KR101367348B1 - Recombinant vector comprising chimeric beta-agarase-B, transformant comprising the same and use of the same - Google Patents

Abstract

The present invention is a marine microorganism ( Zobellia) endo-beta-1,4-glucanase-D derived from Clostridium sp. microorganism at the active site of beta-agarase derived from galactanivorans ) 1,4-D-glucan glucanohydrolase, carboxymethylcellulase, CMCase; EC 3.2.1.4) relates to a vector and Escherichia coli transformant into which the gene synthesized, the recombinant vector into which the gene is inserted And E. coli transformants transformed with the vector to make proteins secreted extracellularly from E. coli.

Description

Recombinant vector comprising chimeric beta-agarase-B, transformant comprising the same and use of the same

The present invention relates to a recombinant vector comprising a chimeric beta-agarase, a transformant thereof, and an application thereof.

Agar (agar) is a polysaccharide mainly found in the cell walls of red algae, a kind of dietary fiber. Agar is a polysaccharide that is difficult to be degraded by digestive enzymes, so it is not provided as a nutrient source, but it is known that it can reduce the absolute amount of cholesterol in the body by inhibiting blood cholesterol and adsorbing and excreted in the form of bile acid, and is widely used as a diet food. . In addition, agar is mainly used as a solid medium for culturing microorganisms, as a stabilizer in confectionery and meat processing, and as a gelling agent in cosmetics and foods.

Agar is composed of about 70% agarose and about 30% agaropectin. The agarose is a neutral polysaccharide, and alternating α-1,3 and β-1,4 bonds between galactose are present in the agarose. On the other hand, agalopectin is an acidic polysaccharide, and the agalopectin is a sulfate, in particular, sulfate, gluconic acid or pyruvate, combined with agarose.

Agarase, an enzyme that decomposes agar, may be classified into alpha-agarase and beta-agarase depending on the site of decomposing bonds. The alpha-agarase decomposes α-1,3 bonds in the galactose polymer of agarose to prepare agaro-oligosaccharides. The agaro-oligosaccharides have apoptosis inducing activity, anticancer activity (Kato, 2000), antiviral activity, It has been reported to have antioxidant activity (Chen et al., 2005; Kato, 2000), immunomodulatory activity (Yoshizawa et al., 1995), antiallergic activity and anti-inflammatory activity. In addition, the beta-agarase decomposes β-1,4 bond in the galactose polymer of agarose to prepare neoagaroligosaccharides, and the neoagaroligosaccharides inhibit bacterial growth (Kono et al., 1989),

It has been reported to have antioxidant activity, starch aging prevention, moisturizing effect (Kobayashi et al., 1997), whitening effect (Kobayashi et al., 1997).

Since the effect of the agar oligosaccharides can be applied to various industries such as cosmetics, pharmaceuticals, and functional foods, researches on the production of low value-added agar into high value-added agar oligosaccharides have been actively conducted using agarase. .

On the other hand, wood-based fibrin is a component of the cell wall of the plant and consists of a complex of cellulose and hemicellulose. Cellulose is a β-1,4-glucose complex and is the most abundant renewable material in nature (Reiter et al. Curr OpinPlant Biol 5 (2002) 536-42). Although the chemical composition is simple, the action of several different enzymes is required to efficiently degrade cellulose (Ximenes et al. Hemicellulases and biotechnology. Recent Res Develop Microbiol 2 (1998) 165-176). Hemicelluloses include xylan, β-1.4-xylose, and glucomannan, β-1,4-glucose and mannose. Anaerobic microorganisms capable of breaking down most cellulose form an enzyme complex called cellulose (Roy H. Doi, The Chemical Record 1 (2001) 24-32). Cellulosomes act on a variety of substrates such as crystalline cellulosic, xylan, mannan, and pectin, and are composed of cellulosomal enzymes and scaffold proteins. The formation of cellulosomes consists of the binding of a dockerine module of one cellosome-forming enzyme and one of several cohisine modules of a support protein. All cellulosome forming enzymes have dockerin modules and those without dockerin modules are non-cellulosome forming enzymes (Bayer et al. Annual Review of Microbiol 58 (2004) 521-554).

The present invention has been made in view of the above necessity, an object of the present invention is to provide a recombinant vector capable of forming agar degrading enzyme complex.

Another object of the present invention is to provide a transformant capable of forming agar degrading enzyme complexes.

Another object of the present invention is to provide a method for forming an agar degrading enzyme complex.

Another object of the present invention is to provide an agar degrading enzyme complex.

In order to achieve the above object, the present invention provides a recombinant vector pET22 (+) cAgaB comprising a chimeric beta-agarase gene comprising a gene of a dockerine module of fibrinase and a beta-agarase gene.

In one embodiment of the present invention, the beta-agarase gene is preferably derived from Zobelia galactoniborane, but is not limited thereto.

In another embodiment of the present invention, the beta-agarase gene is preferably described in SEQ ID NO: 1, but in consideration of the degeneracy of the genetic code of the base sequence described in SEQ ID NO: 1, 80% homology, preferably Preferably, a gene having 85% homology, more preferably 90% homology, most preferably 95% homology, is also included in the beta-agarase gene of the present invention.

In another embodiment of the present invention, the gene of the dockerine module preferably has the nucleotide sequence set forth in SEQ ID NO: 2, but considering the degeneracy of the genetic code, the base sequence of SEQ ID NO: 2 and 80% Genes with homology, preferably 85% homology, more preferably 90% homology, most preferably 95% homology, are also included in the dockerine module gene of the present invention.

In a preferred embodiment of the present invention, the recombinant vector preferably has a cleavage map described in FIG. 2, but is not limited thereto.

The present invention also provides a transformant transformed with the recombinant vector of the present invention.

In one embodiment of the present invention, the transformant is preferably DH5α (cAgaB), but is not limited thereto.

A transformant transformed into E. coli with a recombinant vector comprising a chimeric beta-agarase gene comprising a gene of the above-described fibrinolytic enzyme of the fibrinase and a beta-agarase gene of the present invention was developed. It was deposited with the Korea Microorganism Conservation Center (KCCM) in Yurim Building, Hongje 1-dong, Seodaemun-gu under E. coli DH5α (cAgaB) accession number KCCM11164P on January 7, 2011.

In addition, the present invention provides a cellulose-binding module (CBM) and two of the cellulose-binding protein A, a primary scaffolding subunit of Clostridium-derived cellulose borax. Recombinant vector pET22b (+) mCbpA comprising a small cellulose-binding protein A gene with a Cohesin module is provided.

In one embodiment of the present invention, the gene of the cellulose binding module (Cellulose binding module) (CBM) is preferably having the nucleotide sequence described in SEQ ID NO: 3, in consideration of the degeneracy of the gene code in SEQ ID NO: Genes having 80% homology, preferably 85% homology, more preferably 90% homology, and most preferably 95% homology with the nucleotide sequences described may also be incorporated into the cellulose binding module gene of the present invention. Included.

In one embodiment of the present invention, the genes of the two Cohesin module (Cohesin module) preferably has a nucleotide sequence described in SEQ ID NO: 4 or 5, in consideration of the degeneracy of the gene code SEQ ID NO: 4 Or a gene having 80% homology, preferably 85% homology, more preferably 90% homology, and most preferably 95% homology with the nucleotide sequence of 5; Included in the module genes.

In addition, the present invention provides a chimeric beta-agarase isolated from the transformant of the present invention and a small cellulose-binding protein-A (Mini-Cellulose-binding protein A) isolated from the transformant of the present invention. It provides a method of mixing to form agar degrading enzyme complex.

In addition, the present invention provides a chimeric beta-agarase isolated from the transformant of the present invention and a small cellulose-binding protein-A (Mini-Cellulose-binding protein A) isolated from the transformant of the present invention. Mixing to provide agar degrading enzyme complex.

In the recombinant vector of the present invention, fragments for expression suppression having various functions for suppressing or amplifying or inducing expression, markers for selection of transformants, resistance genes for antibiotics, or out of cells It is also possible to have a gene encoding a signal for secretion.

As a method of culturing the transformant of the present invention, a conventional method used for culturing a host may be used.

As the culture method, any method used for culturing ordinary microorganisms, such as batch type, flow batch type, continuous culture, and reactor type, can be used. Transformants obtained by using bacteria such as Escherichia coli as a host As a medium for culturing, medium or synthetic medium, for example, LB medium, NB medium and the like can be given. In addition, the incubation temperature is accumulated in the above-mentioned temperature range of the above-mentioned temperature, to accumulate and recover the protein of interest in the cells.

The carbon source is required for the growth of microorganisms, and for example, sugars such as glucose, fructose, sucrose, maltose, galactose, and starch; Lower alcohols such as ethanol, propanol and butanol; Polyhydric alcohols such as glycerol; Organic acids such as acetic acid, citric acid, succinic acid, tartaric acid, lactic acid and gluconic acid; Fatty acids such as propionic acid, butanoic acid, pentanic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid and dodecanoic acid can be used.

Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate and ammonium phosphate, as well as those derived from natural products such as peptone, meat juice, yeast extract, malt extract, caseinate and corn steep liquor. Moreover, as an inorganic substance, 1 potassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, etc. are mentioned, for example. You may add antibiotics, such as kanamycin, ampicillin, tetracycline, chloramphenicol, and streptomycin, to a culture liquid.

In addition, when the promoter cultures the microorganism transformed using an inducible expression vector, an inducer suitable for the type of promoter may be added to the medium. For example, isopropyl-D-thiogalactopyranoside (IPTG), tetracycline, indole acrylic acid (IAA), etc. are mentioned as an inducer.

The protein of the present invention can be obtained by centrifuging the cells or supernatants from the cultures obtained, and by individually or appropriately combining cell disruption, extraction, affinity chromatography, cation or anion exchange chromatography, gel filtration, and the like. I can do it.

Confirmation that the obtained purified substance is the target enzyme can be performed by a conventional method, for example, SDS-polyacrylamide gel electrophoresis, western blotting or the like.

As described above, in the present invention, by applying a fibrinolytic enzyme complex (cellulosome) system from Clostridium spp. The chimeric-beta-agarase cAgaB gene linked to the active site of beta-agarase derived from galactanivorans ) was introduced into E. coli. Then, it was confirmed that the chimeric-beta-agarase gene is expressed and secreted at a high level in the E. coli strain. E. coli transformants capable of forming agar degrading enzyme complexes and agar degrading enzymes expressed from the genes according to the present invention may be usefully used in food, cosmetics, and pharmaceutical industries, and in particular, co-glycosylation for bioenergy production using marine biomass. It is expected to be a very useful invention for fermentation fusion process (SSF, simulated saccharification and fermentation or consolidated bioprocessing).

Figure 1 is a schematic diagram showing the design of the chimeric beta-agarase gene and small skeletal protein gene in the present invention, (A) Figure of gene design through the OverlapPCR of Chimeric Agarase
(B) Design schematic showing genes of small skeletal proteins
2 is a schematic diagram of the recombinant vectors pET22 (+) cAgaB and pET22b (+) mCbpA inserted with the chimeric beta-agarase-non gene and the small skeletal protein miniCbpA gene presented in the present invention.
Figure 3 shows the enzymatic secretion into the culture medium by showing the enzymatic activity in the Zymogram through the SDS-PAGA of the colony and culture concentrate of the recombinant vector pET22 (+) cAgaB inserted in the present invention and pET22b (+) SDS-PAGE and His-tag using purified proteins of Escherichia coli cultures containing mCbpA. (A) Zymogram and halo analysis of chimeric agarase cAgaB (AgaB-Doc), (B) Scaffolding His-tag purification of protein miniCbpA and electrophoresis analysis of expression. [M] molecular weight marker, [1-4] elution fraction
Figure 4 is a figure confirming the expression and binding of the enzymes expressed in the present invention by the method of purification using fiber and Western blot.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Example  1: of fibrinase Dockerine  Gene of the module, beta- Agarase  Gene amplification

Base of the dockerine portion of the endo-beta-1,4-glucanase-non gene from the genomic DNA of Clostridium cellulose to clone the gene of the dockerine module of fibrinase for agarase complex formation Referring to the sequence, 5 'of the forward primer is a 10bp sequence of the C-terminal portion of the chimeric beta-agarase AgaB gene derived from Zobelia galactoniboranth, and is a reverse primer. In 5 '), primers were designed and synthesized to insert restriction enzyme Not recognition sequences. Then, PCR was performed using the synthesized primers [5'TGTAGAGAAAGGATCCGCTGGCTCCG3 '(SEQ ID NO: 6) and 5'GCGCGGCCGC TCAATGATGATGATGATGATGTAAAAGCATTTTTTTAAG3' (SEQ ID NO: 7). As a result, a PCR band containing the gene of the dockerine portion of the endo-beta-1,4-glucanase-non gene of 212 bp was identified .

In order to clone the chimeric beta-agarase AgaB gene derived from Zobelia galactoniborane, the forward primer was referred to with reference to the base sequence excluding the signal peptide portion from the genomic DNA of Zobelia galactoniborane. ) 5 'of restriction enzyme SacI, reverse primer (5) of the reverse primer (endo-beta-1,4-glucanase- non-gene gene of the 10-bp sequence of the N-terminal portion of the dockerine portion, respectively) Primers [5'GCGCGAGCTCCGGCGACAATTCAAAATTTGATA3 '(SEQ ID NO: 8) and 5'CAGCGGATCCTTTCTCTACAGGTTTATAGATC3' (SEQ ID NO: 9)] were synthesized. Thereafter, PCR was performed using the synthesized primers. As a result, PCR bands containing beta-agarase AgaB gene derived from 1005 bp Zobelia galactoniborane were identified.

And a Cellulose binding module (CBM) and two cohydration modules of Cellulose-binding protein A, a primary scaffolding subunit of Clostridium-derived cellulose. In order to clone a small cellulose-binding protein-A gene with a Cohesin module, the 5 'of the forward primer is referred to as a base sequence. Reverse primers) were synthesized primers [5'GCGCGAATTCGCAGCGACATCATCAAT3 '(SEQ ID NO: 10) and 5'GCGCGGCCGCTATAGGATCTCCAATATT3' (SEQ ID NO: 11) into which the restriction enzyme NotI recognition sequence was inserted. As a result, the PCR band containing the mCbpA gene which is a part of the cellulose-binding protein-A gene of cellulose boranth cellulose derived from Clostridium of 1647bp was confirmed.

Example 2 Linkage and Cloning of the Gene of the Docker Module of Fibrinase and the Beta-Agarase Gene

The gene and AgaB amplification product of the dockerine module of the fibrinolytic enzyme obtained in Example 1 were electrophoresed on 0.8% agarose gel, and DNA fragments on the agarose gel were recovered using a gel extraction kit (GeneAll).

Thereafter, an overlap PCR reaction was performed using the DNA fragment recovered to connect the gene of the dockerine module of the fibrinolytic enzyme and the beta-agarase gene. From the two DNA fragments recovered, the primer [5'GCGCGAGCTCCGGCGACAATTCAAAATTTGATA3 '(SEQ ID NO: 12) and 5' of the forward primer were inserted so that restriction enzyme SacI was inserted into 5 'of the forward primer, and restriction enzyme NotI recognition sequence was inserted into 5' of the reverse primer. 5'GCGCGGCCGC TCAATGATGATGATGATGATGTAAAAGCATTTTTTTAAG3 '(SEQ ID NO: 13) was designed and synthesized by a PCR reaction resulting in a chimeric beta-derived from Jobelia galactoniboranth, to which the gene of the docker module of 1225bp fibrinase was linked. PCR bands containing the Agarase AgaB gene was confirmed.

The chimeric beta-agarase AgaB gene was then digested with SacI and NotI, and then ligated to pET22b (+), an E. coli expression vector, to Escherichia coli (E. coli, E. coli). coli) was transformed into DH5. The ligation of recombinant plasmid DNA was then isolated from the transformants. The recombinant vector was named pET22 (+) cAgaB and is shown in FIG. 2. The E. coli transformant was named DH5 / cAgaB.

Example  3: E. coli transformants Agarase  Active measurement

In order to confirm the expression of the enzyme protein of the transformant obtained in Example 2, the enzyme activity band (halo) measurement, SDS-PAGE, Zymogram was performed on the medium.

By inducing the expression of E. coli transformants with IPTG, the chimeric AgaB enzyme protein was secreted into the culture medium, and shaken at 30 ° C. for 5 hours, followed by centrifugation to prepare and concentrate the supernatant (Millipore, amicon 10kDa cut off). cAgaB enzyme protein.

 LB agar medium was used as a substrate for the enzymatic reaction to identify the enzyme activity (halo). Colonies of transformed E. coli were inoculated in LB medium and incubated at 37 ° C. for 24 hours, and the enzyme protein obtained by concentrating the culture medium incubated at 37 ° C. for 5 hours in LB liquid medium containing IPTG was placed on LB agar medium. After absorption, the mixture was reacted at 50 ° C for 24 hours. After staining with 0.25% Lugol solution for 5 minutes and rinsing with D.W several times, the enzyme activity band (halo) was confirmed and the results are shown in FIGS. 3-A.

SDS-PAGE was electrophoresed with enzyme protein using 10% poly-acrylamide gel and 0.3% Agarose dissolved poly-acrylamide gel. After that, Zymogram was performed to confirm the expression of enzyme at the correct position. To do this, the renature buffer (100mM Succiniate, 10mM CaCl ₂ , 1mM dTT) was added to the substrate to change the renature buffer every hour for 2 hours. It was made to react at 50 degreeC for 3 hours. After completion of the reaction, the gel was stained with 0.1% Lugol solution for 5 minutes, rinsed several times with DW, and the enzyme activity band (halo) was confirmed at the same position as the cAgaB protein size. This result is shown in FIG. 3-A.

Example  4: small size of yeast transformants Cellulose Binding Protein Expression

In order to confirm the complex formation of the chimeric-agarase to which the dockerine module is linked, protein purification was performed using the interaction between the cellulose-binding module (CBM) and cellulose of the small cellulose-binding protein.

Induce pET22b (+) mCbpA expression to secrete mCbpA protein into the culture medium, inoculate it in LB medium, incubate at 37 ° C for 24 hours, and incubate at 37 ° C for 5 hours in LB liquid medium containing IPTG. One supernatant was prepared and concentrated (Millipore, amicon 10 kDa cut off) to obtain mCbpA small cellulose-binding protein.

Cellulose (Sigmacell Type 50, SIGMA) was added and reacted at room temperature for 1 hour for protein purification using the interaction between cellulose-binding module (CBM) and cellulose. After the reaction, the mixture was rinsed three times with 1 mol sodium chloride 0.02 mol tris buffer (pH 8.0) and eluted with 0.05 mol tris buffer (pH 12.5). SDS-PAGE electrophoresed the enzyme protein using 10% poly-acrylamide gel. As a result, the protein band was confirmed at the 58 kDa position. This result is shown in FIG. 3-B.

Example  5: Confirmation of agar degrading enzyme complex formation

Formation of the complex due to the binding of the small cellulose-binding protein mCbpA to the chimeric beta-agarase cAgaB derived from Jobelia galacttoniborans to which the dockerine module of the endo-beta-1,4-glucanase-non gene is linked In order to confirm that the two proteins were mixed at a low temperature and incubated at a predetermined rate, to confirm the complex formation, first, protein purification using the interaction between the cellulose-binding module (CBM) and cellulose was followed by Western-blotting. Analyzed through. Western-blotting used anti-His-tag primary antibody (ELPIS) and goat anti-rabbit HRP conjugated (Santacruz) secondary antibody and the expression of enzyme protein was confirmed by luminol reagent (Santacruz). This result is shown in FIG.

Korea Microorganism Conservation Center (overseas) KCCM11164P 20110107

Attach an electronic file to a sequence list

Claims (16)

A recombinant vector for preparing an agar degrading enzyme complex comprising a chimeric beta-agarase gene comprising a gene of a dockerine module of fibrinase and a beta-agarase gene. According to claim 1, wherein the beta-agarase gene is a recombinant vector for producing agar degrading enzyme complex derived from Zobelia galactoniborane. The recombinant vector according to claim 1 or 2, wherein the beta-agarase gene has a nucleotide sequence as set forth in SEQ ID NO: 1. The recombinant vector of claim 1, wherein the gene of the dockerine module has a nucleotide sequence of SEQ ID NO: 2. The recombinant vector according to claim 1, wherein the recombinant vector has a cleavage map as described in FIG. The transformant for producing agar degrading enzyme complex transformed with the recombinant vector for producing agar degrading enzyme complex of claim 1. The transformant of claim 6, wherein the transformant is E. coli DH5α (cAgaB) Accession No. KCCM11164P. delete delete delete The basic skeleton subunit (primary scaffolding subunit) of cellulose Robo lance of Agarase with greater loss tree Stadium derived from cellulose - - claim 6 or a chimeric beta separated from the article 7 transformants binding protein-A (Cellulose-binding Agar degrading enzyme complexes were prepared by mixing Cellulose binding module (CBM) and small cellulose-binding protein A with two Cohesin modules in protein A). How to form.
The method of claim 11, wherein the Cellulose binding module (Cellulose binding module; CBM) is characterized in that the protein encoded by the nucleotide sequence of SEQ ID NO.
12. The method of claim 11, wherein the two Cohesin modules are proteins encoded by the nucleotide sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.
Cellulose-binding protein, which is a primary scaffolding subunit of chimeric beta-agarase and Clostridium-derived cellulose boranes isolated from the transformant of claim 6 or 7 A) Agar degrading enzyme complex formed by mixing a cellulose binding module (CBM) and a small cellulose-binding protein A having two cohesin modules.
The agar decomposing enzyme complex according to claim 14, wherein the cellulose binding module (CBM) is a protein encoded by the nucleotide sequence of SEQ ID NO.
The agar decomposing enzyme complex according to claim 14, wherein the two cohesin modules are proteins encoded by the nucleotide sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.

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