KR20120116765A - Novel exoglucanase and the use thereof - Google Patents

Abstract

PURPOSE: A novel exoglucanase which is useful for bio-ethanol production and a manufacturing method of the bio-ethanol are provided to enhance cellulose decomposition activities. CONSTITUTION: A novel exoglucanase is represented by the sequence number 22(SEQ ID NO:22) or by the amino acid sequence of 24. The exoglucanase is revealed from penicillium calcoaceticus. The polynucleotide which codes the exoglucanase is represented by the base sequence of 25 or by the sequence number 23. The expression vector includes the polynucleotide which codes the exoglucanase. The expression vector additionally includes translational fusion partner(TFP). A manufacturing method of the exoglucanase comprises the following steps: cultivating transformant in which the expression vector is introduced to a host cell; and collecting exoglucanase from the culture material or supernatant. A manufacturing method of bio-ethanol comprises the following steps: obtaining transformant in which the expression vector is introduced to the host cell having ethanol fermentation capacity; cultivating the obtained transformant in a culture medium which contains exoglucanase substrate; and collecting the bio-ethanol from the obtained culture materials or supernatant. [Reference numerals] (AA,BB) Own signal

Description

Novel exoglucanase and its use

The present invention is an exoglucanase represented by the amino acid sequence of SEQ ID NO: 22 or 24, a polynucleotide encoding the exoglucanase, an expression vector comprising the polynucleotide, a transformant into which the expression vector is introduced, It relates to a method for producing the exoglucanase using a transformant and a method for producing bio ethanol using the transformant.

Recently, the necessity of sustainable renewable energy is emerging to solve the problem of climate change and energy crisis. Bioethanol, which is a potential green fuel, is being used as an alternative fuel that can reduce dependence on petroleum energy and solve serious environmental problems such as global warming.

Around 80 billion liters of bioethanol is being produced worldwide (Bioenergy Market Analysis and Forecast, Korea Institute of Chemical Economics, 2008), with the United States and Brazil being representative. To date, bioethanol is mainly produced by fermenting glucose from corn and sugar cane, which are food resources, and partially replaces petroleum-derived gasoline, thus reducing gasoline consumption and reducing environmental pollutants. Bioethanol produced using food resources is generally called the first-generation bioethanol fuel, and the sharp increase in demand causes the prices of crops such as corn and sugar cane to soar, and the price of other grains to rise, which leads to energy problems. Bioethanol for resolution is causing another food problem.

Due to these problems, production limits for first-generation bioethanol are expected from 2015 onwards. As a new alternative, so-called second-generation biofuel, cellulosic bioethanol, is in the spotlight. Cellulose ethanol breaks down cellulose, the major constituent of plant cell walls, the most abundant organic substance on the planet, to make glucose and from it to make ethanol. Therefore, unlike the first-generation method of fermenting cornstarch and extracting ethanol, the second-generation cellulose ethanol is a method of producing bioethanol using all tissues such as leaves, stems, and roots of corn that have been left or burned. This method will produce bioethanol from all plant tissues, including corn husks, rice bran, grasses, reeds, silver grass, and wood waste.

The main difference between the process of producing ethanol from cellulose as compared to the process of producing ethanol from starch is the glycosylation process of hydrolyzing glucose, which is a major component of plant cell walls, using an enzyme called cellulase. to be. However, the cellulose of the plant cell wall is not only a polymer material that is bound to a stable structure that is easily decomposed, but also a complex material that is combined with other polymers such as lignin, hemicellulose, and pectin. Decomposition requires several processes. The first process required to produce bioethanol from cellulose is a pretreatment process that separates cellulose fibers from plant resources by applying physical, chemical or biological treatments to the cell walls of the plant. The second process is the saccharification process, which is the process of producing glucose by chemical or biological treatment of the separated cellulose fibers.

In order to hydrolyze cellulose into low molecular sugars such as glucose, an enzyme called cellulase is required. In particular, a highly active cellulase enzyme is essential to effectively break down the fibrous raw material (cellulose) of hard plants. Cellulase is a generic term for enzymes that break down cellulose and can be classified into three enzymes. Endo-glucanase (EC 3.2.1.4), which cuts β-1,4 glycoside bonds of cellulose, and short-chain cellobiose, a glucose dimer, acting on both ends of the cellulose chain Exo-glucanase (EC 3.2.1.91) and β-glucosidase (β-glucosidase) to break down cellobiose to produce glucose.

Exoglucanase is a very important enzyme for effectively breaking down insoluble fiber. Currently, exoglucanase with high activity is required to produce bioethanol economically through fibrin degradation, and thus, studies to increase the activity by improving existing cloned genes to secure this (Percival Zhang et al., Biotechnol Adv. 24, 452, 2006). However, the development of cellulase having excellent resolution is still slow.

Thus, the present inventors identified a fungal penicillium strain having fibrinolytic activity in order to secure a highly active cellulase, and after cloning two exoglucanase genes from the strain, the genes were converted into yeast saccharin. Saccharomyces cerevisiae ) confirmed the presence of exoglucanase activity by recombinant expression in strains, and completed the present invention.

It is an object of the present invention to provide exoglucanase represented by the amino acid sequence of SEQ ID NO: 22 or 24.

Another object of the present invention is to provide a polynucleotide encoding the exoglucanase.

Still another object of the present invention is to provide an expression vector comprising the polynucleotide.

Still another object of the present invention is to provide a transformant into which the expression vector is introduced.

Still another object of the present invention is to provide a method for producing the exoglucanase using the transformant.

Still another object of the present invention is to provide a method for producing bioethanol using the transformant.

As one aspect for achieving the above object, the present invention provides an exoglucanase represented by the amino acid sequence of SEQ ID NO: 22 or 24.

As used herein, the term “exoglucanase” is one of cellulase, an enzyme that breaks down cellulose, and acts on both ends of a cellulose chain to form a short chain cellobiose, a glucose dimer. ) Is a very important enzyme to effectively break down insoluble fiber.

Preferably, the exoglucanase of the present invention may be expressed from Penilillium sp. , More preferably Penylillium genus CF4 strain ( Penilillium) sp . CF4).

In one embodiment of the present invention, after separating and identifying a novel penicillium genus CF4 strain exhibiting cellulase activity (Example 1), SEQ ID NO: 22 showing cellulase activity from the penicillium genus CF4 strain A gene encoding a novel exoglucanase represented by the amino acid sequence of 24 was cloned (Examples 2 and 3), and the gene encoding the exoglucanase was introduced into a strain in Saccharomyces cerevisiae. As a result, it was confirmed that the protein having exoglucanase activity was secreted out of the cells (Example 4 and FIGS. 8 and 9).

In another aspect, the present invention provides a polynucleotide encoding the exoglucanase of the present invention.

As used herein, the term “polynucleotide” refers to a polymer of nucleotides in which nucleotide monomers are long chained by covalent bonds, and are strands of DNA or ribonucleic acid (RNA) or more than a certain length. Polynucleotides encoding the exoglucanase of the invention.

Preferably, the polynucleotide may be a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 23 or 25.

In another embodiment, the invention provides an expression vector comprising a polynucleotide encoding the exoglucanase of the invention.

As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. One type of vector, "plasmid," refers to a circular double stranded DNA loop that can additionally connect DNA fragments therein.

The vector of the present invention can direct the expression of a gene encoding a protein of interest linked operably, such a vector is called an "expression vector." In general, in the use of recombinant DNA techniques, the expression vector is in the form of a plasmid.

According to the host cell, the type of expression vector that can be used may be determined according to the host cell. In the case of using the yeast as a host, for example, YEp13, YCp50, pRS system, pYEX system vector, etc. may be used as the expression vector. . As a promoter, a GAL promoter, an AOD promoter, etc. can be used, for example.

As a method of introducing recombinant DNA into yeast, for example, the electroporation method (Method Enzymol., 194, 182-187 (1990)), the spheroplast method (Proc. Natl. Acad. Sci. USA, 84, 1929-1933 (1978)), lithium acetate method (J. Bacteriol., 153, 163-168 (1983)), and the like.

In addition, the expression vector, fragments for expression inhibition having various functions for inhibiting or amplifying or inducing the expression of a target gene, markers for selection of transformants, genes resistant to antibiotics, and cells outside the 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), the protein fusion factor usable in the present invention is Korean Patent No. 0626753, 0798894, 0975596 It is disclosed in the call.

As used herein, the term “translational fusion partner (TFP)” refers to a gene that is fused with a gene encoding a low expression protein to induce secretory production of a low expression protein. (e. g., E. coli, Pseudomonas (Pseudomonas), and Bacillus subtilis species and the like), fungal (e.g., yeast, Aspergillus (Aspergillus), Penny chamber William (Penicillium), rayijo Perth (Rhizopus), Trichoderma (Trichoderma) species Etc.), from DNA of all prokaryotic or eukaryotic organisms, including plants (e.g., baby poles, corn, tobacco, potatoes, etc.) and animals (e.g., humans, mice, rabbits, dogs, cats, and monkeys). Can be screened.

According to the present invention, the gene inserted into the expression vector is a polynucleotide encoding the above-described exoglucanase, preferably a polynucleotide encoding the exoglucanase represented by the amino acid sequence of SEQ ID NO: 22 or 24. It may be a polynucleotide encoding an exoglucanase represented by the amino acid sequence of SEQ ID NO: 22 or 24 more preferably expressed from a strain of the genus Penicillium.

In another embodiment, the present invention provides a transformant in which the expression vector of the present invention is introduced into a host cell.

As used herein, the term “transformation” means that DNA is introduced into a host cell so that the DNA can be replicated as an extrachromosomal factor or by chromosomal integration.

The host cell used for transformation according to the present invention may be any host cell well known in the art, but has excellent introduction efficiency of the polynucleotide encoding the exoglucanase of the present invention, and introduced polynucleotide. A host having a high expression efficiency may be used, and may include, for example, bacteria, fungi, yeasts, plants or animals (eg, mammals or insects) cells.

Preferably, the host cell may be a cell having an ethanol fermentation ability, more preferably the cell having an ethanol fermentation ability may be Zymonas, yeast, Bacillus.

The yeast is not particularly limited to, Candida (Candida), di berry Oh, my process (Debaryomyces), Hanse Cronulla (Hansenula), Cluj Vero My process (Kluyveromyces), Pichia (Pichia), ski irradiation Caro My process (Schizosaccharomyces) , Yarrowia , Saccharomyces , Saccharomycopsis , Schwanniomyces , Arxula species, more preferably Candida utilis ( Candida utilis ), Candida boidinii ), Candida albicans ), Kluyveromyces lactis ), Pichia pastoris , Pichiastipitis stipitis ), Schizosaccharomyces pombe ), Saccharomyces cerevisiae , Saccharomycopsis Saccharomycopsis fiburigera ), Hansenula polymorpha polymorpha ), Yarrowia lipolytica , Schwanniomyces oxydentalis occidentalis ), Arsula adenimorans adeninivorans ) and the like, and most preferably Kluyveromyces Kluyveromyces maxianus ), Saccharomyces cerevisiae , Saccharomycopsis Saccharomycopsis fiburigera ) can be used.

In one embodiment of the present invention, after transforming the yeast Saccharomyces cerevisiae using an expression vector comprising a polynucleotide encoding the exoglucanase of the present invention, the transformant of the present invention It was confirmed that exoglucanase was produced (Example 4 and FIGS. 8 and 9).

As another embodiment, the present invention provides a method for producing exoglucanase according to the present invention, comprising culturing the transformant of the present invention, and recovering exoglucanase from the culture or the culture supernatant thereof. to provide.

The medium and culture conditions for culturing the transformant of the present invention can be appropriately selected and used depending on the host cell. The nutrient medium preferably contains a carbon source inorganic nitrogen source or an organic nitrogen source necessary for the growth of host cells. Examples of the carbon source include glucose, dextran, soluble starch, sucrose, methanol, and the like. Examples of inorganic or organic nitrogen sources include ammonium salts, nitrates, amino acids, corn steep liquer, peptone, casein, bovine extract, soybean white, and potato extract. If desired, other nutrients such as sodium chloride, calcium chloride, sodium dihydrogen phosphate, inorganic salts such as magnesium chloride, vitamins, and antibiotics (tetracycline, neomasin, ampicillin, and kanamycin) may be included. At the time of culturing, conditions such as temperature, pH of the medium, and incubation time may be appropriately adjusted to be suitable for growing cells and mass production of proteins.

In one embodiment of the present invention, YGaST22-PeCBH1 was produced by using TFP in a vector derived from YEp352, which is a general yeast expression vector having a GAL10 promoter and a GAL7 terminator to express exoglucanase, and TFP was used in the same vector. YGaST13-PeCBH2 was produced. Saccharomyces cerevisiae strains were transformed with each vector, and then cultured in a 30 ° C. YEPD medium (1% yeast extract, 2% peptone, 2% glucose) (Example 6).

The step of recovering the exoglucanase may be isolated and purified by conventional biochemical separation techniques or by an immuno-friendly method using an antibody against one part or another part of the protein. Another method can be purified by an affinity method that adds a tag to the protein sequence and utilizes the antibody or other material with appropriately high affinity for such tag. Conventional biochemical separation techniques include treatment with protein precipitants (salting), centrifugation, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity. Various chromatography, such as chromatography, etc., Usually, these can be combined and used to isolate a high purity protein.

In another embodiment, the present invention provides a method for producing bioethanol, comprising culturing the transformant in the presence of an exoglucanase substrate and recovering the bioethanol therefrom.

Specifically, the production method of the bioethanol of the present invention comprises the steps of: (i) obtaining a transformant in which the expression vector of the present invention is introduced into a host cell having ethanol fermentation ability; (Ii) culturing the obtained transformant in a culture solution containing a substrate of exoglucanase; And (iii) recovering the bioethanol from the culture or culture supernatant obtained in step (ii).

The host cell having the ethanol fermentation ability is not particularly limited thereto, and preferably Zymomonas Mobilis ) strain, yeast strain or Bacillus strain may be used, and more preferably microorganisms expressing or simultaneously expressing endoglucanase or beta-glucosidase, respectively.

The substrate of the exoglucanase is not particularly limited, but may preferably be glucan or polysaccharide that can be degraded by exoglucanase, more preferably starch, dextran, grass It may be at least one selected from the group consisting of lulan and cellulose.

The culture medium containing the substrate of exoglucanase may further include an endoglucanase or beta-glucosidase or a microorganism expressing the same.

The culture method is not particularly limited, but any method used for cultivation of ordinary microorganisms, such as batch type, flow batch type, continuous culture, reactor type, or the like can be used.

Since the novel exoglucanase of the present invention exhibits excellent cellulose degradation activity, it can be widely used in industrial processes such as bioethanol production from woody biomass.

1 is a penicillium isolated from the present invention ( penicillium sp . CF4) is a picture showing the cellulase activity of the strain.
2A is a penicillium isolated from the present invention ( penicillium sp . Fig. 2 is a schematic diagram showing a method for cloning the PeCBH1 gene derived from CF4), Figure 2 B is a schematic diagram showing a method for cloning the PeCBH2 gene, Figure 2 C is a photograph showing the results of electrophoresis of PCR results.
3 is penicillium sp . PeCBH1 from CF4) Amino Acid Sequences of Proteins and Penicillium Oxalicum oxalicum ) is a schematic diagram showing the comparison of the amino acid sequence of the exoglucanase enzyme.
4 is penicillium sp . PeCBH2 from CF4) Amino Acid Sequence of the Protein and Penicillium Decumbens It is a schematic diagram comparing the amino acid sequence of the exoglucanase enzyme derived from decumbens ).
5 is penicillium sp . PeCBH1 from CF4) The amino acid sequence of the protein PeCBH2 It is a schematic diagram comparing the amino acid sequence of a protein.
6 is penicillium sp . PeCBH1 from CF4) Genes and PeCBH2 In connection with the gene for the 24 kinds of yeast protein secreted fusion factor (TFP) and the GAL10 promoter to be introduced into the strain in my process serenity busy in Saccharomyces It is a schematic diagram showing the in vivo recombination process.
7 is penicillium isolated from the present invention ( penicillium sp . PeCBH1 from CF4) Schematic (A) and PeCBH2 representing gene expression vector It is a schematic diagram (B) which shows a gene expression vector.
Figure 8 is an electrophoresis picture showing the results of analysis of PeCBH1 protein expressed in 24 strains in Saccharomyces cerevisiae secreted out of the cell by SDS-PAGE.
Figure 9 is an electrophoresis picture showing the results of SDS-PAGE analysis of PeCBH2 protein expressed in Saccharomyces cerevisiae 24 strains secreted out of the cell.
10 is the protein fusion factor selected for the comparison ratio of PeCBH1 and PeCBH2 according to a secretion signal; each secretion production using (Translational Fusion Partner TFP), its own secretion signal, the yeast mating factor alpha (MFα) secretion signal PeCBH1 Electrophoresis (A) and PeCBH2 showing the results of SDS-PAGE analysis of proteins Electrophoresis picture (B) showing the results of protein analysis by SDS-PAGE.
Figure 11 is an electrophoresis picture (A) showing the results of SDS-PAGE analysis of the medium taken by fermentation of Saccharomyces cerevisiae strain transformed with a recombinant vector containing the PeCBH1 gene in a 5L fermentor fed by fermentation And it is a graph which shows the result of having measured the activity of the cell OD and exoglucanase by each time (B).
Figure 12 is an electrophoresis picture (A) showing the results of SDS-PAGE analysis of the medium taken by fermentation of Saccharomyces cerevisiae strain transformed with a recombinant vector containing the PeCBH2 gene in a 5L fermenter fed by fermentation And it is a graph which shows the result of having measured the activity of the cell OD and exoglucanase by each time (B).
FIG. 13 is a graph showing ion exchange chromatography profiles for recombinant PeCBH1 purification (A of 13) and electrophoresis (B of 13) showing the results of protein SDS-PAGE (B) analysis on the purified fractions.
14 is a graph (A) showing an ion exchange chromatography profile for recombinant PeCBH2 purification and electrophoresis (B) showing the results of protein SDS-PAGE analysis on purified fractions.
15 is purified PeCBH1 In order to confirm the enzyme properties of the protein, a graph showing an optimum pH analysis result (A) and a graph showing an optimum temperature analysis result (B) are shown.
16 is purified PeCBH2 In order to confirm the enzyme properties of the protein, it is a graph (A) showing an optimal pH analysis result and a graph (B) showing an optimum temperature analysis result.
17 shows PeCBH1 Protein and PeCBH2 Photographs (A) showing the TLC results for the standards and photographs (B) showing the TLC results for the reaction products for determining the protein's preferred fibrin substrate end type.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Reference Example  1: used strain and experimental material

Penicillium sp CF4 strain was incubated for 7 days at 25 ° C. using YMA medium (Difco), and transformants in Saccharomyces cerevisiae were yeast nitrogen lacking SD Ura- medium (0.67% amino acid). Base, 2% glucose, 0.5% casamino acid), and then incubated for 40 hours in YPDG (1% yeast extract, 2% bacto peptone, 1% glucose, 1% galactose) medium, 0.6 ml of supernatant 0.4 ml Precipitation with acetone was followed by SDS-PAGE analysis. E. coli DH5α was used for general genetic manipulation.

Reference Example  2: Genetic Engineering and Sequencing Methods

General genetic manipulation was performed according to the technique of Sambrook (Molecular cloning: a laboratory manual, 2nd ed, 1989) and the like, and the gel extraction kit (Viogene) was used to recover the gene from the agarose gel. Al-Samarrai and Schmid (Lett Appl Microbiol. 30, 53-56, 2000) have been according to the method used to extract genomic DNA from a fungal, yeast according to the method such as the technique Gietz (Yeast 11, 355-360, 1995 ) Transformation was performed. E. coli transformation was performed using Inoue (Gene 96, 23-28, 1990). Gene sequencing was performed using Applied Biosystems Model 373A, and primers for polymerase chain reaction were synthesized by Genotech.

Example  One: Cellulase  Isolation and Identification of Fungal Strains Showing Activity

In order to isolate the cellulase-producing fungal strains, various forest soils with decayed fallen leaves and branches were collected at Daejeon suburbs and used as strain isolate sources.

Potato agar medium (potato dextrose agar; PDA, Difco) was used as a basic medium for the isolation of fungal strains. -Sorbose was added at concentrations of 0.1% and 0.4%, respectively, and chloramphenicol was added at a concentration of 0.01% to suppress bacterial development.

In order to perform the fungal separation experiment, 1 g of each of the various soil samples collected was added to 9 ml of sterile saline solution (0.85%, w / w), sufficiently suspended for 20 minutes, and the supernatant was diluted annually. 100 μl of the above-described basic medium was inoculated and plated. Inoculated PDA plates were observed by incubation for 5 to 7 days in a 25 ℃ incubator, each fungal strain was inoculated and incubated in a new PDA medium to inoculate and culture each fungal strain. Cellulase activity verification was inoculated with mold strain to be tested in YMA medium containing 0.5% CMC and incubated for 7 days at 25 ℃, staining the plate cultured with 0.1% congo red solution, After decolorizing with 1M NaCl solution, it was carried out by checking the presence or absence of a clear zone showing cellulase activity and comparing the sizes.

Comparing the activity of four strains (Cf2, Cf4, Cf12 and Cf14 strains) among the isolated fungi, Cf4 strains showing high cellulase activity were formed by forming clear zones of 8 to 12 mm in size for cellulase gene cloning. Were selected (FIG. 1). Figure 1 is a photograph showing the cellulase activity of the penicillium ( penicillium sp. CF4) strain isolated in the present invention.

Sequencing is a method of molecular classification and is widely recognized for rapid and reliable microbial identification. In the case of fungi, sequences of 18S gene, 26S D1 / D2 domain and ITS (Internal Transcribed Spacer region) are widely used for identification. The ITS base sequence has the largest number of databases (DB), and if it shows more than 99% similarity by comparing the base sequence of this region, it is judged as the same species. Therefore, in order to identify Cf4 fungal strains showing cellulase activity, we used a molecular classification method based on the sequencing method, and after analyzing the sequencing of the ITS region, each sequence of the various fungal strains recorded in GenBank Comparison was made with sequencing databases.

The ITS region sequence of 548 bp in length was analyzed from the fungus Cf4 strain.Penicillium decaturense NRRL 28152,Penicillium waksmanii strain NRRL 777,Penicillium canescens NRRL 2147,Penicillium rivolii NRRL 906,Penicillium miczynskii NRRL 1077 andPenicillium manginiiIt showed 99% similarity with NRRL 2134 strains.Penicillium Nucleotide sequence of the ITS region and the various strains belonging to the genus show a similarity of 92-99%Penicillium sp. It was identified as Cf4 strain.

Example  2 : Degenerate Primer  Used Cellulase  Partial amplification of genes

According to the CAZy database (http://www.CAZy.org), a collection of enzyme genes that affect glycoside binding of carbohydrates, the glycoside hydrolase family (GH), which cleaves glycoside binding, It is classified as 112. Examination of the database revealed that the families containing cellulase from eukaryotic cells were GH5, 6, 7, 9, 12, and 61, and the amino acid sequences conserved from each of the selected families were analyzed.

Based on the amino acid sequence, the GH5 family and the GH7 family produced four degenerate primers, respectively, and the GH6, 9, and 12 families produced two degenerate primers, respectively (SEQ ID NOS: 1-14). ).

family primer order SEQ ID NO:
GH5 family GH5F1 5'-TTCTTCYCYGGYAAYGA-3 ' One GH5F2 5'-ATYCCWGTYGGYTANTC-3 ' 2 GH5R1 5'-TTRCARCCNWAYTCRGA-3 ' 3 GH5R2 5'-TARACNADRCCRCCRGA-3 ' 4 GH6 family GH6F 5'-GARCCWGAYWCYHTNGSYAA-3 ' 5 GH6R 5'-GYRCCRTCNCSYTCRCC-3 ' 6
GH7 family GH7F1 5'-GARTTCWCYTTCGAYGT-3 ' 7 GH7F2 5'-TANGGYACYGGYTANTG-3 ' 8 GH7R1 5'-CARCCRTCNGWATCRCA-3 ' 9 GH7R2 5'-AATTGRGTRACRACRGT-3 ' 10 GH9 family GH9F 5'-GGYTGGTANGATGCYGA-3 ' 11 GH9R 5'-CCNTARCCRGTRAYNSARGA-3 ' 12 GH12 family GH12F 5'-GGYATYAARTCYTANCC-3 ' 13 GH12R 5'-CCSGGRGARGARGTRATYTC-3 ' 14

Identified Penicillium sp . 11 primer combinations using the genomic DNA of the Cf4 strain as a template PCR was performed with / GH9R, and GH12F / GH12R). PCR conditions were 95 cycles, 5 cycles, 95 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 30 seconds 30 cycles of the reaction conditions, and further reaction at 72 ℃ for 10 minutes. PCR products were analyzed by electrophoresis on agarose gels.

As a result, fragments of 400 bp and 460 bp were amplified in the GH7F1 / GH7R1 primer combination. Analysis of the nucleotide sequence after cloning into the pGEM-T Easy vector revealed that the two gene fragments had different nucleotide sequences and showed high nucleotide sequence similarity with penicillium-derived exoglucanase, but the same nucleotide sequence was published. It could not be found in the database. Therefore, it was confirmed that the cellulase gene fragment partially amplified by the above method is part of the novel exoglucanase.

Example  3: Genome walking  Technology( Template blocking PCR ) Cellulase  Gene Cloning

Primers were synthesized to secure the entire genes based on the found nucleotide sequences, and then the entire genes were obtained by using genome working technology (Korean Patent Application No. 10-2009-0061895). Cassettes were first prepared in order to use genome working techniques. Cassettes are short double-stranded DNA designed to be linked to restriction enzyme treated genomic DNA. In order to be able to link genomic DNA with one base filled with ddGTP after cleavage with BamHI, BglII, and Sau3A, a CSF primer of SEQ ID NO: 15 and a CSR primer of SEQ ID NO: 16 were synthesized, and 100 μM CSF and 100 μM CSR After mixing 40 μl of the primers each, 250 mM Tris.Cl (pH 8.0), 10 μl of 100 mM MgCl ₂ buffer and 10 μl of distilled water were added thereto, and the mixture was heated in a 95 ° C. constant temperature bath for 5 minutes. It was left to come down.

primer order SEQ ID NO: CSF 5'-GACGCGTAATACGACTCACTATAGGGA-3 ' 15 CSR 5'-ATCTCCCTATAGTGAGTCGTATTACGCGTC-3 ' 16

The cassette produced in this manner has a structure in which one base is smooth and one end protrudes GAT three bases at 5 '. Penicillium to Construct Cassette Library Treated with Two Restriction Enzymes sp . 2 μg of CF4 genomic DNA was digested for 2 hours at 37 ° C. using 10 units of BamHI and BglII, respectively, under 1 × BamHI buffer (Takara) and 1 × BglII buffer (Takara), followed by electrophoresis on agarose gel for complete cleavage. It was confirmed.

After inactivation of the restriction enzyme by treatment in a 70 ° C. water bath for 20 minutes, ddGTP was added to 0.2 mM and reacted with 8 units of Klenow fragment (Klenow fragment, Takara) at 37 ° C. for 30 minutes. DNA was purified using Qiagen's PCR purification kit and then linked using the 40 pmol cassette and DNA ligation kit (Takara). The ligation reaction was carried out at 16 ° C. for 10 hours. The DNA was purified using a PCR purification kit from Qiagen, and then eluted with 20 μl of distilled water to prepare a BamHI cassette library and a BglII cassette library, respectively.

In order to clone the first cellulase gene, cellulase-specific primers [CBH1F (SEQ ID NO: 17) and CBH1R (SEQ ID NO: 18)] were bidirectionally constructed based on the nucleotide sequence of the 0.4 kb PCR product, and then 1 µL of each cassette library. PCR was performed with cassette primer CP (SEQ ID NO: 21) as a template.

primer order SEQ ID NO: CBH1F 5'-GTTTCCAATCTCCCCTGTGGTCTC-3 ' 17 CBH1R 5'-GTGCCTGCATATCGATCAGAGCTG-3 ' 18 CP 5'-ACGCGTAATACGACTCACTATAGGGAGATC-3 ' 21

The reaction composition for cloning the 5 'region of the cellulase gene was as follows: 1 μl of the genome cassette library, 1 pmol of CP, 1 pmol of CBH1R, 5 μl of EX taq buffer (Takara), 5 μl of 2.5 mM dNTP, distilled water. 37 μl, and 1 μl EX taq DNA polymerase (Takara) uses a CBH1F primer instead of the CBH1R primer for cloning the 3 ′ region of the cellulase gene. PCR conditions were 95 ℃ 30 minutes, 95 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 30 minutes of reaction 3 minutes conditions, and further reacted at 72 ℃ for 10 minutes. After the reaction, 10 μl of the reaction solution was subjected to electrophoresis on an agarose gel. As a result of the 5 ′ site cloning reaction using the CP primer and the CBH1R primer, a 2.6 kb fragment was amplified from the BglII cassette library, and the CP primer and the CBH1F primer. In the 3 ′ site cloning reaction using 3.7 kb fragments were amplified from the BamHI cassette library (FIG. 2). Figure 2 A is PeCBH1 derived from penicillium ( penicillium sp. CF4) isolated in the present invention Figure 2 is a schematic diagram showing the method of cloning the gene, Figure 2 B is a schematic diagram showing the method of cloning the PeCBH2 gene, Figure 2 C is a photograph showing the results of electrophoresis of the PCR results, samples of each lane is as follows. are: lane 1: size marker, lane 2: PeCBH1 a result of amplifying the 5 region of a gene, lane 3: as a result of amplifying a third portion of PeCBH1 gene, lane 4: the result of amplifying the 5 parts of the PeCBH2 gene, lane 5: Lane 6: size marker as amplified three sites of PeCBH2 gene.

As a result of cloning each PCR product on pGEM-T Easy vector (Promega), it was confirmed that the region to be cloned was amplified, and the entire sequence of cellulase gene was confirmed. Penicillium cloned by the above method sp . The CF4-derived cellulase gene ( PeCBH1 ) (SEQ ID NO: 23) consists of 1,641 bp base without introns, and the inferred amino acid sequence (SEQ ID NO: 22) consists of 26 secretion signals and a cellulose binding module (cellulose binding module, Exoglucanase consisting of 547 amino acids, including CBM), Penicillium Exoglucanase from oxalicum showed 74.3% amino acid similarity (FIG. 3). Figure 3 is a schematic diagram comparing the amino acid sequence of the PeCBH1 protein derived from penicillium ( penicillium sp. CF4) and the amino acid sequence of the exoglucanase enzyme derived from Penicillium oxalicum .

In order to clone the second cellulase gene, cellulase specific primers [CBH2F (SEQ ID NO: 19) and CBH2R (SEQ ID NO: 20)] were bidirectionally prepared based on the nucleotide sequence of the 0.45 kb PCR product, and then the same method as described above. PCR was performed.

primer order SEQ ID NO: CBH2F 5'-CGCTCTGTACTTCGTCTCCATGGATGC-3 ' 19 CBH2R 5'-CGCACATGGTCTGTCCAGCCTCCTTGC-3 ' 20

In the 5 'site cloning reaction with CP primers and CBH2R primers, 1.5 kb fragments were amplified from the BglII cassette library and in the 3' site cloning reaction with CP primers and CBH2F primers, 4.0 kb fragments were amplified from the BamHI cassette library. Each PCR product was cloned into pGEM-T Easy vector (Promega) to analyze the nucleotide sequence. As a result, it was confirmed that the region to be cloned was amplified, and the entire sequence of the second cellulase gene was confirmed. Penicillium sp . The second cellulase gene derived from CF4 ( PeCBH2 ) (SEQ ID NO: 25) consists of 1,359 bp base containing two introns, and the inferred amino acid sequence (SEQ ID NO: 24) contains 17 secretion signals. Exoglucanase consisting of 452 amino acids, Penicillium Exoglucanase from decumbens showed 78.4% amino acid similarity (FIG. 4). Figure 4 shows the amino acid sequence and the penicillium decumbens ( Penicillium ) of PeCBH2 protein derived from penicillium ( penicillium sp. CF4) It is a schematic diagram comparing the amino acid sequence of the exoglucanase enzyme derived from decumbens ).

PeCBH1 And Comparing the amino acid sequence of the two proteins to confirm the similarity between the PeCBH2 protein, showed a similarity of 71.1% (Fig. 5). Figure 5 shows the amino acid sequence of PeCBH1 protein derived from penicillium ( penicillium sp. CF4) PeCBH2 It is a schematic diagram comparing the amino acid sequence of a protein.

Example  4: Saccharomyces In Serebizaia  Through secretion of the strain PeCBH1 And  PeCBH2  Confirmation of gene activity

PeCBH1 , PeCBH2 isolated in Example 3 An expression vector was prepared to express the gene in Saccharomyces cerevisiae strain. First, in order to express PeCBH1 gene, excluding secretion signal PeCBH1 gene was identified using Penicillium primers SEQ ID NOs: 26 and 27. sp . Amplified from CF4 genomic DNA. In To prepare a vector using in vivo recombination, the PeCBH1 gene amplified by SEQ ID NOs. Type of protein secretion fusion factor (Translational Fusion Partner; TFP, Korean Patent No. 10-0975596) using Y2805 ( Mat a pep4 :: His3 prb1 can1 the his3 -200 ura3 -52) strains in It was introduced through in vivo recombination (FIG. 6). 6 is a penny room Solarium (penicillium sp. CF4) in vivo by connecting PeCBH1 gene and PeCBH2 gene derived from the 24 kinds of yeast protein secreted fusion factor (TFP) and the GAL10 promoter to be introduced into the strain in my process serenity busy in Saccharomyces Schematic diagram showing the recombination process.

Twenty four kinds of transformants were cultured in YPDG medium for 40 hours, centrifuged to remove cells, and 0.6 ml of the remaining culture supernatant was precipitated with 0.4 ml acetone and analyzed on SDS-PAGE (FIG. 8). Figure 8 is an electrophoresis picture showing the results of SDS-PAGE analysis of PeCBH1 protein expressed in Saccharomyces cerevisiae 24 strains secreted out of the cell.

Among 24 protein secretion fusion factors, TFP22 vector (A of FIG. 7) which was excellent in the secretion ability of PeCBH1 was selected. Figure 7 A is PeCBH1 derived from penicillium ( penicillium sp. CF4) isolated in the present invention It is a schematic diagram which shows a gene expression vector.

The vector produced in this way consists of the GAL10 promoter, which is strongly induced by galactose, and TFP22, which induces secretion of the PeCBH1 protein, and the backbone of the YEGα-HIR525 vector (Sohn et al., Process Biochem. 1995, 30, 653). Use In the case of producing a vector using in vivo recombination, there is an advantage that a strain can be obtained in Saccharomyces cerevisiae transformed with a vector to be produced immediately without passing through E. coli. Since the PeCBH2 gene contains two introns, cDNA from which introns were removed was produced for expression in Saccharomyces cerevisiae strain. A primers prepared by SEQ ID NO: 30 / SEQ ID NO: 31, SEQ ID NO: 32 / SEQ ID NO: 33, and SEQ ID NO: 34 / SEQ ID NO: 35 were amplified for each of three exon genes, and then LNK39 (SEQ ID NO: 28) and GT50R (SEQ ID NO: 29). The primers were linked via overlap extension PCR. The subsequent steps Using the same method as in the case of the PeCBH1 Y2805 with 24 types of secretion of the fusion protein factor in After in vivo recombination, each transformant was incubated in YPDG medium for 40 hours, centrifuged to remove cells, and 0.6 ml of the remaining culture supernatant was precipitated with 0.4 ml of acetone and analyzed on SDS-PAGE (FIG. 9). Figure 9 is an electrophoresis picture showing the results of SDS-PAGE analysis of PeCBH2 protein expressed in Saccharomyces cerevisiae 24 strains secreted out of the cell.

Among them, strains were prepared in Saccharomyces cerevisiae transformed with a vector expressing PeCBH2 under the control of a TFP 13 fusion factor having excellent secretion ability of PeCBH2 (FIG. 7B). B of PeCBH2 It is a schematic diagram which shows a gene expression vector.

To confirm the activity of the protein secreted from each transformant, 0.1 ml of enzyme solution was added to 0.9 ml of substrate solution (50 mM Sodium acetate buffer (pH 5.0), 1 mg / ml of p-Nitrophenyl-beta-D- cellobioside) at 50 ° C. for 30 minutes. The reaction solution and the same amount of 2% sodium carbonate were added to stop the reaction, and the absorbance was measured at 410 nm. As a result, 0.92 unit, PeCBH2 was detected in the Saccharomyces cerevisiae strain transformed with the PeCBH1 expression vector. In the Saccharomyces cerevisiae strain transformed with the expression vector, 0.59 unit of exoglucanase activity was confirmed. At this time, the enzyme activity was set to 1 unit of the amount of enzyme capable of producing 1 μM nitrophenol per minute.

Example  5: PeCBH1 , PeCBH2  The gene's own signal, MFa ( mating factor alpha ) Secretion signal, protein secretion Convergence factor ) Expression Comparison Using Fusion Partner

In order to express the PeCBH1 gene using its own secretion signal, a polymerase chain reaction was carried out using SEQ ID NO: 36 / SEQ ID NO: 27, and about 1.7 kb of the fragment was recovered. Plasmids with their own secretion signal genes were obtained from the transformed transformants, and the obtained plasmids were transformed into Y2805 strains to select single colonies. In order to use the MFa secretion signal, the same strain was transformed using TFP No. 6 vector, and single colonies were selected. Selected monomorphs of the self-secretion signal, MFa secretion signal, and TFP22- PeCBH1 were incubated in YPDG medium for 40 hours, centrifuged to remove the cells, and 0.6 ml of the remaining culture supernatant was precipitated with 0.4 ml of acetone. Analysis was performed (A and Table 5 in FIG. 10). 10 is secreted and produced using the protein secretion factor (Translational Fusion Partner (TFP), self-secretion signal, yeast mating factor alpha (MFα) secretion signal selected for comparison of the secretion ratio of PeCBH1 and PeCBH2 according to the secretion signal PeCBH1 Electrophoresis (A of 10) and PeCBH2 showing the results of protein analysis by SDS-PAGE It is an electrophoretic photograph (B of 10) showing the result of protein analysis by SDS-PAGE.

Analysis of Exoglucanase Activity of Strains Selected from PeCBH1 Exoglucanase Activity (u / ml) TFP22 0.974 Self-secreting signal 0.649 MFa secretion signal 0.675

As shown in FIG. 10A, strong bands were observed in the TFP22- PeCBH1 strain, and as shown in Table 5, exoglucanase activity was analyzed using 1 mg / ml of p-Nitrophenyl-beta-cellobioside as a substrate. As a result, when TFP22- PeCBH1 was used, the activity was about 33% higher than its own signal and about 31% higher than MFa.

Meanwhile, in order to express using the secretion signal of the PeCBH2 gene, a 1.3 kb fragment was recovered by polymerase chain reaction with SEQ ID NO: 37 / SEQ ID NO: 35, and the single transformants selected by PeCBH1 were analyzed. (B and Table 6 in FIG. 10).

Analysis of Exoglucanase Activity of Strains Selected from PeCBH2 Exoglucanase Activity (u / ml) TFP13 0.623 Self-secreting signal 0.491 MFa secretion signal 0.473

As a result, as shown in FIG. 10B, TFP13- PeCBH2 A strong band was observed in the strain, and as shown in Table 6 above, TFP13- PeCBH2 was used, which showed about 21% higher activity than the self-secreting signal and about 24% higher than the MFa secretion signal.

Therefore, further studies were performed by selecting TFP22- PeCBH1 and TFP13- PeCBH2 as optimal fusion factors.

Example  6: Exoglucanase  Mass production and refining

Y2805 / ST22- PeCBH1 described above In order to mass-produce cellulase using Y2805 / ST13- PeCBH2 producing strain, fed- batch culture was performed in a 5 L fermenter. Before entering the main culture, the cells were initially incubated in 50 ml YNB (0.67% amino acid-deficient yeast substrate, 0.5% casamino acid, and 2% glucose) medium, and then cultured in 200 ml YEPD liquid medium to activate and inoculated into the culture medium. Incubated at 30 ° C. for 48 hours. SDS-PAGE analysis of the culture supernatant over time to confirm the PeCBH1 exoglucanase secreted in the medium, and confirmed the activity of about 16 unit / ㎖ after fermentation culture for 48 hours (A and B of Fig. 11). FIG. 11 is an electrophoresis photograph showing the results of SDS-PAGE analysis of a medium taken by fermentation of Saccharomyces cerevisiae strain transformed with a recombinant vector containing the PeCBH1 gene in a 5L fermentation vessel over time. A) and a graph showing the results of measuring the activity of cell OD and exoglucanase at each time (B of 11).

PeCBH2 exoglucanase was also subjected to SDS-PAGE analysis of the culture supernatant by time to confirm the enzyme secreted in the medium after fermentation production in the same manner as above, and confirmed the activity of about 13.6 unit / ml after fermentation culture for 48 hours (Fig. 12 A and B). FIG. 12 is an electrophoresis photograph showing the results of SDS-PAGE analysis of a medium taken by fermentation of Saccharomyces cerevisiae strain transformed with a recombinant vector containing the PeCBH2 gene in a 5L fermentation vessel over time. A) and a graph showing the results of measuring the activity of cell OD and exoglucanase at each time (B of 12).

The fermented PeCBH1 protein was concentrated using ultrafiltration (molecular weight 30 kDa cut-off) with 50 mM Tris buffer ( pH8.0 ), and purified by ion exchange chromatography to 50 mM Tris buffer (pH8. The enzyme concentrate was adsorbed onto the HiTrap DEAE column with 0) and the enzyme was eluted with a gradient of increasing NaCl (pH8.0) from 0 to 1M (FIG. 13). FIG. 13 is a graph showing ion exchange chromatography profiles for recombinant PeCBH1 purification (A of 13) and electrophoresis (B of 13) showing the results of protein SDS-PAGE analysis on purified fractions.

Fractions with exoglucanase activity were collected and dialyzed with 50 mM Tris buffer (pH8.0), and then the HiTrap Qff column was selected by secondary ion exchange chromatography to adsorb enzyme dialysate with the same buffer. The fractions having high exoglucanase activity were collected by fusing the enzyme with a gradient of increasing concentration of NaCl (pH 8.0). The fermented PeCBH2 protein was concentrated using ultrafiltration (molecular weight 30 kDa cut-off) in 50 mM Tris buffer ( pH8.0) in the same manner as the PeCBH1, and was purified by ion exchange chromatography. The enzyme concentrate was adsorbed onto the HiTrap DEAE column in buffer (pH8.0) and the enzyme was eluted with a NaCl (pH8.0) rising concentration gradient from 0 to 1M to collect fractions with high exoglucanase activity (Fig. 14). 14 is a graph showing ion exchange chromatography profiles for recombinant PeCBH2 purification (A of 14) and electrophoresis (B of 14) showing the results of protein SDS-PAGE analysis on the purified fractions.

The enzyme was quantified using Bradford method, bovine serum albumin (BSA) was used as a standard, the results of the purification process are shown in Table 7 and Table 8.

Results of Purification of PeCBH1 Way Total protein (mg) Full active (U) Specific activity (U / mg) purchase Drainage Ultrafiltration 58.60 1434.10 24.47 1.00 1.00 DEAE ion exchange chromatography 13.52 527.12 38.99 0.63 1.59 Qff Ion Exchange Chromatography 1.90 84.49 44.47 0.88 1.82

Results of Purification of PeCBH2 Way Total protein (mg) Full active (U) Specific activity (U / mg) purchase Drainage Ultrafiltration 1232.55 1112.40 0.90 1.00 1.00 DEAE ion exchange chromatography 69.78 337.53 4.84 0.19 5.36

Example  7: new Exoglucanase  Enzyme Characterization

Refined PeCBH1 And PeCBH2 enzymes were investigated for their optimum pH and temperature. Sodium citrate (pH 3.0 to 4.5), sodium acetate (pH 4.5 to 5.5), and phosphate (pH 5.5 to 8.0) buffers were used to analyze the optimum pH of the enzyme. The exoglucanase activity described in Example 4 was analyzed using up to ° C. From these results, PeCBH1 showed the maximum activity at pH 5 and 50 ° C (FIG. 15), and PeCBH1 purified from FIG. In order to confirm the enzyme properties of the protein, a graph showing an optimum pH analysis result (A of 15) and a graph showing an optimum temperature analysis result (B of 15) are shown.

PeCBH2 showed maximum activity at pH 5 and 55 ° C. (FIG. 16). 16 is a graph (A of 16) showing an optimum pH analysis result and a graph (B of 16) showing an optimum temperature analysis result in order to confirm the enzyme properties of the purified PeCBH2 protein.

Also refined PeCBH1 And PeCBH2 enzymes were tested by TLC to determine whether each exoglucanase is a reducing or non-reducing end. 0.1 ml of purified enzyme solution was added to 0.9 ml of substrate solution (50 mM sodium acetate buffer (pH 5.0) and 1 mg / ml of p-Nitrophenyl-beta-D-cellotetraoside) at 50 ° C. for 0 to 15 minutes. After reacting at 5 minute intervals, the reaction solution and the same amount of 2% sodium carbonate were added to stop the reaction, and the hydrolyzate of each hour was spotted on TLC glass silica gel and dried. The standard materials used were glucose (G1) and cell Bios (G2), cellotriose (G3), cellotetraoside (G4), pNP-beta-D-glucopyranoside (pNPG1), pNP-beta-D-cellobioside (pNPG2), pNP-beta-D-cellotrioside ( pNPG3), and eight types of pNP-beta-D-cellotetraoside (pNPG4) were used as indicators showing the degradation products of each enzyme over time (Fig. 17A). 17A shows PeCBH1 protein and PeCBH2 A photograph showing the TLC results for a standard for determining the fibrin substrate end type that the protein prefers.

The eluent used for the separation of the standard and the enzyme product over time was such that the ratio of acetonitrile and distilled water was 80:20 (v / v), and 100 ml stock solution (100 ml) was used to detect sugar after drying. Acetone, 1 ml aniline, and 1 g diphenylamine) were sprayed onto silica gel with 10 ml phosphoric acid and dried. T. reesei CBH2, which is known to have cellulase activity at the non-reducing end of fibrin, reacts with the pNP-beta-D-cellotetraoside substrate over time at 120 ° C until the sugar of free silica gel has developed. While cutting with cellobiose and pNP-beta-D-cellobioside, PeCBH1 and PeCBH2 were found to have reducing terminal cellase activity of cutting the substrate used with cellotriose and pNP-beta-D-glucopyranoside (B in FIG. 17B). ). 17B is PeCBH1 Protein and PeCBH2 Photograph showing TLC results for the reaction product to determine the preferred fibrin substrate end type of the protein.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel exoglucanase and the Use <130> PA110209 / KR <160> 37 <170> Kopatentin 1.71 <210> 1 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> GH5F1 primer <400> 1 Thr Thr Cys Thr Thr Cys Tyr Cys Tyr Gly Gly Tyr Ala Ala Tyr Gly   1 5 10 15 Ala     <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> 223 GH5F2 primer <400> 2 Ala Thr Tyr Cys Cys Trp Gly Thr Tyr Gly Gly Tyr Thr Ala Asn Thr   1 5 10 15 Cys     <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <220> 223 GH5R1 primer <400> 3 Thr Thr Arg Cys Ala Arg Cys Cys Asn Trp Ala Tyr Thr Cys Arg Gly   1 5 10 15 Ala     <210> 4 <211> 17 <212> PRT <213> Artificial Sequence <220> 223 GH5R2 primer <400> 4 Thr Ala Arg Ala Cys Asn Ala Asp Arg Cys Cys Arg Cys Cys Arg Gly   1 5 10 15 Ala     <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> 223 GH6F primer <400> 5 Gly Ala Arg Cys Cys Trp Gly Ala Tyr Trp Cys Tyr His Thr Asn Gly   1 5 10 15 Ser Tyr Ala Ala              20 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> GH6R primer <400> 6 Gly Tyr Arg Cys Cys Arg Thr Cys Asn Cys Ser Tyr Thr Cys Arg Cys   1 5 10 15 Cys     <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> GH7F1 primer <400> 7 Gly Ala Arg Thr Thr Cys Trp Cys Tyr Thr Thr Cys Gly Ala Tyr Gly   1 5 10 15 Thr     <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> GH7F2 primer <400> 8 Thr Ala Asn Gly Gly Tyr Ala Cys Tyr Gly Gly Tyr Thr Ala Asn Thr   1 5 10 15 Gly     <210> 9 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> GH7R1 primer <400> 9 Cys Ala Arg Cys Cys Arg Thr Cys Asn Gly Trp Ala Thr Cys Arg Cys   1 5 10 15 Ala     <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> GH7R2 primer <400> 10 Ala Ala Thr Thr Gly Arg Gly Thr Arg Ala Cys Arg Ala Cys Arg Gly   1 5 10 15 Thr     <210> 11 <211> 17 <212> PRT <213> Artificial Sequence <220> 223 GH9F primer <400> 11 Gly Gly Tyr Thr Gly Gly Thr Ala Asn Gly Ala Thr Gly Cys Tyr Gly   1 5 10 15 Ala     <210> 12 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> GH9R primer <400> 12 Cys Cys Asn Thr Ala Arg Cys Cys Arg Gly Thr Arg Ala Tyr Asn Ser   1 5 10 15 Ala Arg Gly Ala              20 <210> 13 <211> 17 <212> PRT <213> Artificial Sequence <220> 223 GH12F primer <400> 13 Gly Gly Tyr Ala Thr Tyr Ala Ala Arg Thr Cys Tyr Thr Ala Asn Cys   1 5 10 15 Cys     <210> 14 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> GH12R primer <400> 14 Cys Cys Ser Gly Gly Arg Gly Ala Arg Gly Ala Arg Gly Thr Arg Ala   1 5 10 15 Thr Tyr Thr Cys              20 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> CSF primer <400> 15 gacgcgtaat acgactcact ataggga 27 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CSR primer <400> 16 atctccctat agtgagtcgt attacgcgtc 30 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CBH1F primer <400> 17 gtttccaatc tcccctgtgg tctc 24 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CBH1R primer <400> 18 gtgcctgcat atcgatcaga gctg 24 <210> 19 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CBH2F primer <400> 19 cgctctgtac ttcgtctcca tggatgc 27 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CBH2R primer <400> 20 cgcacatggt ctgtccagcc tccttgc 27 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <220> CP primer <400> 21 acgcgtaata cgactcacta tagggagatc 30 <210> 22 <211> 546 <212> PRT <213> Artificial Sequence <220> <223> PeCBH1 <400> 22 Met Ala Ala Ala Ile Ser Tyr Arg Ile Tyr Lys Asn Ala Leu Val Leu   1 5 10 15 Ser Ala Phe Leu Val Ala Ala Gln Ala Gln Gln Val Gly Thr Tyr Gln              20 25 30 Thr Glu Thr His Pro Ser Leu Thr Trp Ser Lys Cys Thr Ser Ser Gly          35 40 45 Ser Cys Thr Ser Thr Thr Gly Ser Val Val Ile Asp Ala Asn Trp Arg      50 55 60 Trp Val His Ser Thr Ser Ser Ser Thr Asn Cys Tyr Thr Gly Asn Thr  65 70 75 80 Trp Asp Ala Thr Leu Cys Pro Asp Asp Val Thr Cys Ala Ser Ser Cys                  85 90 95 Ala Val Asp Gly Ala Ser Tyr Ser Ser Thr Tyr Gly Val Thr Thr Ser             100 105 110 Gly Asp Glu Leu Arg Leu Asn Phe Val Thr Thr Ala Ser Gln Lys Asn         115 120 125 Ile Gly Ser Arg Leu Tyr Leu Leu Glu Asp Asp Ser Thr Tyr Glu Met     130 135 140 Phe Gln Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp Val Ser Asn 145 150 155 160 Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val Ser Met Asp Ala                 165 170 175 Asp Gly Gly Thr Ser Arg Phe Pro Thr Asn Lys Ala Gly Ala Lys Tyr             180 185 190 Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe Ile         195 200 205 Ser Gly Glu Ala Asn Val Glu Gly Trp Glu Pro Ser Asp Asn Asp Val     210 215 220 Asn Ser Gly Leu Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp Val 225 230 235 240 Trp Glu Ala Asn Ser Ile Ser Asn Ala Val Thr Pro His Pro Cys Asp                 245 250 255 Thr Pro Thr Gln Thr Glu Cys Thr Thr Asp Ala Cys Gly Gly Thr Tyr             260 265 270 Ser Ser Asp Arg Tyr Ala Gly Thr Cys Asp Pro Asp Gly Cys Asp Phe         275 280 285 Asn Pro Tyr Arg Met Gly Asn Thr Ser Phe Tyr Gly Pro Gly Lys Thr     290 295 300 Val Asp Thr Ser Ser Pro Phe Thr Val Val Thr Gln Phe Ile Thr Asn 305 310 315 320 Asp Gly Thr Ser Ser Gly Thr Leu Ser Glu Ile Lys Arg Phe Tyr Val                 325 330 335 Gln Asp Gly Glu Val Ile Gly Gln Ser Asp Ser Lys Val Ser Gly Val             340 345 350 Thr Gly Asn Ser Ile Thr Thr Asp Phe Cys Thr Ala Gln Lys Lys Ala         355 360 365 Phe Gly Asp Thr Asp Ser Phe Thr Glu His Gly Gly Leu Ala Gly Met     370 375 380 Gly Ala Ala Met Ala Glu Gly Met Val Leu Val Met Ser Leu Trp Asp 385 390 395 400 Asp His Asn Ser Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Thr                 405 410 415 Ala Ser Ser Thr Thr Pro Gly Ala Ala Arg Gly Ser Cys Asp Ile Ser             420 425 430 Ser Gly Asp Pro Asp Thr Val Glu Thr Ala Asp Ala Asn Ser Tyr Val         435 440 445 Ile Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr Tyr Asn Ser     450 455 460 Gly Gly Ser Ser Gly Gly Ser Ser Ser Ser Thr Thr Thr Ser Lys Thr 465 470 475 480 Ser Thr Thr Ser Lys Thr Ser Thr Thr Ser Lys Thr Ser Thr Thr Thr                 485 490 495 Lys Thr Ser Ser Thr Thr Ser Ser Thr Gly Ser Ser Thr Thr Gly Ala             500 505 510 Ala His Tyr Ala Gln Cys Gly Gly Ile Ser Trp Thr Gly Ala Thr Thr         515 520 525 Cys Ala Ser Pro Tyr Thr Cys Thr Lys Gln Asn Asp Tyr Tyr Ser Gln     530 535 540 Cys leu 545 <210> 23 <211> 1641 <212> DNA <213> Artificial Sequence <220> <223> PeCBH1 <400> 23 atggctgccg ctatctctta ccgcatttac aagaatgccc tggtcttgtc tgcctttctc 60 gttgccgctc aggcacaaca ggttggcact tatcagactg aaacgcatcc ttctctgacc 120 tggtccaagt gcaccagcag tggcagttgc acatcgacta ccggcagcgt tgtcatcgat 180 gccaactggc gctgggttca cagcactagc tcatccacca attgctatac aggcaatact 240 tgggatgcaa ctctctgtcc cgacgatgtg acctgtgcat cttcctgtgc ggtcgatggt 300 gcttcatatt caagcaccta cggcgtgact accagcggtg atgagctgcg tctcaacttt 360 gtcaccacgg catcgcagaa gaatatcggc tctcgtcttt acctcttgga ggacgatagt 420 acatacgaaa tgttccaact gctgaaccag gagtttactt tcgatgttga tgtttccaat 480 ctcccctgtg gtctcaatgg agcactctac ttcgtctcaa tggatgccga tggtggcact 540 tctcgattcc ccacgaacaa ggctggcgca aaatacggca ctggttattg tgattcgcag 600 tgccctcgcg atctcaagtt catatctggc gaagccaatg ttgagggctg ggaaccatcc 660 gacaatgacg tgaattctgg ccttggaaac cacggctcct gctgtgctga gatggatgtc 720 tgggaagcca attcaatttc caatgctgtc actccccacc cttgcgacac ccccactcag 780 actgaatgta ccacagatgc ctgtggtggt acctacagct ctgatcgata tgcaggcact 840 tgcgatcctg atggatgtga tttcaaccca taccgtatgg gcaacacctc gttctacgga 900 ccgggcaaga ctgtggacac cagctctccc ttcaccgttg tgactcagtt catcaccaat 960 gatggcactt cttcgggcac tctgagcgag attaaacgct tctacgtgca agatggggag 1020 gtaatcggcc agtccgactc caaggtcagc ggtgtgaccg gcaactccat taccacagat 1080 ttctgcacgg ctcagaagaa ggcctttgga gataccgata gctttactga gcatggtggc 1140 cttgcgggta tgggcgctgc catggccgaa ggaatggtcc tcgtgatgag tctgtgggat 1200 gaccataact ccaacatgct ctggctcgac agcacttatc cgaccacagc ctcctccact 1260 acccccggag ctgcacgcgg ttcctgcgat atctcttcgg gtgatcccga cactgtggag 1320 actgcggatg ctaactcata cgttatctac tcgaacatca aggtcggtcc aattggctca 1380 acttataact ctggtggttc ctccggtgga tcaagctcga gcaccacaac cagcaaaacg 1440 tccactacta gcaaaacatc caccactagc aaaacgtcca ccaccaccaa aacatcctcg 1500 accacttctt ctactggctc gagcactact ggagcggctc actatgccca atgcggtgga 1560 attagctgga ctggtgcgac cacctgcgct agcccataca cctgcaccaa gcagaacgac 1620 tattactctc agtgcctgta g 1641 <210> 24 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> PeCBH2 <400> 24 Met Gln Gln Arg Ala Leu Leu Leu Ser Ala Leu Val Ala Val Ala Arg   1 5 10 15 Ala Gln Gln Ala Gly Thr Gln Thr Ser Glu Thr Lys Pro Ser Leu Thr              20 25 30 Trp Gln Lys Cys Thr Ala Ser Gly Cys Thr Asp Gln Ser Gly Ser Val          35 40 45 Gly Ile Asp Ala Asn Trp Arg Trp Val His Ser Thr Asp Gly Thr Thr      50 55 60 Asn Cys Tyr Thr Gly Asn Glu Trp Asp Ala Asp Leu Cys Pro Asp Asp  65 70 75 80 Glu Thr Cys Ala Thr Asn Cys Ala Leu Asp Gly Ala Asp Tyr Ser Gly                  85 90 95 Thr Tyr Gly Val Glu Ala Asp Gly Asp Ser Leu Ser Leu Thr Phe Lys             100 105 110 Thr Gly Ser Asn Val Gly Ser Arg Leu Phe Leu Met Glu Asp Asp Ser         115 120 125 Thr Tyr Gln Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val     130 135 140 Asp Val Ser Ala Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val 145 150 155 160 Ser Met Asp Ala Asp Gly Gly Leu Ser Lys Tyr Glu Asn Asn Lys Ala                 165 170 175 Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp             180 185 190 Leu Lys Phe Ile Asn Gly Gln Gly Asn Val Asp Gly Trp Glu Pro Ser         195 200 205 Asp Asn Asp Ala Asn Ala Gly Val Gly Gly His Gly Ser Cys Cys Ala     210 215 220 Glu Met Asp Ile Trp Glu Ala Asn Lys Ile Ser Thr Ala Val Thr Pro 225 230 235 240 His Pro Cys Lys Glu Ala Gly Gln Thr Met Cys Glu Gly Asp Ser Cys                 245 250 255 Gly Gly Thr Tyr Ser Ser Thr Arg Tyr Ala Gly Thr Cys Asp Pro Asp             260 265 270 Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn Glu Ser Phe Tyr Gly         275 280 285 Pro Gly Lys Ile Val Asp Thr Ser Ser Lys Met Thr Val Ala Thr Gln     290 295 300 Phe Ile Thr Ser Asp Asn Thr Ala Thr Gly Thr Leu Ser Glu Ile Lys 305 310 315 320 Arg Ile Tyr Val Gln Asn Gly Lys Val Ile Ala Asn Ser Ala Ser Asp                 325 330 335 Val Ser Gly Val Ser Gly Asn Ser Ile Thr Ser Asp Phe Cys Thr Ala             340 345 350 Gln Lys Lys Ala Phe Gly Asp Glu Asp Val Phe Ala Gln Tyr Asn Gly         355 360 365 Met Ser Gly Met Gly Glu Gly Leu Glu Gln Gly Met Val Leu Val Met     370 375 380 Ser Leu Trp Asp Asp His Tyr Ala Asn Met Leu Trp Leu Asp Ser Asn 385 390 395 400 Tyr Pro Thr Asn Ala Thr Ala Ser Asp Pro Gly Val Ala Arg Gly Thr                 405 410 415 Cys Gly Ala Asp Ser Gly Lys Pro Ala Asp Val Glu Ser Ala Ser Ala             420 425 430 Asp Ala Lys Val Val Phe Ser Asn Ile Lys Val Gly Ala Ile Gly Ser         435 440 445 Thr Tyr Ser Ser     450 <210> 25 <211> 1358 <212> DNA <213> Artificial Sequence <220> <223> PeCBH2 <400> 25 atgcagcaac gcgcactcct tctctctgcc cttgtggcag tcgctcgcgc ccagcaggct 60 ggcacgcaga cctcagagac caagccttct ctgacttggc aaaagtgcac tgccagtggc 120 tgcacagatc aatccggatc cgttggtatt gatgctaact ggcgttgggt tcactcgacc 180 gacggaacca ccaactgcta caccggcaac gagtgggatg cggatctctg ccccgacgac 240 gagacttgtg ccacgaactg tgccctggat ggcgcagact actctggcac ctacggcgtg 300 gaggctgacg gcgactctct gtcgctgacc ttcaagaccg gctccaacgt cggttctcgt 360 ctgttcctga tggaggacga cagcacctac cagatgttca agctgctcaa ccaggarttc 420 acctttgacg ttgatgtttc cgctctcccc tgcggtctca atggcgctct gtacttcgtc 480 tccatggatg ccgacggagg tttgtccaag tacgagaata acaaggccgg tgccaagtat 540 ggaactggct actgtgattc gcagtgccct cgtgacctga agttcatcaa cggacagggc 600 aacgtcgacg gctgggagcc ctccgacaac gacgccaacg ccggtgttgg tggccacggc 660 tcttgctgtg cggagatgga tatttgggag gccaacaaga tctcaaccgc cgtgaccccc 720 cacccttgca aggaggctgg acagaccatg tgcgagggcg acagctgcgg tggcacctac 780 tcgtccaccc gctatgccgg tacctgcgac cctgacggat gtgacttcaa cccttaccgc 840 atgggtaacg agagcttcta cggccccgga aagatcgtcg acaccagctc caagatgacc 900 gttgccaccc agttcattac cagcgacaac accgccaccg gtaccctcag cgagatcaag 960 cgtatctatg ttcagaatgg caaggtcatt gccaactcgg cttccgatgt tagcggtgtc 1020 agtggtaact cgatcacccg gacttctgca ctgcgcagaa gaaggccttt ggcgatgagg 1080 atgtcttcgc tcagtacaac ggcatgtctg gtatgggcga gggacttgag cagggtatgg 1140 ttctcgttat gagcctgtgg gatgaccact acgccaacat gctctggttg gacagcaact 1200 accccaccaa tgccactgct tccgatcctg gtgttgctcg tggtacctgc ggtgctgact 1260 ctggcaagcc tgccgacgtc gagtctgcct cggctgatgc caaggtggtg ttctcgaaca 1320 tcaaggttgg tgccattggt tccacctact cttcttaa 1358 <210> 26 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer 26 <400> 26 ctcgccttag ataaaagaca acaggttggc acttatc 37 <210> 27 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer 27 <400> 27 cactccgttc aagtcgactt acaggcactg agagta 36 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> LNK39 primer <400> 28 ggccgcctcg gcctctgctg gcctcgcctt agataaaaga 40 <210> 29 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> GT50R primer <400> 29 gtcattatta aatatatata tatatatatt gtcactccgt tcaagtcgac 50 <210> 30 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer 30 <400> 30 ctcgccttag ataaaagaca gcaggctggc acgcag 36 <210> 31 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer 31 <400> 31 tccgcatccc actcgttgcc ggtgtagcag 30 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer 32 <400> 32 accggcaacg agtgggatgc ggatctctgc 30 <210> 33 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer 33 <400> 33 tcgacgttgc cctgtccgtt gatgaacttc 30 <210> 34 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer 34 <400> 34 tcaacggaca gggcaacgtc gacggctggg 30 <210> 35 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer 35 <400> 35 cactccgttc aagtcgactt aagaagagta ggtggaac 38 <210> 36 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer 36 <400> 36 atttttgaaa attcaagaat tcatggctgc cgctatctct 40 <210> 37 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer 37 <400> 37 gaatttttga aaattcaaga attcatgcag caacgcgcac tc 42

Claims (13)

Exoglucanase represented by the amino acid sequence of SEQ ID NO: 22 or 24.
The exoglucanase of claim 1, wherein the exoglucanase is expressed from a strain of genus Penicillium.
A polynucleotide encoding the exoglucanase of claim 1.
The polynucleotide is a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 23 or 25.
An expression vector comprising the polynucleotide of claim 3.
The expression vector of claim 5, further comprising a translational fusion partner (TFP).
A transformant wherein the expression vector of claim 5 is introduced into a host cell.
The transformant of claim 7, wherein the host cell has ethanol fermentation ability.
A method for producing an exoglucanase according to claim 1 or 2, comprising culturing the transformant of claim 7 and recovering the exoglucanase from the culture or the culture supernatant thereof.
(i) obtaining a transformant in which the expression vector of claim 5 is introduced into a host cell having ethanol fermentation ability;
(Ii) culturing the obtained transformant in a culture solution containing a substrate of exoglucanase; And,
(Iii) recovering bioethanol from the culture or culture supernatant obtained in step (ii).
The method of claim 10, wherein the host cell having ethanol fermentation ability expresses or co-expresses endoglucanase or beta-glucosidase, respectively.
The method of claim 10, wherein the substrate is glucan or polysaccharide.
The method of claim 10, wherein the substrate is at least one selected from the group consisting of starch, dextran, pullulan and cellulose.

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