KR20140146856A - METHOD FOR PREPARING CELLULOSE COMPLEX USING ENDO-β-1,4-GLUCANASE E, XYLANASE B AND MINI CELLULOSE BINDING PROTEIN A FROM CLOSTRIDIUM SP. - Google Patents

Abstract

The present invention relates to a method for preparing a fibrinogenase complex using endo-β-1,4-glucanase E, xylanase B, and mini cellulose binding protein A derived from Clostridium sp. microorganism. More specifically, the present invention relates to a method for preparing (i) a chimeric endo--β-1,4-glucanase E-mini cellulose binding protein A (cCelE-mCbpA) complex, (ii) a xylanase B- mini cellulose binding protein A (XynB-mCbpA) complex, and (iii) a chimeric endo--β-1,4-glucanase E- xylanase B- mini cellulose binding protein A (cCelE-XynB-mCbpA) complex by culturing a recombinant yeast having an ability to produce chimeric Endo��beta;��,4��lucanase E (cCelE), a recombinant yeast having an ability to produce xylanase B (XynB), and a recombinant yeast having an ability to produce mini cellulose binding protein A (miniCbpA). Also, the present invention relates to a method for manufacturing bioethanol using the same.

Description

[0001] The present invention relates to a method for producing a fibrinolytic enzyme complex using endo-beta-1,4-glucanase derived from Clostridium microorganism, xylanase-ratio and small cellulosic binding protein- -1,4-glucanase E, Xylanase B and Mini Cellulose Binding Protein A from Clostridium sp.

The present invention relates to a method for producing a fibrinolytic enzyme conjugate using endo-beta-1,4-glucanase derived from Clostridium genus microorganism, xylanase-bound and small cellulosic binding protein a, Recombinant yeast having the ability to produce chimeric Endo-β-1,4-glucanase E (cCelE), recombinant yeast having the ability to produce xylanase B (XynB) and small cellulosic binding protein (CCelE-mCbpA) complex, (ii) a chimeric endo-beta-1,4-glucanase-small cellulase binding protein a (cCelE-mCbpA) complex, (XynB-mCbpA) complex and (iii) chimeric endo-beta-1,4-glucanase-i-xylanase non-small cellulosic binding protein (cCelE-XynB-mCbpA) ) Complex How to tank and to a method of producing bioethanol using the same.

Concerns over skyrocketing oil prices and energy depletion have heightened interest in alternative energy production. Bioethanol is the most active source of alternative fuels for transportation. This is the initial stage of the development of the electric vehicle or the hydrogen vehicle, which is the final technical objective of the automobile, and it is still necessary to have an alternative energy to replace it. At present, bioethanol can be mixed with gasoline, so it is a representative renewable resource energy in addition to biodiesel. Carbon dioxide that is generated when bioethanol is burned is an exception in the calculation of greenhouse gas defined by the Kyoto Protocol. In addition, unlike other cleaner fuels that require separate infrastructures (such as gas stations) to be deployed, it can be easily deployed in existing infrastructure, making early commercialization easy.

At present, bioethanol can be produced from lignocellulosic biomass such as carbohydrate biomass, mainly sugarcane and corn, cellulosic biomass such as wood, and pulpwood resources, surplus agricultural products and herbaceous crops. Sugarcane and corn starch are currently the representative bioethanol production material. In fact, in Brazil, based on rich sugar cane production, it succeeded in commercialization of ethanol fuel, and by 2004, about 30% With bioethanol. The United States will accumulate substantial commercialization technologies for bioethanol production using abundant corn, replacing 20% of total transport energy demand by bioethanol by 2017.

In the case of carbohydrate-based biomass, which has been used for corn, which has been used in the past, it has been negatively evaluated in terms of raw material supply and price competitiveness due to aggravation due to surging grain prices in early 2008, Is lignocellulosic or lignocellulosic biomass. Unlike conventional carbohydrates, there are no limitations on raw material supply and ethical issues, and various studies have been made to produce bioethanol using these materials. However, most of these raw materials are composed of cellulose, and it is necessary to sufficiently saccharify them for direct ethanol fermentation. However, most of them remain in the form of undissolved oligomers or polymers even after glycosylation.

Clostridium thermosellum Thermocellum is an anaerobic, thermophilic bacterium that hydrolyzes cellulose and produces ethanol. It is known to produce a variety of cellulases and produce cellulosomes, which are enzyme complexes. Combined with the characteristics of bacteria that can grow at very high temperatures, microorganisms have significant advantages as a subject matter for developing processes that industrially produce hydrogen and ethanol from cellulosic biomass resources. In addition, the effective degradation of crystalline fibrin has at least three cellulolytic enzymes: endoglucanase, exoglucanase (or cellobiohydrolase), and beta-glucosidase ) Are required at the same time, and their synergistic action has been reported to increase the fibrinolytic ability greatly (Murashima, K. et al ., & lt ; / RTI & gt ; J Bacteriol , 184 (18): 5088,2002; B Schwarz, Microbiol Biotechnol . , 56: 634, 2001; Shoham, Y. et al ., Trends Microbiol ., 7 (7): 275, 1999).

On the other hand, clostridium celluloborance Cellulovorans is an anaerobically bacterium that is known to produce a variety of cellulases and to produce cellulosomes as enzyme complexes. The basic structure of the dellulosomal complex in these anaerobic bacteria is that the primary scaffolding subunit with a cellulose binding module (CBM) is the main axis and the enzyme of the cellulase or hemicellulase with catalytic module And enzyme subunits are combined. In order to achieve this structure, nine cohesin modules in the basic skeletal subunit are strongly attached to the dockerin module in each enzyme subunit by protein-protein interaction Lt; / RTI >

Accordingly, the present inventors have made intensive efforts to produce an enzyme having excellent fibrinolytic ability. As a result, the present inventors have found that a fibrinolytic enzyme complex composed of endoglucanase, xylanase and small cellulosic binding protein a derived from Clostridium sp. Strain effectively produces bioethanol And the present invention was completed.

It is an object of the present invention to provide an endo-beta-1,4-glucanase which is formed by binding a chimeric endo-beta-1,4-glucanase derived from Clostridium microorganism to a xylanase ratio and a small- (XynB-mCbpA) complex and chimeric endo-beta-1,4-glucanase-i-xylanase non-xylose conjugate protein (cCelE-mCbpA) complex, the xylenese non-small cellulosic binding protein a (CCelE-XynB-mCbpA) complex and a method for producing bioethanol using the resulting cellulolytic enzyme complex.

To achieve the above object, the present invention provides a recombinant vector containing a gene encoding (a) chimeric endo-β-1,4-glucanase E (cCelE) A recombinant yeast transformed with a recombinant vector containing the gene encoding xylanase B (XynB), and a recombinant yeast containing a gene encoding a mini cellulose binding protein A (miniCbpA) Culturing the recombinant yeast transformed with the vector; (b) the chimeric endo-beta-1,4-glucanase produced from each recombinant yeast, the xylanase ratio and the small cellulosic binding protein a, are combined to form (i) chimeric endo- (XynB-mCbpA) complex and (iii) chimeric endo-beta-1,4-glucan complexes, (CCelE-XynB-mCbpA) complex with an agase-free xylenase non-small cellulosic binding protein a (cCelE-XynB-mCbpA) complex; And (d) recovering the formed fibrinolytic enzyme complex. The present invention also provides a method for producing the fibrinolytic enzyme complex.

The present invention also relates to a recombinant vector comprising (a) a recombinant yeast transformed with a recombinant vector containing a gene encoding chimeric endo-beta-1,4-glucanase-1 (cCelE), a recombinant yeast encoding a xylanase B Culturing a recombinant yeast transformed with a gene-containing recombinant vector and a recombinant vector transformed with a recombinant vector containing a gene encoding a mini cellulose binding protein A (miniCbpA); (b) adding biomass to the recombinant yeast cultured in step (a) and further culturing to produce ethanol; And (c) recovering the produced ethanol. The present invention also provides a method for producing ethanol by simultaneous saccharification and fermentation of biomass.

(CCelE-mCbpA) complex prepared by the method of the present invention, (i) a chimeric endo-beta-1,4-glucanase-less cellulosic binding protein a (cCelE- XynB-mCbpA) complex and (iii) chimeric endo-beta-1,4-glucanase-i-xylanase non-small cellulosic binding protein a (cCelE-XynB-mCbpA) complexes have excellent ability to degrade cellulose and xylose (SSF), which can be used for brewing, baking, alcohol production, feed production, and probiotics development, and especially for the decomposition of cellulose and the production of alcohol from degradation products .

1 is a schematic diagram of a pPsADHa vector
FIG. 2 is a diagram showing a gene encoding chimeric endo-β-1,4-glucanase E (cCelE), a gene encoding xylanase B (xylanase B), a gene encoding small celluloses Is a schematic diagram of a recombinant vector pPsADHa into which a gene encoding a mini cellulose binding protein A (miniCbpA) is inserted.
FIG. 3 is a graph showing the results of recombinant expression of the chimeric endo-glucanase (B) gene and the xylanase-free (A) gene in the present invention after culturing in Pichia pastoris , Experimental data.
FIG. 4 is a data showing the enzyme activity in the Zymogram of the colonies and the culture concentrate through the Native-PAGA in order to confirm the yeast enzyme secretion in which the recombinant vectors pPsADHα / cCelE, pPsADHα / XynB and pPsADHα / mCbpA shown in the present invention were inserted .
FIG. 5 is a data showing the Xylenase non-small cellulosic binding protein (XynB-mCbpA) complex formed in the present invention.
FIG. 6 is a graph showing the activity of the fibrinolytic enzyme complex expressed in the present invention.
FIG. 7 is a graph showing the results obtained by using the fibrinolytic enzyme complex expressed in the present invention and comparing the biomass, Miscanthus The results are shown in Fig.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention provides a recombinant vector containing a gene encoding a chimeric endo-β-1,4-glucanase E (cCelE) Containing recombinant vector encoding a recombinant yeast, a recombinant yeast transformed with a recombinant vector containing a gene encoding xylanase B (XynB) and a mini cellulose binding protein A (miniCbpA) Culturing the transformed recombinant yeast; (b) the chimeric endo-beta-1,4-glucanase produced from each recombinant yeast, the xylanase ratio and the small cellulosic binding protein a, are combined to form (i) chimeric endo- (XynB-mCbpA) complex and (iii) chimeric endo-beta-1,4-glucan complexes, (CCelE-XynB-mCbpA) complex with an agase-free xylenase non-small cellulosic binding protein a (cCelE-XynB-mCbpA) complex; And (d) recovering the formed fibrinolytic enzyme complex. The present invention also relates to a method for producing a fibrinolytic enzyme complex.

In the present invention, the recombinant vector of step (a) is introduced with a gene coding for pPsADHa-cCelE and xylanase-ratio in which a gene encoding chimeric endo-beta-1,4-glucanase has been introduced And pPsADH? -MCbpA into which a gene encoding a small cellulosic binding protein-a is introduced.

In the present invention, the gene encoding the xylanase B (XynB) and the gene encoding the mini cellulose binding protein A (miniCbpA) in the step (a) may be selected from the group consisting of Clostridium celluloborance ( Clostridium cellulovorans ).

In the present invention, the (a) step of the key chimeric endo-beta-1,4-glucan azepin-a gene encoding a (cCelE) is Endo of Clostridium Summer selreom (clostridium thermocellum) - beta-1,4 -Glucanase-1 < / RTI > (CelE) and clostridium celluloborance Endo of cellulovorans) - beta-1,4-glucan azepin-ratio (endo-β-1,4-glucanase B; Fig of EngB) can be characterized in that the gene coding for the keorin (dokerin) binding site.

In the present invention, the doCarin module of Clostridium sp. Strain means a module of cellulosomal cellulase protein that forms cellulosome by interaction with Cohesion module, which is part of the cellulose-binding protein of Clostridium sp. do.

In the present invention, the small cellulosic binding protein A (mCbpA) refers to a protein binding to cellulose which forms a cellulosic basic skeletal subunit. The small cellulosic binding protein A used in the present invention is mCbpA, which is cellulose of Clostridium Refers to a small cellulose-binding protein having a cellulose binding module (CBM) and two cohesin modules among cellulose-binding protein-a (CbpA), one of the binding proteins.

In the present invention, "vector" means a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in an appropriate host. The vector may be a plasmid, phage particle or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site into which the foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. Transfection was carried out as described in Sambrook, et . al ., supra , using the calcium chloride method described in section 1.82. Alternatively, electroporation (Neumann, et . al ., EMBO J. , 1: 841, 1982], and can also be used for transformation of these cells.

A nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way as to enable gene expression when a suitable molecule (e. G., Transcriptional activator protein) is attached to the regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; Or a ribosome binding site is operably linked to a coding sequence if positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

When the yeast is used as a host in the present invention, for example, YEp13, YCp50, pRS-based, pYEX-based vectors and the like can be used as an expression vector. As the promoter, for example, GAL promoter, AOD promoter and the like can be used. As a method of introducing the recombinant DNA into yeast, for example, an electroporation method Enzymol . , 194: 182, 1990), the spiroplast method ( Proc . Natl . Acad . Sci . , 84: 1929, 1978) and the lithium acetate method ( J. Bacteriol. , 153: 163, 1983).

The recombinant vector of the present invention can be autonomously replicated in the host cell itself and can also contain a gene encoding a promoter, chimeric endo-beta-1,4-glucanase, xylanase ratio or small cellulosic binding protein a A DNA and a transcription termination sequence which are necessary for expression. In the present invention, although the pPsADHa vector is used, any vector that satisfies the above requirements can be used.

The recombinant yeast according to the present invention can be prepared by inserting the gene on a chromosome of yeast according to a conventional method, or introducing the recombinant vector onto a plasmid of a yeast.

As a method for inserting the gene into the chromosome of the host cell, a commonly known gene manipulation method can be used in the present invention. For example, microinjection (direct insertion of DNA into cells), liposome, directed DNA uptake, receptor ~ mediated DNA transfer, or DNA transport using Ca ⁺⁺ . Is widely used. Examples include retrovirus vectors, adenovirus vectors, adeno-associated viral vectors, herpes simplex virus vectors, poxvirus vectors, or lentiviral vectors. Particularly, retroviruses have a high gene transfer efficiency, gross deletion It can be used in a wide range of cells without binding by host DNA and rearrangement (resulting in alteration of host DNA function by altering the region of the host DNA similar to that of the host DNA).

In the present invention, preferably, Pichia pastoris (Pichia pastoris) to but using, not limited to this, Agrobacterium genus, Aspergillus genus Acetobacter genus Aminobacter, A Agromonas in, Acidphilium genus Bulleromyces genus Bullera, A Brevundimonas genus, Cryptococcus genus, Chionosphaera genus, Candida genus, Cerinosterus genus, Escherichia genus, Exisophiala in, Exobasidium in, Fellomyces in, Filobasidium in, Geotrichum genus, Graphiola genus, Gluconobacter genus, Kockovaella in, Curtzmanomyces in, Lalaria in, Leucospoidium in , Legionella genus, Psedozyma in, Paracoccus genus, Petromyc genus, Rhodotorula genus, Rhodosporidium genus, Rhizomonas in, Rhodobium in, Rhodoplanes in, Rhodopseudomonas in, Rhodobacter genus, Sporobolomyces in, Spridobolus in, Saitoella in, Schizosaccharomyces genus, Sphingomonas genus, Sporotrichum Genus Sympodiomycopsis , Sterigmatosporidium , Tapharina , Tremella , Trichosporon , Tilletiaria , Tilletia , Tol it can be mentioned in yposporium, Tilletiposis genus, genus Ustilago, in Udenlomyce, Xanthophilomyces genus, genus Xanthobacter, genus Paecilomyces, genus Acremonium, Hyhomonus genus, genus Rhizobium and the like.

The present invention relates to a gene encoding a chimeric endo-beta-1,4-glucanase, a gene encoding a xylanase-ratio, and a gene encoding a small cellulase binding protein a, Yeast. In the present invention, yeast is transformed into yeast. However, the present invention is not limited thereto.

In the present invention, a recombinant yeast having a chimeric endo-beta-1,4-glucanase-producing ability, a recombinant yeast having a xylanase-non-producing ability and a recombinant yeast having an ability to produce a small cellulase binding protein were prepared, The endo-beta-1,4-glucanase formed, the xylanase-bound and the small cellulosic binding protein a, is secreted extracellularly from yeast, resulting in the formation of cellulosomes with higher cellulolytic activity Small cellulosic binding protein a (cCelE-mCbpA) complex, (ii) xylanase non-small cellulosic binding protein a (XynB-mCbpA) complex through assembly of (i) chimeric endo- Complex and (iii) chimeric endo-beta-1,4-glucanase-i-xylanase non-small cellulosic binding protein a (cCelE-XynB-mCbpA) complex It was so.

In one embodiment of the present invention, there is provided a recombinant vector comprising (i) a pPsADHa / cCelE recombinant vector into which a chimeric endo-beta-1,4-glucanase- (cCelE) gene is introduced, (ii) a xylanase B gene (B), a pPsADHα / mCbpA recombinant vector having a pPsADHα / XynB recombinant vector introduced therein and (iii) a mini cellulose binding protein A (miniCbpA) gene introduced into a cellulolytic enzyme skeleton protein (FIG. 2) In order to confirm the enzyme activity (halo), experiments were conducted using YPD agar medium and YPD xylan as substrates for the enzyme reaction. As a result, the recombinant xylanase ratio (FIG. 3A) and endo-beta-1, It was confirmed that the activity of 4-glucanase (FIG. 3B) was excellent.

In addition, confirmation of enzyme secretion into a culture medium of recombinant yeast having chimeric endo-beta-1,4-glucanase-producing ability, recombinant yeast having xylanase-non-producing ability and recombinant yeast having ability to produce small cellulase binding protein As a result, it was confirmed that the enzyme activity was observed at the same position as that of chimeric endo-beta-1,4-glucanase, xylanase-bound, small-cellulosic binding protein protein size, (CCelE-mCbpA) complex (Fig. 4 line 4) and (ii) the xylanase non-small cellulosic binding protein < RTI ID = 0.0 > (XynB-mCbpA) complex (line 5 in FIG. 4).

In order to confirm that the chimeric endo-beta-1,4-glucanase-i-xylanase non-small cellulosic binding protein a (cCelE-XynB-mCbpA) complex was formed by the method of the present invention, Unbound protein was removed and the binding of the complex was modified and confirmed by Native-PAGE. As a result, as shown in Fig. 5, the chimeric endo-beta-1,4-glucanase-i-xylanase non-small cellulase binding protein A (cCelE-XynB-mCbpA) was formed (FIG. 5, non denature). Even when the complex binding was modified and confirmed by Native-PAGE, the actual cCelE, XynB and mCbpA could be confirmed in a sample in which cCelE, XynB and mCbpA were mixed at a ratio of 1: 1: 1 (FIG. 5, denature).

(CCelE-mCbpA) complex, xylanase non-small cellulosic binding protein (XynB-mCbpA) complex and chimeric endo-beta-1,4-glucanase- A reducing sugar assay was performed to determine the enzyme activity of the beta-1, 4-glucanase-i-xylanase non-small cellulase binding protein a (cCelE-XynB-mCbpA) complex, It was found that the protein had higher activity when bound (FIG. 6).

In another aspect, the present invention relates to a recombinant yeast transformed with a recombinant vector containing a gene encoding chimeric endo-beta-1,4-glucanase CelE, xylanase B, Culturing the recombinant yeast transformed with the recombinant vector containing the gene encoding the mini-cellulose binding protein A (miniCbpA) and the recombinant yeast transformed with the recombinant vector containing the gene encoding the mini-cellulose binding protein A (miniCbpA); (b) adding biomass to the recombinant yeast cultured in step (a) and further culturing to produce ethanol; And (c) recovering the produced ethanol. The present invention also relates to a method for producing ethanol by simultaneous saccharification and fermentation of biomass.

In the present invention, the biomass is a large aquifer ( Miscanthus can be characterized in that the gianteus). However, not limited to this, the biomass made of a fiber may be variously used as a substrate for ethanol production.

In one embodiment of the present invention, a recombinant yeast having a chimeric endo-beta-1,4-glucanase-producing ability, a recombinant yeast having a xylanase-non-producing ability and a recombinant yeast having a small cellulase binding protein- giant Miscanthus (Miscanthus the medium is cellulose enzyme complex formed gianteus ) was added to the culture medium. As a result, it was found that Pichia pastoris , which is a control group, and Pichia pastoris were smaller than recombinant yeast having a small cellulase- pastoris -pPsADH? -cCelE, Pichia pastoris -pPsADH? -XynB, Pichia It was confirmed that the ethanol production was increased when the Pasteuris-pPsADHa-mini-CbpA was co-cultured (Fig. 7).

When ethanol was produced from the biomass by the method of the present invention, recombinant yeast having chimera endo-beta-1,4-glucanase-producing ability, recombinant yeast having xylanase-non-producing ability and small cellulase binding protein- Beta-1,4-glucanase produced by the recombinant yeast, the xylanase ratio and the small cellulosic binding protein a, are combined to produce (i) a chimeric endo-beta-glucanase- (XynB-mCbpA) complex, and (iii) chimeric endo-beta-1-glucoside-1-glucanase-small cellulosic binding protein a (cCelE-mCbpA) complex, (CCelE-XynB-mCbpA) complex was formed on the surface of the 4-glucanase-i-xylanase non-small cellulase binding protein.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Chimeric  Endo-beta-1,4- Glucanase -this, Jilan Naths  Rain and small Cellulose  Amplification of this gene in binding protein

1-1: pPsADHa Vector production

The pPsADHa vector for cloning the chimeric endo-bter-1, 4-glucanase-1, xylanase ratio and small cellulase binding protein A gene was prepared by the following method.

The N-terminal part of the ADH2 promoter gene (SEQ ID NO: 2) of the restriction enzyme HindIII recognition sequence (AAGCTT) and the Pichia pastoris was inserted into the forward primer (SEQ ID NO: 1) and the reverse primer SEQ ID NO: 3) of the N-terminal portion of the Alpha Factor gene (SEQ ID NO: 4) in the pPICZa vector (INVITROGEN, cat. No. V195-20) and the ADH2 promoter of Pichia pastoris A primer was designed and synthesized to insert the C-terminal portion of the gene (Bioneer, Korea).

pPsADHa primer

[SEQ ID NO: 1] 5'-gcgcaagcttGATCCGAGGGAAAAACCCGGGGACTCAGTTTATCACGTACCGAG-3 '

[SEQ ID NO: 3] 5'-gaaatctcatGATAATTTGGATGGATCGCCAGCACTTATGCAATTGGGT-3 '

Further, in order to prepare another pair of primers, the forward primer (SEQ ID NO: 5) contains the C-terminal portion 10bp sequence (CCAAATTATC) of the ADH2 Promoter gene of Pichia pastoris and the Alpha Factor gene in the pPICZa vector The primers were designed and designed so that the N-terminal part inserts the restriction enzyme EcoRI recognition sequence (GAATTC) and the C-terminal part of the Alpha Factor gene in the pPICZa vector into a reverse primer (SEQ ID NO: 6) Korea).

pPsADHa primer

[SEQ ID NO: 5] 5'-ccaaattatcATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCA TCCTCCGCAT-3 '

[SEQ ID NO: 6] 5'-gcgcgaattcAGCTTCAGCCTCTCTTTTCTCGAGAGATACCCCTTCTTCTTT-3 '

PCR was performed using the two primers thus synthesized. As a result, PCR bands containing the 588 bp ADH2 Promoter gene of Pichia pastoris and the Alpha Factor gene in the 267 bp pPICZa vector were confirmed. Two PCR fragments Overlap PCR techniques (Higuchi R et. Al., Nucleic Acids Res. , 16 (15): 7351, 1988), and the final cloned DNA fragment was digested with Hind III (TAKARA, cat.No.1060A) and EcoR I (TAKARA, cat.No.1040A) restriction enzyme, and inserted into the pPICZa vector to prepare a chimeric pPsADHa vector (Fig. 1).

1-2: Chimeric  Endo-beta-1,4- Glucanase - amplification of this gene

In order to clone the gene of the cytosolic domain of the fibrinolytic enzyme for the formation of the fibrinolytic enzyme complex, the base of the donor-beta-1,4-glucanase-non-gene of the clostridium celluloblastin genomic DNA The plasmid pADHa-cCelE sequence inserted with endo- glucanase CelE gene (chimeric endo-glucanase cCelE; SEQ ID NO: 8) derived from Clostridium thermoshell linked with the sequence (SEQ ID NO: 7) (Korean Patent No. 1120359 The C-terminal portion of the chimeric endo- glucanase cCelE gene and the restriction enzyme Kpn I (GGTACC) recognition sequence are used as a forward primer and the endo-beta-1 (GGTACC) recognition sequence is used as a reverse primer in the reverse primer , 4-glucan azepin-ratio of genes were synthesized by designing the primer so that the N-terminal portion and a restriction enzyme Xba I ((TCTAGA) recognition sequence is inserted into the keorin part (F Pioneer, South Korea).

cCelE primer

[SEQ ID NO: 9] 5'-atatggtaccTCGGGAACAAGCTTTTG-3 '

[SEQ ID NO: 10] 5'-gctctagatcaatgatgatgatgatgatgTAAAAGCATTTTTTTAAGAA-3 '

PCR was then performed using the synthesized primers. As a result, a PCR band including a 1317 bp chimeric endo-beta-1,4-glucanase-ccelE gene (SEQ ID NO: 11) was confirmed. The cloned DNA fragment is Kpn I (TAKARA, cat.No.1068A) and Xba I (TAKARA, cat.No.1093A) treated with a restriction enzyme in the following, pPaADHα vector of Figure 1 prepared in Example 1-1 (Fig. 2C) containing the gene coding for chimeric endo-beta-1,4- glucanase ( cCelE ) was prepared, and the cloned pPaADH? -CCelE was transformed into Pichia pastoris was transformed in pastoris) (Korean Coleection for Type Culture , KCTC 7190), chimeric endo-beta-1,4-glucan kinase was prepared with the recombinant microorganism having a production capability.

1-3: Jilan Naths  Amplification of non-genes

In order to clone a gene (SEQ ID NO: 12) encoding a xylanase-negative (XynB) gene derived from Clostridium celluloborance, a forward primer has an N-termianl portion of the xylanase-ratio (XynB) enzyme EcoR I (GAATTC) recognition sequence, and the reverse primer (reverse primer), the xylan tyrosinase-ratio (XynB) C-termianl portion and a restriction enzyme Kpn I (GGTACC) was synthesized the recognition sequences are respectively inserted primers (Bioneer, Korea). Thereafter, PCR was carried out using the synthesized primers.

XynB primer

[SEQ ID NO: 13] 5'-gcgcgaattcGAAGATGCTCTATTAATAAGTAG-3 '

[SEQ ID NO: 14] 5'-gcggtacctcaatgatgatgatgatgatgGAAACTAGTAATTTGTCCTA-3 '

As a result, it was confirmed that a PCR band of 1821 bp size containing a gene encoding a xylanase-ratio derived from Clostridium celluloborance was confirmed. The cloned DNA fragments EcoR I (TAKARA, cat.No.1040A) and Kpn I (TAKARA, cat.No.1068A) treated with restriction enzymes, and then be inserted into the vector pPaADHα 1, xylan tyrosinase-ratio (XynB (Fig. 2B) containing the gene (XynB) coding for the gene coding for the xylenes (XynB) was prepared, and the cloned pPaADH [alpha] -XynB was transformed into Pichia pastoris , . ≪ / RTI >

1-4: Small Cellulose Binding protein  Amplification of this gene

Cellulose binding module (CBM) and two Cohesin modules (Cellulase-binding protein A) in cellulosic binding protein A, the primary scaffolding subunit of celluloblastine from Clostridium, (SEQ ID NO: 15) with the restriction enzyme EcoR I (GAATT) recognition in the forward primer with reference to the nucleotide sequence to clone the Mini-Cellulose-binding protein A gene And a restriction enzyme Not I (GCGGCCGC) recognition sequence was inserted into the reverse primer (Bioneer, Korea).

mCbp primer

[SEQ ID NO: 16] 5'-gcgcgaattcGCAGCGaCATCATCAAT-3 '

[SEQ ID NO: 17] 5'-gcgcggccgctcaatgatgatgatgatgatgTATAGGATCTCCAATATT-3 '

As a result, it was confirmed that the PCR band containing the mCbpA gene, which is a part of the cellulose-binding protein-A gene of celluloblastine derived from clostridium of 1647 bp, was confirmed. The cloned DNA fragment was treated with EcoR I and Not I restriction enzyme and then inserted into the pPaADH alpha vector of FIG. 1 to obtain a gene ( mCbpA ) encoding a mini-cellulose-binding protein A (Fig. 2A) was prepared, and the cloned pPaADH? -MCbpA was transformed into Pichia pastoris to produce a mini-cellulose-binding protein A production ability . ≪ / RTI >

Activity measurement of recombinant enzyme protein

2-1: Enzyme activity band ( halo ) Measure

In order to confirm the expression level of the enzyme protein in the three kinds of recombinant yeasts prepared in Example 1, the enzyme activity band (halo) of the cCelE, XynB and mini-CbpA enzyme proteins was measured.

 YPD agar medium and YPD xylan were used as substrates for the enzyme reaction to identify the enzyme activity (halo). The recombinant yeast colonies were inoculated on YPD medium, cultured for 24 hours at 30 ° C., cultured in YPD liquid medium for 5 hours at 30 ° C., and the resulting enzyme was concentrated and adsorbed on YPD agar medium and YPD xylan medium Followed by reaction at 50 ° C for 24 hours. After staining with 0.25% congo-red for 5 minutes and washing several times with sodium chloride (NaCl), the enzyme activity (halo) was confirmed (FIG. 3).

As a result, it was confirmed that the recombinant yeast strain had a larger enzyme activity than the wild type P. pastoris , and that the recombinant xylanase-ratio (FIG. 3A) and chimeric endo-glucanase (FIG. 3B) .

2-2: Zymogram ( Zymogram ) Way

Zymogram method was performed to investigate enzyme activity and enzyme complex formation. First, the recombinant yeast was shake-cultured at 30 ° C for 5 hours so that the cCelE, XynB, and mini-CbpA enzyme proteins were secreted into the culture medium. After the culture was centrifuged, the supernatant was concentrated (Millipore, amicon 10 kDa cut off) cCelE, XynB, mini-CbpA enzyme protein.

Native-PAGE was then incubated with 10% polyacrylamide gel (Bio-Rad, cat. No. 161-0156), 0.2% xylan (Sigma aldrich, cat. No. X4252) and CMC (carboxymethylcellulose . No. C4888) was dissolved the poly-acrylamide gel were concentrated in the electrophoretic separation of the enzyme protein by using, renature the substrate is added to _{buffer (100mM Succiniate, 10mM CaCl 2} , 1mM dTT) (sigma aldrich, cat. No. D9779) for 2 hours and then reacted at 50 ° C for 3 hours. After completion of the reaction, the gel was stained with 0.25% congo-red for 5 minutes, washed several times with sodium chloride (NaCl), and the enzyme activity (halo) was confirmed.

As a result, it was found that the reaction between the chimeric endo-glucanase (cCelE) and the small cellulose-binding protein-a (mini-CbpA) and the case of the xylenase- (CCelE-mCbpA) complex (line 4 in FIG. 4) and the xylanase non-small cellulase binding protein (CbpA) were reacted with the chimeric endo-beta-1,4-glucanase- (XynB-mCbpA) complex was formed (line 5 in FIG. 4), and the effect of xylan and CMC (carboxymethylcellulose) on the formed complex was confirmed.

In order to confirm formation of a complex of chimeric endo-beta-1,4-glucanase-i-xylanase non-small cellulosic binding protein a (cCelE-XynB-mCbpA) complex, Protein was removed and the binding of the complex was modified and confirmed by Native-PAGE. As a result, as shown in Fig. 5, the chimeric endo-beta-1,4-glucanase-i-xylanase non- -XynB-mCbpA) was formed (Fig. 5, non denature). Even when the complex binding was modified and confirmed by Native-PAGE, the actual cCelE, XynB and mCbpA could be confirmed in a sample in which cCelE, XynB and mCbpA were mixed at a ratio of 1: 1: 1 (FIG. 5, denature).

2-3: Measurement of reducing sugar ( 감 sugar assay )

(XynB-mCbpA) complex and chymeric endo-beta-1-glucanase-beta-1, 4-glucanase-less cellulosic binding protein a (cCelE-mCbpA) complex, , A reducing sugar assay was performed to determine the enzyme activity of the 4-glucanase-i-xylanase non-small cellulase binding protein a (cCelE-XynB-mCbpA) complex.

Each reaction enzyme solution was mixed at a ratio of 1: 1 to a substrate containing 2% of xylan and carboxymethyl cellulose (CMC), and the enzyme reaction was carried out for 2 hours. Method was used to measure the degraded reducing sugar. As a result, it was found that when the three proteins were bound, they had higher activity (FIG. 6).

Production of bioethanol using recombinant yeast

Pichia pastoris -pPsADHa-cCelE, Pichia pastoris -pPsADHα-XynB and Pichia pastoris- pPsADHα-mini-CbpA were cultured in YPD medium for 24 hours, shaken for 48 hours, and grown in Miscanthus sinensis) ((Note) changhae ethanol) is cultured further in the fermentation medium as a substrate was added. The fermentation broth was cultured at 30 DEG C and 100 rpm, and the culture solution was collected for a predetermined time, and the produced ethanol was measured by gas chromatography. The execution result endo-glucan and xylan tyrosinase kinase insert, as the method Pichia pastoris -pPsADHα / cCelE, Pichia The yields of ethanol production in pastoris -pPsADHα / XynB, Pichia pastoris -pPsADHα / mini-CbpA were higher than those of Pichia pastoris / pPsADH? (Fig. 7).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Korea University Research and Business Foundation <120> Method for Preparing Cellulose Complex Using Endo-b-1,4-glucanase          E, Xylanase B and Mini Cellulose Binding Protein A from          Clostridium sp. <130> P12-B248 <160> 17 <170> Kopatentin 2.0 <210> 1 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> pPsADHa forward primer <400> 1 gcgcaagctt gatccgaggg aaaaacccgg ggactcagtt tatcacgtac cgag 54 <210> 2 <211> 588 <212> DNA <213> Pichia pastoris ADH2 promoter <400> 2 gatccgaggg aaaaaccggg gactcagttt atcacgtacc gagaaattct tcgtttcagc 60 atcatcacca tgttgtccaa ttacagcccg aagcacagtc taatgctgaa ttttgataga 120 gctcatcgtg aacagccaga ttcgaagaaa ggggggatga gatccgggtt catctgcaag 180 agacacagaa aataaaaaac atacgatccg ttcagctacc tggcgcttaa ccaggaaaat 240 cactgctgga gtggccagca tgtcacgagg tggcagaatc cgataatgtg tgattgcgtg 300 tagcatcggc gcaagtcgaa tttcggtcat atccgtgtct ggatattatc cactattttt 360 taatttttca ggttggatgc gattgttccc tttacgtctg gacgatgcct gaagccccag 420 gtatatataa ggggctcgaa agtcctttga ccagctggtt gatttgactt tgtttgttcc 480 tttctttctt tcatctactc atcactcaat tgcattcgca atttcccatt aatacatatt 540 caacttgctc cacatattgc acccaattgc ataagtgctg cgatccat 588 <210> 3 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> pPsADHa reverse primer <400> 3 gaaatctcat gataatttgg atggatcgcc agcacttatg caattgggt 49 <210> 4 <211> 267 <212> DNA <213> Artificial Sequence <220> <223> pPICZa vector Alpha Factor gene <400> 4 atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60 ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120 tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180 aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240 tctctcgaga aaagagaggc tgaagct 267 <210> 5 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> pPsADHa forward primer <400> 5 ccaaattatc atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc 60 at 62 <210> 6 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> pPsADHa reverse primer <400> 6 gcgcgaattc agcttcagcc tctcttttct cgagagatac cccttcttct tt 52 <210> 7 <211> 159 <212> DNA <213> dockerin of endo-b-glucanase B <400> 7 gatgttaaca aagatggaaa ggtaaatgct atcgattatg cagtgcttaa atcaattctt 60 ttaggtacaa atactaacgt tgatttatca gtatcagaca tgaataagga tggtaaagta 120 aatgctttgg atttagctgt tcttaaaaaa atgctttta 159 <210> 8 <211> 1140 <212> DNA <213> endo-b-glucanase E <400> 8 tcgggaacaa agcttttgga tgcaagcgga aacgagcttg taatgagggg catgcgtgat 60 atttcagcaa tagatttggt taaagaaata aaaatcggat ggaatttggg aaatactttg 120 gatgctccta cagagactgc ctggggaaat ccaaggacaa ccaaggcaat gatagaaaag 180 gtaagggaaa tgggctttaa tgccgtcaga gtgcctgtta cctgggatac gcacatcgga 240 cctgctccgg actataaaat tgacgaagca tggctgaaca gagttgagga agtggtaaac 300 tatgttcttg actgcggtat gtacgcgatc ataaatcttc accatgacaa tacatggatt 360 atacctacat atgccaatga gcaaaggagt aaagaaaaac ttgtaaaagt ttgggaacaa 420 atagcaaccc gttttaaaga ttatgacgac catttgttgt ttgagacaat gaacgaaccg 480 agagaagtag gttcacctat ggaatggatg ggcggaacgt atgaaaaccg agatgtgata 540 aacagattta atttggcggt tgttaatacc atcagagcaa gcggcggaaa taacgataaa 600 agattcatac tggttccgac caatgcggca accggcctgg atgttgcatt aaacgacctt 660 gtcattccga acaatgacag cagagtcata gtatccatac atgcttattc accgtatttc 720 tttgctatgg atgtcaacgg aacttcatat tggggaagtg actatgacaa ggcttctctt 780 acaagtgaac ttgatgctat ttacaacaga tttgtgaaaa acggaagggc tgtaattatc 840 ggagaattcg gaaccattga caagaacaac ctgtcttcaa gggtggctca tgccgagcac 900 tatgcaagag aagcagtttc aagaggaatt gctgttttct ggtgggataa cggctattac 960 aatccgggtg atgcagagac ttatgcattg ctgaacagaa aaactctctc atggtattat 1020 cctgaaattg tccaggctct tatgagaggt gccggcgttg aacctttagt ttcaccgact 1080 cctacaccta cattaatgcc gaccccctcg cccacggtga cagcaaatat tttgtacggt 1140                                                                         1140 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> cCelE forward primer <400> 9 atatggtacc tcgggaacaa gcttttg 27 <210> 10 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> cCelE reverse primer <400> 10 gatgttaaca aagatggaaa ggtaaatgct atcgattatg cagtgcttaa atcaattctt 60 ttaggtacaa atactaacgt tgatttatca gtatcagaca tgaataagga tggtaaagta 120 aatgctttgg atttagctgt tcttaaaaaa atgctttta 159 <210> 11 <211> 1298 <212> DNA <213> Artificial Sequence <220> <223> chimeric CelE <400> 11 cgggaacaaa gcttttggat gcaagcggaa acgagcttgt aatgaggggc atgcgtgata 60 tttcagcaat agatttggtt aaagaaataa aaatcggatg gaatttggga aatactttgg 120 atgctcctac agagactgcc tggggaaatc caaggacaac caaggcaatg atagaaaagg 180 taagggaaat gggctttaat gccgtcagag tgcctgttac ctgggatacg cacatcggac 240 ctgctccgga ctataaaatt gacgaagcat ggctgaacag agttgaggaa gtggtaaact 300 atgttcttga ctgcggtatg tacgcgatca taaatcttca ccatgacaat acatggatta 360 tacctacata tgccaatgag caaaggagta aagaaaaact tgtaaaagtt tgggaacaaa 420 tagcaacccg ttttaaagat tatgacgacc atttgttgtt tgagacaatg aacgaaccga 480 gagaagtagg ttcacctatg gaatggatgg gcggaacgta tgaaaaccga gatgtgataa 540 acagatttaa tttggcggtt gttaatacca tcagagcaag cggcggaaat aacgataaaa 600 gattcatact ggttccgacc aatgcggcaa ccggcctgga tgttgcatta aacgaccttg 660 tcattccgaa caatgacagc agagtcatag tatccataca tgcttattca ccgtatttct 720 ttgctatgga tgtcaacgga acttcatatt ggggaagtga ctatgacaag gcttctctta 780 caagtgaact tgatgctatt tacaacagat ttgtgaaaaa cggaagggct gtaattatcg 840 gagaattcgg aaccattgac aagaacaacc tgtcttcaag ggtggctcat gccgagcact 900 atgcaagaga agcagtttca agaggaattg ctgttttctg gtgggataac ggctattaca 960 atccgggtga tgcagagact tatgcattgc tgaacagaaa aactctctca tggtattatc 1020 ctgaaattgt ccaggctctt atgagaggtg ccggcgttga acctttagtt tcaccgactc 1080 ctacacctac attaatgccg accccctcgc ccacggtgac agcaaatatt ttgtacggtg 1140 atgttaacaa agatggaaag gtaaatgcta tcgattatgc agtgcttaaa tcaattcttt 1200 taggtacaaa tactaacgtt gatttatcag tatcagacat gaataaggat ggtaaagtaa 1260 atgctttgga tttagctgtt cttaaaaaaa tgctttta 1298 <210> 12 <211> 1734 <212> DNA <213> xylanase B <400> 12 gaagatgctc tattaataag tagcaccttc gaaggaggta cagatggttg gtcaactatg 60 ggtacaacta ctatttcacc aagtacttta agccatagcg gtaacggaag cctctacgtt 120 tctggtagag ctgcttcatg ggctggacca tgcaatataa gaacagacgt attgaaagca 180 ggaaatacat ataaccttag ttcctatgta atgtacaatg atgctagtgc tgcagatact 240 caacctatta gcttcatgtt aaagtacaca gattctacaa ataaagcttt ctatgtacct 300 atcgcaactg taactgtaaa caaaggtgaa tggacaaaaa tcgaaaatac cgcttataaa 360 atcccagcag aagcaactag tgcaatgatt tattgggaaa ctacaggtac aatgattaac 420 tactatgtcg acgacgttac agcatatggt ccaagcacat ttaatcctaa tgtaactgca 480 gctacaccac ttaaaaacgt atttggaaaa tactttgata taggttgcgc tgcgacacct 540 tctgaagtat ctttacaagt tgctaaagat cttgtaaaaa ctcactacaa caaccttact 600 ataggaaatg agcttaaacc agattatgta ttagataaag ctgcttgcca agcatccggt 660 aacaatgtaa accctcaagt taaattagac agtgcacgta gtcttttaaa atactgtgct 720 gagaacaata tagaagtaag aggccacgtt ttagtatggc atagtcaaac tccttcttgg 780 ttctttaaag aaaacttcag tgacactgga gctactgtat caaaagacgt catgaatcaa 840 cgtcttgaaa actatataaa gaatcttttt gcagcaatta acgcagagtt ccctacatta 900 aagatttatg catgggacgt tgtaaatgaa tgttacctag atggtggtaa tctacgtact 960 gcaggatttc ctgaaactgc tgggaaagaa gcatctgcat ggaatttagt ctatggtgat 1020 gactcataca tagacaatgc tttcacatat gcaagaaaat atgctccagc tggtgttaag 1080 ttattctaca acgactttaa cgaatatata caatcaaaac gtaacgcaat ctacactatg 1140 gcaatgagat taaaatctaa aggtattatc gatggtatcg gtatgcaatc tcacctagat 1200 atgggctttc ctgatacaaa tacttataaa gcagcactaa ctaaattcgg ttcaactggc 1260 ttagaagttc aagttacaga acttgatata actacaagtg atacaagtgc tacaggacta 1320 gcaaatcaag ctactaaata tgcagctatt atgcaaagta ttttagatgc aaaaaaagca 1380 ggtactgcta acattacaaa cgttgtattc tggggtatta ctgatggaac aagctggcgt 1440 gcaacaagat taccattgct attcgatgga aactactatc caaaaccagc tttcgattca 1500 gttgttaaat tagttcctac aagcgactac tatactcctg gttatgcctt aggtgatgta 1560 aacaatgacg gtaaaatcaa tgctctagat cttgcattag tgaaaaaagg tctattaagt 1620 gaatttacag atccagctgc aaaagtagct gcagatgtaa acaaagatgg taatactaat 1680 gctatcgacc ttgcattatt aaagaaatat cttctaggac aaattactag tttc 1734 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> XynB forward primer <400> 13 gcgcgaattc gaagatgctc tattaataag tag 33 <210> 14 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> XynB reverse primer <400> 14 gcggtacctc aatgatgatg atgatgatgg aaactagtaa tttgtccta 49 <210> 15 <211> 1646 <212> DNA <213> Mini-Cellulose-binding protein A <400> 15 cagcgacatc atcaatgtca gttgaatttt acaactctaa caaatcagca caaacaaact 60 caattacacc aataatcaaa attactaaca catctgacag tgatttaaat ttaaatgacg 120 taaaagttag atattattac acaagtgatg gtacacaagg acaaactttc tggtgtgacc 180 atgctggtgc attattagga aatagctatg ttgataacac tagcaaagtg acagcaaact 240 tcgttaaaga aacagcaagc ccaacatcaa cctatgatac atatgttgaa tttggatttg 300 caagcggagc agctactctt aaaaaaggac aatttataac tattcaagga agaataacaa 360 aatcagactg gtcaaactac actcaaacaa atgactattc atttgatgca agtagttcaa 420 caccagttgt aaatccaaaa gttacaggat atataggtgg agctaaagta cttggtacag 480 caccaggtcc agatgtacca tcttcaataa ttaatcctac ttctgcaaca tttgataaaa 540 atgtaactaa acaagcagat gttaaaacta ctatgacttt aaatggtaac acatttaaaa 600 caattacaga tgcaaacggt acagctctaa atgcaagcac tgattatagt gtttctggaa 660 atgatgtaac aataagcaaa gcttatttag caaaacaatc agtaggaaca actacattaa 720 actttaactt tagtgcagga aatcctcaaa aattagtaat tacagtagtt gacacaccag 780 ttgaagctgt aacagctaca attggaaaag tacaagtaaa tgctggagaa acggtagcag 840 taccagttaa cttaacaaaa gttccagcag ctggtttagc aacaattgaa ttaccattaa 900 cttttgattc tgcatcatta gaagtagtat caataactgc tggagatatc gtattaaatc 960 catcagtaaa cttctcttct acagtaagtg gaagcacaat aaaattatta ttcttagatg 1020 atacattagg aagccaatta atcactaagg atggagtttt tgcaacaata acatttaaag 1080 caaaagctat aactggaaca actgcaaaag taacttcagt taaattagct ggaacaccag 1140 tagttggtga tgcgcaatta caagaaaaac cttgtgcagt taacccagga acagtaacta 1200 tcaatccaat cgataataga atgcaaattt cagttggaac agcaacagta aaagctggag 1260 aaatagcagc agtgccagta acattaacaa gtgttccatc aactggaata gcaactgctg 1320 aagcacaagt aagttttgat gcaacattat tagaagtagc atcagtaact gctggagata 1380 tcgtattaaa tccaacagta aacttctctt atacagtaaa cggaaatgta ataaaattat 1440 tattcctaga tgatacatta ggaagccaat taattagtaa agatggagtt tttgtaacaa 1500 taaacttcaa agcaaaagct gtaacaagca cagtaacaac accagttaca gtatcaggaa 1560 cacctgtatt tgcagatggt acattagcag aagtacaatc taaaacagca gcaggtagcg 1620 ttacaataaa tattggagat cctata 1646 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> mCbp forward primer <400> 16 gcgcgaattc gcagcgacat catcaat 27 <210> 17 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> mCbp reverse primer <400> 17 gcgcggccgc tcaatgatga tgatgatgat gtataggatc tccaatatt 49

Claims (7)

A method for producing a fibrinolytic enzyme complex comprising the steps of:
(a) a recombinant yeast transformed with a recombinant vector containing a gene encoding chimeric endo-β-1,4-glucanase E (cCelE), a xylanase- a recombinant yeast transformed with a gene containing a recombinant vector encoding xylanase B (XynB) and a recombinant vector transformed with a recombinant vector containing a gene encoding a mini cellulose binding protein A (miniCbpA) step;
(b) the chimeric endo-beta-1,4-glucanase produced from each recombinant yeast, the xylanase ratio and the small cellulosic binding protein a, are combined to form (i) chimeric endo- (XynB-mCbpA) complex and (iii) chimeric endo-beta-1,4-glucan complexes, (CCelE-XynB-mCbpA) complex with an agase-free xylenase non-small cellulosic binding protein a (cCelE-XynB-mCbpA) complex; And
(d) recovering the formed fibrinolytic enzyme complex.
The recombinant vector of claim 1, wherein the recombinant vector comprises a gene coding for chimeric endo-beta-1,4-glucanase, wherein the gene coding for pPsADH? -CCelE and xylanase- Wherein the pPsADH alpha -XynB and the small cellulosic binding protein a are introduced into the cell line.
The method according to claim 1, wherein the gene encoding the xylanase ratio in step (a) and the gene encoding the small cellulase binding protein a are clostridium celluloborance cellulovorans . &lt; / RTI &gt;
The method according to claim 1, wherein the gene coding for chimeric endo-beta-1,4-glucanase-1 (cCelE) in step (a) is endo-beta-1 of clostridium thermocellum , The gene encoding 4-glucanase and clostridium celluloborance (EngB ) of endo-beta-1,4-glucanase-cellulovorans is bound to a gene encoding a doxorubic region of endo-beta-1,4-glucanase-
The method according to claim 1, wherein the yeast is Pichia pastoris .
A process for the production of ethanol by simultaneous saccharification fermentation of biomass comprising the steps of:
(a) a recombinant yeast transformed with a recombinant vector containing a gene encoding chimeric endo-β-1,4-glucanase E (cCelE), a xylanase- a recombinant yeast transformed with a gene containing a recombinant vector encoding xylanase B (XynB) and a recombinant vector transformed with a recombinant vector containing a gene encoding a mini cellulose binding protein A (miniCbpA) step;
(b) adding biomass to the recombinant yeast cultured in step (a) and further culturing to produce ethanol; And
(c) recovering the produced ethanol;
7. The method of claim 6, wherein the biomass is selected from the group consisting of Miscanthus gianteus ). &lt; / RTI &gt;

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