KR101994252B1 - Recombinant bacillus subtilis strain producing beta agarase and uses thereof - Google Patents

Abstract

The present invention relates to a recombinant Bacillus subtilis strain having the ability to produce beta agarase, and a use thereof, and more particularly, to a strain of Pseudoalteromonas hodoensis sp. Nov strain containing beta agarase and a signal peptide To a transformed recombinant Bacillus subtilis strain and its use.
The recombinant Bacillus subtilis strain according to the present invention is capable of mass production of beta agarase. Beta agarase produced can be used for saccharification of agarose, and saccharification of agarose by beta agarase is useful for biofuels, foods, cosmetics and pharmaceuticals.

Description

RECOMBINANT BACILLUS SUBTILIS STRAIN PRODUCING BETA AGARASE AND USES THEREOF FIELD OF THE INVENTION [0001] The present invention relates to recombinant Bacillus subtilis strains,

The present invention relates to a recombinant Bacillus subtilis strain having the ability to produce beta agarase and its use, and more particularly to a strain of Pseudoalteromonas hodoensis sp. Nov derived beta agarase and a signal peptide To a transformed recombinant Bacillus subtilis strain and its use.

Agarose is a major component of red algae, and is a major constituent of red algae. It contains galactose (D-galactose) and anhydrous galactose (L-AHG) The unit chain agarobiose is repeated and has a linear structure connected with an α-1,3 bond. Since it is possible to synthesize purified compounds using agarose, it can be widely applied to foods, cosmetics and medical industries by using agar or agarose-derived compounds.

In order to saccharify such agarose, an enzyme such as agarase should be used. Agarose is an α-neoagarobiose hydrolase that decomposes into β-agarase acting on β-1,4 bonds or acts on α-1,3 bonds. It is possible to decompose into galactose and anhydrous galactose by using.

Recently, heat-stable beta-agarase AgaA7 has been isolated from Pseudoalteromonas hodoensis sp. Nov. AgaA7 has a molecular weight of about 30 to 35 kDa and exhibits optimal activity at pH 7.0 and 45 ° C. In addition, AgaA7 catalyzes the conversion of agarose into neoagarotetraose, neoagarohexaose, and neoagarooctaose through exolytic and endolytic activities. (WJ Chi, JS Park, DK Kang, SK Hong et al., Appl. Biochem. Biotechnol . 173: 1703-1716, 2014).

In most cases, the isolated agarase has been replicated in E. coli, a Gram negative strain. However, in the process of expressing and purifying a protein, an inclusion body is formed or a protein misfolding problem occurs. On the other hand, Gram positive Bacillus subtilis is a more stable genetic engineering tool than Escherichia coli, and has been frequently used for mass production of proteins. Since it has no extracellular membrane, it can efficiently secrete proteins (JM van Dijl, M. Hecker et al ., Bacillus subtilis: from soil bacterium tosuper-secreting cell factory , Microb. Cell Fact . 12, 2013).

On the other hand, during the process of secretion of the target protein in Bacillus, the translocation process requires an appropriate signal peptide at the amino terminus of the target protein. Signal peptides inhibit the folding of intracellular pre-proteins to complete the translation process and inhibit the activity of deleterious secretory enzymes. (W. Li, X. Zhou, P. Lu et al ., Bottlenecks in the Expression and Secretion of Heterologous Proteins in Bacillus subtilis, Res. Microbiol ., 155: 605-610, 2004).

It is therefore important to introduce the most efficient signal peptide for proper secretion of heterologous proteins.

U. Brockmeier, M. Caspers, R. Freudl, A. Jockwer, T. Eggert et al., Systemacticscreening of all signal peptides from Bacillus subtilis; a strong stratetgy inoptimizing heterologous protein secretion in gram-positive bacteria, J. Mol. Biol. 362: 393-402, 2006.

It is an object of the present invention to provide a recombinant Bacillus subtilis strains capable of mass production of beta agarase and uses thereof.

In order to achieve the above object, the present invention provides a recombinant Bacillus subtilis strain having an ability to produce beta agarase, into which an expression vector containing a gene encoding a beta agarase and a gene encoding a signal peptide is introduced .

The AgaA7 gene encoding the beta agarase may be derived from pseudoalteromonas hodoensis sp. Nov having the nucleotide sequence of SEQ ID NO: 1.

The signal peptide may be composed of an amino acid sequence of any one of SEQ ID NOS: 2 to 7.

The AgaA7 gene encoding the beta agarase and the gene encoding the signal peptide may be introduced in a form inserted into one vector.

The present invention also relates to a method for producing pBE-AgaA7 vector (step a) by introducing AgaA7 gene coding for beta agarase into shuttle vector pBE-S; And a step (b) of transforming the pBE-AgaA7 vector of step a) into bacillus subtilis. The method of producing a recombinant Bacillus subtilis strain having a beta agarase producing ability is also provided.

The shuttle vector pBE-S may comprise an aprE signal peptide derived from Bacillus subtilis, which is composed of the amino acid sequence of SEQ ID NO: 2. The AgaA7 gene coding for beta agarase can be derived from pseudoalteromonas hodoensis sp. Nov. The AgaA7 gene encoding the beta agarase may be composed of the nucleotide sequence of SEQ ID NO: 1.

The present invention also relates to a method for producing a pBE-AgaA7 vector, comprising the steps of: (a) preparing a pBE-AgaA7 vector by introducing an AgaA7 gene encoding a beta agarase into a shuttle vector pBE-S containing a signal peptide; (Step b) of substituting the signal peptide in the pBE-AgaA7 vector of step a) with one signal peptide selected from the group consisting of the amino acid sequence of SEQ ID NO: 3 to SEQ ID NO: 7 and a signal peptide derived from bacillus subtilis, ; And introducing a signal peptide-substituted vector into the bacillus subtilis in step (b) (step c), wherein the recombinant Bacillus subtilis strain has a beta agarase-producing ability.

The signal peptide in the pBE-AgaA7 vector of step b above may be an aprE signal peptide derived from Bacillus subtilis consisting of the amino acid sequence of SEQ ID NO: The AgaA7 gene coding for beta agarase can be derived from pseudoalteromonas hodoensis sp. Nov. The AgaA7 gene encoding the beta agarase may be composed of the nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a method for producing beta agarase comprising culturing the recombinant Bacillus subtilis strain to produce a beta agarase enzyme.

The recombinant Bacillus subtilis strain may be cultured at 25 캜 to 37 캜, more preferably at 37 캜.

The recombinant Bacillus subtilis strain may be cultured in a LB (Luria Bertani Miller) medium, but is not limited thereto.

The present invention also provides an agarose depolymerization method comprising the step of treating agarose with the recombinant Bacillus subtilis strain or a lysate thereof.

The lysate may be prepared by culturing the recombinant Bacillus subtilis strain, followed by centrifugation and ultrasonication, followed by obtaining a lysate in the supernatant. More specifically, (i) culturing the recombinant strain and then centrifuging it; (ii) sonicating the cell pellet of the centrifuged strain; And (iii) centrifuging the treated cells and obtaining a lysate in the supernatant.

 The oligosaccharide can be obtained using the above agarose depolymerization method.

The oligosaccharide can be, but is not limited to, neoagarohexaose, neoagarotetraose, and neoagarobiose.

The recombinant Bacillus subtilis strain according to the present invention is capable of mass production of beta agarase. Beta agarase produced can be used for saccharification of agarose, and saccharification of agarose by beta agarase is useful for biofuels, foods, cosmetics and pharmaceuticals.

Fig. 1 is an image showing the structure of agarose, the hydrolysis activity of AgaA7 beta agarase, and the hydrolysis product.
Figure 2 (A) to form a solid agarose gwangryun in a medium with Lugol's iodine solution B. subtilis pBE-AgaA7 the agar to visualize the hydrolytic activity of the trehalose according to the image, (B) the recombinant according to the time B. subtilis (C) the control and recombinant strains were cultured at 30 ° C for 24 hours, and the concentrated extracellular proteins were subjected to SDS-PAGE and immuno-blotting, .
Figure 3 is a process for screening signal peptides to enhance the secretion of AgaA7 in B. subtilis .
FIG. 4 is a graph showing the relative values of neoagarooligosaccharide secreted per gram of biomass (A) and the relative value of AgaA7 secreted per biomass (AgaA7, parent strain carrying aprE SP was used as a control group, Is indicated in parentheses) and (B) the result of immunoblotting of AgaA7 secreted by the clone.

Hereinafter, the present invention will be described in more detail with reference to Examples. The objects, features and advantages of the present invention will be readily understood through the following examples. The present invention is not limited to the embodiments described herein, but may be embodied in other forms. The embodiments described herein are provided to enable those skilled in the art to fully understand the spirit of the present invention. Therefore, the present invention should not be limited by the following examples.

In one embodiment of the present invention, a gene coding for a signal peptide derived from Bacillus subtilis and an AgaA7 gene coding for beta agarase derived from pseudoalteromonas hodoensis sp. Nov. ( Bacillus subtilis RIK1285) was transformed into a recombinant strain, and it was confirmed that the AgaA7 enzyme could be mass produced by the recombinant strain.

Example 1: B. subtilis  Preparation of pBE-AgaA7 recombinant strain

1-1: Preparation of plasmid and strain

From the pGEX-5X-1 vector (pGEX-5X-1-AgaA7 ) carrying a nucleotide fragment encoding an AgaA7 obtain an agaA7 gene (DY Park et al., Molecular characterization of agarase from the novel marinebacterium Pseudoaltermonas hodoensis sp. Nov , in: Unpublished Master's Thesis, Myongji University, 2013). The shuttle vector pBE-S was purchased from Takara, Korea. pBE-S has pUC-derived ori and ampicillin resistance genes necessary for replication in E. coli and pUB110-derived ori and kanamycin resistance genes necessary for expression and secretion of the desired gene in B. subtilis . In addition, pBE-S has a His-tag sequence at the downstream of the aprE promoter and SP (signal peptide) and MCS upstream of the multi-cloning site (MCS). B. subtilis RIK1285 [ trpC2, lys1, aprE? 3, npR2, nprR2, nprE18 ] was provided as a host for the pBE-S vector (purchased from Takara). E. coli DH5α (Solgent, Daejeon, Korea) was used as a host for the conventional cloning experiments and plasmid maintenance.

1-2:  B. subtilis  Preparation of pBE-AgaA7 recombinant strain

agaA7 gene (GenBank: GQ243743.1) was amplified by polymerase chain reaction (PCR) using pGEX-5X-1-AgaA7 as template DNA. The sequence of the PCR primer is as follows: forward 5'-CGT GAGCTC GCTGATTGGAGCCCTTTTAG-3 '(underlined portion is SacI restriction site, SEQ ID NO: 8) and reverse 5'-CGT TCTAGA CTGGGCTTTATAAACTCGTA-3' Restriction enzyme site, SEQ ID NO: 9) was synthesized in Macrogen, Korea. The PCR product and the pBE-S vector were enzymatically treated with SacI and XbaI (ThermoScientific, Korea). The enzymatically treated DNA fragment was cut into T4 DNA polymerase (ThermoScientific, Korea) and then transformed into E. coli DH5α by heat-shock method. The transformants were selected on Luria Bertani (LB) Miller medium (Sigma Aldrich, USA) containing 1.50% agar and 100 ug kanamycin / l. Suitable plasmids were identified by restriction mapping. The pBE-AgaA7 plasmid prepared by introducing the PCR product AgaA7 gene into the MCS site of the shuttle vector pBE-S containing the aprE signal peptide was transformed into B. subtilis RIK1285 according to the protocol of the manufacturer (Takara, Korea) , And transformants were selected on LB agar medium containing 10 카 kanamycin / l (B. subtilis, Secretory protein expression system product manual, URL http://www.clontech.com/takara/KR/Products/Protein Research / Protein Folding and Expression / B subtilis Secretory Protein Expression System sitex = 10037: 22372: US.). B. subtilis RIK1285 carrying pBE-AgaA7 was also confirmed by restriction mapping.

1-3:  B. subtilis  Identification of strain growth and agarase activity

B. subtilis the pBE-AgaA7 and B. subtilis comprising a vector from the ball pBE 30 ℃ and 37 ℃ for 48 hours and cultured in LB broth (broth). Samples were periodically taken to measure cell growth, extracellular protein concentration and agarase activity. Cell growth was measured at an OD _660nm value based on a standard curve for the dry weight of the cells measured. One OD _660nm unit corresponds to a dry weight (DCW) / l of 0.32 g cells. Protein concentration was measured by the Bradford method. Agarase activity was measured by the dinitrosalicylic acid (DNS) method. A 200 μl aliquot of the supernatant was added to 800 μl 0.25% agarose solution in 50 mM sodium phosphate buffer (pH 7.0). The mixture was incubated at 40 ° C for 1 hour and then 1 ml of DNS reagent (6.5 g DNS in 1 l distilled water, 325 ml 2 M NaOH and 45 ml glycerol) was added. For color development, the solution was placed in a boiling water bath for 10 minutes. After the reaction was terminated by adding the solution to ice, the absorbance was measured at 540 nm with a UV-VIS spectrometer. Thereafter, the amount of oligosaccharides improved was measured on the basis of D-galactose.

To confirm the intracellular agarase activity of the strain, the strain was cultured in LB medium at 30 ° C for 24 hours. The cell extracts were then obtained in cells centrifuged at 4000 rpm for 10 minutes. Cell pellets were resuspended in 1 ml lysis buffer _{(50 mM NaH 2 PO 4,} 300 mM NaCl, pH 8.0). To adjust the final concentration to 1 mg / ml, lysozyme (Sigma Aldrich, USA) was added to the cell suspension, incubated at 4 ° C for 1 hour, and the suspension was mixed every 10 minutes. The suspension was then sonicated for 3 minutes (10% amplitude, 5 second bursts, 10 seconds cool down). The cell slices were separated by centrifugation at 10000 rpm for 10 min at 4 ° C, and supernatants were used for analysis. The protein concentration of the final lysate was measured by the Bradford assay and the agarase activity by the DNS method.

As a result, it was possible to confirm the photoreactivity indicating the production and secretion of AgaA7 in the vicinity of the recombinant strain colony, but the photoreceptor could not be confirmed in the control strain containing the pBE empty vector (FIG. 2A). As a result of the time-dependent growth of the control and recombinant strains, the produced extracellular proteins and the agarase activity, the control strain containing the blank vector at 30 ° C showed an increase in extracellular protein production And the activity of agarase did not appear. In particular, the large amount of extracellular protein produced by B. subtilis pBE-AgaA7 during the log phase indicates that the activity of the AgaA7 agarase secreted in the culture medium is excellent. In addition, B. subtilis pBE-AgaA7 was found to grow better at 37 ° C than at 30 ° C (Fig. 2B).

1-4: Confirmation of production of AgaA7 of the recombinant strain by SDS-PAGE and immunoblotting

Ammonium sulfate was added to precipitate extracellular proteins of B. subtilis pBE-AgaA7 cultured in LB broth at 30 ° C for 24 hours. Cells were removed by centrifugation at 5000 rpm for 30 minutes at 4 < 0 > C. Solid ammonium sulfate was added to the supernatant until saturation of up to 80% and equilibrated by mixing at 4 ° C for 15 minutes. The precipitate was obtained by centrifugation and redissolved in a minimum volume of 50 mM sodium phosphate buffer (pH 7.0). The solution was then dialyzed overnight at 4 ° C for the same buffer. The protein was separated from the 12% acrylamide gel and stained with Coomassie blue. Immunoblotting was performed on Immuno-Blot PVDF (BioRad, Hercules, Calif., USA) membrane by a wet transfer method. The PVDF membrane was treated with blocking buffer and incubated for 2 h with Anti-6X HIS-HRP conjugated with antibody (Rockland, Gilbertsville, PA, USA) at 1: 5000 dilution. Antibody-protein complexes were identified with the OPTI-4CN ™ Substrate Kit (170-8235, BioRad, Hercules, CA, USA).

SDS-PAGE analysis revealed a protein band of 30 kDa corresponding to AgaA7 secreted from the recombinant B. subtilis . Likewise, it was confirmed by immunoblotting that AgaA7 was present in the recombinant strain (Fig. 2C).

Example 2: Signal peptide (SP) is substituted B. subtilis  Preparation of pBE-AgaA7 recombinant strain

2-1: Preparation of SP library and preparation of recombinant strains substituted with signal peptide

To replace the aprE SP of B. subtilis (Takara, Korea) with 173 different signal peptides (Takara, Korea), the pBE-AgaA7 vector was enzymatically treated with MluI and Eco521. In-Fusion reaction solution was prepared by mixing 1X In-Fusion ^® HD enzyme premix (Clontech, Takara, Republic of Korea), in the expanded linear expression plasmid, SP DNA mixture and sterile distilled water (up to 10 μl). Here, the molar ratio of the plasmid to the SP DNA mixture was adjusted to 1: 2. The enzyme-treated signal peptide was attached to the N-terminus of the AgaA7 enzyme. The mixture was incubated at 50 < 0 > C for 15 minutes and then placed on ice. Then, 2 μl of the reaction mixture was introduced into E. coli DH5α (Solgent, Taejon, Korea) and transformed to obtain a plasmid library containing 2000 or more colonies. The transformant colonies were removed from the solid medium and recovered, while the LB broth containing ampicillin was shed. Recombinant plasmids were extracted from the recovered colonies and 1.0 [mu] g of plasmid DNA was introduced into B. subtilis RIK1285 and transformed. At this time, the same method as the protocol used in Example 1 (preparation of B. subtilis pBE-AgaA7 recombinant strain) was applied as the transformation method. 610 B. subtilis colonies were obtained in agar plates with the final transformants. At this stage, approximately 3-8 x 10 ² colonies per μg of plasmid DNA can be obtained (B. subtilis, Secretory protein expression system product manual, URL http://www.clontech.com/takara/KR/Products/ Protein Research / Protein Folding and Expression / B subtilis Secretory Protein Expression System sitex = 10037: 22372: US.).

2-2: SP Screening

To perform the first screening, 610 B. subtilis transformant colonies were transferred to a master plate. Subcultures of the main plate colonies were carried out by spotting onto LB medium containing 1% agarose. Plates were incubated overnight at 30 ° C and stained with Lugol's iodine solution to visualize agarase activity (FIG. 2A and FIG. 3). As a result, the size of the light rings varied, indicating the difference in secretion efficiency between the respective signal peptides (FIG. 3).

Of the clones, 149 clones with the same size and larger halo than the parent strains containing aprE SP were selected. To perform the second screening, clones of relatively identical cell density were transferred to the solid medium. The cell density of each culture was adjusted by diluting to obtain similar OD ₆₆₀ values. Each clone was then incubated in LB broth until it reached the late exponential phase. 5 μl of each cell suspension was transferred to spotted agarose containing LB medium, dried and incubated overnight at 30 ° C. After selection of 38 clones with the same size and the same size of the control as the control in the clones to confirm the extracellular AgaA7 agarase activity, the amount of oligosaccharide released from the clone and the increased extracellular enzyme concentration were determined for the amount of accumulated biomass (FIG. 4A). As a result, c54 of the clones accumulated the largest amount of biomass, but emitted an amount of oligosaccharide similar to that of the control. On the other hand, c382 secreted the greatest amount of AgaA7 because it produced the least amount of oligosaccharide, although it produced the least amount of biomass.

To confirm the amount of agarase secreted from the final clone, immunoblotting was carried out in a slightly modified manner in Examples 1-4. Instead of concentrating the sample with ammonium sulphate precipitate in order to minimize errors due to chemical modification, the extracellular protein was removed by sonication using an Amicon Ultra-15 centrifuge (10 kDa NMWL, EMD Millipore, Korea) After concentration in the supernatant, it was centrifuged at 4000 rpm for 20 minutes at 4 DEG C before immunoblotting. As a result, as shown in FIG. 4B, c382 showed an enhanced band of intensity as compared with parent strain containing aprE SP-AgaA7 fusion protein. This means that the amount of oligosaccharide released and the amount of protein concentrate increased compared to the parent strain. On the other hand, it was confirmed that c54 exhibited a lower intensity band than the parent strain. This is consistent with the result of FIG. 4A.

In addition, to quantify AgaA7 produced and secreted in clones by affinity chromatography, the final clones were cultured in 100 ml of LB medium at an initial cell density of 0.05 AU (absorbance units) Lt; / RTI > The cells were then removed by centrifugation and the supernatant was dialyzed overnight at 4 ° C in LEW buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, pH 8.0). The protein was concentrated in the dialyzed culture supernatant through an ultrasonic treatment method. Purification was completed according to the method of use of ^{Protino ® Ni-TED pre-packed} column (Macherey-Nagel, BMS, Republic of Korea). His-tagged AgaA7 was eluted with LEW buffer containing 20 mM imidazole. Purified samples were identified by SDS-PAGE.

As a result, it was confirmed that parent strain was purified by 0.88 ㎍ AgaA7 / mg DCW while c382 was purified by 1.44 ㎍ AgaA7 / mg DCW which was 1.44 times higher than parent strain in c382.

2-3: Signal Peptide Sequencing and Analysis

The plasmid carried on the final clone was purified and sequenced (Macrogen, Korea). Numerous DNA alignments were performed using the ClustalW2 program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The DNA was translated into RNA via the Expasy tool (http://web.expasy.org/translate/). Signal peptides were identified by homology search using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/). The D-score values of the signal peptides were calculated using a SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP/).

The amino acid sequences and identities of the signal peptides carried on the clones are shown in FIG. 4A and Table 1.

The signal peptide sequence of the clone Clone Signal peptide amino acid sequence D-score value Sympathy Explanation aprE MRSKKLWISLLFALTLIFTMAFSNMSAQA
(SEQ ID NO: 2)
(MRSKKLWISLLFALTLIFTMAFSNMSAQA) 0.38 AprE Serine basic protease c54 MKKWLIIAVSLAIAIVLFMYTKGEAKA
(SEQ ID NO: 3)
(MKKWLIIAVSLAIAIVLFMYTKGEAKA) 0.23 Yvbx Secretory pathway protein c301 MKQGKFSVFLILLLMLMLTLVVAPKGKAEA
(SEQ ID NO: 4)
(MKQGKFSVFLILLLMLTLVVAPKGKAEA) 0.34 YqgA Cell wall binding protein c333 MKKVMLATALFLGLTPAGANA
(SEQ ID NO: 5)
(MKKVMLATALFLGLTPAGANA) 0.35 Pel Pectin hydrolase c382 MKFVKRRIIALVTILMLSVTSLFALQPSAKA
(SEQ ID NO: 6)
(MKFVKRRIIALVTILMLSVTSLFALQPSAKA) 0.47 Lipa Lipase c413 MKQGKFSVFLILLLMLTLVVAPKGKAEA
(SEQ ID NO: 7)
(MKQGKFSVFLILLLMLTLVVAPKGKAEA) 0.34 YqgA Cell wall binding protein

- The sequence in parentheses is derived from the database, and the D-score value is calculated as a 0.45 default D-cutoff value in the SignalP 4.1 server.

C301 and c413, which share the same signal peptide yqgA , showed similar values obtained in the active assay and purification steps. The band intensity in immunoblotting was also similar. On the other hand, c382, the most active clone, was found to contain a lipA signal peptide.

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 thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> MYONGJI UNIVERSITY INDUSTRY AND ACADEMIA COOPERATION FOUNDATION <120> RECOMBINANT BACILLUS SUBTILIS STRAIN PRODUCING BETA AGARASE AND          USES THEREOF <130> P16-0141 <160> 9 <170> KoPatentin 3.0 <210> 1 <211> 854 <212> DNA <213> Artificial Sequence <220> <223> AgaA7 beta-agarase <400> 1 gaattcgctg attggagccc ttttagtatt ccagcacaag caggcgcagg taaaagctgg 60 cagttacaaa gtgtttcaga tgagtttaac tacattgcac aacctaataa taaaccagct 120 gcttttaata atcgttggaa cgcatcttat ataaatgcct ggctaggtcc tggcgataca 180 gaatttagcg ctggccactc atatacaact ggcggggcgc ttggtctgca agcaaccgaa 240 aaagcaggta cgaacaaagt tctctcaggt attatttctt ctaaagcaac ctttacttac 300 cccctatacc tagaggcgat ggtaaaacca acaaacaaca ctatggcaaa tgctgtatgg 360 atgcttagtg ctgattctac ccaagaaatc gatgctatgg agtcatatgg tagcgaccga 420 attggccaag agtggtttga ccaacgtatg catgttagtc atcacgtatt tattcgagac 480 ccatttcaag attaccagcc aaaggacgct ggctcttggg tttacaacaa cggagaaaca 540 taccgcaata aatttcgtag atatggagta cattggaagg atgcgtggaa tttagattac 600 tatatagatg gtgttttggt tcgaagtgtc tctggcccaa atattattga tcctgaaaac 660 tatactaacg gaacaggctt aaacaagcct atgcacataa tactggatat ggaacatcag 720 ccatggcgag acgttaagcc taatgcatct gagcttgcag atcccaataa aagtatattt 780 tgggtagatt ggatacgagt ttataaagcc cagtaatata ttaaaaactt aacttattaa 840 gtgcggaact cgag 854 <210> 2 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> aprE signal peptide <400> 2 Met Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu   1 5 10 15 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala              20 25 <210> 3 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Yvbx signal peptide <400> 3 Met Lys Lys Trp Leu Ile Ile Ala Val Ser Leu Ala Ile Ala Ile Val   1 5 10 15 Leu Phe Met Tyr Thr Lys Gly Glu Ala Lys Ala              20 25 <210> 4 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> YqgA signal peptide of c301 clone <400> 4 Met Lys Gln Gly Lys Phe Ser Val Phe Leu Ile Leu Leu Leu Met Leu   1 5 10 15 Met Leu Thr Leu Val Val Ala Pro Lys Gly Lys Ala Glu Ala              20 25 30 <210> 5 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Pel signal peptide <400> 5 Met Lys Lys Val Met Leu Ala Thr Ala Leu Phe Leu Gly Leu Thr Pro   1 5 10 15 Ala Gly Ala Asn Ala              20 <210> 6 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> LipA signal peptide <400> 6 Met Lys Phe Val Lys Arg Arg Ile Ale Leu Val Thr Ile Leu Met   1 5 10 15 Leu Ser Val Thr Ser Leu Phe Ala Leu Gln Pro Ser Ala Lys Ala              20 25 30 <210> 7 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> YqgA signal peptide of c413 clone <400> 7 Met Lys Gln Gly Lys Phe Ser Val Phe Leu Ile Leu Leu Leu Met Leu   1 5 10 15 Thr Leu Val Val Ala Pro Lys Gly Lys Ala Glu Ala              20 25 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> PGEX-5X-1-AgaA7 template DNA forward PCR primer <400> 8 cgtgagctcg ctgattggag cccttttag 29 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> PGEX-5X-1-AgaA7 template DNA reverse PCR primer <400> 9 cgttctagac tgggctttat aaactcgta 29

Claims (17)

An AgaA7 gene consisting of the nucleotide sequence of SEQ ID NO: 1 encoding a beta agarase derived from pseudoalteromonas hodoensis sp. Nov and an amino acid sequence of any one of SEQ ID NOS: 2 to 7 derived from Bacillus subtilis A recombinant Bacillus subtilis strain having an ability to produce beta agarase, into which an expression vector containing a gene encoding a signal peptide composed of a sequence is introduced.
delete delete delete The method according to claim 1,
Wherein the AgaA7 gene encoding the beta agarase and the gene encoding the signal peptide are introduced in a form inserted into a single vector.
A shuttle vector pBE-S containing a signal peptide derived from Bacillus subtilis composed of the nucleotide sequence of SEQ ID NO: 2 was inserted into a shuttle vector pBE-S containing SEQ ID NO: 1 coding for beta agarase derived from pseudoalteromonas hodoensis sp. Nov (Step a) by introducing the AgaA7 gene consisting of the nucleotide sequence of the pBE-AgaA7 vector into the pBE-AgaA7 vector; And
(B) introducing the pBE-AgaA7 vector of step (a) into bacillus subtilis and transforming the transformed bacillus subtilis strain.
delete delete The method of claim 6,
Wherein the AgaA7 gene coding for beta agarase is composed of the nucleotide sequence of SEQ. ID. NO. 1.
A shuttle vector pBE-S containing a signal peptide derived from Bacillus subtilis composed of the nucleotide sequence of SEQ ID NO: 2 was inserted into a shuttle vector pBE-S containing SEQ ID NO: 1 coding for beta agarase derived from pseudoalteromonas hodoensis sp. Nov (Step a) by introducing the AgaA7 gene consisting of the nucleotide sequence of the pBE-AgaA7 vector;
(Step b) of substituting the signal peptide in the pBE-AgaA7 vector of step a) with one signal peptide selected from the group consisting of the amino acid sequence of SEQ ID NO: 3 to SEQ ID NO: 7 and a signal peptide derived from bacillus subtilis, ; And introducing a signal peptide-substituted vector into bacillus subtilis to transform the bacillus subtilis in step (b) (step c).
delete delete The method of claim 10,
Wherein the AgaA7 gene coding for the beta agarase has the nucleotide sequence of SEQ ID NO: 1.
A method for producing beta agarase comprising the step of culturing a recombinant Bacillus subtilis strain according to any one of claims 1 to 5 to produce a beta agarase enzyme.
15. The method of claim 14,
Wherein the recombinant Bacillus subtilis strain is cultured at 25 캜 to 37 캜.
A method for depolarizing agarose comprising the step of treating an agarose with a recombinant Bacillus subtilis strain of any one of claims 1 to 5 or a lysate thereof.
18. The method of claim 16,
The melt
Culturing the recombinant Bacillus subtilis strain recombinant microorganism of any one of claims 1 or 5, followed by centrifugation and ultrasonication, and then obtaining a lysate in the supernatant, wherein the agarose depolymerization method .

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