KR101481755B1 - Novel endoglucanase and the Use thereof - Google Patents

Abstract

The present invention relates to a polynucleotide comprising an endoglucanase represented by the amino acid sequence of SEQ ID NO: 1, a polynucleotide encoding the endoglucanase, an expression vector comprising the polynucleotide, a transformant into which the expression vector is introduced, The present invention relates to a method for producing endoglucanase by using the transformant and a method for producing bioethanol using the transformant. The novel endoglucanase of the present invention exhibits excellent cellulose decomposing activity and can be widely used in industrial processes such as the production of bioethanol from woody biomass.

Description

Novel endoglucanase and the Use thereof < RTI ID = 0.0 >

The present invention relates to a polynucleotide comprising an endoglucanase represented by the amino acid sequence of SEQ ID NO: 1, a polynucleotide encoding the endoglucanase, an expression vector comprising the polynucleotide, a transformant into which the expression vector is introduced, The present invention relates to a method for producing endoglucanase by using the transformant and a method for producing bioethanol using the transformant.

The need for sustainable renewable energy is emerging for resolving the recent climate and energy crisis. Bioethanol, a potentially viable green fuel, is being used as an alternative fuel to reduce dependence on petroleum energy and to solve serious environmental problems such as global warming at the same time.

Approximately 80 billion liters of bioethanol is produced globally (Analysis and Forecast of Bio-energy Market, Institute of Chemical and Economic Research, 2008). The United States and Brazil are representative. To date, bioethanol production has been mainly obtained by fermenting glucose obtained from corn and sugar cane, which are food resources, to partially replace petroleum-derived gasoline, thereby reducing consumption of gasoline and reducing emissions of environmental pollutants. Bioethanol produced using food resources is generally referred to as first-generation bioethanol fuel. The rapid increase in demand has caused the price of crops such as corn and sugar cane to skyrocket and raise the price of other grains, Bioethanol for resolution is causing another food problem.

Due to these problems, the production limit of first-generation bio-ethanol will be expected from 2015 onwards, and cellulosic bioethanol, a so-called second-generation bio-fuel, is emerging as a new alternative. Cellulose Ethanol is the most abundant organism on the planet. Cellulose, which is a major constituent of plant cell walls, is degraded to make glucose and ethanol. Therefore, second generation cellulose ethanol is a method of producing bioethanol using all tissues such as leaves, stems, and roots of corn left unused or burned, unlike the first generation method of fermenting corn starch and extracting ethanol. This method will enable the production of bioethanol from all plant tissues such as corn husks, rice bran, grass, reed, wheat, and wood waste.

The major difference in the process of producing ethanol from cellulose compared to the process of producing ethanol from starch is that the cellulase, which is the main constituent of the plant cell wall, is hydrolyzed using an enzyme called cellulase to produce glucose to be. However, cellulose in plant cell walls is not only a polymer with a stable structure that is not readily degraded, but also a complex material that is bound together with other polymers such as lignin, hemicellulose, and pectin. Different processes are required to disassemble. The first step required to produce bioethanol from cellulose is a pretreatment process that separates cellulosic cellulose from plant sources by applying physical, chemical, or biological treatments to the cell walls of the plant. The second process is a saccharification process, which refers to the process of producing glucose by chemical or biological treatment of the separated cellulosic fibers.

In order to hydrolyse cellulose to a low molecular weight sugar such as glucose, an enzyme called cellulase is required. In order to effectively decompose a fibrous raw material (cellulose) of a hard plant, a highly active cellulase is essential. Cellulase refers to an enzyme that degrades cellulose and can be classified into three enzymes. Endo-glucanase (EC 3.2.1.4), which cuts the β-1,4 glycosidic bond of cellulose, and short-chain cellobiose, which acts on both ends of the cellulose chain and is a glucose dimer Glucosidase (EC 3.2.1.21), which produces glucose by decomposing cellobiose, and exo-glucanase (EC 3.2.1.9) which makes glucose by hydrolysis of cellobiose.

The endoglucanase is a very important enzyme for effectively decomposing insoluble fibrin. Currently, endoglucanase having high activity is required for economical production of bioethanol through degradation of fibrin. Therefore, in order to secure this, a method of improving the activity of the cloned gene (Percival Zhang et al., Biotechnol Adv. 24 , 452, 2006). However, the development of a cellulase having excellent resolution has not been developed yet.

In order to obtain a highly active cellulase, the present inventors have isolated a yeast strain of Phoma macrostoma Y4 having a fibrinolytic activity from a rotten citrus fruit and then cloned an endoglucanase gene from the strain , The above genes were recombinantly expressed in a yeast Saccharomyces cerevisiae strain to confirm endoglucanase activity, and the present invention was completed.

An object of the present invention is to provide an endoglucanase represented by the amino acid sequence of SEQ ID NO: 1.

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

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

It is still another object of the present invention to provide a transformant having the expression vector introduced therein.

It is still another object of the present invention to provide a method for producing the endoglucanase using the transformant.

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

In one aspect of the present invention, the present invention provides an endoglucanase represented by the amino acid sequence of SEQ ID NO: 1.

The term "endo-1,4-beta-glucanase (EC, 3.2.1.4)" in the present invention is one of the cellulases that are enzymes for degrading cellulose, and acts in the middle of the cellulose chain, Is an enzyme that produces cellobiose which is a glucose polymer or glucose dimer. It is an important enzyme for effectively decomposing insoluble cellulose.

The endoglucanase of the present invention includes not only the endoglucanase represented by the amino acid sequence of SEQ ID NO: 1 but also the functional equivalent of the endoglucanase. Is at least 50%, preferably at least 70%, more preferably at least 90%, even more preferably at least 50%, more preferably at least 90%, more preferably at least 90% Refers to a protein having 95% or more of sequence homology and exhibiting substantially the same physiological activity as the endoglucanase of the present invention.

Preferably, the endoglucanase of the present invention may be an endoglucanase expressed from a Phoma sp. Strain, more preferably an endoglucanase expressed from a Phoma macrostoma Y4 have.

The endoglucanase of the present invention is a temperature showing optimum activity, preferably 45 ° C to 70 ° C, more preferably 50 ° C to 65 ° C, and most preferably 60 ° C. In addition, as the pH showing the optimum activity, it may preferably be an acidic or neutral pH of from pH 2.0 to 8.0, more preferably pH 5.0.

In one embodiment of the present invention, a novel Formoma macrostomy Y4 strain showing cellulase activity was isolated and identified (Example 1), and the amino acid sequence of SEQ ID NO: 1 showing cellulase activity from the Fama macros toma Y4 strain (Examples 2 and 3), and the gene coding for the endoglucanase was introduced into Saccharomyces cerevisiae and introduced into the strain. As a result, it was found that the endoglucanase- It was confirmed that a protein having endoglucanase activity was secreted out of the cell (Example 4, Figs. 6 and 7 and Table 2).

In another aspect, the present invention provides polynucleotides encoding endoglucanases of the present invention.

The term "polynucleotide" in the present invention is a polymer of a nucleotide in which a nucleotide monomer is linked in a long chain by covalent bonds and is a DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) Is a polynucleotide encoding an endoglucanase of the invention.

Preferably, the polynucleotide may be a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 2.

The polynucleotide encoding the endoglucanase of the present invention can be obtained by codon degeneracy, or by considering the codon preference in the organism to which the endoglucanase is to be expressed, the polynucleotide encoding the endoglucanase Various modifications can be made to the coding region within a range that does not change the amino acid sequence of the gene, and various modifications or modifications can be made within a range not affecting the expression of the gene even in the portion excluding the coding region, Those skilled in the art will appreciate that the present invention is also included in the scope of the present invention. That is, as long as the polynucleotide of the present invention encodes a protein having equivalent activity, one or more nucleotide bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention.

In yet another aspect, the present invention provides an expression vector comprising a polynucleotide encoding an endoglucanase of the present invention.

In the present invention, the term "vector" means a nucleic acid molecule capable of carrying other nucleic acid to which it is linked. One type of vector, "plasmid" refers to a circular double-stranded DNA loop in which additional DNA fragments can be ligated.

The vector of the present invention can direct expression of a gene encoding a target protein operatively linked, which is referred to as an "expression vector. &Quot; Generally, in the use of recombinant DNA technology, the expression vector is in the form of a plasmid.

Preferably, the expression vector of the present invention may be an expression vector having the cleavage map of Fig.

The expression vector can be determined depending on the host cell. For example, YEp13, YCp50, pRS, pYEX vectors, etc. can be used as an expression vector when yeast is used as a host . As the promoter, for example, GAL promoter, AOD promoter and the like can be used.

In addition, the expression vector may include a fragment for suppressing expression, having various functions for suppressing, amplifying, or inducing expression of a target gene, a marker for selecting a transformant, a gene resistant to antibiotics, A gene encoding a signal for the purpose of secretion of a secreted protein, and a customized fusion factor suitable for a hung-off protein.

Preferably, a protein fusion factor (TFP) can be used as a custom-made fusion factor suitable for the hatching-prone protein.

As used herein, the term "protein fusion factor" refers to a gene that induces secretory production of a protein having a low expression rate by fusion with a gene encoding a protein having a low expression ratio. Such a protein fusion factor may be a bacterial (for example, Escherichia), Pseudomonas (Pseudomonas) and Bacillus subtilis (Bacillus) species, etc.), fungi (e.g., yeast, Aspergillus (Aspergillus), Penny chamber William (Penicillium), rayijo Perth (Rhizopus), Trichoderma (Trichoderma) species such as ), Plants (e.g., Arabidopsis thaliana, corn, tobacco, potatoes, etc.) and animals (such as humans, rabbits, dogs, cats and monkeys) have.

Preferably, the protein fusion factors which can be used in the present invention can be protein fusion factors disclosed in Korean Patent Nos. 0626753, 0798894 and 0975596, more preferably protein fusion factors of STFP4, STFP9 or STFP13 Can be used,

According to the present invention, the gene inserted into the expression vector is a polynucleotide encoding the above-mentioned endoglucanase, preferably a polynucleotide encoding an endoglucanase represented by the amino acid sequence of SEQ ID NO: 1 , More preferably a polynucleotide encoding an endoglucanase represented by the amino acid sequence of SEQ ID NO: 1 expressed from a strain of phora.

In another embodiment, the present invention provides a transformant obtained by introducing an expression vector of the present invention into a host cell.

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

Transformation methods in the present invention can be carried out by transforming the expression vector of the present invention by methods known in the art, including, but not limited to, transient transfection, microinjection, transduction, Liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroinvasion (e. G. electroporation, sparrowth lysis, or lithium acetate method, and transformed into a host cell. As a method of introducing the recombinant DNA into yeast, for example, an electric invasion method, a spiropalat method, a lithium acetate method, or the like can be used.

The host cell used for the transformation according to the present invention may be any host cell well known in the art, but it has excellent introduction efficiency of the polynucleotide encoding the endoglucanase of the present invention, and the introduced polynucleotide For example, bacteria, fungi, yeast, plants or animals (e. G., Mammalian or insect) cells.

Preferably, the host cell may be an ethanol-producing cell, and more preferably, the ethanol-producing cell may be a zymomonas, a yeast, or a 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 , and Arxula species, and more preferably Candida utilis Candida utilis , Candida boidinii , Candida albicans , Kluyveromyces lactis , Pichia pastoris , Pichia stipitis , Caro My process pombe (Schizosaccharomyces pombe), in my process serenity busy as Saccharomyces (Saccharomyces cerevisiae), a saccharide Mai Cobb cis blood debris galley (Saccharomycopsis fiburigera), Hanse Cronulla Remote fail-fast (Hansenula polymorpha), Yarrow's lipoic poly urticae (Yarrowia lipolytica), the shoe wanni Oh, my process Occidental less (Schwanniomyces occidentalis), are Sula O may be such Denny Nemo lance (Arxula adeninivorans), preferably Cluj Vero Mai It may be Kluyveromyces maxianus , Saccharomyces cerevisiae or Saccharomycopsis fiburigera .

In one embodiment of the present invention, yeast Saccharomyces cerevisiae was transformed with the expression vector of the present invention, and it was confirmed that the endoglucanase of the present invention was produced from the transformant (Example 4 , Figs. 6, 7 and Table 2).

In another embodiment, the present invention relates to a method for producing endoglucanase according to the present invention, comprising culturing the transformant of the present invention and recovering endoglucanase from the culture or the culture supernatant thereof .

The medium for culturing the transformant of the present invention and the culture conditions can be appropriately selected and used depending on the host cell. The nutrient medium preferably contains an inorganic nitrogen source or an organic nitrogen source which is necessary for growth of the host cell. Examples of the carbon source include glucose, dextran, soluble starch, sucrose, and methanol. Examples of the inorganic or organic substance include ammonium salts, nitrates, amino acids, corn steep liquer, peptone, casein, bovine extract, soybean bag, and potato extract. (Nutrients such as sodium chloride, calcium chloride, sodium dihydrogenphosphate, inorganic salts such as magnesium chloride, vitamins, and antibiotics (tetracycline, neomycin, ampicillin, and kanamycin). During the culture, the conditions such as the temperature, the pH of the medium and the incubation time can be appropriately adjusted so as to be suitable for cell growth and mass production of the protein.

In one embodiment of the present invention, YGa-ST13-Y4EGL was prepared using TFP as a vector derived from YEp352, which is a general yeast expression vector having a GAL10 promoter and a GAL7 terminator, in order to express endoglucanase. Each vector was transformed into Saccharomyces cerevisiae and cultured under the conditions of 30 占 폚 YEPD medium (yeast extract 1%, peptone 2%, glucose 2%) (Example 4).

The step of recovering the endoglucanase may be by isolating and purifying the endoglucanase by an immunocompetent method using conventional biochemical separation techniques or using antibodies against one part or another part of the protein. Another method may be purification by affinity tagging with a protein sequence and using antibodies or other materials with a suitably high affinity for these tags. Typical biochemical separation techniques include, but are not limited to, treatment with protein precipitants (salting-out), centrifugation, ultrasound disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, And various chromatographies such as chromatography. Typically, proteins having high purity can be separated by using them in combination.

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

Specifically, the method for producing bio-ethanol of the present invention comprises the steps of (i) introducing an expression vector of the present invention into a host cell having an ethanol-producing ability to obtain a transformant transformed; (Ii) culturing the obtained transformant in a culture medium containing a substrate of endoglucanase; And (iii) recovering the bioethanol from the culture or the culture supernatant obtained in the step (ii).

The ethanol-producing host cell is not particularly limited, but Zymomonas Mobilis strain, yeast strain or Bacillus strain may be used, more preferably exoglucanase or beta-glucoside Microorganisms expressing or simultaneously expressing the respective agents can be used.

The substrate of the endoglucanase may be glucan or polysaccharide which is preferably, but not limited to, endoglucanase, more preferably starch, dextran, May be at least one member selected from the group consisting of ruthenium and cellulose, and more preferably may be cellulose.

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

The culture method is not particularly limited, but any method used for culturing conventional microorganisms such as a batch system, a flow batch system, a continuous culture system, and a reactor system can be used.

The novel endoglucanase of the present invention exhibits excellent cellulose decomposing activity and can be widely used in industrial processes such as the production of bioethanol from woody biomass.

Fig. 1 is a photograph showing cellulase activity of a strain of Phoma macrostoma Y4.
FIG. 2 is a photograph showing partial amplification (A) and Southern blotting (B) of the endoglucanase (EGL) gene of the Fama macros toma Y4 strain using the degenerate primer.
FIG. 3A is a schematic diagram showing a method of cloning an EGL gene derived from Poma macros toma Y4 strain, and FIG. 3B is a photograph showing the result of electrophoresis after PCR. Lane 1: size marker, lane 2: amplification of the 5 'region of the Y4-EGL gene, and the result of amplifying the 3' region of the lane 3: Y4-BGL gene.
FIG. 4 is a graph showing the relationship between the EGL gene derived from Fomar macrostomy Y4 strain and the Aspergillus clavatus NRRL 1, Trichoderma viride , Penicillium marneffei , BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic diagram showing the comparison of homology between the endoglucanase sequences derived from Pyrenophora tritici-repentis Pt-1C-BFP endoglucanase first.
5 is a schematic view showing a method of transforming EGL gene derived from Fomar macrostomy Y4 strain into Saccharomyces cerevisiae as a strain.
FIG. 6 is an electrophoresis image showing the result of SDS-PAGE analysis of endoglucanase expressed in 24 strains of Saccharomyces cerevisiae and secreted out of the cell.
Fig. 7 is an electrophoresis image showing the result of SDS-PAGE analysis before and after Endo-H treatment for comparison of protein sugar chain content.
Fig. 8 is a schematic diagram showing an EGL gene expression vector YGa-ST13-Y4EGL derived from a pomama macros toma Y4 strain.
FIG. 9 shows the result of SDS-PAGE analysis of the medium obtained by fermenting the fermentation broth of Saccharomyces cerevisiae transformed with the recombinant vector containing the EGL gene derived from Poma macros toma Y4 strain in a 5 L fermenter by time course (A) and the graphs showing the results of measuring cell cell growth and activity of endoglucanase at each time (B).
FIG. 10 shows electrophoresis (A) showing the result of SDS-PAGE analysis of the protein of the purified fraction obtained through ion exchange chromatography for purification of recombinant EGL, and measurement results of endoglucanase activity of each fraction Graph (B).
FIG. 11 shows electrophoresis (A) and end-H (endo-H) treatment of the purified fraction obtained by gel filtration chromatography for recombinant EGL purification and electrophoresis (A) FIG. 5B is a graph showing the results of measurement of the endo-endoglucanase activity. FIG.
FIG. 12 is a graph showing the results of analyzing the optimum temperature (A), optimum pH (B), and thermal stability (C) for analyzing enzyme characteristics of the purified recombinant EGL protein.

Hereinafter, the present invention will be described in more detail by way of 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.

Example 1 Screening and Identification of Fungus Strain Showing Cellulase Activity

In order to isolate the cellulase producing fungi, various samples which were corroded were used to cultivate Potato Dextrose Agar (Difco) for 7 days in the basic medium. The mycelia were cut into 5 mm diameter and 100 ml of YM (HEC) supplemented with 0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% dextrose, and incubated at 25 ° C for 5 days with shaking. The cultured mycelium of mycelia was filtered with a filter paper (Watman; 10 μm), and the cellulase activity secreted by the medium was analyzed. 10 μl of the culture supernatant was added to YP (1% yeast extract, 2% peptone) plate medium containing 1% carboxymethylcellulose (CMC) and cultured for 12 hours. The culture medium was stained with 0.1% congo red solution, discolored with 1M NaCl solution, and clear zone formation was confirmed. Thus, fungal strains having excellent cellulase activity were selected. The cellulase activity of Y4 strain isolated from rotten orange, which exhibited the best cellulase activity among the selected strains, is shown in Fig.

In order to identify the Y4 strain having the best cellulase activity, the base sequence of the ITS region was analyzed by a molecular classification method based on the nucleotide sequence analysis method, and a 501 bp long ITS region sequence (SEQ ID NO: 3) was obtained from the Y4 strain These sequence information was compared with the sequence database of various fungal strains in GenBank. As a result, Phoma macrostoma var. incolorata strain CBS 300.36.

Example 2 Isolation and Identification of Cellular Gene from Fomar Macross Tomar Y4

The CAZy database (www.CAZy.org), which contains information on enzyme genes related to the glycosidic bond of carbohydrates to identify the fibrinolytic enzyme gene from the isolated Phoma macrostoma Y4 strain, ), Degenerate primers were prepared and used from GH 5, 6, 7, 9, 12 and 61, which are families containing eukaryotic cell-derived cellulase, based on amino acid sequences having high conservation. The prepared degenerate primer sequences are shown in Table 1.

GH5F2 / GH5R1, GH5F2 / GH5R2, GH6F / GH6R, GH7F1 / GH7R1, GH7F1 / GH5R2, GH7F2 / GH7R1, GH5F1 / GH5R2, GH5F2 / GH5R2, GH5F2 / GH5R2, GH7F2 / GH5R2, GH9F / GH9R, and GH12F / GH12R). The PCR reaction was carried out at 95 ° C for 5 minutes, followed by 30 cycles of 95 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 30 seconds, and further reaction was carried out at 72 ° C for 10 minutes. PCR was performed on the chromosomes of the Foma macroshotoma Y4 strain and Southern blotting was performed using a known cellulase gene complex as a probe. The results are shown in FIG.

As a result, as shown in FIG. 2, a gene fragment was identified at a size of 500 bp in the GH7F1 / GH7R2 primer combination. The amplified fragment was separated from the 0.9% agarose gel at this position and cloned into pGEM-T Easy vector, The sequence was analyzed. As a result of the base sequence analysis, the Fama macros toma Y4 cellulase exhibited high similarity with the endoglucanase derived from Pyrenophora tritici , but the same base sequence could not be found in the published gene database . Therefore, it was confirmed that the partially amplified cellular gene fragment was a part of the novel endoglucanase.

Example 3: Securing endoglucanase gene according to the present invention using template blocking PCR

Genome walking technology (Korean Patent Application No. 10-2009-0061895) was used to secure the entire gene based on the nucleotide sequence information revealed by the method of Example 2. The cassette was first prepared to use the genome walking technique. Cassette means short double stranded DNA designed to be ligated to a restriction enzyme treated genomic DNA.

Genomic DNA of Fomar macrostoma Y4 strain was treated with restriction enzyme BamHI at 37 DEG C for 2 hours and electrophoresed on agarose gel to confirm that it was completely cleaved. Then, restriction enzyme was inactivated by treatment in a constant temperature water bath at 70 DEG C for 20 minutes, After addition of ddGTP to 0.2 mM, 8 units of Klenow fragment (Takara) were added and reacted at 37 DEG C for 30 minutes. The reaction products were purified using a PCR purification kit (Axygen), and then reacted at 16 ° C for 10 hours using a 40 pmol cassette and a DNA ligation kit (Takara). The ligated DNA was purified again using a PCR purification kit, and then eluted with 20 μl of distilled water to prepare a BamHI cassette library.

Based on the nucleotide sequence of the 500 bp PCR product obtained in Example 2 to obtain the endoglucanase gene according to the present invention using the BamHI cassette library, endoglucanase-specific primer MJ 65 (SEQ ID NO: 25 ) And MJ66 (SEQ ID NO: 26) were prepared in bidirection, and 1 μl of each cassette library was used as a template using the cassette primer CSF (SEQ ID NO: 27) and CSR (SEQ ID NO: 28) PCR was performed.

[Genomic cassette library 1 μl, CSF 1 pmol or CSR 1 pmol MJ66 1 pmol or MJ65 1 pmol, EX taq buffer (Takara, Japan) was used to amplify the 5 'and 3' regions of the endoglucanase gene ), 5 μl of 2.5 mM dNTP, 37 μl of distilled water, and 1 μl of EX taq DNA polymerase (Takara)]. The PCR conditions were 95 ° C for 30 minutes, 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 3 minutes Reaction for 30 cycles and further reaction at 72 캜 for 10 minutes was used.

After the PCR reaction, 10 μl of the reaction solution was analyzed by agarose gel electrophoresis. As a result, a 2.5 kb fragment was amplified from the BamHI cassette library in the 5 'site cloning reaction and a 5.0 kb fragment (Fig. 3).

Each of the PCR products was cloned into a pGEM-T Easy vector (Invitrogen), and the nucleotide sequence was analyzed. As a result, it was confirmed that the endoglucanase gene derived from Y4 strain was amplified and the entire sequence of the gene 2) and the deduced amino acid sequence (SEQ ID NO: 1). The Y4-derived endoglucanase gene (Y4-EGL) cloned by the above method was composed of 1227 bp nucleotides and was confirmed to be a protein composed of 408 amino acids. The first 21 amino acid residues at the N-terminal region were presumed to be signal peptides by the online program SignalP analysis (http: // www / cbs / dtu / a / services / SignalP /).

The BLASTX database was searched to compare the amino acid sequence of endoglucanase separated from the present invention with the sequence of other proteins, and the result is shown in FIG.

As shown in FIG. 4, the endoglucanase according to the present invention was confirmed to belong to the glycosyl hydrolase (GH) 7 family, and the endogenous glucanase belonging to the family of pyrethroids 66% with endoglucanase from Tissie-lipopeptide Pt-1C-BFP, 55% with endoglucanase from Humicola insolens , Humicola grisea var. Thermoidea ) Derived endoglucanase and 54% amino acid homology with the resulting endoglucanase.

Example 4. Confirmation of expression and activity of endoglucanase secretion by introduction of protein fusion factor (TFP)

In order to secrete and express the EGL gene (Y4-EGL) derived from the Fomar macrostomy Y4 strain obtained through the method of Example 3 in Saccharomyces cerevisiae by the method shown in Fig. 5, transformation was carried out as follows Respectively.

The Y4-EGL gene except for the secretion signal was reacted at 94 DEG C for 5 minutes using MJ102 (SEQ ID NO: 29) and MJ92 (SEQ ID NO: 30) primers, followed by 94 DEG C for 30 seconds, 55 DEG C for 30 seconds, 72 DEG C for 1 minute and 10 seconds The PCR reaction was carried out at 72 ° C for 7 minutes after 25 cycles. The gene generated after the first PCR was amplified by PCR using 24 STFP vectors as a template and LNK39 (SEQ ID NO: 31) primer and GT50R (SEQ ID NO: 32) primers complementary to the forward primer. Since the PCR product amplified with the LNK39 and GT50R primers contains the same sequence as the vector of 40 bp or more, when introduced into the yeast cell together with the linearized vector, crossing occurs within the cell to produce a circular plasmid vector (see FIG. 5 ). The amplified PCR products were isolated from Escherichia coli strains isolated from E. coli using 24 recombinant DNA fragments, which have the advantage of obtaining a strain of Saccharomyces cerevisiae transformed with the desired vector, Was introduced into strain Y2805 (Mat a pep4 :: HIS3 prb1 can1 his3-200 ura3-52) through a protein fusion factor (Korean Patent No. 0975596) vector. Transformed strains were selected using SC-URA (various amino acids except yeast substrate lacking 0.67% amino acid, 2% glucose and uracil) medium.

Each transformant was cultured in a YPDG medium (1% yeast extract, 2% Peptone, 1% glucose, 1% Galactose) for 40 hours and then centrifuged to remove cells. 0.6 ml of the remaining culture supernatant was suspended in 0.4 ml of acetone And the mixture was allowed to stand at -20 ° C for 2 hours. The protein was centrifuged at 10,000 g for 15 minutes, recovered, developed by SDS-PAGE gel containing 12% glycine at 80 V for about 2 hours, After staining with a staining solution prepared using Phastgel TMBlue R (GE healthcare), decolorization was performed using decolorization buffer (10% Acetic acid, 30% methanol) for 1 hour, and the result is shown in FIG.

 As shown in FIG. 6, it was confirmed that even in a molecular weight portion larger than the expected protein size on SDS-PAGE, the protein was separated into a multi-band form in which the protein was identified. These results indicate that glycosylation occurred at the 3 N-glycosylation trigger sequence sites included on the Y4-EGL protein sequence. Therefore, each protein was subjected to SDS-PAGE analysis after treatment with Endo-H enzyme so as to remove sugars added to the protein by N-glycosylation, and the result is shown in FIG.

As shown in FIG. 7, the endoglucanase protein was identified at the predicted molecular weight region after endo treatment, and the degree of secretion of endoglucanase was found to vary significantly depending on the type of protein secretion fusion factor applied. Among them, the protein secretion fusion factors 4, 9 and 13 showed a high fraction ratio.

Therefore, the endogenous glucanase activity was confirmed by using the transformants to which the protein secretion factor 4, 9 or 13, which showed a high fractional ratio, was applied. Dinitirosalicylic acid (Miller, Anal. Chem, 55: 952-959, 1959) was used to analyze the activity. 100 μl of the substrate solution (100 mM sodium phosphate solution containing 1% carboxymethyl cellulose, pH 7.0) was added to 100 μl of the enzyme solution, reacted at 50 ° C. for 10 minutes, 700 μl of the DNS solution was added, After 5 minutes of treatment, the cells were cooled with cold water, and the absorbance was measured at 540 nm to confirm endoglucanase activity. The results are shown in Table 2. At this time, the enzyme activity was defined as 1 unit of the amount of the enzyme releasing 1 μmol of reducing sugar for 1 minute.

Endoglucanase activity analysis of selected strains Protein secretion factor Endoglucanase activity (u / ml) 4 2.11 9 2.55 13 2.92

As shown in Table 2, it was confirmed that the transformant to which the protein secretion factor 13 was applied exhibited the best endoglucanase activity at 2.92 unit / ml, and was finally selected.

Example 5: Mass production of endoglucanase through fermentation of oil-feeding culture

A recombinant yeast strain transformed with 2805 / YGa-ST13-Y4EGL (FIG. 8) selected through Example 4 was used to perform fed-batch culture in a 5 L fermenter to mass-produce endoglucanase. The cells were cultured for one step in 50 ml of a minimal liquid culture medium (yeast nitrogen substrate lacking 0.67% amino acid, 0.5% casamino acid, 2% glucose) before entering the culture, and further cultured in 200 ml of YPD liquid medium (1% yeast Extract, 2% peptone, 2% glucose), and then inoculated into the culture medium and cultured at 30 ° C for 48 hours. Feeding culture medium (15% yeast extract, 30% glucose) was supplemented according to cell growth rate. After 48 hours of incubation, the cell concentration reached 150 on an OD ₆₀₀ basis. SDS-PAGE analysis was performed by taking the culture supernatant over time to confirm the endoglucanase secreted in the medium. After 48 hours of culture, about 28 units / ml of activity was confirmed (FIG. 9). Proteins produced through fermentation were concentrated using ultrafiltration (molecular weight cut off 30,000) into 50 mM Tris buffer solution (pH 7.5).

Example  6: Endoglucanase  refine

Purification was performed using the enriched endoglucanase enzyme obtained by the method of Example 5 above. As the first step, ion exchange chromatography was carried out using DEAE-Sepharose column. The resin was sufficiently parallelized with a 10 mM ammonium acetate buffer solution (pH 7.0), and 5 ml of the crude enzyme solution was loaded, and the protein was eluted with a gradient of 0 to 1 M NaCl at a flow rate of 0.2 ml / min. The activity of each fraction was measured and only fractions 12 and 13 showing activity were recovered and concentrated using Amicon Ultra-4 (Millipore, 10K NMWL device) for use in the next purification step (FIG. 10).

As a second step, the supernatant (1.6 X 61.5 cm) column, which had been sufficiently equilibrated with a buffer solution containing 100 mM NaCl in 10 mM ammonium acetate, was loaded with 5 ml of the active fraction collected in the previous step and subjected to gel filtration chromatography, And fractionated by size. The activity of each fraction was measured, and fractions 1 to 8 which showed the final activity were collected. The recovered fractions were subjected to SDS-PAGE analysis after treatment with an endo H enzyme to remove sugars added to the protein, and as a result, it was confirmed that a single band of pure endoglucanase protein was purified in fractions 1 to 3 (Fig. 11).

Example 7: Optimal temperature, thermal stability and optimum pH of expressed recombinant endoglucanase

The effect of pH and temperature on enzyme activity was investigated using purified crude enzyme solution to investigate the characteristics of purified endoglucanase through the method of Example 6. In order to investigate the effect of temperature, each activity was measured after reacting in the range of 20 to 80 캜 at intervals of 10 캜, and relative activity was shown based on the highest activity. As a result, it was found that the optimum reaction temperature was 60 ° C, and it was confirmed that the activity was high in the range of 45 ° C to 70 ° C (FIG. 12A).

Further, 50 mM citrate buffer (pH 2.0 to 6.0), 50 mM phosphate buffer (pH 6.0 to 8.0) and 50 mM tris-hydrochloric acid buffer (pH 8.0 to 10.0) were used to investigate the effect of pH on enzyme activity After reacting at 50 ° C, enzyme activity was measured. The relative activity at each pH was found to be maximal at pH 5.0 and was found to be relatively stable even at acidic and neutral pHs of pH 2.0 to 8.0 (FIG. 12B).

In addition, in order to investigate the stability to heat, the crude enzyme solution was left at the respective temperature for each hour, and the residual activity of the remaining enzyme was measured. As a result, the activity of the enzyme was stably maintained at 50 ° C for 1 hour, but the activity of the enzyme decreased at 60 ° C over time (FIG. 12C).

<110> Korea Research Institute of Bioscience and Biotechnology          Korea Institute of Industrial Technology <120> Novel endoglucanase and the Use thereof <130> PA110443KR <160> 32 <170> Kopatentin 1.71 <210> 1 <211> 408 <212> PRT &Lt; 213 > amino acid sequence of endoglucanase derived from Phoma macrostoma Y4 <400> 1 Met Val His Tyr Arg Ile Ile Ser Tyr Ala Leu Leu Ala Val Ala Ser   1 5 10 15 Ala Gln Ser Leu Gly Glu Gly Gln Glu Glu Gln Pro Lys Leu Thr Thr              20 25 30 Tyr Gln Cys Thr Asn Asp Gly Gly Cys Lys Ala Gln Asp Ser Tyr Ile          35 40 45 Val Leu Asp Ser Ala Ser His Asn Ile His Gln Lys Asn Asp Thr Thr      50 55 60 Lys Gly Cys Gly Asn Gly Gly Thr Gly Val Asp Pro Ala Val Cys Pro  65 70 75 80 Asn Gln Glu Thr Cys Ala Gln Asn Cys Ile Leu Glu Pro Ile Ser Asn                  85 90 95 Tyr Gln Asp Tyr Gly Ile Ser Thr Asp Gly Ala Ser Leu Arg Met Asp             100 105 110 Phe Phe Gly Thr Asp Lys Lys Phe Gln Ser Pro Arg Ala Tyr Leu Leu         115 120 125 Asn Glu Asp Lys Lys Asn Tyr Glu Met Leu Gln Leu Thr Gly Lys Glu     130 135 140 Phe Ala Phe Asp Val Asp Val Ser Lys Leu Pro Cys Gly Met Asn Gly 145 150 155 160 Ala Leu Tyr Leu Ser Glu Met Met Ala Asp Gly Gly Arg Ser Ala Leu                 165 170 175 Asn Pro Gly Gly Ala Ser Val Gly Thr Gly Tyr Cys Asp Ala Gln Cys             180 185 190 Phe Val Thr Pro Trp Ile Asn Gly Gln Gly Asn Ile Asn Gly Thr Gly         195 200 205 Val Cys Cys Asn Glu Met Asp Ile Trp Glu Ala Asn Ser Glu Ser Thr     210 215 220 Gln Val Ala Pro His Thr Cys Ser Lys Pro Ser Leu Phe Thr Cys Gln 225 230 235 240 Gly Asp Glu Cys Gly Ala Thr Gly Leu Cys Asp Lys Asn Gly Cys Gly                 245 250 255 Glu Asn Pro Tyr Arg Ser Gly Ala Lys Asp Phe Tyr Gly Pro Gly Leu             260 265 270 Lys Ile Asp Thr Gln Lys Pro Phe Thr Val Val Thr Gln Phe Pro Ala         275 280 285 Glu Asn Gly Val Leu Gln Ser Ile Gly Arg Lys Tyr Val Gln Asn Gly     290 295 300 Val Val Phe Glu Asp Gln Pro Lys Asn Ile Thr Leu Asn Asp Gln Phe 305 310 315 320 Cys Thr Ala Asn Asp Ala Thr Met Phe Met Asn Leu Gly Gly Met Lys                 325 330 335 Gly Met Gly Asp Ala Leu Ser Arg Gly Met Val Leu Ala Leu Ser Val             340 345 350 Trp Trp Asp Glu Ser Lys Phe Met Asn Trp Leu Asp Ser Gly Asn Ala         355 360 365 Gly Pro Cys Asn Ala Thr Glu Gly Asp Pro Lys Asn Ile Val Lys Val     370 375 380 Gln Ala Ala Pro Ser Ala Thr Phe Ser Asn Ile Lys Trp Gly Glu Ile 385 390 395 400 Gly Ser Thr Phe Lys Gly Asn Met                 405 <210> 2 <211> 1227 <212> DNA <213> nucleic acid sequence of endoglucanase derived from Phoma macrostoma Y4 <400> 2 atggtccact acagaattat ctcgtatgct cttttggctg ttgcttctgc tcagtcactc 60 ggcgagggcc aggaggaaca gcccaagttg accacctacc agtgcaccaa tgatggtggc 120 tgcaaggctc aagacagcta catcgttctc gactccgcta gccacaacat ccaccagaag 180 aacgatacca ccaagggctg cggcaatggg ggcactggag tggaccctgc tgtatgcccc 240 aatcaggaga cttgcgccca aaactgcatc ctcgagccca tcagcaacta ccaggactat 300 ggcatctcca ccgacggcgc aagcctacgc atggacttct tcggaacgga caagaaattc 360 cagagcccca gggcgtatct cctcaacgag gacaagaaga attatgagat gctccaattg 420 accggcaagg agttcgcttt cgacgtcgat gtctcgaagc tgccttgcgg catgaatggt 480 gctctgtacc tctctgagat gatggcggat ggtggccgct cagctttgaa cccaggaggt 540 gcgagcgttg gtaccggata ctgcgatgct cagtgcttcg ttactccttg gatcaacggt 600 cagggcaaca ttaatggcac cggtgtttgc tgcaacgaga tggacatctg ggaggccaac 660 agcgaatcta cccaagtcgc tccccacaca tgcagcaagc ccagcctctt cacatgccag 720 ggcgatgagt gcggcgctac cggtctttgc gataagaacg gctgcggcga gaacccctac 780 cgcagcggtg caaaggactt ctatggtcct ggcctcaaga ttgacaccca gaagcccttc 840 accgtcgtca cccagtttcc cgccgagaac ggtgtgcttc aatccattgg gcgcaagtac 900 gttcagaatg gcgttgtttt cgaggatcag cccaagaaca tcaccttaaa cgaccagttc 960 tgcaccgcca atgacgctac tatgttcatg aaccttggag gtatgaaggg catgggcgat 1020 gcgctaagcc gcggtatggt cctggctttg agtgtttggt gggacgagag taaattcatg 1080 aactggcttg acagcggtaa tgctggccca tgcaatgcta ctgagggcga ccccaagaac 1140 attgtcaagg tccaggctgc accttccgcg acgttctcca acatcaagtg gggtgaaatt 1200 ggctcgacct tcaagggcaa catgtga 1227 <210> 3 <211> 501 <212> DNA <213> Artificial Sequence <220> <223> Y4 ITS <400> 3 aaggatcatt acctagagtt gtaggctttg cctgctatct cttacccatg tcttttgagt 60 accttcgttt cctcggcggg tccgcccgcc gattggacaa tttaaaccat ttgcagttgc 120 aatcagcgtc tgaaaaaact taatagttac aactttcaac aacggatctc ttggttctgg 180 catcgatgaa gaacgcagcg aaatgcgata agtagtgtga attgcagaat tcagtgaatc 240 atcgaatctt tgaacgcaca ttgcgcccct tggtattcca tggggcatgc ctgttcgagc 300 gtcatttgta ccttcaagct ctgcttggtg ttgggtgttt gtctcgcctc tgcgcgtaga 360 ctcgcctcga aacaattggc agccggcgta ttgatttcgg agcgcagtac atctcgcgct 420 ctgcactcat aacgacgacg tccaaaagta catttttaca ctcttgacct cggatcaggt 480 agggataccc gctgaactta a 501 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH5F1 primer <400> 4 ttcttcycyg gyaayga 17 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH5F2 primer <400> 5 atyccwgtyg gytantc 17 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH5R1-1 primer <400> 6 tcygartwng gytgyaa 17 <210> 7 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH5R1-2 primer <400> 7 ttrcarccnw aytcrga 17 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH5R2-1 primer <400> 8 tcyggyggyh tngtyta 17 <210> 9 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH5R2-2 primer <400> 9 taracnadrc crccrga 17 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GH6F primer <400> 10 garccwgayw cyhtngsyaa 20 <210> 11 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH6R-1 primer <400> 11 ggygarwgng ayggyrc 17 <210> 12 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH6R-2 primer <400> 12 gyrccrtcnc sytcrcc 17 <210> 13 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH7F1 primer <400> 13 garttcwcyt tcgaygt 17 <210> 14 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH7F2 primer <400> 14 tanggyacyg gytantg 17 <210> 15 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH7R1-1 primer <400> 15 tgygatscng ayggytg 17 <210> 16 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH7R1-2 primer <400> 16 carccrtcng watcrca 17 <210> 17 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH7R2-1 primer <400> 17 acygtygtya cycaatt 17 <210> 18 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH7R2-2 primer <400> 18 aattgrgtra cracrgt 17 <210> 19 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH9F primer <400> 19 ggytggtang atgcyga 17 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GH9R-1 primer <400> 20 tcytwnrtya cyggytangg 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GH9R-2 primer <400> 21 ccntarccrg traynsarga 20 <210> 22 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GH12F primer <400> 22 ggyatyaart cytancc 17 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GH12R-1 primer <400> 23 garatyacyt cytcyccwgg 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GH12R-2 primer <400> 24 ccsggrgarg argtratytc 20 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> MJ65 primer <400> 25 tccttggatc aacggtgttg taagtag 27 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> MJ66 primer <400> 26 cttgaggcca ggaccataga agtcc 25 <210> 27 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CSF primer <400> 27 gacgcgtaat acgactcact ataggga 27 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CSR primer <400> 28 atctccctat agtgagtcgt attacgcgtc 30 <210> 29 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> MJ101 primer <400> 29 ctcgccttag ataaaagaca ggaggaacag cccaag 36 <210> 30 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> MJ92 primer <400> 30 cactccgttc aagtcgactc acatgttgcc cttgaa 36 <210> 31 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> LNK39 primer <400> 31 ggccgcctcg gcctctgctg gcctcgcctt agataaaaga 40 <210> 32 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> GT50R primer <400> 32 tcattattaa atatatatat atatatattg tcactccgtt caagtcgac 49

Claims (13)

Endoglucanase represented by the amino acid sequence of SEQ ID NO: 1.
The endoglucanase according to claim 1, wherein said endoglucanase is expressed from a strain of Phoma.
A polynucleotide encoding an endoglucanase of claim 1.
4. The polynucleotide according to claim 3, wherein the polynucleotide is represented by the nucleotide sequence of SEQ ID NO: 2.
An expression vector comprising the polynucleotide of claim 3.
6. The expression vector of claim 5, further comprising a translational fusion partner (TFP).
A transformant obtained by introducing the expression vector of claim 5 into a host cell.
8. The transformant according to claim 7, wherein the host cell has an ethanol-producing ability.
8. A method for producing endoglucanase according to claim 1, comprising culturing the transformant of claim 7, and recovering endoglucanase from the culture or culture supernatant thereof.
(i) introducing the expression vector of claim 5 into an ethanol-efficient host cell to obtain a transformed transformant;
(Ii) culturing the obtained transformant in a culture medium containing a substrate of endoglucanase; And
(Iii) recovering bio-ethanol from the culture or culture supernatant obtained in step (ii) above.
11. The method for producing bioethanol according to claim 10, wherein the ethanol-producing host cell expresses exoglucanase or beta-glucosidase, respectively, or simultaneously.
11. The method of claim 10, wherein the substrate is glucan or a polysaccharide.
11. The method of producing bioethanol according to claim 10, wherein the substrate is at least one selected from the group consisting of starch, dextran, pullulan, and cellulose.

Images