KR101261852B1 - Agarase expression vector, transgenic organism transformed with the same vector and method for the production of agarase using the same transgenic organism - Google Patents

Abstract

The present invention relates to agarase expression vector comprising a SCO3487 gene derived from Streptomyces coelicolor A3 (2) cloned into a vector capable of replicating in actinomycetes so that transcription is regulated by the ermE promoter. One actinomycetes transformant and agarase production method using the transformant are related.
According to the present invention, it was proved that the Streptomyces coelicolor A3 (2) SCO3487 gene, which was previously unclear, was an Agarez gene, and an expression vector was prepared using the gene. When introduced into, not only an actinomycetes transformant that overexpresses agarases can be prepared, but using these transformants, agares can be produced in a simple and efficient manner.

Description

Agarase expression vector, transgenic organism transformed with the same vector and method for the production of agarase using the same transgenic organism}

The present invention relates to an agarase expression vector, a transformant thereof, and a method for producing agarase using the transformant. Specifically, the SCO3487 gene derived from Streptomyces coelicolor A3 (2) is used in actinomycetes. The present invention relates to an agarez expression vector cloned so that transcription is controlled by a ermE promoter in a replicable vector, an actinomycete transformant transformed with the expression vector, and a method for producing agarase using the transformant.

Galactan in red algae consists of 70% agarose and 30% agaropectin, and agarose consists of D-galactose and anhydrous galactose (3,6-). An agarobiose, in which anhydro-L-galactose) is connected by β (1-> 4) bond, is repeated in units, and these units are in a linear structure connected by α (1-> 3) bonds. . In addition, agalopectin is composed of agarobiose units but is characterized by containing acidic groups such as sulfuric acid groups.

Β-agarase of type 1 breaks down the β (1-> 4) bond of agarose to produce agarotetraose, followed by beta-agarase of type 2 Acts to produce agarobiose from agarotetraose. Agarobiose is finally degraded into galactose and anhydrous galactose by alpha-agarase, which acts on α (1-> 3) binding. In the case of beta-agarese, which breaks down agarose, most of the technology is available from Japan, and there is not enough technology at home and abroad to express a large amount of enzyme at a low cost. Therefore, research to secure the genetic resources of Agarez and develop the expression system is very valuable. In addition, it is also very valuable research to secure a technology for producing agar oligosaccharides having a variety of useful physiological activity using the obtained agarase.

Streptomyces coelicolor A3 (2) is known to secrete agarase extracellularly (Stanier et al., 1942, J. Bacteriol .; Hodgson and Chater, 1981, J. Gen. Microbiol. dagA gene is known as the only beta-agarase gene. In addition, this strain is one of the most studied strains for the molecular biology of actinomycetes, the genome sequence in 2002 is published (Bantley et al., 2002, Nature).

In this regard, the present inventors searched for genes that are expected to be involved in glycolysis due to extracellular secretion from the genome sequence of Streptomyces syphila A3 (2) and select genes showing homology with well-known beta-agarases among them. The purpose of this study was to identify the function and to use it to break down agarose to produce a large amount of agar oligosaccharides. As a result, the present inventors have developed an expression vector of the SCO3487 gene and a transformant thereof, through which the SCO3487 gene has the activity of agares, among which beta-agares, which hydrolyzes agarose. Confirming the production of Neoagarobiose (Neoagarobiose) was to complete the present invention.

Therefore, the main object of the present invention is to secure a new Agarez gene source and to develop an expression system.

A detailed object of the present invention is to select an agarez gene from the genome of Streptomyces coelicolor A3 (2) and to provide an expression vector for expressing this gene in a heterologous strain.

Another detailed object of the present invention is to provide a transformant for producing agarase transformed with the expression vector.

Another detailed object of the present invention is to provide a method for preparing agarase using the transformant.

Still another object of the present invention is to provide agarase prepared by the method of preparing agarase.

Another detailed object of the present invention is to provide a method for preparing aga oligosaccharides using the agarase.

Another detailed object of the present invention is to provide an agar oligosaccharide prepared by the method for preparing aga oligosaccharide.

According to one aspect of the invention, the present invention is agarez expression by cloning the SCO3487 gene derived from Streptomyces coelicolor A3 (2) to regulate the transcription by the ermE promoter to a vector capable of replicating in actinomycetes Provide a vector.

In the genome sequence of Streptomyces Sicala A3 (2), a gene homologous to the beta-agarez gene of the Vibrio strain (Vibrio sp. Strain JT0107) was examined and found to be the SCO3487 gene.

However, it is only possible to predict that the gene will theoretically be similar to the beta-agareze of the heterologous strain, and to determine the function of the actual gene, it is necessary to confirm the function of the protein expressed by the gene.

Actinomycetes-derived genes often form inactive protein bodies even if they are not expressed or expressed in E. coli due to the high frequency of G-C in the gene and the use of rare codons. Therefore, when expressing foreign proteins in E. coli, specific expression conditions for each protein should be established. However, actinomycetes-derived genes do not cause rare codon problems when expressing heterologous proteins containing actinomycetes as host strains, and do not form inactive protein bodies. The actinomycete-derived SCO3487 gene is preferably expressed in actinomycetes.

In order to express the SCO3487 gene in the actinomycetes, the gene must be introduced into the actinomycetes. Therefore, it is preferable to clone it into a vector capable of replicating in the actinomycetes. It is preferable to use a promoter in the efficiency of agarez expression.

A pUWL201PW cloning vector can be used as a vector that can satisfy this. Since this vector has both an origin of actinomycetes and an origin of Escherichia coli, and can be used in both actinomycetes and Escherichia coli, it is useful for cloning using a system of E. coli and subsequently introduced into actinomycetes.

In the present invention, the nucleotide sequence of the Streptomyces coelicolor A3 (2) SCO3487 gene is shown in SEQ ID NO: 1. The gene consists of 2397bp of nucleotide sequence and is expected to produce a protein consisting of 798 amino acids when expressed.

In order to clone the SCO3487 gene as described above, a method of amplifying the gene by polymerase chain reaction (PCR) and then cloning the vector using a restriction enzyme recognition site may be used. The restriction enzyme recognition site to be used for cloning may be one that does not exist in the SCO3487 gene. In some cases, the restriction enzyme recognition site may be applied to the primer when amplifying the gene by PCR.

According to one embodiment of the present invention, PCR is performed using a primer consisting of the nucleotide sequence of SEQ ID NO: 2 and a primer consisting of the nucleotide sequence of SEQ ID NO: 2 as a template and genomic DNA of Streptomyces S. Subsequently, a DNA fragment of about 900bp containing NdeI and XhoI restriction enzyme recognition sites is amplified, including the start codon region of the SCO3487 gene.

In addition, when PCR is performed using a primer consisting of a nucleotide sequence of SEQ ID NO: 4 and a primer consisting of a nucleotide sequence of SEQ ID NO: 5, DNA fragments of about 1,500 bp containing XhoI and BamHI restriction enzyme recognition sites are amplified. Includes the stop codon site of the SCO3487 gene.

These amplification products were treated with restriction enzymes corresponding to the restriction enzyme recognition sites to obtain the fragments. The pUWL201PW vector was treated with NdeI and XhoI restriction enzymes, and then each obtained fragment was conjugated with a DNA ligase. Agarez expression vectors such as pUWL201PW-SCO3487 of FIG. 1 can be prepared.

The agarez expression vector prepared as described above is preferably introduced into actinomycetes to induce expression, and among the actinomycetes, the strain of Streptomyces genus is preferably used as a host strain. Among the strains of the Streptomyces genus, Streptomyces lividans has similar physiological characteristics to Streptomyces syricolor , and the cloning and expression can be performed relatively easily compared to other strains of Streptomyces genus. As such, it is most preferable to use this strain as a host strain.

The transformation method may be selected according to the host strain, but when streptomyces lividans is used as the host strain, it is preferable to transform by a method such as Keither using polyethylene glycol (PEG).

According to the present invention, the transformants transformed with the Agarez expression vector of the present invention were produced and secreted out of the cells.

Therefore, the agarase can be prepared by concentrating or purifying the agarase present in the culture medium by incubating the transformant with a solid and liquid culture.

In the case of the E. coli system, normally expressed proteins are not secreted to the outside of the cell and are present inside the cell, so that the cells have to be broken, and in the process, the sensitive protein may cause a decrease in activity. Therefore, the method of preparing agarez according to the present invention can be said to be a considerably improved and advanced method compared to the expression system using E. coli.

When the agarase prepared according to the method of preparing agarase is reacted with agarose, neoagarobiose may be prepared, wherein the reaction conditions are neutral pH, that is, a condition of pH 6 to 8 and 30 to 45 ° C. It is preferably carried out at the temperature conditions of. More preferably it is carried out at about pH 7 and about 40 ℃.

As described above, according to the present invention, it was proved that the Streptomyces coelicolor A3 (2) SCO3487 gene, which was not previously known, was an Agarez gene. When the introduced expression vector is introduced into the actinomycetes, not only the actinomycetes transformant that overexpresses agarases can be produced, but also the agarases can be produced in a simple and efficient manner by using the transformants.

1 is a genetic map of pUWL201PW-SCO3487.
2 is a photograph taken after the recombinant plasmid is extracted from E. coli introduced with pGEM-I and E. coli introduced with pGEM-II and treated with restriction enzyme EcoRI, followed by electrophoresis.
FIG. 3 is a photograph confirmed by electrophoresis after extracting a recombinant plasmid from Escherichia coli pUWL201PW-SCO3487 was cut with three restriction enzyme combinations (NdeI and BamHI, NdeI and XhoI, XhoI and BamHI).
4 is a photograph showing the agarase enzyme activity in the R2YE solid medium of the pUWL201PW-SCO3487 actinomycetes transformant.
Figure 5 is a photograph confirmed by electrophoresis of SCO3487 protein high expression from the culture medium of pUWL201PW-SCO3487 actinomycetes transformant in SDS-polyacrylamide gel.
Figure 6 is a photograph confirmed by electrophoresis of the SCO3487 protein purified by gel filtration by concentrating the culture medium of pUWL201PW-SCO3487 actinomycetes transformed with sulfate.
7 is a result of measuring the substrate specificity for two types of color substrate (p-Nitrophenyl-α-D-galactopyranoside [pNPαGal] and p-Nitrophenyl-β-D-galactopyranoside [pNPβGal]) of purified SCO3487 protein.
8 is a result of measuring the optimum pH of the SCO3487 protein on pNPβGal substrate.
9 is a result of measuring the optimum reaction temperature and temperature resistance of the pNPβGal substrate of the SCO3487 protein.
10 is a photograph confirming the final degradation product of agarose by SCO3487 protein by thin layer chromatography (TLC).
FIG. 11 shows the results of Direct-Mass analysis of the final decomposition product separated from thin layer chromatography and extracted with methanol.
12 shows the results of nuclear magnetic resonance analysis of the final decomposition product separated from thin layer chromatography and extracted with methanol.

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

Example 1 Acquisition of Agarase Code Gene (SCO3487)

A potential hydrolase code gene homologous to the beta-agarez gene of Vibrio sp. Strain JT0107 from Streptomyces coelicolor A3 (2) American Type Culture Collection (ATCC) BAA-471 (putative hydrolase coding gene) SCO3487 gene (SEQ ID NO: 1) was obtained. This process is described in detail as follows.

Streptomyces Sicicala A3 (2) was shaken in a R2YE liquid medium (Kieser et al., Practical Streptomyces genetics, A laboratory manual, 2000; John Innes Foundation, Norwich, United Kingdom) for 3 days, followed by a Keeser et al. et al., Practical Streptomyces genetics, A laboratory manual, 2000). Polymerization chain reaction (PCR) was performed using the isolated chromosomal DNA as a template to amplify the SCO3487 gene into two parts (fragment-I and fragment-II), which was amplified by 1% agarose gel. (agarose gel) was electrophoresed. In order to prevent PCR error caused by PCR amplification of large genes, SCO3478 gene was divided into two fragments, 900bp and 1500bp, and amplified, respectively. Each of the amplified gene fragments was recovered using an electrophoresis and gel extraction kit (Delbio) and then inserted into pGEM-T easy vector (Promega) to Escherichia coli DH5αF '. Transformed.

PCR was performed using the following 3487-F1 and 3487-R1 primers to obtain a gene fragment (fragment-I) of about 900bp from the start codon of SCO3487.

3487-F1: 5'- gggaCATATGaccgtgcacaagcgcg -3 '(SEQ ID NOs: 2, 26mer)

3487-R1: 5'- gcccgtcgcCTCGAGtcgtggaccg -3 '(SEQ ID NOs: 3, 25mer)

PCR was performed using the following 3487-F2 and 3487-R2 primers to obtain the gene fragment (fragment-II) of the back of SCO3487 (about 1500bp). This fragment contains the stop codon of SCO3487.

3487-F2: 5'- cggtccacgaCTCGAGgcgacgggc -3 '(SEQ ID NOs: 4, 25mer)

3487-R2: 5'- gtctctGGATCCgctactccgctgaacgc -3 '(SEQ ID NOs: 5, 29mer)

Figure 2 is an example of confirming that the amplified gene fragment was inserted by electrophoresis on 1% agarose gel after cleavage of recombinant plasmid from E. coli transformants at 37 ℃ with restriction enzyme EcoRI for 1 hour. The vector into which fragment-I was inserted was named pGEM-I and the vector into which fragment-II was inserted was named pGEM-II. DNA manipulation in Escherichia coli was followed by Sambrook et al. (Sambrook et al., Molecular cloning: a laboratory manual. 2 ^nd Edn. 1989).

※ The PCR reaction conditions of Example 1 were used by TAKARA DNA Polymerase (ExTaq polymerase), and after denature the primer pair and template DNA described above for 5 minutes at 94 ℃, a. 30 seconds at 95 ° C., b. 30 seconds at 63 ° C .; The procedure of polymerization reaction at 72 ° C. for 1 minute (a ~ d) was repeated 30 times, and finally, the reaction was carried out at 72 ° C. for 10 minutes. In addition, by analyzing the nucleotide sequence of the inserted gene fragment of the vector prepared in Example 1, after checking whether there is a PCR error (PCR error) was used only when there is no PCR error.

Example 2. Preparation of high expressing actinomycetes vector (pUWL201PW-SCO3487) of SCO3487

(i) The pGEM-I vector of Example 1 was reacted for 1 hour at 37 ° C. using restriction enzymes NdeI and XhoI, followed by electrophoresis on 1% agarose gel to recover a DNA restriction fragment of about 900 bp.

(ii) The pGEM-II vector of Example 1 was treated with restriction enzymes XhoI and BamHI, followed by electrophoresis on agarose gel to recover a DNA restriction fragment of about 1500 bp.

(iii) pUWL201PW, an E. coli-Streptomyces shuttle vector, was digested with restriction enzymes NdeI and BamHI and recovered after electrophoresis on an agarose gel.

DNA restriction fragments recovered in the above (i), (ii), (iii) were treated with T4 DNA ligase at 16 ° C. for 6 hours or more, and transformed into E. coli to prepare a recombinant expression vector pUWL201PW-SCO3487. (See Figure 1).

Figure 3 shows the recombinant vector from the E. coli transformant to determine whether the DNA restriction fragments of (i) and (ii) were correctly inserted into the pUWL201PW expression vector, and the three restriction enzyme combinations (NdeI and BamHI, NdeI and XhoI, XhoI and BamHI) are the examples of electrophoresis on agarose gel.

PUWL201PW (Doumith et al., 2000, Molecular General Genetics) used in the present invention is an E. coli-actinomycete shuttle vector, which has the advantage of performing genetic manipulations in E. coli instead of the actinomycetes, which are difficult to genetically manipulate, and chromosomal DNA in actinomycetes cells. It is used as an expression vector for high performance actinomycetes that maintains a high copy number.

Example 3 Preparation of SCO3487 High Expression Actinomycetes Transformant Incorporating Recombinant Vector

PUWL201PW-SCO3487 was prepared according to the method of Keizer et al. (Kieser et al., 2000, Practical Streptomyces genetics, A laboratory manual) using Streptomyces lividans TK24 strain to polyethylene glycol (PEG). Transformed. Transformants were selected by overlaying with antibiotic thiostrepton (Sigma-Aldrich) and 50 colonies resistant to antibiotics were passaged at 28 ° C. on new R2YE solid media containing the same antibiotics. . Of the 50 transformants, 10 transformants were inoculated into R2YE liquid medium, shaken and cultured, and centrifuged to recover only the cells. Plasmid DNA (plasmid DNA) was extracted from the recovered cells by a method such as Kidizer and confirmed by electrophoresis on an agarose gel. As a control, a strain transformed with the pUWL201PW vector was used.

Example 4 Analysis of Actinomycetes Transformants

(i) Actinomycetes transformants were inoculated into R2YE solid medium by day, incubated, and stained using Lugol's Iodine solution, and destained with 70% ethanol solution to decompose agars around colonies. (Widdick et al., 2006, Proc. Natl. Acad. Sci. USA).

As a result, agarase activity was observed around the colonies of the actinomycetes transformants into which pUWL201PW-SCO3487 was introduced, while no agarase activity was observed around the colonies of the transformants into which only the vector used as a control was introduced (FIG. 4). Reference).

(ii) The actinomycetes transformants were harvested by shaking culture for 7 days in a liquid medium of R2YE for 7 days, centrifuged at 15,000 rpm for 10 minutes, and the cells were discarded and cultured only. Add 100 µl of 100% Trichloroacetic acid solution to 1 ml of the culture medium from which the cells were removed, mix, leave for 30 minutes on ice, and centrifuge at 15,000 rpm for 10 minutes to discard the supernatant and recover protein pellets. It was. 700 μl of ethanol-ether solution (Ethanol: Ether = 1: 1) was added to the protein pellet, and then washed by centrifugation at 15,000 rpm for 5 minutes, leaving the protein pellet and removing the supernatant. After drying at room temperature to completely remove the ethanol-ether solution was suspended in phosphate buffer solution. Concentrated proteins were identified by electrophoresis on SDS (sodium dodecyl sulfate) -10% polyacrylamide gel to confirm agarez expression.

As a result, a protein band was not observed in the culture medium of the transformant into which only the vector was introduced, whereas an overexpressed SCO3487 protein band of about 88 KDa was observed in the culture medium of the transformant into which the pUWL201PW-SCO3487 was introduced. The overexpressed SCO3487 protein was most expressed on the first day of cultivation, and the expression was gradually decreased toward the late cultivation, which resulted in no expression on the seventh day of cultivation (see FIG. 5).

Example 5 Purification of Recombinant SCO3487 Protein from Actinomycetes Transformants

The transformants were shaken for 24 hours in a 1 L R2YE liquid medium and then centrifuged at 16,000 rpm for 15 minutes to remove the cells and only the culture solution was recovered. Ammonium sulfate was slowly added to the recovered culture so that the final concentration was 75% and centrifuged at 16,000 rpm for 60 minutes to induce protein precipitation. The precipitated protein was eluted with 20 mM Tris-Cl [pH 8.0] buffer, dialyzed for 16 hours using the same buffer, and then protein was purified using a 30 KDa cut off ultrafilter (Satorius). Was concentrated 20 times. 100 μl of the concentrate was purified by gel filtration using a Superdex G200 column. Purified protein was confirmed by electrophoresis on SDS-10% polyacrylamide gel.

As a result, the SCO3487 protein hypersecreted into the culture medium of the transformant was purified by gel filtration to obtain a single protein (see FIG. 6).

Example 6. Characterization of Purified SCO3487 Protein

Enzymatic activity was measured using two types of chromogenic substrates p-Nitrophenyl-α-D-galactopyranoside (pNPαGal) and p-Nitrophenyl-β-D-galactopyranoside (pNPβGal) on purified SCO3487 protein. The enzymatic reaction was performed at 40 ° C. for 24 hours, and the color development of the hydrolyzed substrate was measured at 420 nm.

As a result, enzyme activity was not measured when pNPαGal was used as a substrate, whereas high enzyme activity was measured in an enzyme reaction using pNPβGal as a substrate (see FIG. 7).

In order to determine the optimal pH conditions for the enzyme reaction, the activity after the enzyme reaction with pNPβGal as a substrate was measured under various pH conditions from pH 5.0 to pH 11.0. SCO33487 was observed to show the highest activity in neutral conditions of pH 7.0 (see Figure 8).

The highest enzyme activity was observed at the temperature condition of 40 ° C in the enzyme activity measurement at 20 ° C to 70 ° C under neutral condition of pH 7.0. When enzyme activity was measured after 1 hour of SCO3487 protein at each temperature (20 ~ 70 ℃), enzyme activity is maintained when left at 20 ~ 40 ℃ while deactivation of about 10% at 50 ℃ And it was inactivated by about 80 to 90% at the temperature (60 ~ 70 ℃) condition (see Fig. 9).

Example 7 Analysis of Hydrolysates of Agarose by SCO3487

Using agarose as a substrate, after the enzymatic reaction at pH 7 and 40 ° C, the reaction product was subjected to thin layer chromatography to analyze the hydrolyzate. Silica gel 60 was used as the stationary phase and the reaction was separated by transferring the reaction mixture to a butanol-acetic acid-H ₂ O = 2: 1: 1 mixture. The separated hydrolyzate was observed by inducing a color reaction with 10% sulfuric acid solution (H ₂ SO ₄ : EtOH = 1: 9) and treating it at high temperature. The final degradation product was extracted with 100% methanol solution and commissioned for direct-mass and nuclear magnetic resonance (NMR) analysis (see FIG. 10). Standards include the reaction of beta-agarase and agarose from Sigma, galactose, galactobiose and galactotetraose from Sigma. Was used.

Direct-Mass results of the extracted final degradation product confirmed that the material having a molecular weight of 324.1045, which is estimated to be neoagarobiose or agarobiose, was identified as neoagarobiose by nuclear magnetic resonance analysis. (See FIGS. 11 and 12).

As a result, SCO3487 turned out to be beta-agarase, which produces neoagarobiose as a final degradation product using agarose as a substrate.

<110> Myongji University Industry and Academia Cooperation Foundation <120> Agarase expression vector, transgenic organism transformed with          the same vector and method for the production of agarase using          the same transgenic organism <160> 5 <170> Kopatentin 1.71 <210> 1 <211> 2397 <212> DNA <213> Streptomyces coelicolor A3 (2) <400> 1 atgaccgtgc acaagcgcgc ttgcaccact ccgccgccgc gagccagcag gtcgttccgc 60 gtgaggtggc ctgtcctgat agcggccgcc tgcgccgggt tagtcctggc gaccaccagc 120 cctccggccg tcgcggccgg cgctcatgac ctcggcgacg agaccatgct ctacgacttc 180 caggacggcc tggtaccggc cgaggtcggc ccgtaccagg cgaagacgac aatcgtcggt 240 cgcggcgaca agaagctccg ggtcgatttc caggcccgga agaactacta ctcctcgttc 300 tccgtacgcc ccgagcccgt gtggaactgg tcggcggagg agtccgagtc gctcggcatc 360 gcgatggagc tcacgaaccc gagcgaccgc tccgtccagc tcacgatcga tctggagagc 420 tcgaccggcg tcgccacccg cagtgtcaac gtcccggccg gcggcggtgg cacgtactac 480 ttcgacgtcg acagccccgc gctccaccgc gacaccgggc tgcgcgccga tccgtcctgg 540 ctcgcggaca aggacgtcac ctctgcggtc tggatgtggg gctccaagga gacggacacg 600 agccgcatca gccagctgaa cttctacgtc gccggcctgc tgcacgaccg gtcggtcatc 660 gtggacgaca tccgcgtcgt ccgtgacgcg ccggcagacc ccgattacct caagggcctg 720 gtcgacgcct tcggtcagaa caacaaggtc gactacaagg gaaaggtctc caggacgtcg 780 gagattcttc ggcagcgcgc tgccgaagcc aaggaccttc gcaggcatcc ggttccggag 840 gaccggtcca ggtacggcgg ctggctgaac ggtccacgac tcgaggcgac gggcaacttc 900 cgcgtggaga agtaccaggg gcggtggacc ctggtggacc ctgacggcta cctgttcttc 960 tcaaccggca tcgacaacgc ccgcatgttc gactccccaa ccacgacggg ttacgacttc 1020 gaccatgacg cgatccagga gctgccgccc cccagcctga cggccggcgg ccccgaggac 1080 ctcaaccgcg tccagaagtc ggcgctgccg acccgcacga agatgtccga aacccgcgcc 1140 gacctcttca gcaagttgcc caagtaccgc acccgcgcgg gcgagggctt cggttacgcc 1200 cccgacaccc tggccggtcc cgtcgcgcag ggcgagacct acagcttcta caaggcgaac 1260 gtcgcccgga agtaccccgg cagcaactac atggagcggt ggcgggacaa cacggtcgac 1320 cggatgctca gctggggctt cacctccttc ggcaactgga ccgacccgga gatgtacgac 1380 aacgaccgta tcccgtactt cgcccacggc tggatcaagg gcgacttcaa gacggtgagc 1440 accggccagg actactgggg cccgatgccg gacccgttcg accccgcgtt ctccgacgcc 1500 gcagccagaa ccgcgcgagc agtcgccgac gaggtcgcgg acagcccgtt ggcgatcggc 1560 gtattcatgg acaacgaact gagctggggc aacgccggca gtttcagcac ccgttacggc 1620 gtcgtcatcg acaccatgtc acgtgacgcg gcagagagcc ccaccaagtc ggcgttctcc 1680 gacgaactgg aggagaagta cgggaccatc gacgctctca acgccgcgtg gcagacgaca 1740 gttccgtcat gggaggcact ccgtagcggc agtgccgacc tcggctccga cgagaccgcg 1800 aaggagtccg actactccgc gctcatgacg ctctacgcca ctcagtactt caagacggtc 1860 gacgccgagc tcgacaaggt catgccggac catctctacg cgggttcgag gttcgccagc 1920 tggggccgca caccggaggt cgtcgaggca gcgagcaagt acgtcgacat catgagctac 1980 aacgagtacc gcgagggact gcacccgagc gagtgggcgt ttctcgaaga gctcgacaag 2040 cccagcctca tcggtgagtt ccacatggga acgaccacta ccgggcagcc gcatccgggt 2100 ctcgtctcgg cgggaacgca ggccgagcgg gcacggatgt acgccgagta catggaacag 2160 ctcatcgaca acccgtacat ggtgggcggc cactggttcc agtatgccga ctcgcccgtg 2220 actggcagag cactcgacgg ggagaactac aacattggct tcgtctccgt cacggaccgt 2280 ccctacccgg agatcgtcgc cgctgcccgc gacgtgaacc agcgtctcta tgaccgccga 2340 tacggcaacc tggccacggc cgagggacat tacaccggtc ggcgttcagc ggagtag 2397 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> 3487-F1 primer <400> 2 gggacatatg accgtgcaca agcgcg 26 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 3487-R1 primer <400> 3 gcccgtcgcc tcgagtcgtg gaccg 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 3487-F2 primer <400> 4 cggtccacga ctcgaggcga cgggc 25 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> 3487-R2 primer <400> 5 gtctctggat ccgctactcc gctgaacgc 29

Claims (23)

It contains the SCO3487 gene derived from Streptomyces coelicolor A3 (2), the transcription of the SCO3487 gene is regulated by the ermE promoter, characterized in that for the production of neoagarobiose Expression Vector of Beta-Agares. delete The expression vector of claim 1, wherein the expression vector is represented by pUWL201PW-SCO3487 of FIG. 1. It contains the SCO3487 gene derived from Streptomyces coelicolor A3 (2), the transcription of the SCO3487 gene is regulated by the ermE promoter, characterized in that for the production of neoagarobiose Actinomycetes transformants that are transformed with an expression vector of beta-agarese to produce beta-agarese for neoagarobiose production. delete The actinomycete transformant of claim 4, wherein the expression vector is represented by pUWL201PW-SCO3487 of FIG. 1. 5. The actinomycete transformant according to claim 4, wherein the host of the transformant is Streptomyces revidance. It contains the SCO3487 gene derived from Streptomyces coelicolor A3 (2), the transcription of the SCO3487 gene is regulated by the ermE promoter, characterized in that for the production of neoagarobiose A method for producing beta-agarase for neoagarobiose, characterized by culturing; an actinomycete transformant transformed with a beta-agarase expression vector to produce beta-agarase for neoagarobiose production. delete The method of claim 8, wherein the expression vector is pUWL201PW-SCO3487 of FIG. 1. The method of claim 8, wherein the host of the transformant is Streptomyces revidance. delete delete delete delete It contains the SCO3487 gene derived from Streptomyces coelicolor A3 (2), the transcription of the SCO3487 gene is regulated by the ermE promoter, characterized in that for the production of neoagarobiose Beta-agarese expression vector; actinomycete transformant transformed with beta-agarese for neoagarobiose production; Neoagarobis manufacturing method characterized in that the reaction at 6 to 8 and 30 to 45 ℃. delete 18. The method of claim 16, wherein the expression vector is represented by pUWL201PW-SCO3487 of FIG. 17. The method of claim 16, wherein the host of the transformant is Streptomyces revidance. delete delete delete delete

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