KR20040020100A - An agarase and a gene from Pseudomonas sp. BK1, and a recombinant microbe - Google Patents

Abstract

PURPOSE: Agarase derived from Pseudomonas sp. BK1, a gene thereof, and a recombinant microorganism producing the same are provided, thereby mass-producing the agarase from marine microorganism. CONSTITUTION: A microorganism Pseudomonas sp. BK1 (KFCC 11308) has agarose decomposing activity. Agarase derived from Pseudomonas sp. BK1 has the amino acid sequence set forth in SEQ ID NO: 4 and thermal stability within 10 to 35 deg. C and pH stability within pH 8.5 to 9.5. A gene encoding the agarase has the nucleotide sequence set forth in SEQ ID NO: 2. An expression vector pET-Agr comprises the agarase gene and has a restriction enzyme map shown in figure 5. A recombinant Escherichia coli BL21(DE3) transformed with the expression vector pET-Agr is provided. The agarase is produced by culturing the recombinant Escherichia coli BL21(DE3).

Description

Agarose degrading enzyme derived from Pseudomonas genus ＢＫ1, its gene and recombinant microorganism expressing the same {An agarase and a gene from Pseudomonas sp. BK1, and a recombinant microbe}

The present invention relates to agarose degrading enzymes derived from Pseudomonas genus BK1, its genes and recombinant microorganisms expressing the same. The present invention relates to a recombinant Escherichia coli (Escherichia coli) transformed with an enzyme, a gene encoding the same, an expression vector including an agarose digestion gene, and the previous expression vector, and a recombinant agarose degrading enzyme prepared from the transformant.

Agar, which is abundant in many seaweeds in the ocean, is made up of a mixture of 70% agarose and 30% agaropectin. These components are used to acetylate or methylate agar. When a thing is dissolved in chloroform, agarose dissolves but agalopectin does not dissolve.

Agarose is agarobiose in which beta-di-galactose and 3,6-anhydro-L-galactose are β-1,4 bonds. ) Is a linear polysaccharide in which each agarobiose is bound to α-1,3 as a repeating unit, and is decomposed into oligosaccharides by two kinds of agarases, alpha-agarose degrading enzyme (α). Agaro-oligosaccharides are composed of agarobiose by -agarase, and neo-agarobiose by beta-agarase by β-agarase. Neoagarooligosaccharides (neoagarooligosaccharides) are produced.

To date, agarose degrading enzymes have been isolated from bacteria, actinomycets, and the like, and many studies have been conducted on Pseudomonas sp., But the microorganisms used in the study of the enzymes are mainly land-based microorganisms. .

As for useful enzymes derived from marine microorganisms, alginate degrading enzymes derived from Pseudomonas genus (Korean Patent Publication No. 1999-68710), agarose degrading enzymes derived from basophils (Korean Patent Publication No. 1997-21310) are disclosed. However, the above-described prior art is different from the present invention, and the molecular biology studies using useful enzymes and genes derived from marine microorganisms are rarely performed, including only cell fusion of seaweeds. Only enzymes necessary for hybridization between species are obtained and used.

Therefore, the present inventors have continuously conducted research to solve the above problems, and isolate biochemical and enzymatic characteristics by separating strains having agarose resolution from marine bacteria causing marine algae diseases. Using the powerful agarose decomposing gene secreted from the strain, it was found that the agarose degrading enzyme can be industrially mass-produced and completed the present invention.

Accordingly, an object of the present invention is to provide a novel microorganism having agarose resolution and agarose degrading enzyme derived therefrom.

Another object of the present invention is to provide a gene encoding the agarose degrading enzyme and an expression vector containing the gene.

Still another object of the present invention is to provide a recombinant Escherichia coli transformed with the expression vector and a recombinant agarose degrading enzyme prepared from the transformant, and a method of manufacturing the same.

1 (a) is a green disease of red algae (kim),

Figure 1 (b) is an electron micrograph of the marine bacteria Pseudomonas sp.BK1,

Figure 1 (c) is a state in which the cell wall of green algae (blue) by marine bacteria Pseudomonas sp.

Figure 1 (d) is a state in which the cell wall of red algae (kim) is decomposed by marine bacteria Pseudomonas sp.

Figure 2 shows the activity of agarose degrading enzyme produced by Pseudomonas sp.BK1 according to the present invention,

3 is a systematic location of Pseudomonas sp.BK1,

Figure 4 shows the activity of agarose degrading enzyme produced by E. coli JM109 having a recombinant vector containing agarose degrading gene,

5 is an expression vector pET-Agr used in the present invention,

6 is Pseudomonas sp. BK1, Pseudomonas atlantica, Aeromonas sp. Homology comparison between the β-agarose degrading enzyme amino acid sequences from

Figure 7 (a) is the first purification result by the FPLC system using Mono Q HR 5/5 column of water-soluble recombinant agarose degrading enzyme,

Figure 7 (b) is the secondary purification results by the FPLC system using a superdex 200 column of water-soluble recombinant agarose degrading enzyme,

Figure 7 (c) shows the SDS-PAGE results of the purified products of each step for the recombinant agarose degrading enzyme,

Figure 8 (a) is the optimum temperature and temperature stability of Pseudomonas sp. BK1 derived agarose degrading enzyme,

Figure 8 (b) shows the optimum pH and pH stability of Pseudomonas sp. BK1 derived agarose degrading enzyme.

The present invention provides a novel microorganism Pseudomonas sp. BK1 (KFCC-11308) having agarose resolution as a means for achieving the above object.

The novel microorganism according to the present invention, as shown in Figure 1 (a) in order to isolate a gene producing agarose degrading enzyme, a useful enzyme derived from marine microorganisms, the cell wall of seaweed leaf causing the erythema disease of red algae As a strain having excellent agarose resolution by selecting the site of degradation from the seashore, it was identified as a new strain by morphological and biochemical characterization and named Pseudomonas sp. BK1. It was deposited with the Center on 19 July 2002 and was given accession number KFCC-11308.

The morphological and biochemical properties of the novel strains according to the invention are shown in Table 1.

Pseudomonas sp. Morphological and Biochemical Properties of BK1 division Characteristics Cell shape Rod shape Gram Dyeing voice Arginine dehydrolase production voice Production of lysine dicarboxylase voice Production of ornithine dicarboxylates voice Agarase production positivity Cellulose production positivity U-raise production positivity Catalase Production positivity Oxidation Production positivity Gelatinase Production positivity Glucose fermentation positivity Mannitol fermentation voice Inositol Fermentation voice Sorbitol Fermentation voice Rhamnose fermentation positivity Saccharose fermentation voice Melibiose fermentation positivity Arabinose fermentation positivity

The present invention also provides a high activity agarose degrading enzyme (agarase) derived from the novel strain, the enzyme has a thermal stability in the range of 10 to 35 ℃, preferably 28 to 32 ℃ and pH 8.5 to 9.5 Has a pH stability in the range, preferably pH 9.0, and has the amino acid sequence set forth in SEQ ID NO: 4 of the Sequence Listing.

The present invention provides a gene encoding the agarose degrading enzyme in order to apply the agarose degrading enzyme which is a useful enzyme derived from marine microorganisms having the above characteristics to industrial mass production. The gene is as described in SEQ ID NO: 2 in Sequence Listing.

The present invention also provides an expression vector pET-Agr capable of high expression of the agarose degrading enzyme and recombinant Escherichia coli BL21 (DE3) transformed with the expression vector pET-Agr. It contains the agarose digestion gene and has a cleavage map described in FIG.

From the recombinant E. coli prepared above, the agarose degrading enzyme expressed and purified was analyzed, and the enzyme activity and characteristics were analyzed. As a result, marine microorganism Pseudomonas sp. BK1 secreted the same degree of agarose degrading enzyme. It has been confirmed that it has an activity and characteristics of can be usefully used industrially.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1 Separation of Strains and Confirmation of Agarose Resolution

Approximately 1 g of degrading laver leaves are suspended in 50 ml of sterile seawater, and the solution is diluted 10-fold, 100-fold and 1000-fold with 1% peptone, 0.5% yeast extract, 1% sodium chloride and 1.5% agar. 0.1 ml each of the nutrient medium was added to the plate was incubated for 5 days in an incubator at 25 ℃.

The single strains grown by the above cultures were separated purely, and each strain was added to an aseptically treated seaweed leaf, and then the strains were separated by selecting a group that decomposed the leaf as soon as possible. The photograph taken with is as shown in Fig. 1 (b).

Cell lysis of green algae (green) and red algae (seaweed), which are marine algae, is shown in FIGS. 1 (c) and 1 (d), respectively. As shown, the cells of laver dying to pale green were observed.

In addition, in order to confirm the agarose resolution of the strain isolated by the present invention, after dropping the culture solution on a 1.5% agar plate (agar plate), and then left for 15 hours at 37 ℃ dye KI and I ₂ As a result of adding the containing solution, as shown in FIG. 2, it was confirmed that a clear zone was formed. Therefore, the strain isolated by the present invention was found to have a new specificity with excellent agarose resolution.

Example 2 (Identification of Isolated Strains)

Identification of the strains isolated by the present invention after the morphological and biochemical properties of the isolated bacteria, was identified according to the Bugie's Manual (Krieg, NR & Holt, JG (1984) Bergey's manual of systemic bacteriology) Reconfirmed as 16S ribosomal DNA (ribosomal DNA) nucleotide sequence.

16S ribosomal DNA sequencing method uses extracted DNA and universal primers (Yoon, JH, Lee, ST, Kim, SB, WY, Goodfellow, M. & Park, YH (1997). ) Restriction fragment length polymorphisms analysis of PCR amplified 16S ribosomal DNA for rapid identification of Saccharomonospora strains.Int. J. Syst.Bacteriol 47, 111-114) to amplify 16S RNA. Analyzer 377 (Perkin-Elmer) was used and shown in SEQ ID NO: 1 in the Sequence Listing to which the base sequence was attached.

Molecular systems such as cellular fatty acid composion analysis, quinone (HPLC) analysis, mol% G + C analysis (mol% G + C (HPLC) analysis), etc. As a result of the taxonomic analysis, it was found that the bacteria belong to the gamma-subdivision of Proteobacteria, and the phylogenetic tree in the taxonomic position is shown in Pseudomonas as shown in FIG. sp.) was found to be most similar to ND137 (NCBI accession number; AB052965).

From the above results, the isolated strain according to the present invention was identified as a novel strain and named Pseudomonas sp. BK1, which was deposited on July 19, 2002 at the Korea Microorganism Conservation Center (KCCM) (Accession No. : KFCC-11308).

Example 3 (Culturing of Isolated Strains)

In order to culture the strains isolated and identified in Examples 1 and 2, the culture medium was used by slightly modifying the Zobell medium (Zobell medium; Pseudomonas medium), the composition of which is 5 g of peptone, 1 g of yeast extract, and FePO. ₄ 0.1 g and 2 g of glucose were dissolved in 250 ml of distilled water, 750 ml of sterile seawater was added, and the final pH was adjusted to 7.4, followed by shaking culture at 20 to 25 ° C. for 5 to 7 days in aerobic condition.

Example 4 (Isolation of Chromosome DNA)

Pseudomonas sp. Isolated as described above. Wizard genomic DNA isolation kit (Promega) was used for chromosomal DNA for cloning the useful enzyme secreted from the BK1 strain, namely, agarose.

First, Pseudomonas sp. For general growth of the BK1 strain, shaking culture was carried out under aerobic conditions for 24 hours at 30 ℃, 250 rpm using 3 ml LB medium (1.0% peptone, 0.5% yeast extract, 1.0% sodium chloride, pH 7.0).

After sufficiently growing the cells, the cells obtained by centrifugation for 2 minutes were added with 0.6 ml of cellular nuclei lysis solution, and slowly suspended, incubated at 80 ° C. for 5 minutes, and cooled at room temperature.

0.003 ml RNase was added to the cell suspension, and the mixture was slowly mixed. After culturing at 37 ° C. for 30 minutes, 0.2 ml of protein precipitation solution was added thereto and cooled in ice for 5 minutes.

The pellet was then precipitated by centrifugation at 13,000 rpm for 3 minutes, and the supernatant was transferred to a new 1.5 ml tube, 0.6 ml of isopropanol solution was added to precipitate the DNA, and centrifuged at 13,000 rpm for 2 minutes to discard the supernatant carefully. Next, the DNA was dried and chromosomal DNA was isolated from the dissolved strain by adding TE buffer to the dried DNA.

Example 5 (Preparation of Recombinant Plasmid)

Recombinant DNA technology was based on the method of Sambrook et al. (Sambrook, J., Fritsch, EF & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). To shut-gun cloning the isolated chromosomal DNA, the chromosomal DNA was partially digested with restriction enzyme Sau3A1 and electrophoresed on 0.7% agarose gel.

After recovering DNA fragments corresponding to 3 to 5 kb, the purified fragments were transformed into E. coli JM109 (Takara) by inserting the purified fragment into E. coli expression vector pBlueskript SK (+) (Stratagene) digested with restriction enzyme BamHI. , LB agar plate (1.0% peptone, 0.5% yeast extract, 1.0% sodium chloride, 1.5% agar) and incubated for 15 hours at 37 ℃, and then the colonies where the degradation of agar is observed by naked eye to separate LB liquid medium Diluted with, plated on the LB agar plate and separated pure as a single colony.

In order to finally confirm the activity of agarose degrading enzymes from purely isolated bacteria, the isolated bacteria were incubated in LB liquid medium containing 100 ㎍ / ml of ampicillin (Sigma) for 15 hours at 37 ° C. The cells were collected and crushed by ultrasonic waves, and then the cell extract was dropped on the LB agar plate and left at 37 ° C. for 15 hours.

Then, it was confirmed that a clear zone was formed as shown in FIG. 4 by adding a solution containing KI and I ₂ , which were staining reagents. As a result, it was confirmed that the gene degrading agarose was accurately cloned and expressed. .

Example 6 (Isolation and Subcloning of Recombinant Plasmids)

Plasmid DNA was isolated by alkaline lysis method (Sambrook, J., Fritsch, EF & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Plasmid DNA was extracted from the E. coli bacterium, which was digested with various restriction enzymes (BamHI, BglII, EcoRI, PstI, KhoI, SalI, KpnI, SacI; Takara) and analyzed by electrophoresis on 1% agarose gel. As shown in FIG. 5, it was confirmed that an insertion fragment of about 2.6 kb was present at the BamHI site of the E. coli fermentation vector pBlueskript SK (+) as the sites without the insertion sites SacI, PstI, XhoI, XbaI, and HindIII.

Example 7 Preparation of Recombinant Agarose Degrading Enzyme Expression Recombinant Plasmid pET-Agr

A restriction enzyme map of the cloned gene was generated, and then the open reading frame (ORF) of the enzyme gene was used for the mass expression in Escherichia coli and the pET-3a vector plasmid containing the T7 promotor (Novagen). And mass production by IPTG (Sigma) induction.

To this end, a sense primer (5'-CCCATATGAGAGCACTACTGACGGCCGTTCTCG-3 ') containing an Ndel cleavage site (CATATG) and an antisense primer (5'-TGGATCCCGTTGTTAGTGCCGCAGCCACTGCT-G) containing a BamHI cleavage site (GGATCC) 3 ') was used, and the PCR was performed using 2.6 kb fragment as a template using Ex Taq polymerase (Takara) for 1 minute 30 seconds at 55 ° C, 45 seconds at 55 ° C and 1 minute at 72 ° C. The reaction was performed 35 times to amplify the ORF of 1,014 bp, and then about 1 kb of the amplified DNA fragment was extracted from agarose gel using Gel extraction kit-QiaexII (QIAGEN).

The fragment was inserted into the BamHI / NdeI cleavage site of pET-3a to prepare an agarose degrading enzyme expression vector pET-Agr. (Figure 5)

Example 8 (Sequence Analysis of Cloned Agarose Digestion Gene)

Nucleotide sequences of cloned genes were synthesized by Sanger et al. (Sanger, F., Nicklen, S. & Coulson, AR (1977) DNA sequencing with chain-terminating inhibitors, Proc. Acad. Sci. USA 74, 5463-5467). The ABI PRISM dye terminator cycle sequencing core kit was used to analyze the Applied Biosystems model 373A DNA sequencer.

As a result, it was confirmed that the agarase gene having an open reading frame of 2.6 kb was contained. Pseudomonas sp. The base sequence of the agarose degrading enzyme gene of BK1 is shown in SEQ ID NO: 2 in Sequence Listing.

Pseudomonas sp. According to the present invention. As a result of comparing the amino acid homology of agarose degrading enzymes of the agarose degrading enzyme gene of BK1, as shown in FIG. 6, Pseudomonas atlantica and Aeromonas sp. While the homology of the derived β-agarase was about 61%, the agarose degrading enzyme of the present invention was not found to have a high homology with that of other microorganisms. It was confirmed to belong.

Example 9 Preparation of Recombinant Escherichia Coli and Expression of Agarose Digestion Gene

After the expression vector pET-Agr prepared in Example 7 was transformed into E. coli JM109, the expression vector pET-Agr was separated from the transformants and finally transformed back into E. coli BL21 (DE3).

The transformant obtained above was sterilized with M9H medium (1 g of NH ₄ Cl, 1 g of KH ₂ PO _4, 3 g of Na ₂ HPO ₄ 12H ₂ O, 15 g of tryptone, and 5 g of sodium chloride per liter), followed by glucose 4 g, 1 M MgSO ₄ 1 ml, 50 μg / ml ampicillin), pre-incubated at 37 ° C. for 15 hours, then 1% inoculation of pre-cultured bacteria and incubated at 30 ° C. with absorbance of 0.6 at 600 nm. At this time, IPTG was added to a final concentration of 0.5 mM to induce expression of the agarose digestion gene for 3 hours.

Example 10 (Enzyme Activity Measurement)

To measure the activity of the enzyme, 10 μl of enzyme solution was added to 90 μl of 0.02% agarose substrate solution (pH 9.0 20 mM glycine NaOH buffer solution) and reacted at 30 ° C. for 30 minutes. 0.37 g of CuSO ₄ , 2.4 g of Na ₂ CO _3, 2.4 g of C ₄ H ₄ KNaO ₆ .4H ₂ O, 1.92 g of NaHCO ₃ , 19.2 g of Na ₂ SO ₄ were added and boiled for 10 minutes, followed by cooling to room temperature. Add 100 μl of the reagent ((NH ₄ ) 6Mo ₇ O ₂₄ 4H ₂ O 25 g, H ₂ SO ₄ 42 g, Na ₂ HAsO ₄ 7H ₂ O 3 g) per liter and centrifuge at 14,000 rpm for 2 minutes. 700 μl of a pH 9.0 20 mM glycine NaOH buffer solution was added to the separated supernatant, and the absorbance was measured at 660 nm. The enzyme activity was defined as 1 μmol of reducing sugar per 1 ml.

Example 11 (Isolation and Purification of Recombinase)

Cells were recovered by centrifuging 100 ml of the transformed Escherichia coli BL21 (DE3) containing the expression vector pET-Agr with IPTG (Sigma) to induce the expression of the recombinant protein. The suspension was suspended in pH 9.0), and the cells were disrupted by Sonytion (Vibra cell of Sonic & Materials), followed by centrifugation to separate the supernatant and used for purification of soluble protein.

All steps of enzymatic purification were carried out at 4 ° C., with 90% milk precipitate as first step and centrifugation to precipitate the precipitated protein in 20 mM glycine NaOH buffer (pH 9.0) and sufficiently dialyzed with the same buffer.

In the second step, ion exchange chromatography was performed using a DEAE-sepharose column. First, resin was sufficiently parallelized with 20 mM glycine NaOH buffer (pH 9.0), and then 5 mL of protein was loaded and NaCl concentration gradient (0 ˜0.5 M) was used to measure the activity of each fraction to recover only the fraction in which the activity appeared and used in the next purification step.

In the third step, the active fraction collected in the previous step was sufficiently dialyzed with 20 mM glycine NaOH buffer (pH 9.0), and then, using a Mono Q 5/5 column in a Fast protein liquid chromatography (FPLC) system (Pharmacia, Uppasala, Sweden). Once again, only the active fraction was recovered and recovered as shown in FIG. 7 (a).

Finally, the fraction showing the activity was sufficiently dialyzed with 20 mM glycine NaOH buffer (pH 9.0) and gel filtration using a superdex 200 column.

Then, the resultant electrophoresis resulted in the purification of a single band of pure recombinant agarose degrading enzyme compared to Escherichia coli BL21 (DE3) containing the control vector plasmid (pET 3a). Same as (c).

Figure 7 (c) shows the SDS-PAGE results of each step purified product for the recombinant agarose degrading enzyme, the first lane is the LMW marker (Pharmacia), the second lane is the water-soluble fraction derived from pET-Agr, third Lane is 90% saturated oil precipitate, lane 4 is the product after passing DEAE-Sepharose column, lane 5 is the product after Mono Q column chromatography (FPLC), lane 6 is the product after Superdex 200 column chromatography (FPLC) Means.

Example 12 (Temperature and pH Effect on Purified Enzyme)

As a result of investigating the characteristics of the purified agarose degrading enzyme using the measurement method used in Example 11, as shown in FIG. Showed.

In addition, the optimum pH is 9.0, as shown in Figure 8 (b) showed a pH stability in the range of pH 8.5 to 9.5.

Agarose degrading enzymes and genes derived from marine microorganisms according to the present invention can be applied to the development of high functional foods, pharmaceutical raw materials or feed additives by industrially mass-producing agarose degrading enzymes using the same.

In addition, it is possible to contribute to the development of environment-friendly products by using the useful enzymes derived from marine microorganisms according to the present invention in place of the conventional chemicals used in industrial sites.

In addition, the present invention is meaningful in utilizing marine resources that are not yet widely exploited.

Claims (9)

Novel microorganism Pseudomonas sp. BK1 with agarose resolution (KFCC 11308) Agarose Degrading Enzyme from Microorganism Pseudomonas BK1 of Claim 1 The method of claim 2, Amino acid sequence of the enzyme is characterized by the agarose degrading enzyme, characterized in that <SEQ ID NO: 4> The method of claim 2, Agarose degrading enzymes characterized by thermal stability ranging from 10 to 35 ° C. and pH stability ranging from pH 8.5 to 9.5 The gene encoding the agarose degrading enzyme of claim 2, wherein the agarose degrading gene, characterized in that described by <SEQ ID NO: 2> An expression vector pET-Agr comprising the agarose digestion gene of claim 5 and having a cleavage map as described in FIG. Recombinant Escherichia coli BL21 (DE3) transformed with the expression vector pET-Agr of claim 6 Recombinant agarose degrading enzyme prepared by culturing the recombinant E. coli of claim 7 A method of producing agarose degrading enzyme from the culture by culturing the recombinant E. coli of claim 7.