KR102154123B1 - Alginotytic enzyme complex and method for preparing thereof - Google Patents

Abstract

The present invention relates to an alginate-decomposing enzyme complex and the like. The enzyme complex exhibits a synergistic effect on decomposition of alginates, a main component of brown algae, by using endo- and exo-alginate lyase together with a dockerin and cohesin. The present invention can provide a stable alginate-decomposing enzyme complex by providing a small skeletal protein miniaturizing cellulosome as a skeletal protein. In addition, the small skeletal protein includes a carbohydrate binding module to increase accessibility to a substrate to be decomposed, thereby enabling faster and more efficient alginate decomposition activity. The present invention is expected to be actively used in a field of development and utilization of new bio materials such as biofuels using decomposition products of alginates.

Description

Alginate degrading enzyme complex and method for preparing the same {Alginotytic enzyme complex and method for preparing thereof}

The present invention relates to an alginate degrading enzyme complex, and more specifically, exo-alginate lyase and docurin module; and endo-alginate lyase and docurin module; decomposition of alginate bound to a small skeletal protein including the kohesin module It relates to an enzyme complex and a method for preparing the same.

Brown algae is a group of protozoa that usually belong to marine multicellular algae. About 1,500 species are known worldwide, most of which live in the sea. The main component of brown algae is alginate, which accounts for about 40% of brown algae. Types of brown algae that can be found a lot in Korea include fantail, kelp, seaweed, tot, and hatban.

On the other hand, alginate is composed of mannuronic acid (β-D-mannuronic acid) constituting the cell wall of brown algae and its structural isomer, gluronic acid (α￥α-L-guluronic acid), and is composed of arbitrary repetition of urinate. Has been. In addition, the alginate molecular structural formula is (C6H8O6)n, and in its detailed structure, a portion in which mannuronic acid (M) is repeated is a poly-M block and a portion in which glutonic acid (G) is repeated. Is called a poly-G block, and a part in which mannuronic acid and glutonic acid are repeated is called a poly-MG block.

Alginate lyase is an enzyme that degrades alginate, and is divided into exo- and endo-types according to the part of alginate that the enzyme decomposes, and glycosidic bond through β-elimination. ) To decompose alginate. Exo-alginate lyase cleaves alginate, which is a monomer from the non-reducing end, in an exogenous manner, and endo-alginate lyase cleaves alginate in an endogenous manner.

The uronate monomer finally decomposed by alginate lyase is non-enzymatically 4-deoxy-L-erythro-5-hexoseulose uronic acid (4-deoxy-L-erythro-5). -hexoseulose uronic acid: DEH), and DEH is used as a carbon source for metabolic engineering recombined microorganisms (Yeast, etc.) to provide biofuels such as bioethanol and new biomaterials.

The present inventors have completed the present invention by studying a fast and efficient decomposition method of alginate in order to supply high-quality bio new materials and fuels as decomposition products of alginate abundantly present in brown algae.

KR 10-1120359

The technical problem to be achieved by the present invention is to provide an alginate decomposing enzyme complex in which exo- and endo-type alginate lyase, docurin, and kohesin are bound to a small skeletal protein, and a method of preparing the enzyme complex.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides an alginate degrading enzyme complex bound to a small skeletal protein including an exo-alginate lyase and docurin module; and an endo-alginate lyase and docurin module; and the kohesin module do.

As an embodiment of the present invention, the enzyme complex may include a carbohydrate binding module (CBM).

In addition, the present invention provides a method for preparing an alginate degrading enzyme complex comprising the following steps.

(1) exo-alginate lyase-encoding gene and the first transformant production step injecting a vector containing a gene encoding a docurin module;

(2) a second transformant production step in which a vector including a gene encoding endo-alginate lyase and a gene encoding a docurin module is injected;

(3) preparing a third transformant in which a vector containing a gene encoding a cohesin module and a gene encoding a small skeletal protein is injected;

(4) culturing each of the first to third transformants or in one medium; And

(5) separating the culture supernatant of the transformant.

As an embodiment of the present invention, the gene encoding the exo-alginate lyase in step (1) may be a gene comprising or consisting of the nucleotide sequence of SEQ ID NO: 1.

As another embodiment of the present invention, the gene encoding the endo-alginate lyase of step (2) may include or consist of the nucleotide sequence of SEQ ID NO: 2.

As another embodiment of the present invention, the gene encoding the dockerin module of steps (1) and (2) may include or consist of the nucleotide sequence of SEQ ID NO: 3.

As another embodiment of the present invention, the gene encoding the cohesin module of step (3) may be a gene comprising or consisting of the nucleotide sequence of SEQ ID NO: 4.

In another embodiment of the present invention, the gene encoding the small skeletal protein in step (3) may include or consist of the nucleotide sequence of SEQ ID NO: 5.

As another embodiment of the present invention, the cell used as a host by injection of the vector in the manufacturing method may be Escherichia coli .

As another embodiment of the present invention, the vector is not limited as long as it is actively expressed in the host cell, but may preferably be a pCold II plasmi vector.

The present invention shows a synergistic effect on the decomposition of alginate, the main component of brown algae, by using endo and exo-type alginate lyase together with docurin and kohesin, and by providing a small skeletal protein miniaturizing cellulose as a skeletal protein, stable alginate It is possible to provide a digestive enzyme complex. In addition, the small skeletal protein includes a carbohydrate binding module to increase accessibility to a substrate to be decomposed, thereby enabling a faster and more efficient alginate decomposition activity. The present invention is expected to be actively used in the field of development and utilization of new bio materials such as biofuels using decomposition products of alginate.

1 is a schematic diagram of the preparation of an alginate decomposing enzyme complex.
2A is a schematic diagram of a recombinant vector into which a recombinant exo-alginate lyase gene is inserted, and FIG. 2B is a schematic diagram of a recombinant vector into which an endo-alginate lyase gene is inserted.
Figure 3 is a diagram showing the protein expression of the transformant by performing purification and electrophoresis of the His-tag adhesion protein [(A) BL21/alya1-doc (57 kDa) (B) BL21/alya5-doc (47 kDa) ) (C) BL21/mCbpA (56.8 kDa)].
4 is a view confirming the binding of exo- and endo-alginate lyase with small skeletal proteins using cellulose binding properties.
Figure 5 is a diagram confirming the alginate decomposition activity of exo- and endo- alginate lyase.

The present inventors studied enzyme complex technology that rapidly and efficiently decomposes brown algae, and when endo- and exo-type alginate lyase are simultaneously used, a synergistic effect occurs on the decomposition activity of brown algae, and small skeletal proteins are used. The present invention was completed by confirming through a specific experiment that more active alginate decomposing activity was exhibited by using it as a skeleton protein and providing it as one alginate decomposing enzyme complex.

Accordingly, the present invention provides an exo-alginate lyase and docurin module; and an endo-alginate lyase and docurin module; and an alginate decomposing enzyme complex bound to a small skeleton protein including the kohesin module.

In the present invention, "Alginate lyase" is an alginate degrading enzyme that is the main component of brown algae, and mannuronic acid (β-D-mannuronic acid: M) and glutonic acid (α¥α-L-guluronic acid) constituting alginate : Alginate is decomposed by cleaving the glycosidic bond of G), and it is divided into exo-(exo-) and endo-(endo-) types according to the cleavage site. In the present invention, the alginate lyase gene was exo-(SEQ ID NO: 1) and endo-(SEQ ID NO: 2) genes derived from Zobellia galactanivorans sp. 80% homology, preferably 85% homology, more preferably 90% homology, most preferably 95% homology with the base sequence of the gene in consideration of the degeneracy of It is also possible to use a gene having

In the present invention, "Dokerin" is a function of enhancing the decomposition activity of alginate lyase, and is derived from Clostridium , more specifically Clostridium cellulovorans ( C. cellulovorans ) strain. Although the Dockerin gene (SEQ ID NO: 3) was used, 80% homology with the nucleotide sequence of the gene, preferably 85% homology, more preferably considering the degeneracy of the DNA sequence or the genetic code Preferably, a gene having 90% homology, most preferably 95% homology, may be used.

In the present invention, "Cohesin" interacts with docurin to enhance the decomposition activity of alginate lyase and to bind to small skeletal proteins, and as a result, Clostridium , Specifically, the cohesin gene (SEQ ID NO: 4) derived from the C. cellulovorans strain was used, but in consideration of the degeneracy of the DNA sequence or the genetic code, the nucleotide sequence and the 80 A gene having% homology, preferably 85% homology, more preferably 90% homology, and most preferably 95% homology, may also be used.

On the other hand, cellulosome functions as a skeletal protein of alginate lyase in a state in which it is combined with a cohesin module in nature, but its size is very large, making it difficult to manufacture and use. Accordingly, the present invention aims to provide a skeletal protein of the alginate decomposing enzyme complex by miniaturizing the natural cellulosome, which was named as a small skeletal protein.

Thus, in the present invention, the "small skeleton protein (miniCbpA: mCbpA)" is a protein constituting the skeleton of the enzyme complex of the present invention, and at the same time, the decomposition of the enzyme complex of the present invention including a carbohydrate binding module (CBM) It can perform the function of improving the accessibility of the target substrate, that is, brown algae to alginate. The small skeletal protein was Clostridium , more specifically, a gene (SEQ ID NO: 5) derived from a strain of Clostridium cellulovorans ( C. cellulovorans ), but the DNA sequence or the genetic code was degenerative. In consideration of ), a gene having 80% homology, preferably 85% homology, more preferably 90% homology, and most preferably 95% homology with the base sequence of the gene may be used. have.

In a specific embodiment of the present invention, a recombinant expression vector was prepared by cloning the exo- or endo-alginate lyase and docurin genes into a plasmid, and then injected into E. coli to prepare a transformant.

In addition, in another specific example, a recombinant expression vector obtained by cloning a small skeletal protein gene having a cohesin module into a plasmid was prepared and injected into E. coli to prepare a transformant.

Thus, the exo-alginate lyase and dockerin module conjugate obtained from culturing each of the prepared transformants and obtained from the culture; Endo-alginate lyase and dockerine module conjugate; And a small skeletal protein including a cohesin module to prepare an alginate degrading enzyme complex including exo- and endo-alginate lyase, docurin, kohesin, and small skeletal proteins, and as a result of confirming its decomposition activity, small It was confirmed that the enzyme complex of the present application is more than about 170% superior in alginate decomposition activity than Mixture that does not contain a skeleton protein.

In a specific embodiment of the present invention, the "vector" uses a pColdII plasmid, but any DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host is limited thereto. It is not. Thus, the vector can be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since the plasmid is the most commonly used form of the current vector and is used in specific examples of the present invention, in the specification of the present invention, "plasmid" and "vector" are sometimes interchangeably Used. However, the present invention encompasses other forms of vectors that have functions equivalent to those known or become known in the art.

In addition, in the present specification, "recombinant expression vector" refers to a fragment of double-stranded DNA as a recombinant carrier into which a fragment of a heterogeneous DNA is inserted. Here, heterologous DNA refers to heterologous DNA, which is DNA that is not naturally found in host cells. Once in the host cell, the expression vector can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (heterologous) DNA can be generated.

The vector may include a promoter operatively linked to the gene to be cloned. In the present specification, the term "promoter" promotes expression of the gene to be transfected, and the promoter includes a basic factor required for transcription (Basal element), as well as an enhancer that can be used to promote and regulate expression.

In addition, in the present specification, "transformation" or "transfection" means that DNA is introduced into a host so that the DNA becomes replicable as an extrachromosomal factor or by chromosomal integration completion.

In the present invention, various transformations can be applied and various embodiments can be applied, and specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to a specific embodiment, it is to be understood to include all conversions, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, when it is determined that a detailed description of a related known technology may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

[Example]

Example 1. Securing alginate lyase and docurin genes

With reference to the alya1 and alya5 genes derived from Zobellia galactanivorans, the alginate lyase gene was amplified using a primer set having nucleotide sequences of SEQ ID NOs: 6-7 and 8-9. In the primer set having the nucleotide sequences of SEQ ID NOs: 6 and 8, the forward primers (SEQ ID NOs: 6 and 8) were designed to recognize the 10 bp upper end at the position where the amplification product gene will be inserted into the vector, and the reverse primer (SEQ ID NO: 7 And 9) was designed and manufactured to recognize the 10bp lower end at the above position (using Bioneer's Cloning Kit).

In addition, the sequence of the endo-beta-1,4-glucanase-ratio (Endo-β-1,4-Glucanase-B) gene from the genome of Clostridium cellulovorans as a reference sequence The docurin gene was amplified using primer sets having base sequences of numbers 10-11 and 12-13. The amplification product was confirmed to be a gene of a region encoding a Dockerin domain having a size of 159 bp by performing a southern bolt.

Subsequently, overlap PCR was performed to produce a DNA fragment to which the obtained alginate lyase gene and dockerin gene are linked. Specifically, from the two DNA fragments recovered, the primer was designed so that a recognition sequence of 10 bp of the forward binding site of the pColdII vector and 10 bp of the rear binding site of the pColdII vector is inserted into the 5'of the forward primer. And synthesized.

As a result, 1540 bp (endo-type), 1250 bp (exo-type) of the Docorin domain gene linked to the recombinant alginate lyase gene could be amplified.

Example 2. Cloning for endo-alginate lyase and docurin domain fusion

The gene of the docurin domain of the fibrinolytic enzyme obtained in Example 1 and the amplification product of the endotype alginate gene were electrophoresed on a 0.8% agarose gel, and the DNA fragment on the agarose gel was recovered using a Gel extraction Kit (GeneAll). I did.

Subsequently, the recovered DNA fragment and the pCold II vector were digested with sacI and kpnI restriction enzymes, and ligated in a 50°C bath for 15 minutes using a bioneer kit to prepare a recombinant plasmid (pColdIIalya1-doc).

The plasmid vector was injected into E. coli BL21 to prepare a transformant (BL21 Rosetta alya1-doc), and cultured to separate the recombinant plasmid (pColdIIalya1-doc) from the culture (see FIG. 2A).

Example 3. Exo-alginate lyase and docurin expression vector and transformant construction

As in Example 2, the gene of the docurin domain of the fibrinolytic enzyme and the amplification product of the exotype alginate gene obtained in Example 1 were electrophoresed on a 0.8% agarose gel, and the DNA fragment on the agarose gel was gel extraction kit. It was recovered using (GeneAll).

Subsequently, the recovered DNA fragment and pCold II vector were digested with sacI and kpnI restriction enzymes, and ligated in a 50°C bath for 15 minutes using a bioneer kit to prepare a recombinant plasmid (pColdIIalya5-doc).

The plasmid vector was injected into E. coli BL21 to prepare a transformant (BL21 Rosetta alya5-doc), and the recombinant plasmid (pColdIIalya5-doc) was again isolated from the culture by culturing it (see Fig. 2b).

Example 4. Construction of cohesin and small skeletal protein expression vectors and transformants

4-1. Securing Kohesin and small skeletal protein genes

Among Cellulose-binding protein A, a primary scaffolding subunit of cellulose derived from Clostridium , a carbohydrate binding module (CBM) and two noses In order to clone the Mini-Cellulose binding protein A gene with a Hesin module, referring to the nucleotide sequence, 5'of the forward primer (SEQ ID NO: 14) has a restriction enzyme BamHI recognition sequence ( ggatcc) and reverse primers (SEQ ID NO: 15) were synthesized with a restriction enzyme XhoI recognition sequence (ctcgag), respectively.

As a result, a PCR band containing a small skeletal protein gene (mCbpA) (SEQ ID NO: 5), which is a part of the 1659 bp Clostridium-derived cellulose-binding protein-A gene, was confirmed.

4-2. Construction of cohesin and small skeletal protein expression vectors and transformants

As in Examples 2 and 3, the small bone tissue protein gene amplification product containing the cohesin gene obtained in Example 4-1 was electrophoresed on an agarose gel, and DNA fragmentation was performed using a Gel exraction Kit (GeneAll). Recovered.

Then, the recovered mCbpA gene and pET22b(+) vector were digested with BamHI and XhoI restriction enzymes, and ligated in a 50° C. bath for 15 minutes using a bioneer kit to prepare a recombinant plasmid (pET22b-mCbpA).

The plasmid vector was injected into E. coli BL21 to prepare a transformant (BL21/mCbpA), cultured, and the recombinant plasmid (pET22b-mCbpA) was again isolated from the culture.

Example 5. Confirmation of expression of recombinant alginate lyase protein in E. coli transformants

In order to confirm the expression of the enzyme protein of the transformants prepared in Examples 2 and 3, purification and SDS-PAGE were performed using His-Tag. Conditions for inducing protein expression of BL21/alya1-doc, BL21/alya5-doc, and BL21/mcbpA recombinant strains were prepared, inoculated into LB medium, cultured at 37°C for 24 hours, and 500 μ\μM IPTG, ampicillin, and chloramphenicol Cells were obtained by incubating for 12 hours in LB liquid medium containing and centrifuging.

Subsequently, the obtained cells were disrupted by ultrasonic waves, and the supernatant obtained by centrifugation was concentrated and purified using His-tag linked to the N-terminal of the protein. Thereafter, 10% SDS-PAGE electrophoresis was performed, and it was confirmed that the result of staining through Coomassie Blue appeared at the same location as the expected protein size (see FIG. 3).

Example 6. Recombinant endo- and exo-type alginate lyase protein assembly confirmed complex

In order to confirm the formation of the enzyme protein complex of the transformants obtained in Examples 4 and 5, the formation of the enzyme complex was observed using the interaction between the cellulose binding module and cellulose.

In order to observe the formation of the three types of enzyme complexes obtained in Examples 4 and 5 above, protein purification using the interaction between a carbohydrate binding module (CBM) and cellulose containing a small skeletal protein was performed. For this, cellulose (Sigmacell Type 50, SIGMA) was added and reacted at room temperature for 1 hour. After the reaction, the mixture was rinsed three times with 1 M sodium chloride 20 mM Tris buffer (pH 8.0) and eluted with 50 mM Tris buffer (pH 12.5). For its SDS-PAGE, the enzyme protein was electrophoresed using 12% poly-acrylamide gel (see FIG. 4).

Example 7. Exo- and endo-alginate lyase complex activity assay

In order to confirm the activity due to the combination of endo- and exo-type alginate lyase, an analysis of alginate decomposition activity using alginate as a substrate was performed. The enzyme activity analysis method is to use 0.1% alginate dissolved in a solvent of 100 mM Tris-Hcl as a substrate, and react with 100 mM Tris-Hcl (pH 7.0) as a reaction buffer for 15 minutes at 30° C., and then use the decomposed alginate monomer absorber. The absorbance at a wavelength of 235 nm was analyzed.

As a result, when polymerized with a combination of endo- and exo-type alginate lyase, the activity was measured higher than that of a single enzyme, and when a complex of endo- and exo-type alginate lyase was formed, the amount of decomposed DEH was higher than that of other cases. It was confirmed that the increase was further 170% (see FIG. 5).

As described above, a specific part of the present invention has been described in detail, and for those of ordinary skill in the art, it is obvious that this specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> Korea University Research and Buisness Foundation <120> Alginotytic enzyme complex and method for preparing thereof <130> DHP19-189 <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 1059 <212> DNA <213> Zobellia galactanivorans <400> 1 atgaaaaaaa cagtattagc tacagttacc gtattcgtcc ttttcgcctg taaagacaaa 60 cctaaggcca ctacggacag tgcatcggaa tcggccgttg caatgaaaaa caccactaaa 120 tacccaagtg agcttatccc tcaaatggat gaatggaaaa ttctcttagg cgatggcacc 180 cataaagaag accttgtgaa ttatgcgaag gatgactttt tttatgttga acatgaaaat 240 gaaacagatt gggtagtatt caaaacaccg aactcgggca ttacctcaag gacctcaagt 300 aataccagaa cggaattagg gcagaaaaaa cactggattc ccgaaacagg agggaagttg 360 aatgcaacct taaaggttca acacgtctcc acttcgggcg atgctagggt tgccgcgtca 420 tactctgtcg tggtcggcca aattcacagc gatgaaggac acgaaaacga accgataaaa 480 atcttttaca aaaaatttcc aggccataca aaaggctccg ttttttggaa ttacgagatt 540 aacaccaagg gtgataattc aaagcgatgg gattactcca cggcagtttg gggatacgat 600 atgtcggtcg ttggcccaac ggctacctca taccccgaag aaccggaaga cggtatagcc 660 ttgggggagg agtttagtta tgaaatcaac gtttatgaag gtatcatgta cctcactttt 720 tctagtgaag gccacaagac cattaaattc accaaaaacc tattaaaatc caattttacc 780 aagaagtccg atattcctca acagataaag acgctgtatg cttcaatagg tcgtgacggt 840 atcgaacgtg aaaatgccta tgccggggaa atacaatact ttaagctagg agcctacaac 900 caaaccaacg gaaaatcccc agaggataat ctggtatgga gtacaggggc cgacgtttac 960 gatggtgaca ttgccaagca atatgcaaat ggaagctatg ccgaggtatg gttcaaagaa 1020 gcgacgcttg gaagtggaag cgccccagaa gcccagtga 1059 <210> 2 <211> 1341 <212> DNA <213> Zobellia galactanivorans <400> 2 atgaaaaaaa atgtatttac gacgcttcgc acggtcgtca acggagacat tatgtggaaa 60 cttattcccg tatttttctt ggccttatgc ctaggaagtt gttcggaaga accagtcgac 120 ccagaggaag aagcggtcct taccaaactt tccgccaatt caacggccat tggaatttct 180 tcggtctcgg ccagcacctc acaatcaccg aatgtagcca gtaataccct agacggatct 240 acgtctaccc gttggtcagg ttatggtgat ggggcttcca tcacatacga ccttggcagt 300 tcggccaata ttgattacgt gaaaatcgct ttctataaag gagacagcag aaagaccaag 360 tatgaagttt gggtaggtaa ttctacttct agcctaacga aaatcaaatc gaaaaccagt 420 tcaggttcta cttcagatta cgaaactatt gacctcccca actctaccgc ccgttatatt 480 aggattgtcg gtaagggcta tgtacttaac agcggtggaa gcaccgtttt atggaacagc 540 attaccaaat tccaagcatg gggctccggt ggttcgtcca cccttcccat tagcggaaac 600 tcacctgcct cagttctagg aatcacggca aacacttgga aaataaacag ttttatagga 660 agtccaggct catctgccac ctattacgat gacattaccg atgccagtgg catttcgtac 720 aatacctatt ccgatgacaa ttacttctat accgatggcg aatgggtata ctttaaatgc 780 tacagaggtt tgggcggttc ggccaattca caaaacccaa gggtagagct acgtgagatg 840 gacaacggca acttggcaag ttggaccggc gatagcggca cccataccat ggaatggacc 900 gtacaggtaa accagctccc acaagatacc gacggcgatg gcggggtact ctgctttggt 960 caaatacatg ggccaagtaa aaattcagac ggcgttgaag tcgatgatgt agtaagggtt 1020 cagttcattg gtgaagaaaa ccaatcatcg ggatcggtaa aactaaagat cagcggctat 1080 gttaccgaag aacaaggagg cagccaaacc ttcagcggct acagtcttga caccacttac 1140 aactgtaaac tggtatattc cggaggttac gtagaactct ttatgaacgg ctctagcgta 1200 ttcagaaaga aaatggaagt tgacgatcta tctgaaaact attttaaagt cggtaactac 1260 ttgcaatccg ttaaaggcgc aagttatacg ggatcctacg gtttggttcg tatcaaaaac 1320 ctaagcgtaa cccacaatta a 1341 <210> 3 <211> 159 <212> DNA <213> Clostridium cellulovorans <400> 3 gatgttaaca aagatggaaa ggtaaatgct atcgattatg cagtgcttaa atcaattctt 60 ttaggtacaa atactaacgt tgatttatca gtatcagaca tgaataagga tggtaaagta 120 aatgctttgg atttagctgt tcttaaaaaa atgctttta 159 <210> 4 <211> 407 <212> DNA <213> Clostridium cellulovorans <400> 4 gtaacagcta caattggaaa agtacaagta aatgctggag aaacggtagc agtaccagtt 60 aacttaacaa aagttccagc agctggttta gcaacaattg aattaccatt aacttttgat 120 tctgcatcat tagaagtagt atcaataact gctggagata tcgtattaaa tccatcagta 180 aacttctctt ctacagtaag tggaagcaca ataaaattat tattcttaga tgatacatta 240 ggaagccaat taatcataag gatggagttt ttgcaacaat aacatttaaa gcaaaagcta 300 taactggaac aactgcaaaa gtaacttcag ttaaattagc tggaacacca gtagttggtg 360 atgcgcaatt acaagaaaaa ccttgtgcag ttaacccagg aacagta 407 <210> 5 <211> 1647 <212> DNA <213> Clostridium cellulovorans <400> 5 gcagcgacat catcaatgtc agttgaattt tacaactcta acaaatcagc acaaacaaac 60 tcaattacac caataatcaa aattactaac acatctgaca gtgatttaaa tttaaatgac 120 gtaaaagtta gatattatta cacaagtgat ggtacacaag gacaaacttt ctggtgtgac 180 catgctggtg cattattagg aaatagctat gttgataaca ctagcaaagt gacagcaaac 240 ttcgttaaag aaacagcaag cccaacatca acctatgata catatgttga atttggattt 300 gcaagcggag cagctactct taaaaaagga caatttataa ctattcaagg aagaataaca 360 aaatcagact ggtcaaacta cactcaaaca aatgactatt catttgatgc aagtagttca 420 acaccagttg taaatccaaa agttacagga tatataggtg gagctaaagt acttggtaca 480 gcaccaggtc cagatgtacc atcttcaata attaatccta cttctgcaac atttgataaa 540 aatgtaacta aacaagcaga tgttaaaact actatgactt taaatggtaa cacatttaaa 600 acaattacag atgcaaacgg tacagctcta aatgcaagca ctgattatag tgtttctgga 660 aatgatgtaa caataagcaa agcttattta gcaaaacaat cagtaggaac aactacatta 720 aactttaact ttagtgcagg aaatcctcaa aaattagtaa ttacagtagt tgacacacca 780 gttgaagctg taacagctac aattggaaaa gtacaagtaa atgctggaga aacggtagca 840 gtaccagtta acttaacaaa agttccagca gctggtttag caacaattga attaccatta 900 acttttgatt ctgcatcatt agaagtagta tcaataactg ctggagatat cgtattaaat 960 ccatcagtaa acttctcttc tacagtaagt ggaagcacaa taaaattatt attcttagat 1020 gatacattag gaagccaatt aatcactaag gatggagttt ttgcaacaat aacatttaaa 1080 gcaaaagcta taactggaac aactgcaaaa gtaacttcag ttaaattagc tggaacacca 1140 gtagttggtg atgcgcaatt acaagaaaaa ccttgtgcag ttaacccagg aacagtaact 1200 atcaatccaa tcgataatag aatgcaaatt tcagttggaa cagcaacagt aaaagctgga 1260 gaaatagcag cagtgccagt aacattaaca agtgttccat caactggaat agcaactgct 1320 gaagcacaag taagttttga tgcaacatta ttagaagtag catcagtaac tgctggagat 1380 atcgtattaa atccaacagt aaacttctct tatacagtaa acggaaatgt aataaaatta 1440 ttattcctag atgatacatt aggaagccaa ttaattagta aagatggagt ttttgtaaca 1500 ataaacttca aagcaaaagc tgtaacaagc acagtaacaa caccagttac agtatcagga 1560 acacctgtat ttgcagatgg tacattagca gaagtacaat ctaaaacagc agcaggtagc 1620 gttacaataa atattggaga tcctata 1647 <210> 6 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Endo-Alginate lyase_primer_F <400> 6 gcgcgagctc atgaaaaaaa atgtatttac gacgctt 37 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Endo-Alginate lyase_primer_R <400> 7 cagcggatcc attgtgggtt acgcttaggt 30 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Exo-Alginate lyase_primer_F <400> 8 gcgcgagctc atgaaaaaaa cagtattagc tacagttacc 40 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Exo-Alginate lyase_primer_R <400> 9 cagcggatcc ctgggcttct ggggc 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Dokerin_primer_F <400> 10 aacccacaat ggatccgctg gctcc 25 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Dokerin_primer_F <400> 11 gcgcgcggta cctcataaaa gcattttttt aagaacagct 40 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Dokerin_primer_R <400> 12 agaagcccag ggatccgctg gctcc 25 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Dokerin_primer_R <400> 13 gcgcgcggta cctcataaaa gcattttttt aagaacagct 40 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Mini-cellulose binding protein A_primer_F <400> 14 ggatccgcag cgacatcatc aa 22 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Mini-cellulose binding protein A_primer_R <400> 15 gcgcctcgag gctataggat ctccaatatt tat 33

Claims (10)

Exo-Alginate lyase and Dockerin module; and Endo-Alginate lyase and Dockerin module; This Kohesin module As an alginate degrading enzyme complex bound to a small skeletal protein containing,
The enzyme complex is characterized in that it further comprises a carbohydrate binding module (carbohydrate binding module: CBM), alginate degrading enzyme complex.
delete Method for producing an alginate degrading enzyme complex comprising the following steps:
(1) Including a gene encoding an exo-alginate lyase containing the nucleotide sequence of SEQ ID NO: 1 and a gene encoding a Dockerin module containing the nucleotide sequence of SEQ ID NO: 3 The first transformant manufacturing step in which the vector is injected;
(2) Including a gene encoding Endo-Alginate lyase containing the nucleotide sequence of SEQ ID NO: 2 and a gene encoding a Dockerin module containing the nucleotide sequence of SEQ ID NO: 3 A second transformant manufacturing step in which the vector is injected;
(3) A third trait injected with a vector containing a gene encoding a cohesin module containing the nucleotide sequence of SEQ ID NO: 4 and a gene encoding a small skeletal protein containing the nucleotide sequence of SEQ ID NO: 5 Conversion body manufacturing step;
(4) culturing the first to third transformants in a medium; And
(5) separating the culture supernatant of the transformant.
delete delete delete delete delete The method of claim 3,
The vector of steps (1) to (3) is a pCold II plasmid vector, characterized in that, the method for producing an alginate degrading enzyme complex.
The method of claim 3,
The transformant in steps (1) to (3) is Escherichia coli , characterized in that, the method for producing an alginate decomposing enzyme complex.

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