JP5392942B2 - Novel alkaline alginate lyase and its utilization - Google Patents

Description

  The present invention relates to a novel alkaline alginate lyase that is a component contained in brown algae and that can degrade alginic acid used in food additives and the like to lower the molecular weight, and a gene encoding the same.

  Alginic acid is a polysaccharide that is abundant in seaweeds such as brown algae and red algae, and is a kind of dietary fiber. Commercially available sodium alginate has been extracted from brown algae such as kelp and giant kelp. It is also known that bacteria such as Pseudomonas and Azotobacter produce alginic acid.

  In Japan, sodium alginate is an existing additive, and propylene glycol alginate and sodium alginate are designated as food additives. These are used in the food industry as thickening stabilizers and gelling agents for soups, beverages, and jelly, and are also incorporated in diet foods as dietary fibers. Besides foods, it is used in the medical field such as dental materials as a stabilizer for cosmetics, fibrous gel as a surgical thread, and calcium alginate gel as a wound dressing. Alginates are also used in the paper and textile industries, and calcium alginate is also used for immobilizing and encapsulating cells and enzymes, and is used in the fermentation and chemical industries.

  Such widely used alginic acid is known to vary in viscosity and various properties depending on its molecular weight (sugar chain length). It is known that a method using an alginate-degrading enzyme is effective for adjusting the sugar chain length (for example, see Patent Document 1). Alginic acid is also known to be the main component of biofilms produced by Pseudomonas aeruginosa (Pseudomonas aeruginosa), and it has also been proposed to develop alginate lyase as an enzyme agent for refractory infections involving biofilms. (For example, refer to Patent Document 2).

In order to obtain such alginic acid, a method of extracting alginic acid from various algae under alkaline conditions is generally performed. Therefore, if there is an enzyme that operates under a more alkaline condition than the conventional alginate lyase, it is considered that extraction, decomposition, and fractionation operations can be simplified. In addition, it can be easily estimated that the removal of biofilm is highly effective under alkaline conditions. However, the conventionally known alginate lyase is significantly different from the pectate lyase class, and it is well known that the optimum reaction pH is in the vicinity of neutrality. Non-Patent Document 1). Among the alginate lyases produced by bacteria, the only one with the most alkaline optimum reaction pH is Bacillus no. Alginate lyase produced by M-2 strain, and its optimum pH is around 9. This enzyme has been reported to desorb and degrade the β-1,4 bond of polymannuronic acid (see Non-Patent Document 2). In addition, alginate lyase encoded by chlorella virus (CVN1) has been reported to have an optimum reaction pH of 10.5. This enzyme has a molecular weight of 36800 and requires calcium ions for the reaction, but it is not clear which of polyguluronic acid and polymannuronic acid is cleaved (Non-patent Document 3).
Therefore, an enzyme that degrades highly alkaline alginate lyase produced by bacteria and works well under high alkaline conditions, in particular mannuronic acid guluronic acid block and polyguluronic acid, has been desired.

Japanese Patent No. 2926249 JP-A-9-9962 Wong et al., “Annu Rev Microbiol”, 54, 289-340, 2000 Horikoshi and Akiba, “Alkalophilic microorganisms, A new microbial world”, Japan Scientific Societies Press, p.137, 1982 Suda et al. FEMS Microbiol. Lett., 180, 45-53, 1999

  The present invention provides an alginate-degrading enzyme having activity under high alkaline conditions produced by bacteria in view of the current situation regarding alginic acid-degrading enzymes as described above, that is, a novel having an optimum reaction pH in a high alkaline region. An object of the present invention is to provide an alginate degrading enzyme and a gene encoding the same.

  The inventors of the present invention conducted intensive research to achieve the above-mentioned object, obtained a strain that produces a novel alkaline alginate lyase from various marine samples such as deep-sea bottom mud, and further obtained the alkaline alginate. The lyase was purified, various properties were examined, and a gene encoding this enzyme was successfully obtained, thereby completing the present invention.

That is, the present invention provides a novel alkaline alginate lyase having the following enzymological properties.
(1) Action:
It acts favorably on the mannuronic acid guluronic acid block and polyguluronic acid constituting alginic acid, and decomposes alginic acid by β elimination.
(2) Optimal reaction pH:
PH 10 in 50 mM glycine sodium hydroxide buffer and pH 9 in the presence of 0.2 M sodium chloride.
(3) The optimum reaction temperature is around 30 ° C.
(4) Molecular weight:
The molecular weight by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis is about 30,000.
(5) pH stability:
It is stable at pH 6-9 when it is kept at 30 ° C. for 60 minutes.

The present invention also includes an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or an amino acid sequence in which one or more amino acids in this sequence are deleted, substituted, or added, or SEQ ID NO: in the sequence listing. The above-mentioned novel alkaline alginate lyase having an amino acid sequence having 60% or more identity with the amino acid sequence represented by 1 is provided.

  The present invention also provides the novel alkaline alginate lyase described above, which is derived from Agariblance SP JAM-A1m.

Furthermore, the present invention includes the following groups (a) to (d):
(A) DNA encoding a protein having the amino acid sequence shown in SEQ ID NO: 1,
(B) DNA encoding a protein having an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 1 have been deleted, substituted, or added;
(C) DNA having the nucleotide sequence shown in SEQ ID NO: 2, or (d) encoding a protein that hybridizes with the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions and has alkaline alginate lyase activity DNA,
A gene encoding an alkaline alginate lyase selected from the group consisting of:

  The present invention also provides a recombinant vector containing any of the above genes and a microorganism transformed with the vector.

  Furthermore, the present invention also relates to the enzymatic property or the amino acid sequence, characterized in that Agariblance SP JAM-A1m or the transformed microorganism is cultured, and alkaline alginate lyase is collected from the culture solution. The present invention provides a method for producing an alkaline alginate lyase having

  The novel alkaline alginate lyase of the present invention has a high alginic acid decomposing activity in a high alkali range of pH 9-10, and particularly preferred to decompose manuronic acid guluronic acid block and polyguluronic acid that constitute alginic acid. It has characteristic properties not found in alginate-degrading enzymes.

  The alkaline alginate lyase of the present invention is an alginate-degrading enzyme having an enzymatic property as described above, particularly an optimum reaction pH in a high alkaline region of pH 9-10. Alginate is a polysaccharide composed of polyguluronic acid, polymannuronic acid, and a manuronic acid guluronic acid block which is a mixture of manuronic acid and guluronic acid. Divided into acid lyases. The alkaline alginate lyase of the present invention acts on the manuronic acid guluronic acid block and polyguluronic acid constituting the alginic acid, and also acts slightly on the polymannuronic acid, thereby decomposing alginic acid by β elimination. Among them, it is considered that it mainly acts on the α-1,4 bond of polyguluronic acid because of its high degradation rate for guluronic acid block and polyguluronic acid which is a polymer of guluronic acid.

  In addition, the alkaline alginate lyase of the present invention has an alginate decomposing activity in a high alkaline region of pH 9 to 10 in a 50 mM glycine sodium hydroxide buffer, and exhibits maximum activity at pH 10. In addition, by adding sodium chloride, the alginic acid decomposing activity is greatly increased to 20 times or more in the wide range of pH 7 to 10 when no addition is made, and the optimum pH is increased from 10 to 9 by the addition of sodium chloride. shift.

The alkaline alginate lyase of the present invention is active at a reaction temperature of 5 to 40 ° C., but the optimum reaction temperature is around 30 ° C. The addition of sodium chloride shows a 2.5-5 fold increase in decomposition activity at each temperature, but the optimum reaction temperature remains unchanged at 30 ° C.
The molecular weight of the alkaline alginate lyase of the present invention is about 30,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

  Further, the alkaline alginate lyase of the present invention has a stable activity in a relatively wide pH range from pH 6 to around pH 9 under the condition of constant temperature at 30 ° C. for 60 minutes. This pH stability shows a phenomenon that it decreases conversely by adding sodium chloride, and the activity is reduced to about 60% at the minimum compared to the case where no sodium chloride is added, and the stable range is narrowed from pH 5 to pH 8. .

Such an alkaline alginate lyase of the present invention is not particularly limited to the method shown below. For example, as an alginate lyase-producing bacterium, among the enzymes produced by certain species of microorganisms belonging to the genus Agaribolance. It was found to be found.
That is, as an example of microorganisms screened from various marine samples such as deep-sea bottom mud by the present inventors, Agariblance SP JAM-A1m strain, which is a bacterium isolated from sea-floor mud near Kagoshima Bay, can be mentioned. .
One of the characteristics of this JAM-A1m strain is that it grows as a white small colony on marine agar and produces a very powerful agar-degrading enzyme, which dissolves the agar medium during growth and storage in a refrigerator. As one.

As a result of specifying this strain on the basis of the base sequence encoding the 16S rRNA gene of this JAM-A1m strain, it was found to be 98.6% (1453 bases in 1473 bases match) with the 16S rRNA gene sequence of Agariblance Albus MKT187 strain, Agariblance SP JAMB-A11 strain and 98.4% (1411 bases match 1434 bases), Agariblance Albus MKT89 16S rRNA gene sequence and 99.9% (1377 bases match 1377 bases), Agariblance Albus MKT82 Although the sequence of the strain and 99.6% (1373 bases out of 1379 are identical) match each other, none of them completely matched, and it was proved to be a novel strain. Therefore, this fungus as Agariboransu sp JAM-A1m Ltd. (Agarivorans sp.JAM-A1m), National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center (Location: zip code 305-8566, Ibaraki Prefecture, Higashi 1-Chome, Tsukuba-shi Deposited in the middle 6) as deposit number FERM P-21397.

The alkaline alginate lyase of the present invention comprises a microorganism belonging to the genus Agaribolas, preferably the agaribrans sp. JAM-A1m strain, which is an alkaline alginate lyase-producing bacterium described above, and an alginate containing an assimilable carbon source, nitrogen source and other essential nutrients. It can culture | cultivate using the added culture medium and can obtain from the culture supernatant. However, since the alkaline alginate lyase of the present invention is an inducing enzyme, alginic acid or an acid hydrolyzate of alginic acid is essential as a carbon source necessary for the culture. Further, assimilating glucose, sucrose, maltose and the like can be added to promote growth. Moreover, as a nitrogen source which can be utilized, fish meat extract, polypeptone, tryptone, casamino acid, skim milk, corn gluten meal, corn steep liquor, soybean powder, NZ amine, and the like can be used.
The culture temperature is preferably 5 to 30 ° C., particularly preferably 30 ° C., and the pH is preferably 6 to 9, particularly preferably 7 to 8.5.

  Further, the alkaline alginate lyase of the present invention can be produced in large quantities by cloning from the chromosomal DNA of Agariblance sp. JAM-A1m strain and using an appropriate vector and host fungus.

  The alkaline alginate lyase of the present invention thus obtained is the amino acid sequence of SEQ ID NO: 1 in the sequence listing, or an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence. It is a protein that still retains alkaline alginate lyase activity. The number of amino acid deletions is preferably in the range of 1 to 10, and the addition of amino acids includes those with 1 to several amino acids added to both ends.

Further, the alkaline alginate lyase of the present invention includes a protein comprising an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 1 and retaining alkaline alginate lyase activity.

In addition, these equivalent amino acid sequences can be performed by comparing amino acid sequences using a known algorithm such as Lipman-Pearson method (Science, 227, 1435, 1985). Specifically, the identity can be obtained using a GENTYTYX-MAC (software development) search homology or maximum matching program.

  The base sequence encoding these equivalent amino acid sequences can be prepared using a known technique such as site-directed mutagenesis. For example, it can be prepared by introducing a mutation using a mutation introduction kit (Mutan-super Express Km kit: manufactured by Takara Bio Inc.) using site-directed mutagenesis.

According to the above method, when compared with the amino acid sequence of alkaline alginate lyase having the amino acid sequence shown in SEQ ID NO: 1 of the present invention and other known alkaline alginate lyase, alginate lyase produced by Klebsiella pneumonia And 53.2%, alginate lyase obtained from genome analysis of Vibrio spelendidus strain 12B01 ( Vibrio spelendidus 12B01) and 52%, obtained from genome analysis of Saccharophagus degradans strain 2-40 ( Saccharophagus degradans 2-40) It showed only 49.6% identity with the obtained alginate lyase. Moreover, it was less than 25% identity with the alkaline alginate lyase encoded by the previous chlorella virus (CVN1).

From these results, alkaline alginate lyase consisting of the amino acid sequence shown in SEQ ID NO: 1 of the present invention is judged to be a novel enzyme. That is, the present invention includes those having 60% identity or more and having alkaline alginate lyase activity when the corresponding sequence in the amino acid sequence shown in SEQ ID NO: 1 is appropriately aligned. Further, the alkaline alginate lyase of the present invention more preferably has an identity in the amino acid sequence shown in SEQ ID NO: 1 of 70% or more. More preferably it contains 80% or more identity , most preferably 90% or more identity .

  Next, the alkaline alginate lyase gene of the present invention may be any protein as long as it encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or a protein consisting of an amino acid sequence equivalent to this amino acid sequence. 2 or a gene containing a DNA encoding an alkaline alginate lyase hybridized under stringent conditions with the DNA consisting of the base sequence shown in SEQ ID NO: 2 is preferable.

  The gene encoding the alkaline alginate lyase of the present invention is cloned from, for example, a microorganism belonging to the genus Agaribolas, preferably an agariblance SP JAM-A1m strain by using, for example, the shotgun method or PCR method can do.

In order to produce alkaline alginate lyase using the gene of the present invention, the above-described alkaline alginate lyase gene is incorporated into any vector suitable for expressing the gene in the target host, and this recombinant vector is used. A host is transformed, the resulting transformant is cultured, and alkaline alginate lyase is collected from the culture solution.
The culture may be carried out by inoculating a medium containing a carbon source, a nitrogen source and other essential nutrients that can be assimilated by the host microorganism, and following a conventional method.

  Collection and purification of alkaline alginate lyase from the culture obtained by culturing can be performed according to a general method. The alkaline alginate lyase of the present invention accumulates in the culture supernatant, and the cells are removed from the culture solution by centrifugation or filtration, and the remaining culture supernatant solution is usually used for ammonium sulfate precipitation, ultrafiltration and the like. The target enzyme can be concentrated by means of the method. The enzyme solution thus obtained can be used as it is or after drying it as a powder. Furthermore, these enzyme solutions can be purified by a combination of various column chromatography, and further crystallized and granulated by known methods.

  More specifically, separation and purification of this crude enzyme solution can be performed, for example, as necessary, by separation means such as salting-out method, precipitation method, ultrafiltration method, ion exchange chromatography, isoelectric point chromatography, hydrophobicity. That have been further separated and purified by a combination of known methods such as affinity chromatography, gel filtration chromatography, adsorption chromatography, affinity chromatography, and reverse phase chromatography can be used.

  Next, the present invention will be described in more detail with reference to examples. In the examples, unless otherwise noted, “%” is mass%.

In the following Examples, the activity of alkaline alginate lyase was determined by the following method.
That is, an enzyme was added to a reaction solution consisting of 100 mM glycine-sodium hydroxide buffer (pH 10) and 0.1% sodium alginate (total amount 0.5 mL), and the reaction was performed at 30 ° C. for 15 minutes. Thereafter, 2 mL of 5 mM hydrochloric acid was added to stop the reaction, and the absorbance of this reaction solution at 235 nm was measured. One unit (1 U) of enzyme was an amount that increased the absorbance by 0.01 per minute under the above conditions. The protein was measured using a protein assay kit (Bio-Rad) with bovine serum albumin as a standard.

Example 1: Screening for Alginate-Degrading Enzyme Producing Bacteria 25 kinds of depths collected from around Kagoshima Bay in a medium in which 0.2% w / v sodium alginate (manufactured by Wako Pure Chemical Industries ) was added to marine agar 2216 (Difco) A certain amount of seabed mud was smeared and cultured in a thermostatic bath at 30 ° C. A soft agar solution composed of 0.2% sodium alginate, 0.8% agar, and 50 mM morpholinopropanesulfonic acid-sodium hydroxide (hereinafter referred to as “MOPS”) buffer (pH 7) is poured onto the plate on which colonies appeared, and solidified. And then left at 30 ° C. for 24 hours. Alginate lyase-producing bacteria were poured with a 0.2% cetylpyridinium chloride solution to select colonies that formed halos. The selected strain was purified by passing the selected colony to marine agar several times. Next, the selected strain was inoculated into a test tube containing marine broth containing 0.2% sodium alginate, cultured at 30 ° C. for 1 to 3 days, and then enzyme activity was measured. For the culture solution that clearly showed activity, the activity was measured at pH 5 (acetate buffer solution), pH 7 (MOPS buffer solution), pH 9 (glycine-sodium hydroxide buffer solution) with the buffer solution, and the most in the alkaline region. The A1m strain was selected as an enzyme producing bacterium exhibiting high activity.

Example 2: Determination of 16S rRNA gene sequence of JAM-A1m strain The 16S rRNA gene of JAM-A1m strain obtained by the screening of Example 1 was determined as follows. That is, a single colony grown on marine agar was picked with a toothpick and suspended in 25 μL of a PCR buffer solution to prepare a template. During DNA thermal cycler (Gene Amp PCR system 9700: manufactured by ABI PRISM) using 27F (5'-AGAGTTTGATCCCTGCTCAG-3 ') and 1492R (5'-GGCTACTCTGTTACGACTT-3') designed from bacterial 16S rRNA gene consensus sequence PCR was performed using LA Taq DNA polymerase (manufactured by Takara Bio Inc.). The reaction was conducted at 96 ° C. for 2 minutes, followed by 30 cycles of reaction at 96 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 1.5 minutes, and finally held at 72 ° C. for 7 minutes. The PCR product is purified by treatment with shrimp alkaline phosphatase and exonuclease (manufactured by GE Healthcare), and the nucleotide sequence is obtained using a DYDynamic ET Terminal Sequencing kit (manufactured by GE Healthcare) and a DNA sequencer (Mega BACE 1000: manufactured by GE Healthcare). It was determined. The obtained base sequence is shown in SEQ ID NO: 3.

Example 3 Purification of Alkaline Alginate Lyase The JAM-A1m strain selected in Example 1 was treated with 0.5% polypeptone S (Nippon Pharmaceutical), 0.5% yeast extract, 2% sodium chloride, 0.02% sulfuric acid. The mixture was cultured with shaking in a liquid medium (pH 7) supplemented with magnesium heptahydrate, 0.002% manganese chloride, 0.01% monopotassium phosphate and 0.2% sodium alginate for 24 hours. The culture supernatant (2 L) is concentrated with hollow fiber, further diluted with 10 mM Tris-HCl buffer (pH 7.5), concentrated, and then equilibrated with 50 mM Tris-HCl buffer (pH 7.5). The enzyme was eluted by a concentration gradient elution method using potassium chloride. The active fractions were collected, added to ammonium sulfate to a final concentration of 2M, and attached to a butyl Toyopearl column (Tosoh) equilibrated with 10 mM Tris-HCl buffer (pH 7.5) containing 2M ammonium sulfate. It was. The enzyme was eluted with a concentration gradient from 2M to 0.5M ammonium sulfate. This purification operation was repeated, and one protein band was obtained by SDS-polyacrylamide gel electrophoresis. The result is shown in FIG.

Example 4 Enzymatic Properties of Alkaline Alginate Lyase (1) Action Alginate is a polysaccharide composed of polyguluronic acid, polymannuronic acid and guluronic acid block, and guluronic acid lyase depending on which alginate-degrading enzyme degrades it. And mannuronic acid lyase. There are also lyases that can be degraded by both. Thus, alginic acid was acid hydrolyzed according to the method of Haug et al. (Acta Chem. Scand., 20, 183-190, 1966) and separated into polyguluronic acid and polymannuronic acid fractions. The fraction of guluronic acid block of mannuronic acid was prepared according to another method by Haug et al. (Acta Chem. Scand., 21, 691-704, 1967).
According to the enzyme activity measurement method shown in paragraph No. [0037], the enzyme reaction was performed with 0.1% of each substrate in accordance with the enzyme activity measurement method shown in paragraph [0037]. The performance was evaluated. As a result, assuming that the degradation activity for sodium alginate is 100%, the degradation activities for guluronic acid block, polyguluronic acid, and polymannuronic acid are 131.7 ± 16.5%, 83.3 ± 16.9%, and It was 27.3 ± 13.4%. Therefore, it was found that the alkaline alginate lyase of the enzyme of the present invention is an enzyme that prefers the glucuronic acid block and polyguluronic acid block and decomposes by β-elimination. From this, it is considered that this enzyme is likely to act on the α-1,4 bond of the mannuronic acid guluronic acid block and polyguluronic acid constituting alginic acid.

(2) Molecular Weight As can be seen from SDS-polyacrylamide gel electrophoresis of the purified enzyme obtained in Example 3, the molecular weight of the enzyme of the present invention was about 30000. As a molecular weight marker, Precision Plus Protein Standards (manufactured by Bio-Rad) was used.

(3) Amino terminal amino acid sequence After SDS-polyacrylamide gel electrophoresis of the purified enzyme, the enzyme was blotted onto a PVDF membrane (Imobron P; manufactured by Millipore). The blotted protein portion was subjected to an amino acid sequencer (497HT type; manufactured by Applied Biosystems), and the amino acid sequence was determined. As a result, the amino terminal amino acid sequence was Ala-Thr-Thr-Thr-Pro-Ala-Glu-Val-Leu-Asp-Leu-Ser.

(4) Optimum reaction pH
In various buffer solutions of 100 mM MOPS buffer (pH 6-8), 100 mM Tris-HCl buffer (pH 7-9), 100 mM glycine-sodium hydroxide buffer (pH 8-11), 30 ° C., 15 minutes, The alginic acid decomposition reaction by the enzyme of the present invention was performed, and the enzyme activity was measured. The results are shown in FIG. 2 as relative activity with the maximum enzyme activity as 100%. In FIG. 2, “●: solid line” indicates a case where only various buffer solutions of 100 mM are used, and “◯: dotted line” indicates a case where 0.2 M sodium chloride is added to the 100 mM buffer solution.
The alkaline alginate lyase of the present invention had activity in a pH range of 9 to 10 in 100 mM glycine-sodium hydroxide buffer, and showed the highest activity at pH 10. In addition, when 0.2 M sodium chloride was added to the reaction solution, the enzyme was activated in the range of pH 7 to 10, and the activity increased greatly by a maximum of 20 times, and the pH was highest with 100 mM glycine-sodium hydroxide buffer at pH 9. The activity was high and the optimum reaction pH value shifted to pH9.

(5) Optimum reaction temperature Activity was measured at various temperatures from 5 to 45 ° C in a 100 mM glycine-sodium hydroxide buffer (pH 10). The results are shown in FIG. 3 as relative activity with the maximum enzyme activity as 100%. In FIG. 3, “●: solid line” indicates the case where only 100 mM glycine-sodium hydroxide buffer is used, and “◯: dotted line” indicates the case where 0.2 M sodium chloride is added to the 100 mM glycine-sodium hydroxide buffer. .
As can be seen from the results, the alkaline alginate lyase of the present invention was active at a temperature of 5 to 40 ° C., and the optimum reaction temperature was 30 ° C. Similarly, in 100 mM glycine-sodium hydroxide buffer (pH 9) containing 0.2 M sodium chloride, significant enzyme activation was observed, and the activity at 30 ° C. increased by about 2.5 times. The temperature was not changed at 30 ° C.

(6) pH stability The enzyme of the present invention was added to a system to which 0.2 M sodium chloride was added or not added in various 20 mM buffers, and the mixture was incubated at 30 ° C. for 60 minutes, followed by rapid cooling in ice water. Thereafter, the residual activity was measured by the method shown in paragraph [0037].
However, the following buffer solutions were used for each pH:
pH 4-6: acetate buffer,
pH 6-8: MOPS buffer,
pH 7-9: Tris-HCl buffer,
pH 9-11: Glycine-sodium hydroxide buffer,
pH 11 to 12.5: phosphate-sodium hydroxide buffer,
The results are shown in FIG. 4 as relative activity with the maximum residual activity as 100%. In FIG. 4, “●: solid line” indicates a case of various buffer solutions of 20 mM, and “◯: dotted line” indicates a case of addition of 0.2 M sodium chloride to various buffer solutions of 20 mM.

  As can be seen from the results, the alkaline alginate lyase of the present invention was stable up to around pH 6-9 in the system without sodium chloride. On the other hand, stability was not unexpectedly maintained in the presence of sodium chloride, and the residual activity showed only about 60% of the activity in the system without sodium chloride even in a narrow range of pH 6-7. It has been found that sodium chloride contributes to enzyme activation but does not contribute to stability.

Example 5: Cloning of Alginate Lyase Gene and Determination of Nucleotide Sequence From Agariblance SP JAM-A1m strain cultured with marine broth 2216 at 30 ° C for 1 day, Saito and Miura (Biochim. Biophys. Acta, 72, 619-629, 1963), chromosomal DNA was prepared. This was completely digested with the restriction enzyme Sau 3AI (Roche) to obtain a DNA fragment of 0.75 kb to 3 kb. These fragments were ligated to a vector pUC18 that had been cleaved with restriction enzyme Bam HI (manufactured by Roche) in advance to prepare a gene library, and then Escherichia coli DH5α was transformed. The transformed strain was grown on an LB medium containing 0.1% alginic acid and 100 ppm ampicillin in the LB medium.

  The expression of the enzyme of the present invention was confirmed by the multilayer method shown below. That is, after the transformant was grown, a soft agar solution composed of 0.2% alginic acid, 50 mM glycine-sodium hydroxide (pH 9), 0.2M sodium chloride and 0.8% agar was overlaid and solidified, It was left for 1 day at night. A 0.1% cetylpyridinium chloride solution was poured, and a strain having a dissolution spot due to alginic acid decomposition around the colony was selected. The selected transformant was planted and purified in an LB medium containing 100 ppm of ampicillin, liquid culture was performed, and a plasmid was extracted using a High Pure Plasmid Isolation kit (Roche). Primer A (SEQ ID NO: 5′-CAAGGCGATTATAAGTTGGTAACG-3 ′) and primer B (SEQ ID NO: 5′-CTTCCCGCTCGTATGTTGTTGTG-3 ′) designed based on the sequence outside the multicloning site of pUC18 in order to obtain an insert fragment ) Was used to amplify the inserted fragment by PCR using the plasmid extracted using (as a template after heat denaturation at 96 ° C. for 2 minutes, 96 ° C. for 20 seconds and 68 ° C. for 3 minutes for 30 cycles). Cloning was performed (TA cloning kit: manufactured by Invitrogen). Escherichia coli TOP10 was transformed with the obtained plasmid, grown on an LB medium containing X-gal and ampicillin, and a transformant was selected using β-galactosidase production as an indicator. The obtained transformant was subjected to colony PCR using the above primers A and B, and the amplified gene fragment was purified by treatment with shrimp alkaline phosphatase and exonuclease (manufactured by GE Healthcare), and the DYDynamic ET Terminal Sequencing kit ( The base sequence was determined using a DNA sequencer (manufactured by GE Healthcare) and a DNA sequencer (Mega BACE 1000: manufactured by GE Healthcare).

  Using the primers designed on the basis of the obtained base sequence, the base sequence of the fragment amplified using the chromosomal DNA of Agariblance SP JAM-A1m as a template is determined, and the sequence encoding the alkaline alginate lyase containing the signal sequence therein is determined. An open reading frame consisting of 930 base pairs shown in SEQ ID NO: 2 in the column table was found. The amino acid sequence shown in SEQ ID NO: 1 in the sequence listing was determined from this base sequence. In the determined sequence consisting of 309 amino acids, the N-terminal amino acid sequence of the purified enzyme obtained in Example 4 (2) (21st to 32nd amino acids in SEQ ID NO: 1) was found. It was done.

Example 6 Production of Alkaline Alginate Lyase by Escherichia coli Transformant The upstream part of the gene encoding alkaline alginate lyase determined in Example 5 is represented by primer C (SEQ ID NO: 6′-AAT GGATCC ATCGGGTCTAATCCAGTTTGATGTTC-3 ′, underlined Part shows newly added Bam HI site), primer D (SEQ ID NO: 7: 5′-AAT GGATCC TTCCGCTTTAGCGTTGGTAAAACGC-3 ′, underlined shows newly added Bam HI site), and Agariblance SP JAM- PCR was performed using the chromosomal DNA of the A1m strain as a template. PCR conditions were heat denaturation at 96 ° C. for 2 minutes, 30 cycles of 96 ° C. for 20 seconds and 68 ° C. for 3 minutes. The obtained PCR amplified fragment was purified with Wizard SV Gel and PCR Clean-up system (manufactured by Promega), digested with restriction enzyme Bam HI, and then ligated to the similarly treated vector pUC18 to prepare a recombinant plasmid. did. This was used to transform E. coli DH5α strain. The obtained transformed strain was grown on an LB medium containing 100 ppm of ampicillin, and then production of alkaline alginate lyase was confirmed by the multilayer method shown in Example 5.

A comparison of the properties of the enzyme of the present invention as described above and a conventionally known alginate lyase is as follows. That is, the highest identity in the amino acid sequence Klebsiella Noimonia subsp aerogenes type25 (Klebsiella pneumonia subsp. Aerogenes) production to alginate lyase (Non-Patent Documents 3 and 4) has a molecular weight of 31400, de polyguluronic acid β The optimum reaction pH is around 7, and the optimum reaction temperature is around 36 ° C. In addition, it has been shown that this enzyme is also activated by salt, but the degree of activation is only about 1.6 times in the presence of 0.3 M salt. In contrast, the enzyme of the present invention has a molecular weight of about 30,000, an optimum reaction pH of 10, and an optimum reaction temperature of 30 ° C. The enzyme of the present invention is activated 10 times or more at pH 9 by adding 0.2 M sodium chloride.
Moreover, although the high alkali alginate lyase derived from the chlorella virus (CVN1) described above has a molecular weight of 36800 and an optimum reaction pH of 10.5, it requires calcium for the reaction. In the reaction, calcium is not required, and no identity is recognized in both amino acid sequences, and it is considered that the enzyme is essentially different from the enzyme of the present invention.
As described above, the enzyme of the present invention is a novel enzyme whose properties are significantly different from those of conventionally known alginate lyases.

  Unlike the conventionally known alginic acid lyase, the alkaline alginate lyase of the present invention has a high alginic acid decomposing activity in an alkaline region of pH 9-10. Alginic acid is generally produced from seaweed by extraction under alkaline conditions. However, by utilizing the enzyme of the present invention, alginic acid can be decomposed and reduced in molecular weight under alkaline conditions. , Decomposition and fractionation can be performed more easily. For this reason, in various food fields, cosmetics / medical fields, etc., the viscosity and other properties of alginic acid used can be adjusted by using the enzyme of the present invention, which is useful.

It is a figure which shows the result of SDS-PAGE of the enzyme of the present invention. It is a figure which shows the relationship between the enzyme activity of this invention enzyme, and pH. It is a figure which shows the relationship between the enzyme activity of this invention enzyme, and temperature. It is a figure which shows the relationship between enzyme activity and pH for evaluating the pH stability of this invention enzyme.

Claims (6)

It has the following enzymatic properties and has an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or an amino acid sequence in which 1 to 10 amino acids in this sequence are deleted, substituted, or added. Alternatively, a novel alkaline alginate lyase having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing:
(1)
Action:
It acts favorably on the mannuronic acid guluronic acid block and polyguluronic acid constituting alginic acid, and decomposes alginic acid by β elimination.
(2)
Optimum reaction pH:
PH 10 in 50 mM glycine sodium hydroxide buffer and pH 9 in the presence of 0.2 M sodium chloride.
(3)
The optimum reaction temperature is around 30 ° C.
(Four)
Molecular weight:
The molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is about 30,000.
(Five)
pH stability:
It is stable at pH 6-9 when it is kept at 30 ° C. for 60 minutes. The novel alkaline alginate lyase according to claim 1, which is derived from Agariblance SP JAM-A1m. A group consisting of the following (a) to (d):
(A) DNA encoding a protein having the amino acid sequence shown in SEQ ID NO: 1,
(B) a DNA encoding a protein having an amino acid sequence in which 1 to 10 amino acids of the amino acid sequence shown in SEQ ID NO: 1 have been deleted, substituted or added;
(C) DNA having the nucleotide sequence shown in SEQ ID NO: 2, or (d) encoding a protein that hybridizes with the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions and has alkaline alginate lyase activity DNA,
A gene encoding an alkaline alginate lyase selected from   A recombinant vector containing the gene according to claim 3.   A microorganism transformed with the vector according to claim 4. Agariboleans sp JAM-A1m, or the transformed microorganism according to claim 5, wherein the alkaline alginate lyase is collected from the culture solution, and the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, Alternatively , a method for producing an alkaline alginate lyase having an amino acid sequence in which 1 to 10 amino acids in this sequence are deleted, substituted, or added .