KR20200013439A - A mutant lipase having enhanced transesterification activity and its application for biodiesel production - Google Patents

Abstract

The present invention relates to a lipase secretion expression cassette comprising nucleic acid encoding lipase, an expression vector comprising the lipase secretion expression cassette, a transformed microorganism containing the expression vector, a method for preparing lipase by culturing the microorganism, a method for converting vegetable waste oil to biodiesel using the lipase, and mutant lipase.

Description

Mutant lipase having enhanced ester transfer efficiency and biodiesel conversion technology using the same {A mutant lipase having enhanced transesterification activity and its application for biodiesel production}

The present invention relates to a lipase secretion expression cassette, comprising a nucleic acid encoding a lipase, comprising an expression vector comprising the lipase secretion expression cassette, a transformed microorganism containing the expression vector, and culturing the microorganism to produce a lipase. A method, a method for converting vegetable waste oil into biodiesel using the lipase, and a mutant lipase.

Lipases hydrolyze ester bonds in neutral lipids to produce fatty acids and glycerol and have varying substrate specificities depending on the carbon number of the fatty acids. In addition to hydrolysis, lipase can perform various reactions such as esterification reaction, ester bond transfer reaction, amino decomposition, acid decomposition, and alcohol decomposition, and can be easily obtained in large quantities by genetic engineering technology. Research into industrial applications in the industrial and pharmaceutical industries is constantly expanding.

Biodiesel is a fatty acid methyl ester produced by esterification of copper and vegetable oils with methanol.They have very similar properties to existing petroleum-based diesel oils. If used, the demand and necessity of the advanced alternative clean energy that can drastically reduce the pollution of automobiles, which is the main cause of air pollution, have been greatly increased. The production of biodiesel in various oils requires two stages of chemical reactions. First, fatty acids and glycerol are produced by hydrolysis of lipids, followed by esterification of fatty acids and alcohols (methanol) to methyl ester compounds, that is, biodiesel. To produce.

Biodiesel production mainly uses chemical catalytic methods using strong bases, etc., but has a disadvantage of causing secondary environmental pollution due to complex reaction processes, by-product generation, and generation of wastewater. In particular, the use of vegetable oil as a biodiesel raw material not only causes food problems, but also has a high price of biodiesel since the price is high. To this end, non-edible low-cost waste oil, drinking oil or sludge oil, etc. are used as raw materials for biodiesel. However, these waste oils have a higher acid value (free fatty acid content) and water content than purified pure oils, and thus, the production efficiency is very low due to saponification reaction when using a conventional base catalyst. Therefore, the production cost is increased because it has to go through complicated steps such as pretreatment with acid catalyst. On the other hand, biocatalytic lipases can perform both the basic catalyst for the transesterification reaction and the acid catalyst for the fatty acid ester reaction simultaneously under the same reaction conditions. Step biodiesel conversion is possible. Therefore, the development of biocatalytic biodiesel production technology is very active in an effort to utilize waste cooking oil as a raw material worldwide, and many commercial plants are in operation in the United States and China, and commercialization is also in progress in Brazil, Israel and Peru.

 Enzyme biodiesel technology was first reported by Nelson in 1996 that biodiesel production was possible through lipase in organic solvents and reported that biodiesel can be produced with high efficiency by immobilized lipase in China, Japan and Israel. Recently, Novozyme, a global enzyme company, is also producing and supplying enzymes for biodiesel. However, since the cost of biocatalysts is higher than that of conventional chemical catalysts, high activity lipases have been developed to secure economic feasibility of biodiesel production.

Accordingly, the present inventors have made a diligent effort to develop an economical biocatalytic lipase. As a result, Thermomyces ranusinosus ( Thermomyces) lanuginosus ) -derived lipases were selected for mutant lipases with improved ester transfer activity by at least 50% through a directed evolutionary technique, and the protein secretion fusion factor technology of yeast Saccharomyces cerevisiae (Korean Patent No. 0975596) Was used to mass produce lipase. The lipase thus produced was used to develop a biodiesel conversion optimization technology to complete the present invention.

One object of the invention is a lipase secretion expression cassette, comprising a nucleic acid encoding a lipase, wherein the lipase is one of the 46th, 108th, 155th, and 270th amino acids from the N-terminus in the amino acid sequence of SEQ ID NO: 1. It is to provide a lipase secretion expression cassette in which one or more amino acids are substituted with other amino acids.

Another object of the present invention to provide an expression vector comprising the lipase secretion expression cassette.

Still another object of the present invention is to provide a transformed microorganism comprising the expression vector.

Still another object of the present invention is the step of culturing the transgenic microorganism; And (ii) recovering the lipase from the cultured microorganism or the culture supernatant thereof.

Still another object of the present invention is to provide a method for converting vegetable waste oil into biodiesel using lipase produced from the transformed microorganism.

Another object of the present invention is a mutant lipase having increased ester transfer activity and secretory expression capacity, wherein the 46th, 108th, 155th, and 270th amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 1 are substituted with other amino acids. To provide.

This will be described in detail below. Meanwhile, each of the descriptions and the embodiments disclosed in the present invention may be applied to each of the other descriptions and the embodiments. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention is not to be limited by the specific description described below.

One aspect of the present invention for achieving the above object is to provide a lipase secretion expression cassette comprising a nucleic acid nucleic acid encoding a lipase. In addition, the lipase secretion expression cassette may further include a nucleic acid encoding a protein secretion fusion factor, but is not limited thereto.

As used herein, the term “lipase” is known to produce fatty acids and glycerol by hydrolyzing ester bonds of neutral lipids and having various substrate specificities depending on the carbon number of the fatty acids. Lipase is used in various fields of the industry because it can perform various reactions such as esterification reaction, ester bond transfer reaction, amino decomposition, acid decomposition, alcohol decomposition in addition to hydrolysis reaction.

For the purpose of the present invention, a lipase secretion expression cassette comprising a nucleic acid encoding a lipase and a nucleic acid encoding a protein secretion fusion factor is used to prepare a mutant lipase having improved ester transfer reaction efficiency, and a large amount of lipase is produced through a directed evolution technique. Produced. Specifically, the lipase of the present invention may be a lipase derived from Thermomyces lanuginosus , but is not limited thereto. In addition, the lipase of the present invention in other terms Thermomyces lanuginosus Lipase, TLL.

In a specific embodiment, the lipase secretion expression cassette, comprising a nucleic acid encoding a lipase of the present invention and a nucleic acid encoding a protein secretion fusion factor, comprises a lipase secretion expression cassette comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1. A lipase secretion expression cassette comprising a nucleic acid encoding a lipase and a nucleic acid encoding a protein secretion fusion factor, wherein the lipase is 46, 108 from the N-terminus in the amino acid sequence of SEQ ID NO: 1. It is to provide a lipase secretion expression cassette in which the second, 155th, and 270th amino acids are substituted with other amino acids. In addition, the lipase is a lipase secretion expression cassette in which the amino acid sequence of SEQ ID NO: 1 is substituted with asparagine from the N-terminus to the 46th amino acid, the 108th amino acid is vaginated, the 155th amino acid is glycine, and the 270th amino acid is substituted with aspartame. It may be, but is not limited thereto.

In addition, even if it is described in the present invention as a lipase secretion expression cassette, substituted with an amino acid sequence described by a specific sequence number, if it has the same or corresponding activity as a lipase secretion expression cassette consisting of the amino acid sequence of the sequence number It is apparent that proteins having amino acid sequences in which some sequences have been deleted, modified, substituted or added may also be used in the present invention. For example, in the case of having the same or corresponding activity as that of the lipase secretion expression cassette, in addition to the specific mutation that imparts specific activity, a meaningless sequence before or after the amino acid sequence of the corresponding SEQ ID number or a naturally occurring mutation, or its potential It is not intended to exclude sexual mutations, and it is obvious that even within the scope of the present application, even if such sequences are added or mutated.

Specifically, the lipase secretion expression cassette in which the amino acid of SEQ ID NO: 1 is substituted with another amino acid in the lipase secretion expression cassette may include the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80% homology or identity thereto, It is not limited to this. Specifically, the lipase secretion expression cassette of the present invention is a lipase secretion having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with SEQ ID NO: 1 and SEQ ID NO: 1. Expression cassettes. In addition, if there is an amino acid sequence having such homology or identity and corresponding efficacy to the protein, in addition to the amino acid sequence at the 46th, 108th, 155th, and 270th positions, an amino acid sequence in which some sequences are deleted, modified, substituted, or added It is obvious that a protein having a protein is included within the scope of the present invention.

The term “homology” refers to the percent identity between two polynucleotide or polypeptide moieties. It means the degree of coincidence with a given amino acid sequence or nucleotide sequence and can be expressed as a percentage. In this specification, homologous sequences thereof having the same or similar activity as a given amino acid sequence or base sequence are designated as "% homology". Homology between sequences from one moiety to another can be determined by known art. In addition, "identity" refers to the degree of sequence relevance between amino acid or nucleotide sequences, and in some cases determined by the match between strings of such sequences. For example, using standard software that calculates parameters such as score, similarity, etc., specifically using BLAST 2.0, or by comparing sequences by Southern hybridization experiments under defined stringent conditions. And suitable hybridization conditions that are defined are within the skill of the art and are 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.

In addition, it is obvious that a nucleic acid encoding a lipase secretion expression cassette consisting of SEQ ID NO: 1 or a lipase having homology with the homology thereof and a nucleic acid encoding a protein secretion fusion factor may also be included by codon degeneracy. In addition, a lipase having the activity of a lipase secretion expression cassette consisting of SEQ ID NO: 1 is hydrided under stringent conditions with a complementary sequence to all or part of the nucleotide sequence, for example, a probe that can be prepared from a known gene sequence. Any nucleic acid sequence encoding a nucleic acid encoding and a protein secretion fusion factor can be included without limitation.

In a specific embodiment of the present invention as a result of improving the TLL through the directional evolution, I108V, N270D in the first generation strain, additionally R155G in the second generation strain, it was analyzed that the amino acid mutation of K46N additionally in the third generation strain (Table 3 Finally, the third-generation strains (I108V, N270D, R155G, K46N) obtained were 68.7% increased compared to wild type (FIG. 10), and hydrolytic activity increased 78.2% (FIG. 11) and ester transfer activity increased 58.7%. It was confirmed (Fig. 12).

As used herein, the term protein fusion factor refers to a peptide which is fused with a gene encoding a low expression protein to induce secretion of a low expression protein and a gene encoding the same. It is disclosed in registered Patent No. 10-0975596.

The protein secretion fusion factor may be one protein fusion factor selected from the group consisting of SEQ ID NOs: 8 to 31, which may be represented as TFP 1 to TFP 24 in the present invention (Table 2).

Specifically, the protein secretion fusion factor TFP 6 consisting of the amino acid sequence of SEQ ID NO: 13, TFP 8 consisting of the amino acid sequence of SEQ ID NO: 15, TFP 11 consisting of the amino acid sequence of SEQ ID NO: 18, consisting of the amino acid sequence of SEQ ID NO: 20 It may be selected from the group consisting of TFP 13 and TFP 16 consisting of the amino acid sequence of SEQ ID NO: 23, but is not limited thereto.

In a specific embodiment of the present invention, the expression of the enzyme was compared and analyzed using a total of 25 transformed microorganisms to which one protein containing a self-secreting peptide and 24 protein secretion fusion factors were applied to express a lipase. As a result, it was confirmed that the secretion expression ability is excellent when using a protein secretion fusion factor than when using a self-secreting peptide.

As another embodiment, the present invention provides an expression vector comprising the expression cassette.

As used herein, the term "vector" refers to a nucleic acid encoding a target lipase and a nucleic acid encoding a protein secretion fusion factor operably linked to a suitable regulatory sequence to enable expression of the target lipase secretion expression cassette in a suitable host. DNA preparations containing sequences are meant. The regulatory sequence may comprise a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation. After being transformed into a suitable host cell, the vector can be replicated or function independent of the host genome and integrated into the genome itself.

The vector to be used in the present invention is not particularly limited as long as it can replicate in a host cell, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as a phage vector or cosmid vector. As a plasmid vector, pBR, pUC, and pBluescriptII systems are used. , pGEM system, pTZ system, pCL system and pET system can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pACY Duet-1 vectors and the like can be used.

For example, a nucleic acid encoding a desired lipase and a nucleic acid encoding a protein secretion fusion factor in the chromosome may be replaced with a mutated polynucleotide through an intracellular chromosome insertion vector. Insertion of the polynucleotide into the chromosome can be made by any method known in the art, such as, but not limited to, homologous recombination. The method may further include a selection marker for checking whether the chromosome is inserted. Selection markers are used to screen for cells transformed with a vector, i.e., to confirm the insertion of a desired nucleic acid molecule, and to select a phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface variant lipases. Markers that impart a may be used. In an environment treated with a selective agent, only cells expressing a selection marker survive or exhibit different expressing traits, so that transformed cells can be selected.

In addition, the expression vector, fragments for expression suppression having various functions for inhibiting or amplifying or inducing the expression of a target gene, markers for selection of transformed microorganisms, genes for resistance to antibiotics, and out of cells It may further include a gene encoding a signal for the purpose of secretion, a custom fusion factor suitable for the non-expressing protein, and the like. In particular, as a custom fusion factor suitable for the poorly expressed protein, it is preferable to use a translational fusion partner (TFP).

As another aspect of the present invention, the present invention provides a transformed microorganism wherein the expression vector of the present invention is introduced into a host cell.

In another embodiment, the present invention provides a microorganism that produces a lipase having increased reducing activity, including a nucleic acid encoding the lipase and a nucleic acid encoding a protein secretion fusion factor. Specifically, a microorganism comprising a nucleic acid encoding a lipase and a nucleic acid encoding a protein secretion fusion factor and / or a nucleic acid encoding the lipase and a nucleic acid encoding a protein secretion fusion factor may contain a nucleic acid encoding a lipase and a protein secretion fusion factor. It may be a microorganism produced by transformation with a vector containing a nucleic acid encoding, but is not limited thereto.

The term "transformation" in the present invention means introducing a vector comprising a polynucleotide encoding a target protein into a host cell to enable expression of the protein encoded by the polynucleotide in the host cell. The transformed polynucleotides may include all of them, as long as they can be expressed in the host cell, either inserted into the chromosome of the host cell or located outside the chromosome. The polynucleotide also includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be introduced into and expressed in a host cell. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self-expression. The expression cassette may include a promoter, a transcription termination signal, a ribosomal binding site, and a translation termination signal, which are typically operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self replication. In addition, the polynucleotide may be introduced into the host cell in its own form and operably linked with a sequence required for expression in the host cell, but is not limited thereto.

In addition, the term "operably linked" means that the gene sequence is functionally linked with a promoter sequence for initiating and mediating the transcription of a polynucleotide encoding a variant polypeptide of interest of the present invention.

Specifically, the host cell may be a cell having an ethanol fermentation ability, more specifically, the cell having an ethanol fermentation ability may be Zymonas, yeast, Bacillus. More specifically, the cell having the ethanol fermentation ability may be a strain Y2805G (Mat a pep4 :: HIS3 prb1 can1 his3-200 ura3-52 gal80).

The yeast is not particularly limited, but Candida , Debaryomyces , Hansenula , Kluyveromyces , Pichia , Schizosaccharomyces , Yarrowia , Saccharomyces , Saccharomycopsis , Schwanniomyces , Arxula species, more specifically Candida utilis ( Candida utilis), Candida seems dini (Candida boidinii), Candida albicans (Candida albicans ), Kluyveromyces lactis , Pichia pastoris pastoris , Pichia stipitis , Schizosaccharomyces pombe ), Saccharomyces cerevisiae ), Saccharomycopsis fiburigera , Hansenula polymorpha ( Hansenula) polymorpha ), Yarrowia lipolytica ), Schwanniomyces occidentalis , Arxula adeninivorans , etc., and most specifically Kluyveromyces maxianus , Saccharomyces ceres Saccharomyces cerevisiae , Saccharomycopsis fiburigera ) can be used.

In one embodiment of the present invention, yeast Saccharomyces cerevisiae Y2805G (Mat a pep4 :: HIS3 prb1 can1 his3-200 ura3-52) using an expression vector comprising a polynucleotide encoding a lipase of the present invention. gal 80) After transforming the strain, it was confirmed that each enzyme was produced from the transforming microorganism, and compared with the activity of the ribase produced from the microorganism, the secreting ability of the protein was better than when using a secreted peptide When secretion fusion factors 6, 8, 11, 13, 16 were used, it was confirmed that the secretion expression ability is better (Fig. 4).

As another aspect of the present invention, the method comprises the steps of: (i) culturing the transgenic microorganism; And (ii) recovering the lipase from the cultured microorganism or the culture supernatant thereof. Lipases prepared for the purposes of the present invention may be ones having improved secretion capacity in microorganisms with improved lipase ester transfer activity.

In the above method, the step of culturing the microorganism is not particularly limited, but may be performed by a known batch culture method, continuous culture method, fed-batch culture method and the like. At this time, the culture conditions are not particularly limited thereto, but using a basic compound (eg, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg, phosphoric acid or sulfuric acid), an appropriate pH (eg, pH 5 to 9, specifically Can adjust pH 6 to 8, most specifically pH 6.8), and maintain aerobic conditions by introducing oxygen or oxygen-containing gas mixture into the culture. The culture temperature may be maintained at 20 to 45 ℃, specifically 25 to 40 ℃, can be incubated for about 10 to 160 hours, but is not limited thereto. The 5'-inosinic acid produced by the culture may be secreted into the medium or remain in the cell.

In addition, the culture medium used may include sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seeds) as carbon sources. Oils, peanut oils and coconut oils), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (e.g. glycerol and ethanol) and organic acids (e.g. acetic acid) may be used individually or in combination. This is not restrictive. Nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate) and the like can be used individually or in combination, but is not limited thereto. As a source of phosphorus, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, a corresponding sodium-containing salt, and the like may be used individually or in combination, but is not limited thereto. The medium may also include essential growth-promoting substances such as other metal salts (eg magnesium sulfate or iron sulfate), amino acids and vitamins.

The method for recovering the lipase produced in the culturing step of the present application may collect the desired lipase from the culture using a suitable method known in the art according to the culture method. For example, centrifugation, filtration, anion exchange chromatography, crystallization and HPLC can be used, and the desired lipase can be recovered from the medium or microorganism using any suitable method known in the art.

In addition, the recovery step may include a purification process, it may be carried out using a suitable method known in the art. Thus, the recovered lipase may be a microbial fermentation broth containing purified form or lipase.

In another embodiment, the present invention provides a method for converting vegetable waste oil to biodiesel using lipase produced from a transforming microorganism.

The term "biodiesel" is a fatty acid methyl ester produced by esterification of copper and vegetable oils with methanol, and has very similar properties to those of conventional petroleum-based diesel oils. When used in combination with diesel, it is an advanced alternative clean energy that can drastically reduce automobile pollution, which is the main cause of air pollution.

In the present invention, the raw material of biodiesel may be non-edible low-cost cooking oil, lean waste oil or sludge oil as vegetable waste oil, but is not limited thereto, and waste oil having high water content or high free fatty acid content (acid value) may also be included. have. In addition, waste oils as well as low-cost vegetable oils can be used without limitation.

For the purpose of the present invention, the biodiesel can be produced by converting vegetable waste oil using lipase mass-produced using the lipase secretion expression cassette of the present invention. In a specific embodiment, it was confirmed that the lipase obtained by culturing the improved microorganisms through directional evolution increased all of the expression amount, hydrolytic activity, and ester transfer activity compared to the wild type (FIGS. 10 to 12).

In addition, the biodiesel conversion rate with the lipase CALB derived from Candida antarctica , which is most representatively used in the biodiesel conversion process, was compared. Specifically, as a result of the reaction of the substrate solution containing soybean oil and water by adding TLL, CALB was found to show a very low conversion efficiency compared to the TLL by the activity inhibition phenomenon according to the water content (Fig. 13). Through the above results, it can be seen that the lipase produced in the present invention can be effectively used even when producing biodiesel using a substrate having water such as waste cooking oil or waste oil.

Another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 1, the 46-, 108-, 155, and 270th amino acid substitution from the N-terminal substitution, the increased transesterification activity and secretion expression capacity is increased It is to provide type lipase.

Specifically, the lipase is a variant lipase in which the 46th amino acid is asparagine, the 108th amino acid is vaginated, the 155th amino acid is glycine, and the 270th amino acid is substituted with aspartame in the amino acid sequence of SEQ ID NO: 1. It may be, but is not limited thereto.

The transformed strain of the present invention can produce lipase having improved ester transfer activity with high efficiency, and can be usefully used in all processes requiring ester transfer reaction.

1 is a schematic diagram showing the in vivo recombination process of introducing a lipase gene into a strain of Saccharomyces cerevisiae by linking it to a GAL10 promoter vector containing 24 yeast protein secretion fusion factors (TFP).
Figure 2 shows the results of SDS-PAGE analysis of lipase protein expressed in 24 strains of Saccharomyces cerevisiae secreted out of the cell.
3 is a result of Western blot analysis of lipase protein expressed in Saccharomyces cerevisiae 24 strains secreted out of the cell.
Figure 4 shows the results of analyzing the lipase activity of the culture medium in 24 strains of Saccharomyces cerevisiae.
5 is a result of analyzing the lipase protein secreted by SDS-PAGE by culturing three single strains from four strains selected first from 24 strains of TFP.
6 is a result of Western blot analysis of the secreted lipase protein by culturing three single strains from four strains selected first from 24 strains of TFP.
FIG. 7 shows the results of analyzing lipase activity of a single strain culture medium selected three from four strains selected first from 24 strains of TFP.
Fig. 8 is a schematic diagram of YGa-ST11-TLL-6H expression and secretion vector containing secretion fusion factor TFP 11 and lipase gene.
9 is a schematic diagram showing a process for selecting TLL with improved activity by a directional evolution method.
10 is a result of analyzing the secretion amount of the lipase improved activity step by step through the SDS-PAGE.
11 shows the results of analyzing the hydrolytic activity of lipase with improved activity step by step.
12 is a result of analyzing the ester transfer reaction activity of the lipase with improved activity step by step.
13 shows the results of comparing the biodiesel conversion ability of the final improved TLL and CalB.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are intended to illustrate the present invention by way of example, the scope of the present invention is not limited by these examples, it will be apparent to those of ordinary skill in the art.

Example  One. Thermomyses Ranuzino  origin Lipase  Gene synthesis and improvement

Thermomyces lanuginosus ) -derived lipase gene (TLL, SEQ ID NO: 1) was synthesized by BIONIA Co., Ltd., a gene to which yeast codon optimization was applied based on sequence information of NCBI GenBank: AAC08588.1.

Primary PCR using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 as a template (95 ℃ 5 minutes once; 95 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minutes 25 repetitions; 72 ℃ 7 minutes In a single reaction, mature TLL (mature form) genes were amplified. The gene amplified by the first PCR was again amplified by LNK39 (SEQ ID NO: 4) and HisGT50R (SEQ ID NO: 5) primers and recombined into cells with a vector containing protein secretion fusion factor (TFP) (in vivo recombination). Recombinant gene was introduced, and a recombinant gene containing a histidine tag to be used as an index at the carboxy terminus was obtained.

The amplified gene is a yeast strain Y2805G (Mat a pep4 :: HIS3 prb1 can1 his3-200 ura3-) with a linear vector containing 24 kinds of protein secretion fusion factors (Korean Patent No. 0975596) that help the expression of protein secretion. 52, gal80) were introduced together to form a transforming microorganism through intracellular recombination (FIG. 1).

Since there is a secreted peptide of thermomyses ranusinos lipase itself, the synthesized gene was induced in the same manner as the reaction conditions using the primers of SEQ ID NO: 6 and SEQ ID NO: 3 to induce the expression of recombinant lipase by the secreted peptide. First PCR reactions were performed. The first PCR reaction product was again amplified twice with GAL47 (SEQ ID NO: 7) and HisGT50R (SEQ ID NO: 5) and introduced into yeast strain Y2805G together with a linearized vector without TFP and transformed through intracellular recombination. Microorganisms were allowed to form.

SEQ ID NO: Primer Name Sequence (5'-3 ') 2 TLL-STFP-F CTCGCCTTAGATAAAAGAAGTCCTATTCGTCGAGAGG 3 TLL-STFP-R TTAGTGATGGTGATGGTGATGAAGACATGTCCCAATTAAC 4 LNK39 GGCCGCCTCGGCCTCTGCTGGCCTCGCCTTAGATAAAAGA 5 HisGT50R GTCATTATTAAATATATATATATATATATTGTCACTCCGTTCAAGTCGACTTAGTGATGGTGATGGTGATG 6 TLL-Self-F GAATTTTTGAAAATTCAAGAATTCATGAGGAGCTCCCTTG 7 GAL47 GCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTCAAGAATTC

Transformed microorganisms undergoing intracellular recombination were selected in SD-Ura plate media (yeast substrate lacking 0.67% amino acid, nutritional supplement lacking 0.77% uracil, 2% glucose, 2% agarose). The amino acid sequence of the protein secretion fusion factor used in the present invention is as follows (Table 2).

SEQ ID NO: Protein secretion fusion factor Sequence (5'-3 ') 8 TFP 1 MFNRFNKFQAAVALALLSRGALGDSYTNSTSSADLSSITSVSSASASATASDSLSSSDGTVYLPSTTISGDLTVTGKVIATEAVEVAAGGKLTLLDGEKYVFSSEAASASAGLALDKR 9 TFP 2 MTPYAVAITVALLIVTVSALQVNNSCVAFPPSNLRGKNGDGTNEQYATALLSIPWNGPPESSRDINLIELEPQVALYLLENYINHYYNTTRDNKCPNNHYLMGGQLGSSSDNRSLNEAASASAGLALDKR 10 TFP 3 MQFKNVALAASVAALSATASAEGYTPGEPWSTLTPTGSISCGAAEYTTTFGIAVQAITSSKAKRDVISQIGDGQVQATSAATAQATDSQAQATTTATPTSSEKMAASASAGLALDKR 11 TFP 4 MRFAEFLVVFATLGGGMAAPVESLAGTQRYLVQMKERFTTEKLCALDDKAASASAGLALDKR 12 TFP 5 MFNRFNKFQAAVALALLSRGALGAPVNTTTEDETALIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVAASASAGLALDKR 13 TFP 6 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVAASASAGLALDKR 14 TFP 7 MVFGQLYALFIFTLSCCISKTVQADSSKESSSFISFDKESNWDTISTISSTADVISSVDSAIAVFEFDNFSLLDSLMIDEEYPFFNRFFANDVSLTVHDDSPLNISQSLSPIMEQFTVDELPESASDLLYEYSLDDKSIVLFKFTSDAYDLKKLDEFIDSCLSFLEDKSGDNLTVINS 15 TFP 8 MLQSVVFFALLTFASSVSAIYSNNTVSTTTTLAPSYSLVPQETTISYADDLAASASAGLALDKR 16 TFP 9 MKFSTAVTTLISSGAIVSALPHVDVHQEDAHQHKRAVAYKYVYETVVVDSDGHTVTPAASEVATAATSAIITTSVLAPTSSAAAADSSASIAVSSAALAKNEKISDAAASATASTSQGASSSSYLAASASAGLALDKR 17 TFP 10 MNWLFLVSLVFFCGVSTHPALAMSSNRLLKLANKSPKKIIPLKDSSFENILAPPHENAYIVALFTATAPEIGCSLCLELESEYDTIVASWFDDHPDAKSSNSDTSIFFTKVNLEDPSKTIPKAFQFFQLNNVPRLFIFKLNSPSILDHSVISISTDTGSERMKQIIQASFSFS 18 TFP 11 MKFSSVTAITLATVATVATAKKGEHDFTTTLTLSSDGSLTTTTSTHTTHKYGKFNKTSKSKTPLAASASAGLALDKR 19 TFP 12 MASFATKFVIACFLFFSASAHNVLLPAYGRRCFFEDLSKGDELSISFQFGDRNPQSSSQLTGDFIIYGPERHEVLKTVRELAASASAGLALDKR 20 TFP 13 MQYKKTLVASALAATTLAAYAPSEPWSTLTPTATYSGGVTDYASTFGIAVQPISTTSSASSAATTASSKAKRAASQIGDGQVQAATTTASVSTKSTAAAVSQIGDGQIQATTKTTAAAVSQIGDGQIQATTKTTSAKTTAAAVSQISDGQIQATTTLA 21 TFP 14 MQFKNALTATAILSASALAANSTTSIPSSCSIGTSATATAQATDSQAQATTTAPLAASASAGLALDKR 22 TFP 15 MVSKTWICGFISIITVVQALSCEKHDVLKKYQVGKFSSLTSTERDTPPSTTIEKWWINVCEEHNVEPPEECKKNDMLCGLTDVILPGKDAITTQIIDFDKNIGFNVEETESALTLTLNGATWGANSFDAKLEFQCNDNMKQDELAASASAGLALDKR 23 TFP 16 MKLSALLALSASTAVLAAPAVHHSDNHHHNDKRAVVTVTQYVNADGAVVIPAATTATSAAADGKVESVAAATTTLSSTAAAATTLAASASAGLALDKR 24 TFP 17 MKLSTVLLSAGLASTTLAQFSNSTSASSTDVTSSSSISTSSGSVTITSSEAPESDNGTSTAAPTETSTEAPTTAIPTNGTSTEAPTTAIPTNGTSTEAPTDTTTEAPTTALPTNGTSTEAPTDTTTEAPTTGLPTNGTTSAFPPTTSLPPSNTTTTPPYNPSTDYTTDYTVVTEASAGLDCPKRAS 25 TFP 18 MRFSTTLATAATALFFTASQVSAIGELAFNLGVKNNDGTCKSTSDYETELQALKSYTSTVKVYAASDCNTLQNLGPAAEAEGFTIFVGVWPLAASASAGLALDKR 26 TFP 19 MRLSNLIASASLLSAATLAAPANHEHKDKRAVVTTTVQKQTTIIVNGAASTPVAALEENAVVNSAPAAATSTTSSAASVATAASSSENNSQVSAAASPASSSAATSTQSSLAASASAGLALDKR 27 TFP 20 MQFSTVASIAAVAAVASAAANVTTATVSQESTTLVTITSCEDHVCSETVSPALVSTATVTVDDVITQYTTWCPLTTEAPKNGTSTAAPVTSTEAPKNTTSAAPTHSVTSYTGAAAKALPAAGALLAASASAGLALDKR 28 TFP 21 MKFSSALVLSAVAATALAESITTTITATKNGHVYTKTVTQDATFVWGGEDSYASSTSAAESSAAETSAAETSAAATTSAAATTSAAETSSAAETSSADEGSGSSITTTITATKNGHVYTKTVTQDATFVWTGEGSSNTWSPSSTSTSSEAATSSASTTATTLLAASASLALAGA 29 TFP 22 MKFQVVLSALLACSSAVVASPIENLFKYRAVKASHSKNINSTLPAWNGSNSSNVTYANGTNSTTNTTTAESSQLQIIVTGGQVPITNSSLTHTNYTRLFNSSSALNITELYNVARVVNETIQDNLAASASAGLALDK 30 TFP 23 MRAITLLSSVVSLALLSKEVLATPPACLLACVAQVGKSSSTCDSLNQVTCYCEHENSAVKKCLDSICPNNDADAAYSAFKSSCSEQNASLGDSSGSASSSVLAASASAGLALDKR 31 TFP 24 MKLSTVLLSAGLASTTLAQFSNSTSASSTDVTSSSSISTSSGSVTITSSEAPESDNGTSTAAPTETSTEAPTTAIPTNGTSTEAPTTAIPTNGTSTEAPTDTTTEAPTTALPTNGTSTEAPTDTTTEAPTTGLPTNGTTSAFPPTTSLPPSNTTTTLAASASAGLALDKR

A total of 25 transformed microorganisms containing one secreted peptide and 24 protein secreted fusion factors were incubated in YPD medium (1% yeast extract, 2% peptone, 2% glucose) for 40 hours and then centrifuged. Cells were removed, and the culture supernatant was used to confirm the expression of TLL by SDS-PAGE analysis (FIG. 2) and Western blot using an anti-His antibody (FIG. 3).

As a result, when the protein secretion fusion factors 6, 8, 11, 13, and 16 were used than the secretion peptide itself, it was confirmed that the secretion ability is excellent.

Simultaneous confirmation of secretion expression, Thermomyces ranusinos derived lipase produced from each transgenic microorganism using tributyrin as a substrate Butyric acid produced by lanuginosus lipase (TLL) was titrated with sodium hydroxide to compare lipase activity.

As a result, the activity of protein secretion fusion factors 6, 8, 11, 13, 16, which had good secretion ability, was higher than that of the self-secreting peptide (Fig. 4).

Since the strains selected by the above method are analyzed by mixing the transformed microorganisms of each secreted fusion factor, three single colony cells per secreted fusion factor 6, 8, 11, 13, 16 are isolated for accurate analysis. Secretory expression and activity of TLL were analyzed. SDS-PAGE analysis (Fig. 5), secretion expression was confirmed by Western blot using a sample cultured in the same medium and conditions (Fig. 6), the activity was analyzed by pH titration (Fig. 7).

As a result, it was confirmed that 11 of the selected TFP was the most excellent expression and activity of secretion TLL. As a result, YGaST11-TLL-6H containing TFP 11 as a protein secretion fusion factor was prepared as an expression vector for high secretion production of thermomyses ranusinosus-derived lipase (FIG. 8).

Example  2. Through Directional Evolution Of TLL  improvement

Error-prone PCR was performed using primers of SEQ ID NO: 7 and SEQ ID NO: 5 with the YGaST11-TLL-6H vector as a template. The composition of the PCR reaction was performed by adjusting the concentration of MnSO ₄ and dGTP so that 7-9 bases per 1 kb of genes could be randomly substituted.

PCR was performed once at 94 ° C. for 30 seconds; Repeating 94 DEG C for 30 seconds and 68 DEG C for 1 minute and 25 times; It was carried out at 68 ℃ 1 min once conditions. The amplified gene was introduced into the yeast strain Y2805G (Mat a pep4 :: HIS3 prb1 can1 his3-200 ura3-52, gal80) together with the linearized vector as described above to induce transformation of microorganisms through intracellular recombination. Transgenic microorganisms are plated in SD-Ura + tributyrin plate medium (yeast substrate lacking 0.67% amino acid, nutritional supplement lacking 0.77% uracil, 2% glucose, 2% agarose, 1% tributyrin) It was then selected based on the size of the clear ring generated around the colonies by recombinant lipase (FIG. 9).

In order to confirm the activity of the primary screened variant according to the size of the transparent ring in SD-Ura + tributyrin medium, the colonies were inoculated in YPD liquid medium and cultured for 40 hours to verify the expression and activity using the culture supernatant. It was. The expression level was analyzed by the Image J program the intensity of the band formed on the SDS-PAGE. Activity was verified hydrolysis activity and ester transfer reaction activity, respectively. The hydrolytic activity was analyzed by the amount of 4-nitrophenol produced by lipase using 4-nitrophenyl palmitate as a substrate. The ester transfer activity was 9 ml of soybean oil and methanol as a substrate. 1 ml of the culture supernatant was added to 1 ml, and the amount of fatty acid methyl ester formed after the reaction at 45 ° C. for 1 hour was analyzed by gas chromatography.

By repeating the same method three times with the first generation variant genes obtained through the above process, variants 2G and 3G with increased activity were prepared in stages.

As a result of recovering the TLL gene from each strain of each generation strain, it was analyzed that I108V, N270D, 2nd generation strain R155G, and 3rd generation strain K46N additional amino acid mutation in the first generation strain (Table 3). Most of these mutations corresponded to the lid (108) and nucleophilic elbow regions (155) corresponding to the active region of the lipase, and were also identified as mutations adjacent to the active centers (168, 223, 280).

Variation Sequence Information of TLL Improved Strains through Directional Evolution Strain Variant sequence WT (wild type) - 1G I108V, N270D 2G I108V, N270D, R155G 3G I108V, N270D, R155G, K46N

As a result of comparing the expression levels of the strains selected at each stage using SDS-PAGE, the final generation of the third-generation strains showed an increase of 68.7% compared to the wild type (FIG. 10) and a 78.2% increase in hydrolytic activity (FIG. 11). Ester transition activity increased by 58.7% (FIG. 12).

Example 3 Comparison of Biodiesel Conversion Efficiency Using Immobilized TLL

Biodiesel conversion was compared with Candida antarctica- derived lipase CALB, the most representative biodiesel conversion process.

Specifically, the reaction conditions were 1g of immobilized enzyme to the substrate solution to which 4% water was added to 9ml of soybean oil and 14% methanol concentration was added to the substrate solution to react for 3 hours at 40 ℃, 200 rpm Samples were identified by gas chromatography.

As a result, it was confirmed that CALB shows a very low conversion efficiency compared to the TLL due to the activity inhibition phenomenon according to the moisture content (Fig. 13).

Through the above results, it can be seen that the lipase produced in the present invention can be effectively used even when producing biodiesel using a substrate having water such as waste cooking oil or waste oil.

From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> A mutant lipase having enhanced transesterification activity and          its application for biodiesel production <130> KPA180743-KR <160> 55 <170> KoPatentIn 3.0 <210> 1 <211> 291 <212> PRT <213> Thermomyces lanuginosus <220> <221> PEPTIDE (222) (1) .. (291) <223> lipase <400> 1 Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu   1 5 10 15 Ala Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe              20 25 30 Asn Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn          35 40 45 Asp Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro      50 55 60 Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser  65 70 75 80 Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys                  85 90 95 Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile             100 105 110 Gly Asn Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly         115 120 125 Cys Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp     130 135 140 Thr Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr 145 150 155 160 Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val                 165 170 175 Ala Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser             180 185 190 Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr         195 200 205 Val Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile     210 215 220 Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro 225 230 235 240 Glu Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp                 245 250 255 Ile Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro             260 265 270 Asn Ile Pro Asp Ile Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gly         275 280 285 Thr cys leu     290 <210> 2 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> TLL-STFP-F <400> 2 ctcgccttag ataaaagaag tcctattcgt cgagagg 37 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> TLL-STFP-R <400> 3 ttagtgatgg tgatggtgat gaagacatgt cccaattaac 40 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> LNK39 <400> 4 ggccgcctcg gcctctgctg gcctcgcctt agataaaaga 40 <210> 5 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> HisGT50R <400> 5 gtcattatta aatatatata tatatatatt gtcactccgt tcaagtcgac ttagtgatgg 60 tgatggtgat g 71 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> TLL-Self-F <400> 6 gaatttttga aaattcaaga attcatgagg agctcccttg 40 <210> 7 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> GAL47 <400> 7 gcgtccatcc aaaaaaaaag taagaatttt tgaaaattca agaattc 47 <210> 8 <211> 118 <212> PRT <213> Unknown <220> <223> TFP 1 <400> 8 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 Leu 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> 9 <211> 130 <212> PRT <213> Unknown <220> <223> TFP 2 <400> 9 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 Pro 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> 10 <211> 117 <212> PRT <213> Unknown <220> <223> TFP 3 <400> 10 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> 11 <211> 62 <212> PRT <213> Unknown <220> <223> TFP 4 <400> 11 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> 12 <211> 97 <212> PRT <213> Unknown <220> <223> TFP 5 <400> 12 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 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> 13 <211> 93 <212> PRT <213> Unknown <220> <223> TFP 6 <400> 13 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 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> 14 <211> 226 <212> PRT <213> Unknown <220> <223> TFP 7 <400> 14 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 Ala Asp Val Ile 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> 15 <211> 64 <212> PRT <213> Unknown <220> <223> TFP 8 <400> 15 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> 16 <211> 138 <212> PRT <213> Unknown <220> <223> TFP 9 <400> 16 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 Val Val Leu Ala Pro Thr Ser  65 70 75 80 Ser Ala 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> 17 <211> 199 <212> PRT <213> Unknown <220> <223> TFP 10 <400> 17 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 Pro 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> 18 <211> 77 <212> PRT <213> Unknown <220> <223> TFP 11 <400> 18 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 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> 19 <211> 94 <212> PRT <213> Unknown <220> <223> TFP 12 <400> 19 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> 20 <211> 174 <212> PRT <213> Unknown <220> <223> TFP 13 <400> 20 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 Ala 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 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> 21 <211> 68 <212> PRT <213> Unknown <220> <223> TFP 14 <400> 21 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 Thr Ala Pro Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala      50 55 60 Leu Asp Lys Arg  65 <210> 22 <211> 157 <212> PRT <213> Unknown <220> <223> TFP 15 <400> 22 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> 23 <211> 98 <212> PRT <213> Unknown <220> <223> TFP 16 <400> 23 Met Lys Leu Ser Ala Leu Leu 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 Thr Ala Thr Ser Ala Ala Ala Asp Gly Lys      50 55 60 Val Glu Ser Val Ala 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> 24 <211> 195 <212> PRT <213> Unknown <220> <223> TFP 17 <400> 24 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 Ile Ser Thr Ser Ser Gly 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 Le Le Pro Pro Ser Asn Thr 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> 25 <211> 105 <212> PRT <213> Unknown <220> <223> TFP 18 <400> 25 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> 26 <211> 124 <212> PRT <213> Unknown <220> <223> TFP 19 <400> 26 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 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 Thr Ser Ser Ala Ala Ser Val Ala  65 70 75 80 Thr Ala Ala Ser Ser Ser Glu Asn Asn Ser Gln Val Ser Ala Ala Ala                  85 90 95 Ser Pro Ala Ser 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> 27 <211> 138 <212> PRT <213> Unknown <220> <223> TFP 20 <400> 27 Met Gln Phe Ser Thr Val Ala Ser Ile Ala Ala Val Ala Ala Val Ala   1 5 10 15 Ser Ala 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 Ser 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 Lys Ala Leu Pro Ala Ala Gly Ala Leu Leu Ala Ala Ser         115 120 125 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg     130 135 <210> 28 <211> 176 <212> PRT <213> Unknown <220> <223> TFP 21 <400> 28 Met Lys Phe Ser Ser Ala Leu Val Leu 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 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 Ser Glu Ala Ala Thr Ser Ser Ala Ser Thr Thr 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> 29 <211> 138 <212> PRT <213> Unknown <220> <223> TFP 22 <400> 29 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> 30 <211> 115 <212> PRT <213> Unknown <220> <223> TFP 23 <400> 30 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 Ser Val Leu Ala Ala Ser Ala Ser Ala Gly Leu Ala Leu             100 105 110 Asp Lys Arg         115 <210> 31 <211> 170 <212> PRT <213> Unknown <220> <223> TFP 24 <400> 31 Met Lys Leu Ser Thr Val Leu Leu Ser Ala Gly Leu Ala Ser Thr Thr   1 5 10 15 Leu Ala Gln Phe Ser Asn Ser Thr Ser Ala Ser Ser Thr Asp Val Thr              20 25 30 Ser Ser Ser Ser Ile Ser Thr Ser Ser Gly 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 Thr Leu Ala Ala Ser 145 150 155 160 Ala Ser Ala Gly Leu Ala Leu Asp Lys Arg                 165 170 <210> 32 <211> 354 <212> DNA <213> Unknown <220> <223> TFP 1 <400> 32 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> 33 <211> 390 <212> DNA <213> Unknown <220> <223> TFP 2 <400> 33 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> 34 <211> 351 <212> DNA <213> Unknown <220> <223> TFP 3 <400> 34 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> 35 <211> 186 <212> DNA <213> Unknown <220> <223> TFP 4 <400> 35 atgagatttg cagaattctt ggtggtattt gccacgttag gcggggggat ggctgcaccg 60 gttgagtctc tggccgggac ccaacggtat ctggtgcaaa tgaaggagcg gttcaccaca 120 gagaagctgt gtgctttgga cgacaaggcc gcctcggcct ctgctggcct cgccttagat 180 aaaaga 186 <210> 36 <211> 291 <212> DNA <213> Unknown <220> <223> TFP 5 <400> 36 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> 37 <211> 279 <212> DNA <213> Unknown <220> <223> TFP 6 <400> 37 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> 38 <211> 678 <212> DNA <213> Unknown <220> <223> TFP 7 <400> 38 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> 39 <211> 192 <212> DNA <213> Unknown <220> <223> TFP 8 <400> 39 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> 40 <211> 414 <212> DNA <213> Unknown <220> <223> TFP 9 <400> 40 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> 41 <211> 597 <212> DNA <213> Unknown <220> <223> TFP 10 <400> 41 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> 42 <211> 231 <212> DNA <213> Unknown <220> <223> TFP 11 <400> 42 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> 43 <211> 282 <212> DNA <213> Unknown <220> <223> TFP 12 <400> 43 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> 44 <211> 522 <212> DNA <213> Unknown <220> <223> TFP 13 <400> 44 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> 45 <211> 204 <212> DNA <213> Unknown <220> <223> TFP 14 <400> 45 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> 46 <211> 471 <212> DNA <213> Unknown <220> <223> TFP 15 <400> 46 atggtatcga 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> 47 <211> 294 <212> DNA <213> Unknown <220> <223> TFP 16 <400> 47 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> 48 <211> 585 <212> DNA <213> Unknown <220> <223> TFP 17 <400> 48 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> 49 <211> 315 <212> DNA <213> Unknown <220> <223> TFP 18 <400> 49 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> 50 <211> 372 <212> DNA <213> Unknown <220> <223> TFP 19 <400> 50 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> 51 <211> 414 <212> DNA <213> Unknown <220> <223> TFP 20 <400> 51 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> 52 <211> 528 <212> DNA <213> Unknown <220> <223> TFP 21 <400> 52 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> 53 <211> 414 <212> DNA <213> Unknown <220> <223> TFP 22 <400> 53 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> 54 <211> 345 <212> DNA <213> Unknown <220> <223> TFP 23 <400> 54 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> 55 <211> 510 <212> DNA <213> Unknown <220> <223> TFP 24 <400> 55 atgaaattat caactgtcct attatctgcc ggtttggcct cgactacttt ggcccaattt 60 tccaacagta catctgcttc ttccaccgat gtcacttcct cctcttccat ctccacttcc 120 tctggctcag taactatcac atcttctgaa gctccagaat ccgacaacgg taccagcaca 180 gctgcaccaa ctgaaacctc aacagaggct ccaaccactg ctatcccaac taacggtacc 240 tctactgaag ctccaaccac tgctatccca actaacggta cctctactga agctccaact 300 gatactacta ctgaagctcc aaccaccgct cttccaacta acggtacttc tactgaagct 360 ccaactgata ctactactga agctccaacc accggtcttc caaccaacgg taccacttca 420 gctttcccac caactacatc tttgccacca agcaacacta ccaccactct ggccgcctcg 480 gcctctgctg gcctcgcctt agataaaaga 510

Claims (12)

A lipase secretion expression cassette comprising a nucleic acid encoding a lipase, wherein the lipase replaces one or more of the 46, 108, 155, and 270th amino acids from the N-terminus with another amino acid in the amino acid sequence of SEQ ID NO: 1 Lipase secretion expression cassette.
The method of claim 1,
The lipase secretion expression cassette is a lipase secretion expression cassette, which further comprises a nucleic acid encoding a protein secretion fusion factor.
The method of claim 1,
The lipase is a lipase secretion expression cassette in which the amino acid sequence of SEQ ID NO: 1 is substituted with asparagine from the N-terminal to the 46th amino acid, 108th amino acid valine, 155th amino acid glycine, and 270th amino acid aspartame.
The method of claim 2,
The protein secretion fusion factor is TFP 6 consisting of the amino acid sequence of SEQ ID NO: 13, TFP 8 consisting of the amino acid sequence of SEQ ID NO: 15, TFP 11 consisting of the amino acid sequence of SEQ ID NO: 18, TFP 13 consisting of the amino acid sequence of SEQ ID NO: 20 and Lipase secretion expression cassette, which is selected from the group consisting of TFP 16 consisting of the amino acid sequence of SEQ ID NO: 23.
An expression vector comprising the lipase secretion expression cassette according to any one of claims 1 to 4.
Transgenic microorganism comprising the expression vector of claim 5.
The method of claim 6,
The transforming microorganism is a yeast, transgenic microorganism.
According to claim 7,
Wherein the yeast is Candida (Candida), di berry Oh, my process (Debaryomyces), Hanse Cronulla (Hansenula), Cluj Vero My process (Kluyveromyces), Pichia (Pichia), ski irradiation Caro My process (Schizosaccharomyces), Yarrow's (Yarrowia) , Saccharomyces , Schwanniomyces and Arsula ( Arxula ) is a transgenic microorganism selected from the group consisting of.
(i) culturing the transgenic microorganism of any one of claims 6 to 8; And
(ii) recovering the lipase from the cultured microorganism or the culture supernatant thereof.
A method for converting vegetable waste oil into biodiesel using a lipase produced from the transformed microorganism of claim 6.
A variant lipase having increased ester transfer activity and secretory expression capacity, wherein the 46th, 108th, 155th, and 270th amino acids from the N-terminus in the amino acid sequence of SEQ ID NO: 1 are substituted with other amino acids.
The method of claim 11,
Wherein the other amino acids are asparagine, valine, glycine, and aspartame, mutant lipase with increased ester transfer activity and secretion expression ability.

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