTGATCAGTATTTCCACTGATACTGGCTCAGAAAGAATGAAGCAAATCATACAAGCCATTAAGCAGTTCTCGCAAGTAAACGACTTCTCTTTACACTTACCTGTGGGTCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 71) TFP 11 ATGAAGTTCTCTTCTGTTACTGCTATTACTCTAGCCACCGTTGCCACCGTTGCCACTGCTAAGAAGGGTGAACATGATTTCACTACCACTTTAACTTTGTCATCGGACGGTAGTTTAACTACTACCACCTCTACTCATACCACTCACAAGTATGGTAAGTTCAACAAGACTTCCAAGTCCAAGACCCCCCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 72) TFP 12 ATGGCCTCATTTGCTACTAAGTTTGTCATTGCTTGCTTCCTGTTCTTCTCGGCGTCCGCCCATAATGTCCTTCTTCCAGCTTATGGCCGTAGATGCTTCTTCGAAGACTTGAGTAAGGGTGACGAGCTCTCCATTTCGTTCCAGTTCGGTGATAGAAACCCTCAATCCAGTAGCCAGCTGACTGGTGACTTTATCATCTACGGGCCGGAAAGACATGAAGTTTTGAAAACGGTTAGGGAACTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 73) TFP 13 ATGCAATACAAAAAGACTTTGGTTGCCTCTGCTTTGGCCGCTACTACATTGGCCGCCTATGCTCCATCTGAGCCTTGGTCCACTTTGACTCCAACAGCCACTTACAGCGGTGGTGTTACCGACTACGCTTCCACCTTCGGTATTGCCGTTCAACCAATCTCCACTACATCCAGCGCATCATCTGCAGCCACCACAGCCTCATCTAAGGCCAAGAGAGCTGCTTCCCAAATTGGTGATGGTCAAGTCCAAGCTGCTACCACTACTGCTTCTGTCTCTACCAAGAGTACCGCTGCCGCCGTTTCTCAGATCGGTGATGGTCAAATCCAAGCTACTACCAAGACTACCGCTGCTGCTGTCTCTCAAATTGGTGATGGTCAAATTCAAGCTACCACCAAGACTACCTCTGCTAAGACTACCGCCGCTGCCGTTTCTCAAATCAGTGATGGTCAAATCCAAGCTACCACCACTACTTTAGCCCCTCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 74) TFP 14 ATGCAATTCAAGAACGCTTTGACTGCTACTGCTATTCTAAGTGCCTCCGCTCTAGCTGCTAACTCAACTACTTCTATTCCATCTTCATGTAGTATTGGTACTTCTGCCACTGCTACTGCTCAAGCCACCGATAGTCAAGCCCAAGCTACTACTACCGCACCCCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 75) TFP 15 ATGGTATCGAAGACTTGGATATGTGGCTTCATCAGTATAATTACAGTGGTACAGGCCTTGTCCTGCGAGAAGCATGATGTATTGAAAAAGTATCAGGTGGGAAAATTTAGCTCACTAACTTCTACGGAAAGGGATACTCCGCCAAGCACAACTATTGAAAAGTGGTGGATAAACGTTTGCGAAGAGCATAACGTAGAACCTCCTGAAGAATGTAAAAAAAATGACATGCTATGTGGTTTAACAGATGTCATCTTGCCCGGTAAGGATGCTATCACCACTCAAATTATAGATTTTGACAAAAACATTGGCTTCAATGTCGAGGAAACTGAGAGTGCGCTTACATTGACACTAAACGGCGCTACGTGGGGCGCCAATTCTTTTGACGCAAAACTAGAATTTCAGTGTAATGACAATATGAAACAAGACGAACTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 76) TFP 16 ATGAAATTATCCGCTCTATTAGCTTTATCAGCCTCCACCGCCGTCTTGGCCGCTCCAGCTGTCCACCATAGTGACAACCACCACCACAACGACAAGCGTGCCGTTGTCACCGTTACTCAGTACGTCAACGCAGACGGCGCTGTTGTTATTCCAGCTGCCACCACCGCTACCTCGGCGGCTGCTGATGGAAAGGTCGAGTCTGTTGCTGCTGCCACCACTACTTTGTCCTCGACTGCCGCCGCCGCTACAACCCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 77) TFP 17 ATGAAATTATCAACTGTCCTATTATCTGCCGGTTTAGCCTCGACTACTTTGGCCCAATTTTCCAACAGTACATCTGCTTCTTCCACCGATGTCACTTCCTCCTCTTCCATCTCCACTTCCTCTGGCTCAGTAACTATCACATCTTCTGAAGCTCCAGAATCCGACAACGGTACCAGCACAGCTGCACCAACTGAAACCTCAACAGAGGCTCCAACCACTGCTATCCCAACTAACGGTACCTCTACTGAAGCTCCAACCACTGCTATCCCAACTAACGGTACCTCTACTGAAGCTCCAACTGATACTACTACTGAAGCTCCAACCACCGCTCTTCCAACTAACGGTACTTCTACTGAAGCTCCAACTGATACTACTACTGAAGCTCCAACCACCGGTCTTCCAACCAACGGTACCACTTCAGCTTTCCCACCAACTACATCTTTGCCACCAAGCAACACTACCACCACTCCTCCTTACAACCCATCTACTGACTACACCACTGACTACACTGTAGTCACTGAATATACTACTTACTGTCCGGAACGGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 78) TFP 18 ATGCGTTTCTCTACTACACTCGCTACTGCAGCTACTGCGCTATTTTTCACAGCCTCCCAAGTTTCAGCTATTGGTGAACTAGCCTTTAACTTGGGTGTCAAGAACAACGATGGTACTTGTAAGTCCACTTCCGACTATGAAACCGAATTACAAGCTTTGAAGAGCTACACTTCCACCGTCAAAGTTTACGCTGCCTCAGATTGTAACACTTTGCAAAACTTAGGTCCTGCTGCTGAAGCTGAGGGATTTACTATCTTTGTCGGTGTTTGGCCACTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 79) TFP 19 ATGCGTCTCTCTAACCTAATTGCTTCTGCCTCTCTTTTATCTGCTGCTACTCTTGCTGCTCCCGCTAACCACGAACACAAGGACAAGCGTGCTGTGGTCACTACCACTGTTCAAAAACAAACCACTATCATTGTTAATGGTGCCGCTTCAACTCCAGTTGCTGCTTTGGAAGAAAATGCTGTTGTCAACTCCGCTCCAGCTGCCGCTACCAGTACAACATCGTCTGCTGCTTCTGTAGCTACCGCTGCTTCCTCTTCTGAGAACAACTCACAAGTTTCTGCTGCCGCATCTCCAGCCTCCAGCTCTGCTGCTACATCTACTCAATCTTCTCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 80) TFP 20 ATGCAATTTTCTACTGTCGCTTCTATCGCCGCTGTCGCCGCTGTCGCTTCTGCCGCTGCTAACGTTACCACTGCTACTGTCAGCCAAGAATCTACCACTTTGGTCACCATCACTTCTTGTGAAGACCACGTCTGTTCTGAAACTGTCTCCCCAGCTTTGGTTTCCACCGCTACCGTCACCGTCGATGACGTTATCACTCAATACACCACCTGGTGCCCATTGACCACTGAAGCCCCAAAGAACGGTACTTCTACTGCTGCTCCAGTTACCTCTACTGAAGCTCCAAAGAACACCACCTCTGCTGCTCCAACTCACTCTGTCACCTCTTACACTGGTGCTGCTGCTAAGGCTTTGCCAGCTGCTGGTGCTTTGCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 81) TFP 21 ATGAAATTCTCTTCCGCTTTGGTTCTATCTGCTGTTGCCGCTACTGCTCTTGCTGAGAGTATCACCACCACCATCACTGCCACCAAGAACGGTCATGTCTACACTAAGACTGTCACCCAAGATGCTACTTTTGTTTGGGGTGGTGAAGACTCTTACGCCAGCAGCACTTCTGCCGCTGAATCTTCTGCCGCCGAAACTTCTGCCGCCGAAACCTCTGCTGCCGCTACCACTTCTGCTGCCGCTACCACTTCTGCTGCTGAGACTTCTTCTGCTGCTGAGACTTCTTCTGCTGATGAAGGTTCTGGTTCTAGTATCACTACCACTATCACTGCCACCAAGAACGGTCACGTCTACACTAAGACTGTCACCCAAGATGCTACTTTTGTCTGGACTGGTGAAGGCAGCAGCAACACCTGGTCTCCAAGTAGTACCTCTACCAGCTCAGAAGCTGCTACCTCTTCTGCTTCAACCACTGCAACCACCCTGCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 82) TFP 22 ATGAAGTTCCAAGTTGTTTTATCTGCCCTTTTGGCATGTTCATCTGCCGTCGTCGCAAGCCCAATCGAAAACCTATTCAAATACAGGGCAGTTAAGGCATCTCACAGTAAGAATATCAACTCCACTTTGCCGGCCTGGAATGGGTCTAACTCTAGCAATGTTACCTACGCTAATGGAACAAACAGTACTACCAATACTACTACTGCCGAAAGCAGTCAATTACAAATCATTGTAACAGGTGGTCAAGTACCAATCACCAACAGTTCTTTGACCCACACAAACTACACCAGATTATTCAACAGTTCTTCTGCTTTGAACATTACCGAATTGTACAATGTTGCCCGTGTTGTTAACGAAACGATCCAAGATAACCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 83) TFP 23 ATGCGTGCCATCACTTTATTATCTTCAGTCGTTTCTTTGGCATTGTTGTCGAAGGAAGTCTTAGCAACACCTCCAGCTTGTTTATTGGCCTGTGTTGCGCAAGTCGGCAAATCCTCTTCCACATGTGACTCTTTGAATCAAGTCACCTGTTACTGTGAACACGAAAACTCCGCCGTCAAGAAATGTCTAGACTCCATCTGCCCAAACAATGACGCTGATGCTGCTTATTCTGCTTTCAAGAGTTCTTGTTCCGAACAAAATGCTTCATTGGGCGATTCCAGCGGCAGTGCCTCCTCATCCGTTCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 84) TFP 24 ATGAAATTATCAACTGTCCTATTATCTGCCGGTTTGGCCTCGACTACTTTGGCCCAATTTTCCAACAGTACATCTGCTTCTTCCACCGATGTCACTTCCTCCTCTTCCATCTCCACTTCCTCTGGCTCAGTAACTATCACATCTTCTGAAGCTCCAGAATCCGACAACGGTACCAGCACAGCTGCACCAACTGAAACCTCAACAGAGGCTCCAACCACTGCTATCCCAACTAACGGTACCTCTACTGAAGCTCCAACCACTGCTATCCCAACTAACGGTACCTCTACTGAAGCTCCAACTGATACTACTACTGAAGCTCCAACCACCGCTCTTCCAACTAACGGTACTTCTACTGAAGCTCCAACTGATACTACTACTGAAGCTCCAACCACCGGTCTTCCAACCAACGGTACCACTTCAGCTTTCCCACCAACTACATCTTTGCCACCAAGCAACACTACCACCACTCTGGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA (SEQ ID NO: 85)

Since the polymerase chain reaction products amplified with the primers LNK39 and GT50R primers contain the same sequences of more than 40 base pairs with the linearized vector ends, when introduced into yeast cells together with the linearized vector, crossing occurs in the cells, and a circular plasmid vector (FIG. 4). Transformants transformed through intracellular recombination were grown on a selective medium without uracil (0.67% amino acid-deficient yeast substrate, 0.77% uracil deficient nutritional supplement, 2% glucose), thereby selecting transformants . Expression of all 8 selected lipase genes was carried out on YPT medium and primary genes were selected. As a result, it was confirmed that only three genes of CBLIP2 , CBLIP5 , and CBLIP7 among the eight lipases form a transparent ring in the tributyrin medium to exhibit lipase activity. Therefore , in order to confirm the activity and the protein of the three genes ( CBLIP2, CBLIP5 , and CBLIP7 ) , the cells were cultured in YPDG (1% yeast extract, 2% peptone, 1% glucose, 1% galactose) for 40 hours, centrifuged, . 0.6 ml of the supernatant was precipitated with 0.4 ml of acetone and subjected to SDS-PAGE analysis (Figs. 5 to 7). FIGS. 5 to 7 are electrophoresis images showing the result of SDS-PAGE analysis of the culture supernatant of 24 transformants formed by introducing lipase into yeast strain Y2805. As a result, Bands of different sizes were observed. This was a significant difference from the protein size deduced from the lipase gene size, which contained N-glycosylation inducing sequences on all three lipase protein sequences, presumably due to the sugar chain addition. Thus, lipase expressed from each of the transformants was treated with an endo-enzyme, which is a sugar chain-removing enzyme, to form an expected size protein (FIGS. 5 to 7). However, it was confirmed that CBLIP2 was expressed at a size larger than the expected size, and the size was differently expressed in each TFP (FIG. 5). This seems to be due to the fact that TFP is not isolated properly but secreted in a fused form. However, the expression of CBLIP2 was found to be the best among the three genes.

To confirm the activity of the secreted protein from each transformant, 20 μl of the enzyme solution was added to 0.98 ml of a substrate solution (50 mM Tris-HCl buffer (pH 7.5): ethanol: 50 mM p-nitrophenyl butyrate (PNPP) or palmitate (PNPP) = 380: 19: 1) at 30 ° C for 5 minutes, and then the absorbance at 410 nm was measured. As a result, CBLIP2 showed good activity at TFP 11, 13, 19, and 23, among which TFP 19 was the best, and single colonies were selected therefrom. Among the three substrates, the short-length PNPB activity was the best, and the PNPD activity was the worst (Fig. 8). CBLIP5 had good activity at TFP 8, 9, 13, and 19, among which TFP 8 was the best, and single colony selection was performed therefrom. CBLIP5 was found to have the best activity of PNPD among the three substrates (Figure 9). CBLIP7 had good activity at TFP 4, 8, 13, 20, 21, 22, 23, and 24, among which TFP 8 was the best, and single colony selection was performed from this. CBLIP7 was found to have the best activity of PNPP, the longest of the three substrates (Fig. 10). The enzyme activity was defined as 1 unit of enzyme capable of producing 1 μM nitrophenol at about 1 minute.

Example  4: CBLIP2 , CBLIP5 And CBLIP7  The self-signal of the gene, MFα  (mating factor alpha) secretion signal and protein secretion factor as fusion partners

In order to confirm the lipase secretion inducing ability of the protein secretion factor selected in the present invention, the expression level of the protein expressed by MFα, the most widely used yeast secretory signal and yeast, was compared. CBLIP2 , CBLIP5 And In order to express the gene using self-secretion signal of CBLIP7 gene, each gene was subjected to polymerase chain reaction with primers of SEQ ID NOS: 86 to 88 / SEQ ID NOS: 89 to 91 (Table 6) Respectively.

primer Base sequence SEQ ID NO: CBSPlip2-F 5'-tccaaaaaaaaagtaagaatttttgaaaattcaaggatccatgatctgttcattctttag-3 ' 86 CBSPlip5-F 5'-tccaaaaaaaaagtaagaatttttgaaaattcaaggatccatgattttaacgaatctt-3 ' 87 CBSPlip7-F 5'-tccaaaaaaaaagtaagaatttttgaaaattcaaggatccatgttcaaacagcttttc-3 ' 88 CBSPlip2-R 5'-aatatatatatatatattgtcactccgttcaagtcgacctattcacttctagtagaat-3 ' 89 CBSPlip5-R 5'-aatatatatatatatattgtcactccgttcaagtcgacttaatagcccaaattttg-3 ' 90 CBSPlip7-R 5'-aatatatatatatatattgtcactccgttcaagtcgacctaattgatatggaagaa-3 ' 91

Then, the above-mentioned fragment was transformed into YEGα-HIR525 vector and Y2805 strain digested with BamH1 / Sal1 to generate a circular vector through in vivo recombination, and a single vector with well-generated vector was selected on UD medium . To use the MFa secretion signal, the same strain was transformed with the TFP 6 vector and single colonies were screened. (TFP19- CBLIP2 , TFP8- CBLIP5 , and TFP8- CBLIP7 ) were cultured in YPDG medium for 40 hours after single transfection using self-secretory signal or MFa secretion signal and the highest secreted and selected single transformants The cells were removed by centrifugation and a culture supernatant was obtained. To confirm the activity of the secreted protein from each transformant, 20 μl of the enzyme solution was added to 0.98 ml of a substrate solution (50 mM Tris-HCl buffer (pH 7.5): ethanol: 50 mM p-nitrophenyl butyrate (PNPP) or palmitate (PNPP) = 380: 19: 1) at 30 ° C for 5 minutes, and then the absorbance at 410 nm was measured. As a result, As shown in Table 7, expression using all TFPs was significantly better than that expressed by self-secretory signal or MFa secretion signal. Since CBLP2 activity was the best among these , CBLIP2 was purified and analyzed for protein characteristics.

CBLIP2 Lipase activity
(U / min / ml) CBLIP5 Lipase activity
(U / min / ml) CBLIP7 Lipase activity
(U / min / ml) TFP19 810 TFP8 550 TFP8 210 Self-secretory signal 630 Self-secretory signal 370 Self-secretory signal 65 MFa secretion signal 150 MFa secretion signal 50 MFa secretion signal 60

Example  5: Oil prices Pear form  Lipase through fermentation ( CBLIP2 ) Mass production

Using the Example 4 in which the recombinant yeast strain transformed with the selected Y2805 / YGa-ST19- CBLIP2 through to mass production of the lipase was to perform a fed-batch culture in a 5 L fermenter. However, in order to express the GAL10 promoter in the wild-type yeast strain, galactoside, which is an expensive inducer, is required. Therefore, a gal80 mutant strain, Y2805Δgal80, which can induce expression by glucose alone, was used. Thus, the lipase gene was replaced with Y2805Δgal80 ( Mat α pep4 :: HIS3 gal80 :: Tc190 , pRB1 ura3 can1 his3-200 -52) separated from the strain obtained in Example 4 for expression of the plasmid in the strain, YGaST19- CBLip2 vector transformants after a lipase activity to give a good transformant in Y2805Δgal80. The cells were cultured for one step in 50 ml of a minimal liquid medium (yeast nitrogen substrate lacking 0.67% amino acid, 0.5% casamino acid, 2% glucose) before entering the present culture, and then 200 ml of YPD liquid medium (1% yeast Extract, 2% peptone, 2% glucose) and then inoculated into the culture medium and cultured at 30 ° C for 48 hours. (15% yeast extract, 30% glucose) according to the cell growth rate. After 48 hours of incubation, the cell concentration reached approximately 180 on an OD ₆₀₀ basis. To identify the lipase secreted in the medium, the supernatant was taken by time, analyzed by SDS-PAGE, and the protein activity was checked at intervals of 12 hours. As a result, the activity reached about 230 KU / L / min at 48 hours (FIG. 11). Proteins produced by fermentation were concentrated twice by ultrafiltration (molecular weight cut off 30,000) into 50 mM Tris buffer solution (pH 7.5).

Example  6: Lipase ( CBLIP2 ) Tablets

The fermentation concentrate obtained by the method of Example 5 was purified using an ion exchange method. The protein of CBLIP2 was purified by using an anion exchange column (Q FF column) because the protein was negatively charged when using 20 mM sodium acetate buffer (pH 5.5) at a pH of 4.6. The resin was sufficiently parallelized with 20 mM sodium acetate buffer (pH 5.5), and 6 ml of the fermentation concentrate was loaded and the protein was eluted with a gradient of 1 M NaCl at 0 ° C at a flow rate of 2 ml / min 12a). Proteins of each fraction were confirmed by SDS-PAGE, and the purified lipase was confirmed in fractions 8, 9, and 10 (FIG. 12B). The activity of each fraction was measured, and only fractions 8, 9 and 10 (FIG. 12C) showing high activity were recovered. After desalting using a PD-10 desalting column, the activity of the purified solution thus obtained was measured by PNPP And the activity and protein content of the fermented concentrate were compared. As a result, the purified protein was reduced to about 3-fold and the amount of protein was reduced by 5-fold, but the activity was not significantly reduced, and the purified product was found to have a high specific activity (Table 8).

Purification step Total volume
(Ml) Enzyme activity
(U / ml) Protein concentration (mg / ml) Specific activity
(U / mg) Refining factor
(ship) yield
(%) Fermented concentrate 6 196964 15.9 12387.7 One 100 Q FF 5.5 147179.9 3.8 38731.5 3.1 68.5

Example  7: Biochemical characterization of expressed recombinant lipase

7-1: Investigation of effect of pH and temperature

The effect of pH and temperature on enzyme activity was investigated using purified enzyme solutions to investigate the properties of the purified lipase by the method of Example 6. In order to investigate the effect of temperature, each activity was measured after reacting in the range of 20 to 80 캜 at intervals of 10 캜, and relative activity was shown based on the highest activity. As a result, it was confirmed that it exhibited the highest activity at 50 to 60 ° C (FIG. 13).

To investigate the effect of pH on the enzyme activity, sodium citrate buffer (pH 5.0), potassium phosphate buffer (pH 6.0 and 7.0), Tris-HCl buffer (pH 8.0 ), Glycine-NaOH buffer solution (pH 9.0 and 10.0) at 50 ° C, and enzyme activity was measured. Relative activity at pH was found to be high at basic pH 8.0, 9.0 and 10.0, and the highest activity was observed at pH 8.0. Whereas it exhibited relatively low activity at acidic and neutral pH (Fig. 14A).

In addition, to examine the stability against pH, the purified enzyme solution was allowed to stand at each pH for 1 hour, and residual activity of the remaining enzyme was measured. As a result, it was confirmed that activity was not affected at most pH (FIG. 14B).

7-2: Metal ion , Inhibitors, surfactants and solvents

The effect of metal ion, inhibitor, surfactant and solvent on enzyme activity was investigated using the purified enzyme solution used above.

A first metal ion of ^{1 mM (Ba 2 +, Ca} 2 +, Mg 2 +, K +, Li +, Hg 2 +, Na +) was added to 1 hour to the back, left residual activity of the enzyme was allowed to stand for Were measured. As a result, most of the metal ions were not affected (FIG. 15A).

Second, the effects of inhibitors and surfactants were investigated. (EDTA, SDS, Tween 20, Tween 80, Triton X-100, PMSF, beta-mercaptoethanol) was added in the same manner as in the above metal ion effect measurement method, After standing, the residual activity of the remaining enzyme was measured. As a result, it was confirmed that the activity of Tween 20, Tween 80, and Triton X-100 was greatly affected, and enzyme activity was inhibited (FIG. 15B).

Finally, the effect of solvent was investigated. 30% of each solvent (acetone, ethanol, methanol, 2-propanol, butanol) was added and left for 1 hour, and remaining activity of the remaining enzyme was measured. As a result, 2-propanol and butanol were highly inhibited, and the remaining solvents were not affected (FIG. 15C).

7-3: Investigation of substrate specificity

The purified enzyme solution used above was used to investigate the substrate specificity. 4-Nitrophenyl hexanoate (C 8), C 8 (PNP) 4-Nitrophenyl decanoate (C 8), C 12 (PNPL 4-Nitrophenyl laurate) C14 (PNPM: 4-NItrophenyl myristate) and C16 (PNPP: 4-Nitrophenyl palmitate) were dissolved in acetonitrile to a concentration of 50 mM. 10 μl of the enzyme solution and 50 μl of each substrate (50 mM): 50 mM Tris-HCl (pH 7.5): ethanol in the ratio of 0.1: 1.9: 38 were mixed and incubated at 30 ° C. for 5 minutes. The substrate specificity of the lipolytic enzyme was confirmed. As a result, the activity was high in short length substrates C4 and C6, and relatively similar activities were shown in C8 to C16 (FIG. 16). From these results, it was judged to have specificity for a substrate having a short carbon number.

Example  8: CTG  Codon ( codon ) Influence investigation

According to the published study, some Candida species translate the CTG codon into non-leucine serine, which when expressed in Saccharomyces cerevisiae yeast, translates to the original amino acid leucine, not serine, (Nt J Mol Sci. 2011; 12 (6): 3950-65. Doi: 10.3390 / ijms12063950. Epub 2011 Jun 14.A novel cold-active lipase from Candida albicans: cloning, expression and characterization of the recombinant enzyme Lan DM1, Yang N, Wang WK, Shen YF, Yang B, Wang YH). To investigate the effect of abnormal CTG codons on the previously expressed three proteins CBLIP2 , CBLIP5 and CBLIP7 . It has three types of CTG codons of the protein was the only CBLIP5 and had three CTG codon (Fig. 17). The activity of the substituted protein (SEQ ID NO: 92) was confirmed by replacing CTG with the common serine codon TCT. The gene of CBLIP5 (SEQ ID NO: 93) in which CTG was replaced with TCT was transformed into Saccharomyces cerevisiae cerevisiae) primer STFPL5-F (Table 3, SEQ ID NO: 24), Lip5CTG1 ~ 3-F ( Table 9, SEQ ID NO: 94, 96 and 98) and STFPL5-R (Table 3, SEQ ID NO: 32) to express in, Lip5CTG1 (Table 3, SEQ ID NO: 36) and GT50R (Table 3, SEQ ID NO: 36) and 3-R (Table 9, SEQ ID NOS: 95, 97 and 99) 37) was used to perform a second overlapping extension PCR to construct a vector and a gene capable of in vivo recombination.

gene order SEQ ID NO: CbLIP5 MILTNLLVYFSLIACTICAPMTILKPSEDDFYTAPDGFEDEKLGTILKWRKTPYQIKSIYFPVNIKNSWQLLVRSEDAIGNPVAVTASLFEPYNGNTSRLVSYQVAEDSASFDCAPSYSFVGGGYHTVVAKAEMILIQGALDQGYYVVAPDYEGPNAAFTAGITSGMSTINVLRAVLSDQRSNETRIDSDAEVVLWGYSGGTIPSGWAASMAPWYAKDINDNLKGAAMGGWVTNITATAEIVEGSLFEGLVAAAINGLAQQYEEINEALDVYLVPDKLETFRSAKNECVIDVVFSFAYQSAFSGSDPYTSDGWGVLEDPKVKKIVNQNTLGLNVTEKFKPEIPLFVYHGILDEIVPFKDAQRVYDVWCKEGINSFEFAVSNSTGHLLEVLEGSGAALKWISDRFDGVAPTKGCHRQTRLSNIFYPGSFTGISDILSALVRNVMGSPIGLYDEQVEKRSDVTEDEKLLKRSFAYTDEEIFRRILDNSYPDMAQNLGY 92 CbLIP5 atgattttaacgaatcttcttgtatatttcagcttgatagcgtgcacaatttgtgctccaatgacgatcttgaaaccttctgaagatgacttttatactgctccagatggctttgaggatgaaaagttaggtactattcttaaatggagaaagactccttatcagattaagagtatctacttcccagttaacattaagaatagttggcaattgcttgtgagatctgaagatgcaatcggtaatccagttgcagtcactgcttccctttttgaaccgtacaatggtaacacatccaggttggtctcataccaagttgcagaggatagtgcttcgttcgactgtgcaccgtcatattcatttgtaggaggaggttaccatacagtggttgcaaaagctgagatgattttgatacaaggagcattagatcaaggttattacgttgttgcaccagactatgaaggacccaatgctgcatttactgccgggataacatccggcatgagcaccattaatgttcttcgagcagtcttaagtgatcagaggagtaatgaaacgagaattgactcagatgcagaagtggttctttggggatatagtggaggcacaatccctagtggttgggcagcatctatggctccatggtatgctaaagatatcaatgataatcttaaaggagcagcaatgggtggatgggtaactaatattactgccacggctgaaattgtagaagggtcccttttcgaaggattggttgcagcagccatcaatggattagcacaacaatatgaagaaattaatgaggcacttgatgtttatcttgtacctgataagctagagacatttagatcagcaaagaatgaatgcgttattgatgtagtat
tttcctttgcttatcaaagtgcattcagtggaagtgatccttatacatctgatggatggggtgtattagaagacccaaaagtgaagaagattgtcaaccaaaatacccttggattaaatgtgactgaaaaattcaaaccagagatcccactatttgtttatcatggaattcttgatgaaattgttcctttcaaagatgcacagagagtttatgatgtttggtgtaaagaaggcattaattcatttgaatttgccgtctctaattctactggccatcttcttgaggtacttgaaggcagtggagcagcgttgaagtggatatctgacagattcgatggagttgcaccaaccaaaggatgtcatagacaaacaagattaagtaatatcttctatccaggttcttttactggaatatctgacattttatctgctttagtcagaaatgtcatgggaagccctattggactttatgatgaacaagttgaaaagagatcagatgtaaccgaagacgaaaaattattgaaaaggtcttttgcatacacagatgaagaaatctttagaagaatacttgacaacagctatccagatatggctcaaaatttgggctattaa 93 primer order Lip5CTG1-F AGTGATCCTTATACA TCT GATGGATGGGGTGTA 94 Lip5CTG1-R TACACCCCATCCATC AGA TGTATAAGGATCACT 95 Lip5CTG2-F ATCTTCTATCCAGGT TCT TTTACTGGAATATCT 96 Lip5CTG2-R AGATATTCCAGTAAA AGA ACCTGGATAGAAGAT 97 Lip5CTG3-F AAATTATTGAAAAGG TCT TTTGCATACACAGAT 98 Lip5CTG3-R ATCTGTGTATGCAAA AGA CCTTTTCAATAATTT 99

The expression vector containing the amplified gene was transformed into a yeast strain Y2805 ( Mat alpha pep4 :: HIS3 prb1 ( SEQ ID NO: 2)) together with a vector containing 24 protein secretion fusion factors (Table 5) can1 his3 ura3 was -200 to -52) to form body is introduced in the strain transformed by the recombinant cells. Transformants formed through intracellular recombination were spotted on UDGT medium (yeast substrate lacking 0.67% amino acid, nutritional supplement lacking 0.77% uracil, 2% glucose, 1% galactose, 1% tributyrin) Four TFPs with large transparent rings were selected. As a result, the activities of TFP 8, 9, 13 and 19 were good, and CBLIP5 substituted with TCT containing four TFPs selected was compared with CBLIP5 containing the same TFP before substitution. Likewise, the cells were cultured in YPDG (1% yeast extract, 2% peptone, 1% glucose, 1% galactose) for 40 hours and centrifuged to obtain supernatant. 0.6 ml of the supernatant was precipitated with 0.4 ml of acetone and subjected to SDS-PAGE analysis (Fig. 18 (a)). As a result, it was confirmed that the expression level of the protein before and after the substitution was not significantly different. However, it was confirmed that the activity of the four TFPs after substitution was about three times higher than that before substitution (Fig. 18B). This suggests that the abnormal CTG codon of Candida bauterii SH14 affects the lipase activity.

Example 9: Immobilization of CBLIP2 and effect of biodiesel conversion

In order to develop the immobilized catalyst for biodiesel production using recombinant mass produced CBLIP2, immobilization was carried out using Lewatit VP OC 1600 (Bayer) as a fixed adsorption carrier. Before fixing the enzyme, 30 g of the carrier was washed with 75 ml of methanol for 60 minutes and then washed again with 300 ml of 50 mM Tris buffer solution (pH 7.6) at 25 for 1 hour. The enzyme solution was immobilized in a shaking incubator at 25 ° C and 200 rpm for 15 hours by adding 2000 units per gram of resin. The immobilization time was compared with the initial enzyme activity present in the supernatant after the elapse of a certain time, and the immobilization reaction was performed until the residual activity was no longer changed (FIG. 19). In order to confirm the amount of protein remaining in the supernatant by reaction time, the amount of CBLIP2 enzyme remaining in the supernatant was determined without taking the sample by immobilization time. The immobilized carrier was recovered by using a filter paper and a glass funnel, and the carrier immobilized with 300 ml of the buffer solution was repeatedly washed. The washed immobilized enzyme was dried at room temperature using a vacuum pump and stored at 4 ° C.

As described above, CBLIP2 was immobilized on an adsorbent carrier to compare the conversion efficiencies of biodiesel conversion (0, 5, 10 and 20%) by moisture content. 0.5 ml of methanol was added to 10 ml of vegetable oil (soybean oil), 10% (w / v) of each immobilized enzyme was added, and water was added to each concentration. Were analyzed by HPLC (High performance liquid chromatography, Agilent). To achieve 100% conversion of fatty acid methyl ester to triacylglyceride (TG) and methanol, about 11% (v / v) of methanol is needed. However, since methanol at high concentration deactivates the enzyme, 0.5 ml of methanol was added at the initial stage of the reaction, and 0.5 ml of methanol was further added after 10 hours.

For the analysis of the converted biodiesel, 200 μl of the sample was collected over time, and the supernatant was recovered by centrifugation at 13,000 × g for 5 minutes. The supernatant was recovered and diluted to an appropriate ratio with acetone. 5 μl of the recovered biodiesel was subjected to high performance liquid chromatography , Agilent). Analytical conditions were a stationary phase column (C18 4.6 x 150 mm) and mobile phase acetone, acetonitrile. The analytical method was 100% acetonitrile for 20 minutes after initial analysis, 50% for 20 minutes to 30 minutes, and 100% for 30 minutes to 40 minutes. After the analysis, the peak area at the target position was integrated and the conversion rate was measured by comparing with the standard curve. Standard curves were obtained by purchasing commercially available fatty acid methyl esters (Sigma) and analyzing them under the same conditions. Standard curves were prepared using the peak area of each fatty acid methyl ester concentration. As a result of the measurement, no reaction was observed in the absence of moisture, but the conversion was excellent when 10% of moisture was added at 5%, and the reaction was decreased at 10% when 20% was added (FIG. This enzyme has a characteristic lipase activity that increases the activity at the interface of oil and water. This property provides the advantage of being able to convert to biodiesel using a substrate containing a large amount of water such as waste cooking oil .

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 scope of the present invention as defined by the appended claims.

Korea Biotechnology Research Institute KCTC18455P 20160304

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> Novel lipase from yeast Candida butyri SH-14 and the use thereof <130> KPA160011-KR <160> 99 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ITS1 <400> 1 tccgtaggtg aacctgcgg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS4 <400> 2 tcctccgctt attgatatgc 20 <210> 3 <211> 584 <212> DNA <213> Artificial Sequence <220> <223> ITS region of Candida butyri SH-14 <400> 3 agaaaaaata ttcttgccgc gcttactgcg cggcgagaat aacaccttac acacagtgga 60 ttttttttaa aagaactatt gcgtttggct tggctaacgc tgggccagag gattcagtaa 120 acttcaattt ttaattgaat tgttatttta atatttttgt caatttgttt gattaaattc 180 aaaataatct tcaaaacttt caacaacgga tctcttggtt ctcgcatcga tgaagaacgc 240 agcgaaatgc gataagtaat atgaattgca gattttcgtg aatcatcgaa tctttgaacg 300 cacattgcgc cttatggtat tccataaggc atgcctgttt gagcgtcatt tctctctcaa 360 acctttgggt ttggtattga gtgatactct tagtcggact aggcgtttgc ttgaaaagta 420 ttggcaagag tgtactggat agtactaact ggttattcaa tgtattaggt ttatccaact 480 cgttgaagtg ctggtggtaa atttctagta acggctcggc cttacaacaa caaacaagtt 540 tgacctcnaa tcaggtaaga atacccgctg aacttaagca tatc 584 <210> 4 <211> 338 <212> PRT <213> Artificial Sequence <220> <223> LIP1 <400> 4 Met Lys Leu Ser Thr Phe Leu Ser Ala Ser Ile Ile Phe Trp Arg Ala   1 5 10 15 Thr Glu Thr Ala Ser Thr Pro Leu Ser Pro Arg Ser Gly Asp Asn Val              20 25 30 Pro Lys Asp Tyr Gln Lys Leu Lys Asp Tyr Ala Asn Leu Val Ser Phe          35 40 45 Ala Tyr Cys Lys Glu Leu Ile Pro Ser Lys Leu Gly Ser Asn Ser Ser      50 55 60 Asn Cys Pro Ile Leu Arg Cys Gln Glu Glu Glu Tyr Lys Asn Ile Glu  65 70 75 80 Val Leu Asn Leu Phe Asp Phe Asn Asp Phe Gly Ser Ile Gly Ser Gly                  85 90 95 Tyr Thr Gly Ile Asp His Gln Asn Lys Arg Ile Ile Leu Ala Phe Arg             100 105 110 Gly Thr Ser Thr Asn Arg Asp Trp Leu Ala Asn Ile Asn Val Pro Phe         115 120 125 Lys Lys Tyr Asn Pro Leu Ser Asn Leu Gln Leu Lys Ala Asp Ile Glu     130 135 140 Cys Glu Gly Cys Lys Val His Lys Gly Phe Tyr Glu Phe Ile Glu Lys 145 150 155 160 His Cys Val Ala Leu Ile Lys Gln Val Ala Gln Leu Lys Gln Lys Tyr                 165 170 175 Pro Asp Tyr Gln Leu Val Val Leu Gly His Ser Leu Gly Gly Val Phe             180 185 190 Ala Leu Leu Ser Gly Met Glu Phe Leu Leu Met Asp Phe Gln Pro Leu         195 200 205 Val Ile Thr Tyr Ala Ser Pro Lys Val Gly Asn Lys Ala Met Val Arg     210 215 220 Phe Ile Asp Gln Leu Phe Glu Thr Ser Lys Val Ala Gln Gln Ser Gln 225 230 235 240 Asn Asp Tyr Asp Phe Asn His Gly Tyr Ile Arg Val Val His Asn Tyr                 245 250 255 Asp Met Val Pro Met Leu Pro Pro Arg Glu Leu Gly Tyr Tyr His Gly             260 265 270 Gly Val Glu Tyr His Ile Glu Lys Ser Asn Leu Pro His Asn Pro Asp         275 280 285 Ser Val His Asn Arg Gly Gln Asn His Phe Ser Ser Asn Lys Asp Glu     290 295 300 Gly Pro Val Glu Lys Leu Glu Arg Ile Ala Thr Ser Leu Val Leu Val 305 310 315 320 Phe Thr Ser Lys Glu His Thr Gln Tyr Phe Ile Pro Ile Thr Gly Cys                 325 330 335 Lys Asp         <210> 5 <211> 468 <212> PRT <213> Artificial Sequence <220> <223> LIP2 <400> 5 Met Ile Cys Ser Phe Phe Arg Leu Leu Thr Ile Val Thr Leu Val Ile   1 5 10 15 Ala Ala Pro Thr Thr Leu Val Pro Pro Thr Glu Asp Pro Phe Tyr Thr              20 25 30 Ala Pro Lys Gly Phe Glu Ser Ala Glu Leu Gly Thr Val Leu Ala Tyr          35 40 45 Arg Asn Thr Pro Ala Pro Ile Arg Ser Ile Tyr Phe Glu Val Asn Ile      50 55 60 Lys Asn Ser Trp Gln Leu Leu Val Arg Ser Ser Asp Ser Phe Gly Asn  65 70 75 80 Pro Ser Val Val Thr Thr Val Phe Glu Pro Phe Asn Ala Asp Pro                  85 90 95 Ser Lys Leu Val Ser Tyr Gln Val Ala Gln Asp Ser Ala Tyr Leu Asp             100 105 110 Cys Ser Pro Ser Tyr Ser Phe Met Asn Gly Gly Gly Leu Ser Thr Ile         115 120 125 Asn Asn Gln Ile Glu Thr Val Leu Ile Gln Thr Ala Leu Asp Gln Gly     130 135 140 Tyr Tyr Val Ser Ser Pro Asp Tyr Glu Gly Leu Lys Ser Ala Phe Thr 145 150 155 160 Gly Gly Ile Gln Ala Gly His Gly Thr Leu Asp Ser Ile Arg Gly Ala                 165 170 175 Leu Ser Ser Ser Asn Ile Thr Gly Val Lys Lys Asp Ala Asp Thr Ile             180 185 190 Leu Trp Gly Tyr Ser Gly Gly Ser Leu Ala Ser Gly Trp Ala Ala Ala         195 200 205 Leu Gln Pro Thr Tyr Ala Pro Glu Leu Ala Ser Asn Leu Leu Gly Val     210 215 220 Ala Leu Gly Gly Trp Val Thr Asn Ile Thr Ala Thr Ile Thr Ser Val 225 230 235 240 Ser Gly Thr Ile Phe Ser Gly Leu Gly Ala Met Gly Met Ala Gly Leu                 245 250 255 Ser Asn Glu Tyr Thr Asp Leu Tyr Gly Tyr Leu Lys Thr Ala Met Pro             260 265 270 Ala Asp Lys Tyr Glu Glu Phe Thr Lys Ala Tyr Ser Ile Cys Ala Ala         275 280 285 Glu Ala Leu Ile Glu Tyr Asn Phe Asp Asp Trp Phe Glu Gly Glu Asp     290 295 300 Arg Tyr Phe Thr Asp Gly Phe Lys Val Leu Asn Glu Glu Pro Thr Tyr 305 310 315 320 Ser Ile Ile Arg Asn Asn Thr Leu Gly Leu Ile Ala Gly Gln Met Pro                 325 330 335 Glu Ile Pro Val Phe Val Tyr His Gly Thr Leu Asp Gln Ile Val Pro             340 345 350 Tyr Asp Gln Ala Glu Arg Val Tyr Asp Ile Trp Cys Asp Ala Gly Ile         355 360 365 Lys Ser Phe Glu Phe Ala Thr Asp Leu Thr Ala Gly His Ile Thr Glu     370 375 380 Leu Val Gln Gly Ser Gly Ala Ala Phe Gly Trp Ile Lys Gly Met Phe 385 390 395 400 Glu Gly Thr Lys Lys Pro Val Ser Gly Cys Arg Lys Thr Pro Arg Ile                 405 410 415 Ser Asn Leu Leu Tyr Pro Gly Thr Val Arg Ser Val Thr Asp Val Val             420 425 430 Gly Ala Leu Leu Asp Asn Ile Leu Gly Phe Asp Ile Gly Pro Asn Gly         435 440 445 Glu Asn Leu Ile Val Glu Asn Asn Ser Val Ile Ser Lys Ala Asn Ser     450 455 460 Thr Arg Ser Glu 465 <210> 6 <211> 345 <212> PRT <213> Artificial Sequence <220> <223> LIP3 <400> 6 Met Ala Met Leu Met Leu Leu Phe Phe Leu Phe Glu Ile Ala Cys Gly   1 5 10 15 Asn Ile Trp Val Tyr Pro Gly Ser His Gln Lys Ser Gly Glu Glu Glu              20 25 30 Arg Thr Pro Val Ile Asp Ile Glu Val Tyr Arg Asn Met Phe Thr          35 40 45 Tyr Ala His Leu Ile Asp Ile Ser Tyr Cys Ile Ser Ser Thr Thr Arg      50 55 60 Leu Glu Glu Pro Phe Asn Cys Asp Leu Asn Cys Glu Lys Arg Phe Pro  65 70 75 80 Asn Val Thr Leu Val Tyr Gln Trp Tyr Phe Asp Ser Val Cys Gly                  85 90 95 Tyr Ile Ala Thr Thr Tyr Ser Asn Ile Phe Asn Tyr Glu Ser Glu Glu             100 105 110 Gly Lys Gly His Lys Lys Thr Ile Ile Val Ser Leu Arg Gly Thr Arg         115 120 125 Ser Ile Phe Asp Ser Tyr Thr Asp Ile Lys Val Asp Met Val Asn Tyr     130 135 140 Tyr Asn Tyr Gly Ser Asn Ile Gln Glu Cys Gly Thr Asp Cys Lys Val 145 150 155 160 His Arg Gly Phe Tyr Lys Tyr Tyr Ile Asn Thr Leu Leu Lys Ile Glu                 165 170 175 Gly Ile Leu Arg Asn Glu Leu Gln Thr Asp Asp Asp Tyr Glu Leu Leu             180 185 190 Ile Val Gly His Ser Leu Gly Aly Val Gly Leu Leu Leu Gly Leu         195 200 205 Tyr Tyr Leu Asp Glu Gly Phe Asp Lys Ile Thr Leu Val Thr Met Gly     210 215 220 Gln Pro Leu Val Gly Asn Lys Gln Phe Ala Glu Phe Val Asp Asn Val 225 230 235 240 Met Gly Ser Arg Leu Pro Ile Glu His Asn Thr Phe Asn Arg Lys Phe                 245 250 255 Phe Arg Val Ile His Lys Asp Asp Ile Val Ala Thr Ile Pro Ser Asn             260 265 270 Asn Arg Ile Leu Asp Ser Tyr Ser Gln Phe Asp Asn Gln Ile Tyr Leu         275 280 285 Asn Cys Leu Ala Ser Asp Thr Met Pro Ser Leu Glu Gln Val Leu Asp     290 295 300 Cys Phe Asp Gly Asp Asn Pro Gln Cys Ile Ser Gly Asp Ile Glu Asn 305 310 315 320 Tyr Leu Leu Ser His Asn Tyr Leu Gln Ile His Thr Thr Tyr Phe Arg                 325 330 335 Ser Met Gly Leu Cys Gly Ile Arg Ile             340 345 <210> 7 <211> 513 <212> PRT <213> Artificial Sequence <220> <223> LIP4 <400> 7 Met Met Ile Thr Thr Arg Ile Leu Val Cys Phe Ala Leu Ile Ala Leu   1 5 10 15 Thr Phe Ala Ala Pro Leu Thr Val Leu Lys Pro Ser Glu Asp Glu Phe              20 25 30 Tyr Ala Ala Pro Asp Gly Phe Glu Asp Glu Lys Leu Gly Thr Ile Leu          35 40 45 Lys Trp Arg Lys Thr Pro Tyr Gln Ile Lys Ser Ile Tyr Phe Pro Val      50 55 60 Asn Ile Lys Asn Ser Trp Gln Leu Leu Val Arg Ser Glu Asp Ala Ile  65 70 75 80 Gly Asn Pro Val Ala Val Thr Ala Ser Leu Phe Glu Pro Tyr Asn Gly                  85 90 95 Asn Thr Ser Arg Leu Val Ser Tyr Gln Val Ala Glu Asp Ser Ala Ser             100 105 110 Phe Asp Cys Ala Pro Ser Tyr Ser Phe Val Gly Gly Gly Tyr His Thr         115 120 125 Val Val Ala Lys Ala Glu Met Ile Leu Ile Gln Gly Ala Leu Asp Gln     130 135 140 Gly Tyr Tyr Val Val Ala Pro Asp Tyr Glu Gly Pro Asn Ala Val Phe 145 150 155 160 Thr Ala Gly Val Thr Ser Gly Met Ser Thr Ile Asn Val Leu Arg Ala                 165 170 175 Val Leu Gly Glu Gly Arg Arg Asn Glu Thr Arg Ile Asp Pro Asp Ala             180 185 190 Glu Val Val Leu Trp Gly Tyr Ser Gly Gly Thr Ile Pro Ser Gly Trp         195 200 205 Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa     210 215 220 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 225 230 235 240 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala                 245 250 255 Glu Ile Val Asp Gly Thr Leu Phe Ser Gly Leu Val Pro Ala Ala Leu             260 265 270 Asn Gly Leu Ser Ala Gln Tyr Glu Glu Met Asp Glu Ala Ile Asp Lys         275 280 285 Phe Leu Lys Pro Asn Lys Leu Glu Lys Phe Arg Ser Gly Arg Asn Glu     290 295 300 Cys Ala Ile Asp Val Val Phe Ser Phe Ala Tyr Gln Asp Thr Phe Ser 305 310 315 320 Gly Phe Asp Thr Tyr Ser Lys Asp Gly Trp Thr Ile Leu Asp Asp Pro                 325 330 335 Asp Val Arg Asp Ile Leu Ala Glu Asn Thr Leu Gly Val Asn Val Thr             340 345 350 Lys Lys Phe Lys Pro Glu Ile Pro Leu Phe Val Tyr His Gly Ile Leu         355 360 365 Asp Glu Ile Val Pro Phe Lys Asp Ala Gln Arg Val Tyr Asp Val Trp     370 375 380 Cys Glu Glu Gly Tyr Asp Ser Phe Glu Phe Ala Val Ser Asn Ser Thr 385 390 395 400 Gly His Ile Leu Glu Val Ile Glu Gly Ser Ala Gly Leu Lys Trp                 405 410 415 Ile Ser Asp Arg Phe Asp Gly Val Gln Pro Thr Lys Gly Cys His Arg             420 425 430 Gln Thr Arg Leu Thr Asn Leu Phe Tyr Pro Gly Leu Phe Thr Gly Val         435 440 445 Thr Asp Ile Leu Ser Ala Leu Met Arg Asn Ile Phe Gly Ser Pro Ile     450 455 460 Gly Leu Tyr Asp Glu Gln Ile Glu Lys Arg Ser Asp Met Ser Glu Asp 465 470 475 480 Glu Lys Leu Leu Lys Arg Leu Phe Glu Phe Thr Asp Ser Glu Ile Phe                 485 490 495 Lys Arg Ile Val Gln His Ser Tyr Pro Glu Met Ala Lys Lys Leu Asp             500 505 510 Leu     <210> 8 <211> 496 <212> PRT <213> Artificial Sequence <220> <223> LIP5 <400> 8 Met Ile Leu Thr Asn Leu Leu Val Tyr Phe Ser Leu Ile Ala Cys Thr   1 5 10 15 Ile Cys Ala Pro Met Thr Ile Leu Lys Pro Ser Glu Asp Asp Phe Tyr              20 25 30 Thr Ala Pro Asp Gly Phe Glu Asp Glu Lys Leu Gly Thr Ile Leu Lys          35 40 45 Trp Arg Lys Thr Pro Tyr Gln Ile Lys Ser Ile Tyr Phe Pro Val Asn      50 55 60 Ile Lys Asn Ser Trp Gln Leu Leu Val Arg Ser Glu Asp Ala Ile Gly  65 70 75 80 Asn Pro Val Ala Val Thr Ala Ser Leu Phe Glu Pro Tyr Asn Gly Asn                  85 90 95 Thr Ser Arg Leu Val Ser Tyr Gln Val Ala Glu Asp Ser Ala Ser Phe             100 105 110 Asp Cys Ala Pro Ser Tyr Ser Phe Val Gly Gly Gly Tyr His Thr Val         115 120 125 Val Ala Lys Ala Glu Met Ile Leu Ile Gln Gly Ala Leu Asp Gln Gly     130 135 140 Tyr Tyr Val Val Ala Pro Asp Tyr Glu Gly Pro Asn Ala Ala Phe Thr 145 150 155 160 Ala Gly Ile Thr Ser Gly Met Ser Thr Ile Asn Val Leu Arg Ala Val                 165 170 175 Leu Ser Asp Gln Arg Ser Asn Glu Thr Arg Ile Asp Ser Asp Ala Glu             180 185 190 Val Val Leu Trp Gly Tyr Ser Gly Gly Thr Ile Pro Ser Gly Trp Ala         195 200 205 Ala Ser Met Ala Pro Trp Tyr Ala Lys Asp Ile Asn Asp Asn Leu Lys     210 215 220 Gly Ala Ala Met Gly Gly Trp Val Thr Asn Ile Thr Ala Thr Ala Glu 225 230 235 240 Ile Val Glu Gly Ser Leu Phe Glu Gly Leu Val Ala Ala Ala Ile Asn                 245 250 255 Gly Leu Ala Gln Gln Tyr Glu Glu Ile Asn Glu Ala Leu Asp Val Tyr             260 265 270 Leu Val Pro Asp Lys Leu Glu Thr Phe Arg Ser Ala Lys Asn Glu Cys         275 280 285 Val Ile Asp Val Val Phe Ser Phe Ala Tyr Gln Ser Ala Phe Ser Gly     290 295 300 Ser Asp Pro Tyr Thr Leu Asp Gly Trp Gly Val Leu Glu Asp Pro Lys 305 310 315 320 Val Lys Lys Ile Val Asn Gln Asn Thr Leu Gly Leu Asn Val Thr Glu                 325 330 335 Lys Phe Lys Pro Glu Ile Pro Leu Phe Val Tyr His Gly Ile Leu Asp             340 345 350 Glu Ile Val Pro Phe Lys Asp Ala Gln Arg Val Tyr Asp Val Trp Cys         355 360 365 Lys Glu Gly Ile Asn Ser Phe Glu Phe Ala Val Ser Asn Ser Thr Gly     370 375 380 His Leu Leu Glu Val Leu Glu Gly Ser Gly Ala Ala Leu Lys Trp Ile 385 390 395 400 Ser Asp Arg Phe Asp Gly Val Ala Pro Thr Lys Gly Cys His Arg Gln                 405 410 415 Thr Arg Leu Ser Asn Ile Phe Tyr Pro Gly Leu Phe Thr Gly Ile Ser             420 425 430 Asp Ile Leu Ser Ala Leu Val Arg Asn Val Met Gly Ser Pro Ile Gly         435 440 445 Leu Tyr Asp Glu Glu Val Glu Lys Arg Ser Asp Val Thr Glu Asp Glu     450 455 460 Lys Leu Leu Lys Arg Leu Phe Ala Tyr Thr Asp Glu Glu Ile Phe Arg 465 470 475 480 Arg Ile Leu Asp Asn Ser Tyr Pro Asp Met Ala Gln Asn Leu Gly Tyr                 485 490 495 <210> 9 <211> 491 <212> PRT <213> Artificial Sequence <220> <223> LIP6 <400> 9 Met Leu Ile Thr Thr Lys Ile Leu Val Tyr Phe Ala Leu Ile Ile Leu   1 5 10 15 Thr Ser Ala Ala Pro Met Thr Ile Leu Lys Pro Ser Glu Asp Glu Phe              20 25 30 Tyr Ala Ala Pro Asp Gly Phe Glu Asp Glu Lys Leu Gly Thr Ile Leu          35 40 45 Lys Trp Arg Lys Thr Pro Tyr Gln Ile Lys Ser Ile Tyr Phe Pro Val      50 55 60 Asn Ile Lys Asn Ser Trp Glu Leu Leu Val Arg Ser Glu Asp Ala Ile  65 70 75 80 Gly Asp Pro Val Ala Val Thr Ala Thr Ile Phe Glu Pro Tyr Asn Ala                  85 90 95 Asn Ser Ser Arg Leu Val Ser Tyr Gln Val Ala Glu Asp Ser Pro Ser             100 105 110 Phe Asn Cys Ser Pro Ser Tyr Ala Phe Val Gly Gly Gly Tyr Pro Thr         115 120 125 Val Val Thr Lys Ala Glu Met Ile Leu Ile Gln Ser Ala Leu Asn Glu     130 135 140 Gly Tyr Tyr Val Val Val Pro Asp Tyr Glu Gly Pro Asn Ala Val Phe 145 150 155 160 Gly Val Gly Val Thr Ser Ala Met Ser Thr Ile Asn Val Leu Arg Ala                 165 170 175 Val Leu Ser Asp Gln Arg Arg Asn Glu Thr Arg Ile Asp Ser Asp Ala             180 185 190 Glu Val Val Leu Trp Gly Tyr Ser Gly Gly Thr Ile Pro Ser Ser Trp         195 200 205 Ala Ala Ser Met Ala Pro Trp Tyr Ala Lys Asp Ile Asn Gln His Leu     210 215 220 Ile Gly Ala Ala Leu Gly Gly Trp Val Ser Asn Leu Thr Ala Leu Met 225 230 235 240 Glu Ile Val Glu Gly Ser Leu Phe Glu Gly Leu Val Pro Asn Ile Ile                 245 250 255 Asn Gly Leu Ser Gln Gln Phe Glu Glu Ile Asn Asp Ala Phe Asp Asp             260 265 270 Tyr Leu Tyr Pro Ser Lys Arg Glu Lys Phe Arg Ser Thr Lys Lys Gln         275 280 285 Cys Val Phe Asp Ala Cys Leu Ser Phe Ala Phe Gln Ser Thr Phe Leu     290 295 300 Gly Asp His Pro Tyr Val Lys Gly Lys Trp Ser Val Leu Glu Glu Ala 305 310 315 320 Lys Val Lys Asn Ile Leu Glu Asn Asn Thr Leu Gly Leu Lys Val Thr                 325 330 335 Lys Lys Phe Lys Pro Glu Ile Pro Ile Phe Ala Phe His Gly Ile Lys             340 345 350 Asp Glu Ile Val Pro Phe Lys Asp Ser Gln Arg Leu Tyr Asp Val Trp         355 360 365 Cys Glu Glu Gly Ile Asn Ser Phe Glu Phe Ala Val Ser Ser Ser Ser     370 375 380 Gly His Leu Leu Glu Val Val Glu Gly Ser Gly Ala Ala Leu Ala Trp 385 390 395 400 Ile Ser Asp Arg Phe Arg Gly Ile Pro Ala Val Lys Gly Cys Glu Arg                 405 410 415 Lys Glu Arg Leu Thr Asn Leu Phe Tyr Pro Gly Leu Phe Ser Ser Val             420 425 430 Ser Asn Ile Leu Met Ala Leu Ile Arg Asn Ile Met Gly Ser Pro Ile         435 440 445 Gly Leu Tyr Asp Glu Glu Ala Lys Lys Arg Ser Gly Ser Thr Glu Glu     450 455 460 Glu Ile Ile Met Lys Arg Leu Leu Glu Tyr Thr Asp Glu Glu Ile Tyr 465 470 475 480 Arg Arg Ile Ile Glu Thr His Ser Asp Met Phe                 485 490 <210> 10 <211> 463 <212> PRT <213> Artificial Sequence <220> <223> LIP7 <400> 10 Met Phe Lys Gln Leu Phe Leu Leu Phe Ser Leu Leu Ile Phe Ala Leu   1 5 10 15 Thr Leu Pro Thr Gly Leu Val Pro Ser Ser Gln Asp Ser Phe Tyr Ser              20 25 30 Ala Pro Thr Gly Phe Glu Ser Ala Glu Leu Gly Thr Ile Leu Lys Phe          35 40 45 Arg Pro Ser Pro Ala Pro Ile Arg Ser Val Tyr Phe Lys Val Lys Ile      50 55 60 Lys Ser Ser Trp Gln Leu Leu Val Arg Ser Ser Asp Ser Phe Gly Asn  65 70 75 80 Pro Ser Val Ile Val Thr Thr Val Phe Glu Pro Phe Asn Ala Asp Pro                  85 90 95 Ser Lys Leu Ile Ser Tyr Gln Val Ala Gln Asp Ser Ala Ser Asn Asp             100 105 110 Cys Ser Pro Ser Tyr Ala Phe Met Asp Gly Gly Gly Phe Glu Thr Val         115 120 125 Thr Ser Gln Ala Glu Met Leu Leu Ile Gln Thr Ala Leu Asp Gln Gly     130 135 140 Tyr Tyr Val Val Ser Pro Asp Tyr Glu Gly Leu Lys Ala Val Tyr Thr 145 150 155 160 Gly Gly Ile Gln Ser Gly His Gly Thr Leu Asp Ser Leu Thr Ala Ala                 165 170 175 Leu Thr Ser Lys Asn Ile Thr Gly Ile Asn Ser Asp Ala Lys Ser Ile             180 185 190 Leu Trp Gly Tyr Ser Gly Gly Ala Leu Ala Ala Gly Trp Ala Ala Ala         195 200 205 Leu Gln Pro Thr Tyr Ala Pro Asp Leu Lys Gln Ser Leu Leu Gly Ala     210 215 220 Ala Leu Gly Gly Phe Val Thr Asn Val Thr Ala Thr Val Thr Ala Val 225 230 235 240 Asn Gly Gly Pro Phe Ser Gly Leu Val Gly Met Gly Ile Ala Gly Leu                 245 250 255 Ser Asn Glu Tyr Pro Glu Leu Lys Asp Tyr Leu Lys Glu Gln Met Tyr             260 265 270 Pro Asp Lys Tyr Glu Gln Phe Gln Glu Ile Tyr Gly Leu Cys Thr Ala         275 280 285 Glu Gly Ala Leu Lys Phe Ala Phe Thr Asp Tyr Phe Asn Gly Pro Asn     290 295 300 Lys Tyr Val Asn Gly Ile Gly Val Leu Ala Asn Glu Pro Ala Ala Ser 305 310 315 320 Ile Leu Lys Asn Asn Thr Leu Gly Leu Val Ser Ser Gln Val Pro Asp                 325 330 335 Ile Pro Leu Phe Val Tyr His Gly Val Ile Asp Ser Leu Val Pro Tyr             340 345 350 Lys Glu Thr Glu Arg Val Tyr Asn Thr Trp Cys Asp Glu Gly Ile Glu         355 360 365 Ser Phe Glu Leu Ala Ala Asp Leu Thr Ala Gly His Leu Thr Glu Val     370 375 380 Leu Gln Gly Ser Gly Ala Ala Phe Gly Trp Ile Thr Lys Arg Phe Asp 385 390 395 400 Gly Lys Ala Pro Ile Ser Gly Cys Arg Lys Thr Lys Arg Leu Thr Asn                 405 410 415 Leu Leu Tyr Pro Gly Thr Val Gly Ser Val Thr Asn Leu Ile Ser Ala             420 425 430 Leu Phe Thr Asn Val Leu Gly Gly Asp Ile Gly Pro Asn Gly Glu Ser         435 440 445 Met Lys Pro Gly Asn Ser Glu Leu Asp His Phe Phe His Ile Asn     450 455 460 <210> 11 <211> 646 <212> PRT <213> Artificial Sequence <220> <223> LIP8 <400> 11 Met Met Met His Ile Asp Glu Pro Val His Lys Tyr Leu Ser Pro Met   1 5 10 15 His Lys His Pro Phe Pro Met Thr Phe Ile Pro Phe Leu Val Ala Phe              20 25 30 Phe Met Thr Tyr Glu Cys Thr Val Ser Ser Ser Tyr Gln Phe Leu Val          35 40 45 Tyr Thr His Lys Pro Thr Phe Glu Lys Met Thr Gln Asn Leu Asn Leu      50 55 60 Arg Asn His His Lys Gln Met Thr Lys Asn Asn Glu Arg Trp Ser Pro  65 70 75 80 Ile Arg Trp Val Leu Gly Leu Ala Leu Val Thr Thr Val Val Leu Tyr                  85 90 95 Ile His Gly Gly Asn Ile Phe Lys Glu Ser Glu Asp Tyr Ile Ser Asn             100 105 110 Gln Val Met Glu Ala Ala Asp Leu Gln Gly Asp Ser Phe Arg Leu Lys         115 120 125 His Ile Phe Gln His Gly Ala Gly Ser Glu Tyr Tyr Arg Val His Arg     130 135 140 Arg Leu Asp Ile Thr Glu Glu Tyr Leu Ser Arg Thr Gly Leu Asp Glu 145 150 155 160 Val Ala Met Glu Ala Asp Thr Thr Asp Val Ser Ser Asn Ser Leu Glu                 165 170 175 Asp Val Tyr Ala Gln Asn Asp Trp Pro Leu Ala Phe Gln Gly His Asn             180 185 190 Pro Trp Asn Met Lys Met Pro Val Arg Ser Ser Asn His Lys Ala Lys         195 200 205 Ile Arg Arg Leu Thr Glu Arg His Thr Pro Gly Phe Leu Asp Ser Tyr     210 215 220 Leu Asp Tyr Ala Ile Glu Val Lys Gly Asn Pro Gln Lys Leu Asn Met 225 230 235 240 Ile Asn Leu Gln Trp Asp Glu Glu Glu Asp Leu Ile Leu Pro Asp Val                 245 250 255 His Asp Lys Glu Ser Leu Val Met Leu Ala Leu Met Ser Ser Asn Ala             260 265 270 Tyr Val Lys Phe Pro Lys Asp Asp Asn Glu Lys Lys Lys Ser Asp Trp         275 280 285 Arg Asp Val Gly Glu Pro Trp Glu Pro Asp Glu Asn Asn Thr Asp Ile     290 295 300 Glu Phe Gly Trp Asp Gly Asp Gly Val Arg Gly His Val Phe Val Asn 305 310 315 320 Glu Val Asn Asn Thr Val Val Ile Ala Leu Lys Gly Thr Ser Gly Ala                 325 330 335 Gly Ile Pro Gly Ala Gly Glu Asp Glu Thr Thr Asn Asn Asp Lys Leu             340 345 350 Asn Asp Asn Leu Leu Phe Leu Cys Cys Cys Ala Arg Val Ser Tyr Leu         355 360 365 Trp Thr Thr Val Cys Asp Cys Tyr Glu Lys Ala Tyr Thr Cys Asn Gln     370 375 380 Asp Cys Leu Glu Lys Glu Leu Thr Arg Lys Asp Arg Tyr Tyr Gln Ala 385 390 395 400 Ala Leu Glu Ile Tyr Arg Asn Val Ser Ser Ile Tyr Pro Ser Asn Gln                 405 410 415 Tyr Lys Ile Trp Leu Thr Gly His Ser Leu Gly Gly Ser Leu Ala Ser             420 425 430 Leu Val Gly Arg Thr Tyr Gly Leu Pro Val Val Ala Phe Glu Ala Pro         435 440 445 Gly Glu Leu Leu Ala Thr Gln Arg Leu His Leu Pro Gln Pro Pro Gly     450 455 460 Tyr Pro Lys Tyr Met Glu His Ile Tyr His Ile Gly Asn Thr Ala Asp 465 470 475 480 Pro Ile Phe Met Gly Val Cys Asn Gly Val Ser Ser Thr Cys Asn Ala                 485 490 495 Ala Gly Tyr Ala Met Glu Thr Ala Cys His Thr Gly Lys Leu Cys Val             500 505 510 Tyr Asp Val Val Thr Asp Arg Gly Trp Ser Val Asn Val Leu Asn His         515 520 525 Arg Ile His Thr Val Val Asp Glu Ile Ile Leu Lys Tyr Asn Thr Thr     530 535 540 Ala Glu Cys Val Glu Gln Pro Pro Cys Arg Asp Cys Phe Asn Trp Arg 545 550 555 560 Tyr Val Thr His Asp Asp Asp Glu Asp Asp Glu Pro Lys Leu Pro Asn                 565 570 575 Pro Leu Ile Pro His Ile Ser His His Ser Thr Ala Thr Lys Ser Gln             580 585 590 Gly Pro Ser His Thr Ala Tyr Asp Ser Ile Ser Ser Ser Ser Ser         595 600 605 Gly Ser Ser Ser Ser Ser Ser Ser Lys Leu Pro Glu Lys Gln Lys Cys     610 615 620 Leu Lys Arg Thr Trp Tyr Gly Trp Cys Arg Glu Trp Gly Pro Ala Asp 625 630 635 640 Gly Glu Gly Asp Gln Phe                 645 <210> 12 <211> 1017 <212> DNA <213> Artificial Sequence <220> <223> LIP1 <400> 12 atgaaactat ctacctttct ttctgctagt ataatattct ggagagccac cgaaacagcc 60 agtaccccat tgagtccacg ttctggtgac aatgttccca aagattatca aaaattaaaa 120 gattatgcta atcttgtatc ttttgcatac tgcaaagaat tgataccatc gaaacttgga 180 agataatgata gtaattgtcc aatattgaga tgccaagaag aagagtacaa aaatatagaa 240 gtattgaatc tttttgattt caatgatttt gggagtattg gatcaggcta tactggaata 300 gaccatcaaa ataaaagaat aattctagct ttccgtggga caagtacaaa ccgagactgg 360 ctagcaaata taaatgtacc ttttaagaaa tataatcctt tgtctaacct tcaactgaaa 420 gcagatatag aatgtgaagg ctgtaaagtg cataaaggat tctatgaatt cattgaaaaa 480 cattgtgtag ctctcattaa acaagttgct caattaaaac aaaagtatcc ggattatcaa 540 ttggtggttt taggtcattc tttaggagga gtgtttgctt tattaagtgg gatggagttt 600 ctgcttatgg atttccaacc acttgtaatt acatatgcaa gtcctaaagt tggaaataaa 660 gcaatggtta ggtttattga tcagttattc gaaacatcca aagtcgccca acaatctcaa 720 aatgattacg acttcaacca tggttatatt cgtgtagtgc ataactatga catggttcct 780 atgttacccc caagagaatt gggatattat catggaggtg ttgaatatca tattgagaaa 840 agtaatttac cacataatcc cgatagtgtt cacaatagag gacaaaacca ttttagctcc 900 aataaagatg aaggtcctgt tgaaaagctt gagagaattg ctacatcgtt ggttctggta 960 tttacgtcta aagaacatac tcaatacttt attccaatca ctggttgcaa agattga 1017 <210> 13 <211> 1407 <212> DNA <213> Artificial Sequence <220> <223> LIP2 <400> 13 atgatctgtt cattctttag actattgacc attgtaactt tggttattgc tgctcctacc 60 actttagttc ctccaactga agatcctttc tatactgcac caaagggctt cgaatcagca 120 gagttaggta ctgttttggc ttatagaaac actccagctc caatcagaag tatttatttt 180 gaagttaata tcaaaaactc atggcaattg ttagtcaggt cttctgattc atttggtaat 240 ccttcagtag ttgtaactac tgtttttgaa ccatttaatg ctgatccttc caagttagtt 300 tcttatcaag ttgctcaaga ttctgcatat cttgattgtt caccatcata ttccttcatg 360 aatggaggtg gtctttctac tattaacaat caaattgaga ctgttttaat tcaaacagca 420 ttagaccaag gttattatgt tgtttctcca gattatgaag gattgaaatc ggctttcacc 480 ggtggtattc aagctggtca tggtacattg gattccatta gaggtgcttt atccagtagt 540 aacatcactg gtgttaaaaa ggacgcagat actattcttt ggggttattc tggaggttct 600 ttagctagtg gatgggctgc agctttacaa ccaacttatg caccagaatt ggcttccaac 660 ttacttggtg ttgctttagg tggatgggtt accaatatta ctgctactat aacaagtgtt 720 agtggtacca tattctctgg attgggtgct atgggaatgg ctggtttaag taatgagtac 780 accgatttat acggttacct taagactgct atgccagcag ataaatatga agaattcact 840 aaagcttatt caatatgtgc tgctgaagct cttattgaat ataattttga tgattggttt 900 gaaggcgaag atagatactt taccgatggc tttaaagttt tgaatgaaga accgacatat 960 tccatcattc gtaacaatac attgggttta attgctggtc agatgccaga aattcctgtt 1020 ttcgtttacc atggaactct cgaccagatc gtaccatatg atcaagctga aagggtttat 1080 gatatttggt gtgatgctgg tattaaatct tttgaatttg ctactgattt aactgctggt 1140 catatcactg agcttgtaca aggtagtggt gcagcctttg gatggatcaa aggcatgttt 1200 gaaggaacta aaaaaccagt ttctggttgt agaaaaacac ctagaatttc aaacttactt 1260 tacccaggta ctgtaagatc tgttactgac gttgttggtg ctttgcttga taatatctta 1320 ggatttgata ttggtccaaa tggtgaaaac cttattgttg aaaacaacag cgtcatcagt 1380 aaagctaatt ctactagaag tgaatag 1407 <210> 14 <211> 1038 <212> DNA <213> Artificial Sequence <220> <223> LIP3 <400> 14 atggcaatgt taatgttatt gttcttccta ttcgaaatag catgtggaaa tatttgggtt 60 tatcctggaa gtcatcagaa atcaggagag gaagaacgta cgccagtacc cattgatata 120 gaagtgtatc gtaatatgtt cacttatgca catcttattg atatatcata ttgtatatct 180 tcaacaacaa ggctcgaaga accgtttaac tgtgacttga attgtgaaaa gcgatttcct 240 aacgttactt tggtgtacca atggtatttt gacgattcag tttgtggtta tattgcaacc 300 acatattcta atatttttaa ttatgaatca gaagagggaa aaggtcacaa gaaaacaatc 360 atagtttctt tgagaggaac aagatcaata ttcgattcgt atactgacat taaagttgat 420 atggtaaatt actataacta tggaagtaat attcaagaat gtggaaccga ttgtaaagtt 480 catcgagggt tctataaata ttatattaat actcttctta agatagaagg aattctacga 540 aatgaattac aaaccgatga tgattatgag ctattgatag tgggccattc tttgggcggt 600 gt; gtaacaatgg gtcaacctct agtagggaat aagcaatttg ctgaatttgt ggataatgtt 720 atggggagca gattgcctat tgaacataat actttcaata gaaaattttt cagagttatt 780 cataaggatg atattgttgc aactattccc agtaataata gaatattgga ttcatactct 840 caatttgata atcaaatcta cttgaattgt ctggcttccg acacgatgcc ttctttagag 900 caggtgttgg actgttttga tggtgataat ccccagtgca ttagtggcga cattgagaat 960 tatctcttgt ctcataacta tttacaaatt catactactt atttccgctc aatgggttta 1020 tgtggaataa gaatctaa 1038 <210> 15 <211> 1542 <212> DNA <213> Artificial Sequence <220> <223> LIP4 <400> 15 atgatgatta ccacgagaat cttggtatgt tttgctttaa tagcgctcac ttttgctgca 60 ccattgacag ttttaaaacc atccgaagat gaattttatg ctgccccaga tggttttgaa 120 gatgagaagt taggtactat tcttaaatgg agaaagactc cttatcagat aaagagtatc 180 tacttcccag ttaacattaa gaatagttgg caattgcttg tgagatctga agatgcaatc 240 ggtaatccag ttgcagtcac tgcttccctt tttgaaccgt acaatggtaa cacatccagg 300 ttggtctcat accaagttgc agaggatagt gcttcgttcg actgtgcacc gtcatattca 360 tttgtaggag gaggttacca tacagtggtt gcaaaagctg agatgatttt gatacaagga 420 gcattagatc aaggttatta cgttgttgca ccagattatg aaggacccaa tgctgtattt 480 actgctggtg taacatctgg gatgagtacc atcaatgttc tcagagctgt attaggcgaa 540 ggtaggagaa atgagaccag aattgaccca gatgcagaag tggttctttg gggatatagt 600 ggaggcacaa tccctagtgg ttgggcagnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 660 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 720 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnagcaga gattgtcgat 780 ggtactttat tcagtgggtt agttcctgcg gcactcaatg gattatcagc acagtatgaa 840 gaaatggatg aagctattga taagtttctt aaacctaaca aactagaaaa attcagatct 900 ggaagaaatg agtgtgctat agatgtggta ttttcttttg catatcaaga tacttttagt 960 gggtttgata cttattccaa agatggatgg actattttgg atgacccaga tgtcagagat 1020 attcttgccg aaaataccct tggagtaaat gtgaccaaaa agttcaaacc agagattcca 1080 ttatttgttt atcatggcat tcttgatgaa attgttcctt tcaaagacgc acagagagtt 1140 tatgatgttt ggtgtgaaga aggttatgat tcttttgaat ttgcagtttc taattctact 1200 ggacacattc ttgaagttat tgaaggtagt gcagccggat taaaatggat atcggacaga 1260 ttcgatggag tacaaccaac caaaggatgt catagacaaa caagattaac taatcttttc 1320 tatccaggtc tgtttacagg agtcacagac atcttatctg cattaatgag aaatatattc 1380 ggaagtccaa ttggtctcta cgatgaacaa attgaaaaga gatcagatat gtccgaagac 1440 gaaaaattat tgaagagact gtttgaattc actgattctg aaatattcaa aagaattgtt 1500 caacacagtt accctgaaat ggccaaaaaa ttagacttat aa 1542 <210> 16 <211> 1491 <212> DNA <213> Artificial Sequence <220> <223> LIP5 <400> 16 atgattttaa cgaatcttct tgtatatttc agcttgatag cgtgcacaat ttgtgctcca 60 atgacgatct tgaaaccttc tgaagatgac ttttatactg ctccagatgg ctttgaggat 120 gaaaagttag gtactattct taaatggaga aagactcctt atcagattaa gagtatctac 180 ttcccagtta acattaagaa tagttggcaa ttgcttgtga gatctgaaga tgcaatcggt 240 aatccagttg cagtcactgc ttcccttttt gaaccgtaca atggtaacac atccaggttg 300 gtctcatacc aagttgcaga ggatagtgct tcgttcgact gtgcaccgtc atattcattt 360 gtaggaggag gttaccatac agtggttgca aaagctgaga tgattttgat acaaggagca 420 ttagatcaag gttattacgt tgttgcacca gactatgaag gacccaatgc tgcatttact 480 gccgggataa catccggcat gagcaccatt aatgttcttc gagcagtctt aagtgatcag 540 aggagtaatg aaacgagaat tgactcagat gcagaagtgg ttctttgggg atatagtgga 600 ggcacaatcc ctagtggttg ggcagcatct atggctccat ggtatgctaa agatatcaat 660 gataatctta aaggagcagc aatgggtgga tgggtaacta atattactgc cacggctgaa 720 attgtagaag ggtccctttt cgaaggattg gttgcagcag ccatcaatgg attagcacaa 780 caatatgaag aaattaatga ggcacttgat gtttatcttg tacctgataa gctagagaca 840 tttagatcag caaagaatga atgcgttatt gatgtagtat tttcctttgc ttatcaaagt 900 gcattcagtg gaagtgatcc ttatacactg gatggatggg gtgtattaga agacccaaaa 960 gtgaagaaga ttgtcaacca aaataccctt ggattaaatg tgactgaaaa attcaaacca 1020 gagatcccac tatttgttta tcatggaatt cttgatgaaa ttgttccttt caaagatgca 1080 cagagagttt atgatgtttg gtgtaaagaa ggcattaatt catttgaatt tgccgtctct 1140 aattctactg gccatcttct tgaggtactt gaaggcagtg gagcagcgtt gaagtggata 1200 tctgacagat tcgatggagt tgcaccaacc aaaggatgtc atagacaaac aagattaagt 1260 aatatcttct atccaggtct gtttactgga atatctgaca ttttatctgc tttagtcaga 1320 aatgtcatgg gaagccctat tggactttat gatgaacaag ttgaaaagag atcagatgta 1380 accgaagacg aaaaattatt gaaaaggctg tttgcataca cagatgaaga aatctttaga 1440 agaatacttg acaacagcta tccagatatg gctcaaaatt tgggctatta a 1491 <210> 17 <211> 1476 <212> DNA <213> Artificial Sequence <220> <223> LIP6 <400> 17 atgttgatta ccacgaaaat cttggtatat tttgctttga taatcttgac tagtgctgct 60 ccaatgacaa tcttgaaacc ttctgaagat gaattctatg ctgccccaga tggctttgaa 120 gatgagaagt taggcactat tcttaaatgg agaaagactc cttatcagat taagagtatc 180 tacttcccag ttaacattaa gaatagttgg gaattgcttg ttagatctga agatgcgatt 240 ggagatccag ttgcagttac tgctactatc tttgagcctt ataatgccaa ttcatctagg 300 ttggtttcat accaagttgc agaggacagt ccttcattca actgttcacc atcatatgca 360 tttgtaggtg gtggctaccc gacagtagta accaaggctg agatgatatt aatccagtcc 420 gctttgaatg agggctatta cgttgtagtt cctgattacg agggccctaa tgctgtattt 480 ggggtaggtg ttacttctgc aatgagcacc attaatgttc ttagagcagt tttgagtgat 540 cagagacgta atgaaaccag aattgactca gacgcagaag tggttctttg gggatatagt 600 ggaggaacaa ttccaagcag ttgggctgca tctatggctc catggtatgc taaagatatc 660 aaccaacatc ttattggagc agcattggga ggatgggtta gcaatcttac ggcgcttatg 720 gagattgttg aaggatctct ctttgaaggt ttagttccaa atattataaa tggattgagt 780 caacaattcg aagaaataaa tgatgcattt gatgactatc tttatccaag taaaagagag 840 aaatttagat caacaaagaa gcaatgtgta tttgatgcct gtttgtcatt tgcattccaa 900 agtactttcc taggcgatca tccttacgta aaggggaaat ggagtgtctt ggaggaagca 960 aaagtgaaga atatcctcga aaataataca cttggattga aagtgactaa gaaattcaag 1020 ccagaaattc ctatatttgc atttcatgga ataaaagatg agatagttcc atttaaagac 1080 tctcaaagac tttatgatgt ttggtgtgaa gaagggataa attcatttga gtttgctgtt 1140 tccaaatcaa gtggacacct tcttgaagtt gttgaaggaa gtggggccgc tttggcatgg 1200 atatctgata gattcagggg tatacctgct gttaaaggct gtgaaaggaa agaaagatta 1260 accaatttgt tctatcctgg tctgttttct tctgtttcta atattttaat ggctcttatt 1320 agaaatatta tgggaagtcc aattggtctc tatgatgaac aagcaaaaaa gagatcaggc 1380 tctaccgaag aagagataat aatgaaaaga ctgcttgaat acactgacga agaaatctac 1440 aggagaataa tcgaaacaca ttcagatatg ttttag 1476 <210> 18 <211> 1392 <212> DNA <213> Artificial Sequence <220> <223> LIP7 <400> 18 atgttcaaac agcttttctt attatttagc ttacttattt ttgctttgac tcttccaact 60 ggtttggttc ctcctagtca agattctttt tattctgcac caactggatt cgagtctgca 120 gaattaggta ctatcttgaa gtttagacct tcgccagctc caattagaag tgtttacttc 180 aaagtcaaga ttaagagctc ctggcaatta ttggtcagat cttccgattc atttggtaat 240 ccttctgtta ttgtcaccac tgtctttgaa ccattcaatg ctgatccttc caaattaatt 300 tcttaccaag ttgctcaaga tagtgcctcc aatgattgtt ctccatctta tgctttcatg 360 gatggtggag gatttgaaac cgtgacttcc caagcagaaa tgcttttaat tcaaactgca 420 ttagatcaag gttattatgt tgtttctcca gattatgaag gattaaaggc tgtttatact 480 gggggtattc aatctggtca tggtactttg gattcactta ctgcagcttt aactagtaaa 540 aatattacag ggattaatag tgatgctaaa tctattcttt ggggttattc tggtggcgct 600 ttagctgctg gatgggctgc tgctttacaa ccaacttatg ctccagactt gaaacaaagt 660 ttacttggtg ctgcacttgg tggatttgtt actaatgtta ctgctacggt tactgcagtt 720 aatggtggtc cattttctgg tttagttggt atgggtattg ctgggttgag taacgaatat 780 cctgaattga aagactattt aaaagaacaa atgtacccag ataaatacga gcaatttcaa 840 gagatttatg gactttgtac agcagaaggt gcacttaagt ttgctttcac cgactacttc 900 aatggaccaa ataagtacgt taatggtatt ggagttttag ctaacgaacc cgctgctagt 960 attcttaaaa ataatacgct tggacttgtt ccatcccaag ttccagatat tccacttttt 1020 gtttatcacg gtgtaattga ttcccttgtt ccttataagg aaacagaaag agtgtataat 1080 acttggtgtg acgaaggaat tgagtcgttt gaattggccg ctgacttaac tgccgggcac 1140 cttaccgagg ttcttcaagg aagtggtgct gcttttggat ggattacgaa gagattcgat 1200 gggaaagccc caatttcagg ctgcagaaaa accaaaagat taactaattt attataccca 1260 ggaacagtgg gatccgttac caatttgatt tcagccttat tcaccaatgt tcttggcggt 1320 cialis catatcaatt ag 1392 <210> 19 <211> 1941 <212> DNA <213> Artificial Sequence <220> <223> LIP8 <400> 19 atgcatatgc atatcgatga acctgtgcat aaatatctat ctccgatgca taaacaccca 60 tttccaatga cttttatacc gtttttggta gctttcttca tgacatatga atgtactgtc 120 agccactctt atcagttttt ggtatataca cacaagccaa cttttgagaa aatgacccaa 180 aacctgaatc tcagaaatca ccacaagcaa atgaccaaaa acaacgagag atggagcccc 240 atacgctggg tattggggct ggcacttgta acaacggtag ttctttacat tcacggggga 300 aatatattta aagagtcaga ggattatata agcaaccaag tcatggaggc tgcagatctt 360 cagggtgata gctttcggct taagcatatc ttccagcatg gggccggatc tgagtattat 420 cgggttcata gacgtcttga tatcacggag gaatacttgt cccgtacggg gcttgacgaa 480 gtggccatgg aagctgacac aactgacgtt agcagcaata gtttagaaga tgtctatgct 540 caaaacgact ggccattggc atttcaaggc cataatccat ggaatatgaa gatgcctgtg 600 cgtagcagta accacaaggc gaagatccgg cggttgacag agcgccatac gcctggattt 660 ttagattcat acttggatta cgccattgaa gtcaagggga atcctcaaaa acttaatatg 720 attaatttac aatgggatga agaggaagac ttgatacttc cagatgtgca tgataaagag 780 tcattggtaa tgttggcatt gatgtcttcc aatgcgtatg tgaagtttcc aaaggatgat 840 aatgaaaaga aaaagagtga ctggcgagat gtaggagaac catgggaacc agatgaaaac 900 aatactgaca ttgaatttgg atgggatgga gatggggtcc ggggacatgt atttgtcaat 960 gaagttaaca atacagtggt tattgcattg aaaggtacga gtggagcagg cattcctggt 1020 gcaggagaag acgaaactac caataatgat aaacttaatg ataatttatt attcctgtgt 1080 tgttgtgctc gtgtaagtta cttgtggaca acggtctgtg actgttatga gaaggcttac 1140 acttgtaatc aggattgctt ggagaaagaa ttgacccgta aagatagata ttatcaagca 1200 gcattagaaa tctatcgaaa tgttagtagt atctatcctt ctaatcagta taaaatatgg 1260 ttaacaggcc attcattagg gggatcattg gcttcattag tggggagaac ttatggactc 1320 cctgtagtag cttttgaagc accaggagag ctattagcta ctcaaagact tcatttacct 1380 caacctccag gatatcccaa atatatggaa catatttacc atattggtaa tactgcagac 1440 cctattttca tgggggtatg taatggagtt tcttcaacat gtaatgcagc gggttatgca 1500 atggaaactg cttgtcatac tggaaaatta tgtgtttatg atgttgtcac tgatagagga 1560 tggagtgtta atgtattaaa tcacaggatc cacactgtgg tggatgaaat tatcttaaag 1620 tacaatacaa cagcagaatg tgttgaacaa ccaccatgta gagattgctt caattggaga 1680 tatgttactc atgatgatga tgaggatgat gagccaaaat tgcccaaccc tttaattcct 1740 catatttcac accacagtac tgctacgaag tcacaaggac catcacatac agcttatgat 1800 agtatcagta gtagtagcag cagcggtagc agcagtccta gtagtagtaa acttccagag 1860 aaacaaaaat gtttgaagag aacatggtac ggctggtgca gagaatgggg accagctgac 1920 ggagaaggtg atcaattttg a 1941 <210> 20 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> STFPL1-F <400> 20 ctcgccttag ataaaagagc cagtacccca ttgagtc 37 <210> 21 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> STFPL2-F <400> 21 ctcgccttag ataaaagacc taccacttta gttcct 36 <210> 22 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> STFPL3-F <400> 22 ctcgccttag ataaaagaaa tatttgggtt tatcct 36 <210> 23 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> STFPL4-F <400> 23 ctcgccttag ataaaagagc accattgaca gtttta 36 <210> 24 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> STFPL5-F <400> 24 ctcgccttag ataaaagagc tccaatgacg atcttg 36 <210> 25 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> STFPL6-F <400> 25 ctcgccttag ataaaagagc tccaatgaca atcttgaa 38 <210> 26 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> STFPL7-F <400> 26 ctcgccttag ataaagact tccaactggt ttggttcc 38 <210> 27 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> STFPL8-F <400> 27 ctcgccttag ataaaagaat gcatatgcat atcgat 36 <210> 28 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> STFPL1-R <400> 28 cactccgttc aagtcgactt aatctttgca accagtg 37 <210> 29 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> STFPL2-R <400> 29 cactccgttc aagtcgactt attcacttct agtaga 36 <210> 30 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> STFPL3-R <400> 30 cactccgttc aagtcgactt agattcttat tccaca 36 <210> 31 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> STFPL4-R <400> 31 cactccgttc aagtcgactt ataagtctaa ttttttgg 38 <210> 32 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> STFPL5-R <400> 32 cactccgttc aagtcgactt aatagcccaa attttgag 38 <210> 33 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> STFPL6-R <400> 33 cactccgttc aagtcgactt aaaacatatc tgaatgtg 38 <210> 34 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> STFPL7-R <400> 34 cactccgttc aagtcgactt aattgatatg gaagaagt 38 <210> 35 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> STFPL8-R <400> 35 cactccgttc aagtcgactt aaaattgatc accttctc 38 <210> 36 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> LNK39 <400> 36 ggccgcctcg gcctctgctg gcctcgcctt agataaaaga 40 <210> 37 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> GT50R <400> 37 gtcattatta aatatatata tatatatatt gtcactccgt tcaagtcgac 50 <210> 38 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> TFP1 <400> 38 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> 39 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> TFP2 <400> 39 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> 40 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> TFP3 <400> 40 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> 41 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> TFP4 <400> 41 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> 42 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> TFP5 <400> 42 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> 43 <211> 93 <212> PRT <213> Artificial Sequence <220> <223> TFP6 <400> 43 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> 44 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> TFP7 <400> 44 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> 45 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> TFP8 <400> 45 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> 46 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> TFP9 <400> 46 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> 47 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> TFP10 <400> 47 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> 48 <211> 77 <212> PRT <213> Artificial Sequence <220> <223> TFP11 <400> 48 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> 49 <211> 94 <212> PRT <213> Artificial Sequence <220> <223> TFP12 <400> 49 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> 50 <211> 174 <212> PRT <213> Artificial Sequence <220> <223> TFP13 <400> 50 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> 51 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> TFP14 <400> 51 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> 52 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> TFP15 <400> 52 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> 53 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> TFP16 <400> 53 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> 54 <211> 195 <212> PRT <213> Artificial Sequence <220> <223> TFP17 <400> 54 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> 55 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> TFP18 <400> 55 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> 56 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> TFP19 <400> 56 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> 57 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> TFP20 <400> 57 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> 58 <211> 176 <212> PRT <213> Artificial Sequence <220> <223> TFP21 <400> 58 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> 59 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> TFP22 <400> 59 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> 60 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> TFP23 <400> 60 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> 61 <211> 170 <212> PRT <213> Artificial Sequence <220> <223> TFP24 <400> 61 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> 62 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> TFP1 <400> 62 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> 63 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> TFP2 <400> 63 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> 64 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> TFP3 <400> 64 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> 65 <211> 186 <212> DNA <213> Artificial Sequence <220> <223> TFP4 <400> 65 atgagatttg cagaattctt ggtggtattt gccacgttag gcggggggat ggctgcaccg 60 gttgagtctc tggccgggac ccaacggtat ctggtgcaaa tgaaggagcg gttcaccaca 120 gagaagctgt gtgctttgga cgacaaggcc gcctcggcct ctgctggcct cgccttagat 180 aaaaga 186 <210> 66 <211> 291 <212> DNA <213> Artificial Sequence <220> <223> TFP5 <400> 66 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> 67 <211> 279 <212> DNA <213> Artificial Sequence <220> <223> TFP6 <400> 67 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> 68 <211> 678 <212> DNA <213> Artificial Sequence <220> <223> TFP7 <400> 68 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> 69 <211> 192 <212> DNA <213> Artificial Sequence <220> <223> TFP8 <400> 69 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> 70 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> TFP9 <400> 70 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> 71 <211> 597 <212> DNA <213> Artificial Sequence <220> <223> TFP10 <400> 71 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> 72 <211> 231 <212> DNA <213> Artificial Sequence <220> <223> TFP11 <400> 72 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> 73 <211> 282 <212> DNA <213> Artificial Sequence <220> <223> TFP12 <400> 73 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> 74 <211> 522 <212> DNA <213> Artificial Sequence <220> <223> TFP13 <400> 74 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> 75 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> TFP14 <400> 75 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> 76 <211> 471 <212> DNA <213> Artificial Sequence <220> <223> TFP15 <400> 76 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> 77 <211> 294 <212> DNA <213> Artificial Sequence <220> <223> TFP16 <400> 77 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> 78 <211> 585 <212> DNA <213> Artificial Sequence <220> <223> TFP17 <400> 78 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> 79 <211> 315 <212> DNA <213> Artificial Sequence <220> <223> TFP18 <400> 79 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> 80 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> TFP19 <400> 80 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> 81 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> TFP20 <400> 81 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> 82 <211> 528 <212> DNA <213> Artificial Sequence <220> <223> TFP21 <400> 82 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> 83 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> TFP22 <400> 83 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> 84 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> TFP23 <400> 84 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> 85 <211> 510 <212> DNA <213> Artificial Sequence <220> <223> TFP24 <400> 85 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 <210> 86 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> BCSPlip2-F <400> 86 tccaaaaaaa aagtaagaat ttttgaaaat tcaaggatcc atgatctgtt cattctttag 60                                                                           60 <210> 87 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> BCSPlip5-F <400> 87 tccaaaaaaa aagtaagaat ttttgaaaat tcaaggatcc atgattttaa cgaatctt 58 <210> 88 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> BCSPlip7-F <400> 88 tccaaaaaaa aagtaagaat ttttgaaaat tcaaggatcc atgttcaaac agcttttc 58 <210> 89 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> BCSPlip2-R <400> 89 aatatatata tatatatatt gtcactccgt tcaagtcgac ctattcactt ctagtagaat 60                                                                           60 <210> 90 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> BCSPlip5-R <400> 90 aatatatata tatatatatt gtcactccgt tcaagtcgac ttaatagccc aaattttg 58 <210> 91 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> BCSPlip7-R <400> 91 aatatatata tatatatatt gtcactccgt tcaagtcgac ctaattgata tggaagaa 58 <210> 92 <211> 496 <212> PRT <213> Artificial Sequence <220> <223> CbLIP5 codon modification <400> 92 Met Ile Leu Thr Asn Leu Leu Val Tyr Phe Ser Leu Ile Ala Cys Thr   1 5 10 15 Ile Cys Ala Pro Met Thr Ile Leu Lys Pro Ser Glu Asp Asp Phe Tyr              20 25 30 Thr Ala Pro Asp Gly Phe Glu Asp Glu Lys Leu Gly Thr Ile Leu Lys          35 40 45 Trp Arg Lys Thr Pro Tyr Gln Ile Lys Ser Ile Tyr Phe Pro Val Asn      50 55 60 Ile Lys Asn Ser Trp Gln Leu Leu Val Arg Ser Glu Asp Ala Ile Gly  65 70 75 80 Asn Pro Val Ala Val Thr Ala Ser Leu Phe Glu Pro Tyr Asn Gly Asn                  85 90 95 Thr Ser Arg Leu Val Ser Tyr Gln Val Ala Glu Asp Ser Ala Ser Phe             100 105 110 Asp Cys Ala Pro Ser Tyr Ser Phe Val Gly Gly Gly Tyr His Thr Val         115 120 125 Val Ala Lys Ala Glu Met Ile Leu Ile Gln Gly Ala Leu Asp Gln Gly     130 135 140 Tyr Tyr Val Val Ala Pro Asp Tyr Glu Gly Pro Asn Ala Ala Phe Thr 145 150 155 160 Ala Gly Ile Thr Ser Gly Met Ser Thr Ile Asn Val Leu Arg Ala Val                 165 170 175 Leu Ser Asp Gln Arg Ser Asn Glu Thr Arg Ile Asp Ser Asp Ala Glu             180 185 190 Val Val Leu Trp Gly Tyr Ser Gly Gly Thr Ile Pro Ser Gly Trp Ala         195 200 205 Ala Ser Met Ala Pro Trp Tyr Ala Lys Asp Ile Asn Asp Asn Leu Lys     210 215 220 Gly Ala Ala Met Gly Gly Trp Val Thr Asn Ile Thr Ala Thr Ala Glu 225 230 235 240 Ile Val Glu Gly Ser Leu Phe Glu Gly Leu Val Ala Ala Ala Ile Asn                 245 250 255 Gly Leu Ala Gln Gln Tyr Glu Glu Ile Asn Glu Ala Leu Asp Val Tyr             260 265 270 Leu Val Pro Asp Lys Leu Glu Thr Phe Arg Ser Ala Lys Asn Glu Cys         275 280 285 Val Ile Asp Val Val Phe Ser Phe Ala Tyr Gln Ser Ala Phe Ser Gly     290 295 300 Ser Asp Pro Tyr Thr Ser Asp Gly Trp Gly Val Leu Glu Asp Pro Lys 305 310 315 320 Val Lys Lys Ile Val Asn Gln Asn Thr Leu Gly Leu Asn Val Thr Glu                 325 330 335 Lys Phe Lys Pro Glu Ile Pro Leu Phe Val Tyr His Gly Ile Leu Asp             340 345 350 Glu Ile Val Pro Phe Lys Asp Ala Gln Arg Val Tyr Asp Val Trp Cys         355 360 365 Lys Glu Gly Ile Asn Ser Phe Glu Phe Ala Val Ser Asn Ser Thr Gly     370 375 380 His Leu Leu Glu Val Leu Glu Gly Ser Gly Ala Ala Leu Lys Trp Ile 385 390 395 400 Ser Asp Arg Phe Asp Gly Val Ala Pro Thr Lys Gly Cys His Arg Gln                 405 410 415 Thr Arg Leu Ser Asn Ile Phe Tyr Pro Gly Ser Phe Thr Gly Ile Ser             420 425 430 Asp Ile Leu Ser Ala Leu Val Arg Asn Val Met Gly Ser Pro Ile Gly         435 440 445 Leu Tyr Asp Glu Glu Val Glu Lys Arg Ser Asp Val Thr Glu Asp Glu     450 455 460 Lys Leu Leu Lys Arg Ser Phe Ala Tyr Thr Asp Glu Glu Ile Phe Arg 465 470 475 480 Arg Ile Leu Asp Asn Ser Tyr Pro Asp Met Ala Gln Asn Leu Gly Tyr                 485 490 495 <210> 93 <211> 1491 <212> DNA <213> Artificial Sequence <220> <223> CbLIP5 codon modification <400> 93 atgattttaa cgaatcttct tgtatatttc agcttgatag cgtgcacaat ttgtgctcca 60 atgacgatct tgaaaccttc tgaagatgac ttttatactg ctccagatgg ctttgaggat 120 gaaaagttag gtactattct taaatggaga aagactcctt atcagattaa gagtatctac 180 ttcccagtta acattaagaa tagttggcaa ttgcttgtga gatctgaaga tgcaatcggt 240 aatccagttg cagtcactgc ttcccttttt gaaccgtaca atggtaacac atccaggttg 300 gtctcatacc aagttgcaga ggatagtgct tcgttcgact gtgcaccgtc atattcattt 360 gtaggaggag gttaccatac agtggttgca aaagctgaga tgattttgat acaaggagca 420 ttagatcaag gttattacgt tgttgcacca gactatgaag gacccaatgc tgcatttact 480 gccgggataa catccggcat gagcaccatt aatgttcttc gagcagtctt aagtgatcag 540 aggagtaatg aaacgagaat tgactcagat gcagaagtgg ttctttgggg atatagtgga 600 ggcacaatcc ctagtggttg ggcagcatct atggctccat ggtatgctaa agatatcaat 660 gataatctta aaggagcagc aatgggtgga tgggtaacta atattactgc cacggctgaa 720 attgtagaag ggtccctttt cgaaggattg gttgcagcag ccatcaatgg attagcacaa 780 caatatgaag aaattaatga ggcacttgat gtttatcttg tacctgataa gctagagaca 840 tttagatcag caaagaatga atgcgttatt gatgtagtat tttcctttgc ttatcaaagt 900 gcattcagtg gaagtgatcc ttatacatct gatggatggg gtgtattaga agacccaaaa 960 gtgaagaaga ttgtcaacca aaataccctt ggattaaatg tgactgaaaa attcaaacca 1020 gagatcccac tatttgttta tcatggaatt cttgatgaaa ttgttccttt caaagatgca 1080 cagagagttt atgatgtttg gtgtaaagaa ggcattaatt catttgaatt tgccgtctct 1140 aattctactg gccatcttct tgaggtactt gaaggcagtg gagcagcgtt gaagtggata 1200 tctgacagat tcgatggagt tgcaccaacc aaaggatgtc atagacaaac aagattaagt 1260 aatatcttct atccaggttc ttttactgga atatctgaca ttttatctgc tttagtcaga 1320 aatgtcatgg gaagccctat tggactttat gatgaacaag ttgaaaagag atcagatgta 1380 accgaagacg aaaaattatt gaaaaggtct tttgcataca cagatgaaga aatctttaga 1440 agaatacttg acaacagcta tccagatatg gctcaaaatt tgggctatta a 1491 <210> 94 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Lip5CTG1-F <400> 94 agtgatcctt atacatctga tggatggggt gta 33 <210> 95 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Lip5CTG1-R <400> 95 tacaccccat ccatcagatg tataaggatc act 33 <210> 96 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Lip5CTG2-F <400> 96 atcttctatc caggttcttt tactggaata tct 33 <210> 97 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Lip5CTG2-R <400> 97 agatattcca gtaaaagaac ctggatagaa gat 33 <210> 98 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Lip5CTG3-F <400> 98 aaattattga aaaggtcttt tgcatacaca gat 33 <210> 99 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Lip5CTG3-R <400> 99 atctgtgtat gcaaaagacc ttttcaataa ttt 33

Claims (21)

Candida butyri strain with accession number KCTC18455P, having lipase activity.
delete SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 92.
4. The lipase according to claim 3, wherein the lipase is encoded by any one of nucleotide sequences selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO:
A lipase secretion expression cassette comprising a nucleic acid encoding the lipase of claim 3 or 4 and a nucleic acid encoding a translational fusion partner (TFP).
6. The method according to claim 5,
TFP 3 of SEQ ID NO: 40, TFP 4 of SEQ ID NO: 41, TFP 5 of SEQ ID NO: 42, TFP 6 of SEQ ID NO: 43, TFP 7 of SEQ ID NO: 44, SEQ ID NO: TFP 9 of SEQ ID NO: 47, TFP 9 of SEQ ID NO: 47, TFP 11 of SEQ ID NO: 48, TFP 12 of SEQ ID NO: 49, TFP 13 of SEQ ID NO: 50, TFP 14 of SEQ ID NO: 51, SEQ ID NO: 52, TFP 16 of SEQ ID NO: 53, TFP 17 of SEQ ID NO: 54, TFP 18 of SEQ ID NO: 55, TFP 19 of SEQ ID NO: 56, TFP 20 of SEQ ID NO: 57, TFP 21 of SEQ ID NO: 58, SEQ ID NO: 59 A TFP 23 of SEQ ID NO: 60, and a TFP 24 of SEQ ID NO: 61.
6. The method of claim 5, wherein the lipase has an amino acid sequence of SEQ ID NO: 5 and is linked to TFP 11 of SEQ ID NO: 48, TFP 13 of SEQ ID NO: 50, TFP 19 of SEQ ID NO: 56, or TFP 23 of SEQ ID NO: , A lipase secretion expression cassette.
6. The method of claim 5, wherein the lipase has the amino acid sequence of SEQ ID NO: 8 and is linked to TFP8 of SEQ ID NO: 45, TFP 9 of SEQ ID NO: 46, TFP 13 of SEQ ID NO: 50, or TFP 19 of SEQ ID NO: Lipase secreted expression cassette.
6. The method of claim 5, wherein the lipase has an amino acid sequence of SEQ ID NO: 10, TFP4 of SEQ ID NO: 41, TFP8 of SEQ ID NO: 45, TFP13 of SEQ ID NO: 50, TFP20 of SEQ ID NO: 57, TFP 21 of SEQ ID NO: 59, TFP 23 of SEQ ID NO: 60, or TFP 24 of SEQ ID NO: 61.
6. The method of claim 5, wherein the lipase has the amino acid sequence of SEQ ID NO: 92 and is linked to TFP8 of SEQ ID NO: 45, TFP 9 of SEQ ID NO: 46, TFP 13 of SEQ ID NO: 50, or TFP 19 of SEQ ID NO: Lipase secreted expression cassette.
6. The lipase secretion expression cassette according to claim 5, wherein the nucleic acid encoding the lipase and the nucleic acid encoding the protein secretion fusion factor are linked to each other by a linker.
12. The cassette of claim 11, wherein said linker comprises a nucleic acid encoding a protease recognition sequence or an affinity tag.
6. The cassette of claim 5, wherein the secretion expression cassette further comprises a nucleic acid encoding an affinity tag that is linked to the carboxy terminal or amino terminus of the lipase.
14. The method of claim 13, wherein the affinity tag is selected from the group consisting of GST, MBP, NusA, thioredoxin, ubiquitin, FLAG, BAP, HIS, STREP, CBP, CBD, , A lipase secretion expression cassette.
A vector comprising the lipase secretion expression cassette of claim 5.
A transforming microorganism comprising the lipase of claim 3 or 4, or a nucleic acid encoding said lipase and a nucleic acid encoding a protein secretion fusion factor.
17. The transforming microorganism according to claim 16, wherein the transforming microorganism is yeast.
18. The method of claim 17, wherein the yeast is selected from the group consisting of Candida , Debaryomyces , Hansenula , Kluyveromyces , Pichia , Schizosaccharomyces , Wherein said strain is selected from the group consisting of Yarrowia , Saccharomyces , Schwanniomyces and Arxula .
18. The method of claim 17, wherein the yeast is selected from the group consisting of Candida butyri , Candida utilis , Candida boidinii), Candida albicans (Candida albicans), Cluj Vero Mai Seth lactis (Kluyveromyces lactis , Pichia pastoris , Pichia stipitis ), skiing investigation Schizosaccharomyces pombe ), Saccharomyces cerevisiae , Hansenula ( Hansenula polymorpha , Yarrowia lipolytica , Schwanniomyces occidentalis , and Arxula adeninivorans . &lt; Desc / Clms Page number 13 &gt;
(i) culturing the transformed microorganism of claim 16; And
(ii) recovering the lipase from the cultured microorganism or culture supernatant thereof.
4. A method for producing biodiesel comprising the steps of: maintaining a lipase of claim 3 or 4 and reacting it with an alcohol.

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