KR101608977B1 - Protease of sequence 4 having algicidal activity, gene encoding the same and algicidal formulation comprising the same - Google Patents

Abstract

The present invention relates to a germicidal active protein degrading enzyme, a gene encoding the germicidal active protein degrading enzyme, and a fungicide containing the same, and the proteinase of the present invention is excellent in fungicidal activity, .

Description

An algicidal activity protease comprising SEQ ID NO: 4, a gene encoding the same, and an algicide comprising the same {PROTEASE OF SEQUENCE 4 HAVING ALGICIDAL ACTIVITY, GENE ENCODING THE SAME AND ALGICIDAL FORMULATION COMPRISING THE SAME}

The present invention relates to a protease that inhibits the growth of red tide organisms and a gene encoding the same.

The causative species that cause red tides in the ocean are composed of flagella algae such as Bacillariophyceae, Raphidophyceae, and Dinophyceae, and flagella algae in particular occupy most of them. Diatoms can grow on agar-containing medium, whereas flagella algae cannot grow, so studies of algicidal bacteria using the soft-agar overlay method have been limited.

Alteromonas isolated from marine and coastal environments are Alteromonas, Bacillus. ( Bacillus ), Cowlobacter ( Caulobacter ), Cytopaga ( Cytophaga ), Flavobacterium ( Flavobacterium ), Pseudo Alteromonas ( Pseudoalteromonas ), Pseudomonas ( Pseudomonas ), Saphorospira ( Saporospira ), vibrio ( Vibrio ) The genus is the main-slave. The saljo bacteria separated from the ocean is classified haksang Flavobacterium-Saito wave complex (complex) and proteobacteria acids (Proteobacteria branch), for example, Alteromonas, Pseudomonas, Pseudomonas Alteromonas, Spira and Vibrio Sapporo It can be classified as In general, species of the flavobacterium-cytopaga complex, which have the ability to degrade various biomacromolecules, can use the cell walls of algae and the excrements released from them as a nutrient source. It is thought to have a close relationship with plankton.

According to the results of the studies so far, the killing mechanisms of acaricides can be divided into two categories. The first mechanism is a contact mechanism by which algicidal bacteria attach to the surface of red tide organisms and energize red tide organisms. Bell and Alteromonas Paper is known as a representative example. The second mechanism is a mechanism by which algicidal bacteria secrete algicidal substances to the outside of cells to inhibit or aid the growth of red algal organisms, and most of the algicidal bacteria are included therein. Most algicidal bacteria isolated from the ocean have a mechanism to produce extracellular substances, but there are few reports on the separation, identification and mechanism of these substances.

The present inventors isolated the Kordia algicida OT-1 strain, an acaricide that kills Skeletonema costatum , which is a red tide organism, and as of April 24, 2008, the organisms within the Korea Research Institute of Bioscience and Biotechnology. It has been deposited with the Resource Center (KCTC, Korean Collection for Type Cultures) (accession number KCTC 11320BP; accession date 2008.4.8).

In addition, as a result of investigating the similarity of 16S rRNA with strains registered in NCBI (national center for biotechnology information), and analyzing the base sequence of 16S rRNA, it was confirmed that the strain is a novel strain belonging to Flavobacteria. (Sohn et al., Int. J. Syst. Evol. Microbiol ., 54:675-680, 2004).

An object of the present invention is to provide a novel protein capable of inhibiting the growth of red tide organisms and a gene encoding the same.

Another object of the present invention is to provide an algicide containing the protein as an active ingredient.

Another object of the present invention is to provide a method of treating a red tide area using the above formulation.

The present invention provides an algicidal activity protease having an amino acid sequence of any one of SEQ ID NOs: 1, 4 to 7, 26 and 27, and a gene encoding the protease.

In addition, the present invention provides an algicide containing the proteolytic enzyme as an active ingredient.

In addition, the present invention provides a method for controlling red tide, including the step of treating the algicide in the area where the red tide phenomenon has occurred. In more detail , using a Cordia algicida (Kordia algicida ) OT1 strain (KCTC 11320BP), the step of producing any one or more proteases selected from the group consisting of SEQ ID NOs: 1, 7 and 27; And it provides a method for controlling red tide, comprising the step of introducing an algicide containing the produced protease as an active ingredient into the sea. The algicide is introduced at the same time as the Cordia algicida (Kordia algicida ) OT1 strain (KCTC 11320BP) producing the protease, or from the culture of the Cordia algicida (Kordia algicida ) OT1 strain (KCTC 11320BP) Pure or partially isolated protease may be added as an active ingredient. During the culture, a medium containing 0.5-6% NaCl, 0.05g-1.2g/l ^{Mg 2+ , and 0.05-1.0g/l Ca 2+} may be used to make it suitable for seawater conditions.

As described above, since the algicidal activity protein of the present invention effectively inhibits the growth of red tide organisms, it can be usefully used for controlling red tide in a red tide occurrence area.

Figure 1 is a result of separating the protein of the algicidal bacteria Kordia algicida OT-1 strain (KCTC 11320BP) by non-denaturing (native)-PAGE (12%).
2 is a result of separating the proteins of band Nos. 3 and 10 showing algicidal activity among proteins isolated from the Cordia aljishida OT-1 strain (KCTC 11320BP) by SDS-PAGE under denaturing conditions.
Figure 3 shows the structure of the protein KAOT1_10476 having the amino acid sequence of SEQ ID NO: 1.
4 is a schematic diagram of various genetic constructs for characterizing the KAOT1_10476 protein.
5 is a result of confirming the expression level by SDS-PAGE after expressing the 10476_1 to 10476_9 proteins.
6 shows the results of refolding to solubilize the 10476_1 and 10476_6 to 10476_9 proteins.
7 is a result of confirming the proteolytic activity of proteins 10476_1 and 10476_6 to 10476_9.
8 is a result of observing the processing of the 10476_8 protein for one week.
9 and 10 are results confirming the proteolytic activity of 10476_7 protein according to temperature and pH.
11 and 12 are results confirming the proteolytic activity of 10476_7 protein according to metal ions (zinc ions or calcium ions).
13 and 14 show KAOT1_10476 protein and various marine microorganisms ( Flavobacterium johnsoniae ) UW101, Croceibacter atlanticus HTCC2559, Leeuwenhoekiella blandensis MED217 and This is the result of comparing the homology of Cordy.
FIG. 15 is a result of amplifying a gene for expressing an algicidal protein of marine microorganisms having homology to KAOT1_10476 protein by PCR.
16 is a result of confirming the expression of the algal activity protein of marine microorganisms having homology to the KAOT1_10476 protein.

The present invention provides an algicidal activity protease having the amino acid sequence of SEQ ID NO: 1 isolated from the algicidal bacterium Cordia algicida (Kordia algicida) OT-1 strain (KCTC 11320BP).

In the amino acid sequence of SEQ ID NO: 1, amino acids 1 to 27 correspond to a signal peptide, amino acids 28 to 98 correspond to a processing peptide, and amino acids 99 to 278 correspond to a mature protein ( mature protein), and the mature protein includes a metal binding site (200 to 217 amino acids) ( FIG. 3 ).

The amino acid sequence of the protein may be substituted, added or deleted within a range that does not affect the function of the protease, and only a part of the protein may be used depending on the purpose, and the amino acid sequence modified as described above may also be used in the present invention. It is included in the scope of. Accordingly, the present invention includes a polypeptide having an amino acid sequence substantially identical to that of the protein and fragments thereof.

Accordingly, the present invention provides the amino acid sequence of SEQ ID NO: 27, which does not include a signal peptide in the protein, and does not include a signal peptide and a processing peptide, an algicidal proteolytic enzyme having the amino acid sequence of SEQ ID NO: 26. It also includes an algicidal activity protease.

The gene encoding the protein has various modifications to the coding region within a range that does not change the amino acid sequence of the protein due to the degeneracy of the codon or considering the preferred codon in the organism to express the protein. This can be made, and various modifications or modifications can be made within a range that does not affect the expression of the protein even in portions other than the coding region, and such modified genes are also included in the scope of the present invention. Accordingly, the present invention includes a polynucleotide having substantially the same sequence as the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 and a fragment thereof, and preferably includes a polynucleotide having the nucleotide sequence of SEQ ID NO: 2 do.

In addition, polynucleotides having the nucleotide sequences of SEQ ID NOs: 28 and 29, which encode algicidal proteases having the amino acid sequences of SEQ ID NOs: 26 and 27, are also included.

The proteolytic enzyme of SEQ ID NO: 1 has better proteolytic activity when it additionally includes a processing peptide in addition to the mature protein portion, and the protein including the processing peptide and the mature protein is self-cleaving over time. ) Is converted into a mature protein ( FIGS. 7 and 8 ), and it can be seen that the C-terminus of the mature protein plays a very important role in the processing of the protein.

In addition, the proteolytic enzyme of the present invention is at a pH of 6.5 to 8, preferably at a pH of 7 to 8, and at a temperature of 18 to 28°C, preferably at 20 to 23°C, most preferably at 20°C; And 80 to 100 mM of calcium ions exhibited optimal activity in the presence of calcium ions ( FIGS. 9, 10 and 12 ), and it was confirmed that zinc ions inhibit the activity of proteases ( FIG. 11 ).

In addition, the protease of the present invention having the amino acid sequence of SEQ ID NO: 1 is Cochlodinium polykrikoides , Thalassiosira sp., Heterosigma akashiwo , skeletal shows the retrograde nematic Costa lithium (Skeletonema costatum), Alexandria Solarium (Alexandrium sp), caviar Toshio Ross (Chaetoceros cuvisetus), luggage saljo excellent activity against algae, such as titanium nodi (Gymnodium sp).

The protease having the amino acid sequence of SEQ ID NO: 1 is Flavobacterium johnsoniae UW101 (ZP_01247095, SEQ ID NO: 4), Croceibacter atlanticus HTCC2559 (ZP_00951344, SEQ ID NO: : 5) and Leeuwenhoekiella blandensis MED217 (ZP_01059780, SEQ ID NO: 6) shows 40% or more identity in a gene coverage of 80% or more, and in particular, a mature protease after the processing peptide Portions show a high homology of at least 50% ( FIG. 13 ). In addition, it also shows high homology with the KAOT1_11562 gene (SEQ ID NO: 7) of Kordia algicida (FIG. 14 ).

In addition, the expression form and activity of these proteins are almost similar to those of the protein having the amino acid sequence of SEQ ID NO: 1 ( FIG. 16 ).

As mentioned above, since the gene encoding each protein can be modified or modified within a range that does not change the amino acid sequence of the protein, the present invention encodes the amino acid sequence of SEQ ID NOs: 4 to 7 And polynucleotides having substantially the same sequence as each of the nucleotide sequences and fragments thereof, and these are preferably polynucleotides having the nucleotide sequences of SEQ ID NOs: 8 to 11.

Since the algicidal proteolytic enzyme of the present invention exhibits excellent algicidal activity, the protein and the gene encoding it can be usefully used for red tide control.

Accordingly, the present invention also provides an algicide containing as an active ingredient a protein having an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1 and 4 to 7. The active ingredient may be included in about 1 to 99% by weight based on the weight of the total composition.

The present invention also provides a method for controlling red tide by treating the algicide in seawater where red tide has occurred.

In the method of the present invention, the amount of the algicide used is variable depending on the degree of accumulation of organic matter, the degree of occurrence of red tide, and the like.

The method of using the formulation may be applied to various methods such as spraying on the sea level or spraying on the seafloor with a pipe, but is not limited thereto. In this case, the method includes the state of the algicide and seawater to which the algicide is applied. Considering the condition of the lake, it can be used appropriately.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Example 1 : Culture of algicide

The algicidal bacteria Kordia algicida OT-1 strain (KCTC 11320BP) was prepared in ZoBell 2216e solid medium (Oppenheimer, CH, et al., J. Mar. Res., 11:10-18 , 1952]), and then inoculate 100 ml of Zobel 2216e liquid medium (see Oppenheimer, CH, et al., 1952) in an amount of 3% (v/v) and inoculate 150 at 25°C. A pre-culture solution was prepared by shaking culture for 1 day at rpm. The culture solution was added at 3% (v/v) to a 5 liter pot fermentor (Korea Fermentation Engineer) containing 3 liter of Zobel 2216e liquid medium, and then cultured under conditions of 25° C., 300 rpm and 0.5 vvm.

Example 2 : Preparation of red tide creatures

The host organisms for the search for algicidal bacteria are Cochlodinium polykrikoides , Thalassiosira sp., Heterosigma akashiwo , and Skeletonema costatum . , Alexandria Solarium (Alexandrium sp), caviar Toshio Ross (Chaetoceros cuvisetus), luggage nodi titanium was used a (Gymnodium sp), each bird a F / 2 broth _{(NaNO 3 0.075 g, NaH 2} PO 4? H 2 O 0.005 g, Na ₂ SiO ₃ ?9H ₂ O 0.030 g, 1 ml F/2 trace metal solution, 1 ml F/2 vitamin solution and 1,000 ml seawater; Stein, Handbook of phycological methods, Cambridge Univ. Press, 1973). After culturing until the logarithmic growth phase by using, it was prepared as follows for exploration.

Red tide organisms are diluted with F/2 liquid medium so that the final density becomes a fluorescence intensity of 0.05 (excitation wavelength; 434 ㎚, emission wavelength; 670 ㎚) using a fluorescent photometer (Hitachi, Japan), and then a test tube (110 ㎜× 10 mm) to prepare a test tube for the MPN (most probable number) method by dispensing 2.7 ml each.

Example 3 : Selection of algicidal protein

<3-1> Purification and isolation of algicide protein

In order to separate the agicidal activity protein of the agicidal bacteria cultured in Example 1, the algicidal bacteria cultured in Example 1 were cultured for 30 hours using a 5 liter fermentor, and then an ultrafiltration kit (Ultrafiltration kit). ) Was used to concentrate a material of 10 kDa or more, and then the protein was separated by performing a non-denaturing (native)-PAGE (12%), and the results are shown in FIG. 1. In FIG. 1 , lane 1 is a size marker, and lane 2 is a bacterium protein.

<3-2> Measurement of algicidal activity of the isolated protein

The protein isolated as described in <3-1> was subjected to non-denaturing electrophoresis for 100 minutes on a 10% polyacrylamide gel at a constant current of 20 mA, and then part of the gel was Coomassive brilliant blue. By staining with R-250, the position of the band was confirmed. The remaining gel was divided into 13 bands according to the band pattern and sectioned into small sizes, and then 1.5 ml of 20 mM Tris-HCl buffer solution (pH 8.0) was added and left in a refrigerator at 4° C. for 2 hours. After centrifuging the solution to precipitate the gel, the supernatant was concentrated using Ultracon (cut-off size, 10 kDa), and the concentrated sample was Ron (Skeletonema costatum ) using ron ( lawn) The algal activity was measured according to the analysis method. At this time, a culture medium of algicidal bacteria was used as a positive control, and Zobel 2216e liquid medium, F/2 medium, and 20 mM Tris-HCl (pH 8.0) buffer solution were used as the negative control according to the conditions of the sample.

Specifically, skeletonema costatium was cultured for 1 to 2 weeks under the conditions of 20° C., photoperiod 14L (5000 lux)/10D. The activity was compared by measuring the diameter (mm) of the clear zone created in the lawn of Skeletonema Costatium. If there is no transparent area, it is "-", and the size of the transparent area is 1 mm. If the back side is “+”, if it is 2 mm, it is “++”, if it is 3 mm, it is “+++”, and if it is 5 mm, it is “++++”, and the results are shown in Table 1 below.

Band number in Figure 1 Algicidal activity 13 - 12 - 11 + 10 +++ 9 + 8 - 7 - 6 - 5 - 4 - 3 ++ 2 + One - Negative control - Positive control ++++

As shown in Table 1, it can be seen that the proteins of bands 3 and 10 have excellent algicidal activity.

In addition, SDS-PAGE was performed on the proteins of bands 3 and 10 selected above under denaturing conditions (refer to [Laemmli, Nature, 227:680-685, 1970]) to confirm the expression of the protein of band number 10. ( FIG. 2 ; Lane 1: size marker, lane 2: algicidal protein, lane 3: protein of band number 3 and lane 4: protein of band number 10), as a result of analyzing the amino acid sequence of the N-terminus of the protein, The amino acid sequence of SEQ ID NO: 3 having 20 amino acids was obtained.

Thereafter, the amino acid sequence was searched for similarity using protein sequence databases such as NCBI Blast, PDF, and SWISSPROT, and as a result, it was found that there was no similarity to the previously reported protein sequence.

Example 4 : Identification of ORF (open reading frame) of algicidal protein

After analyzing the genomic sequence of the algicidal bacterium Cordia aljishida OT-1 strain (KCTC 11320BP), data mining was performed using the N-terminal amino acid sequence of SEQ ID NO: 3, SEQ ID NO: KAOT1_10476 protein (SEQ ID NO: 1) in which 18 of the 20 amino acids of 3 matched was identified.

Example 5 : Construction of an algicidal protein expression vector

The KAOT1_10476 protein of SEQ ID NO: 1 is composed of a signal peptide (1 to 27 amino acids), a processing peptide (28 to 98 amino acids) and a maturation region (99 to 278 amino acids), as shown in FIG. 3. It was predicted to be a metallo protease, and the N-terminal sequence isolated from the wild-type strain was expected to be a mature protease from which the processing peptide was removed by secreting extracellularly.

Therefore, in order to confirm the algicidal activity of the protein, each expression vector was constructed as follows so that various proteins as shown in FIG . 4 were expressed.

First, the primer combination of Table 2 below designed to have Nde I and Xho I restriction sites, using the gene of SEQ ID NO: 2 and TLA1 polymerase (Bioneer, Korea) as a template, the KAOT1_10476 protein of SEQ ID NO: 1 or Various DNAs encoding their fragments were amplified by PCR. The PCR was repeated 30 times for 2 minutes at 95°C, 1 minute at 55°C, and 1 minute at 72°C.

PCR product primer Sequence number 10476_1 OT1m_t 12 OT1m_bc 13 10476_2 OT1p_t 17 OT1m_bc 13 10476_3 OT1m_t 12 OT1delta5_bc 15 10476_4 OT1m_t 12 OT1delta26_bc 16 10476_5 OT1m_t 12 OT1m_bH 14 10476_6 OT1p_t 17 OT1m_bH 14 10476_7 OT1m_t 12 OT1m_bc 13 10476_8 OT1p_t 17 OT1m_bc 13 10476_9 OT1p_t 17 OT1delta5_bc 15

Thereafter, the pET24a expression vector was digested by treatment with restriction enzymes Nde I and Sal I, and then purified using a plasmid purification kit (Qiagen), ligated with each of the PCR products obtained above, and transformed into E. coli DH5α. . After purifying the DNA from the transformant obtained above using a plasmid purification kit, and then sequencing it to confirm that the complete DNA without mutation or frame shift was cloned, the DNA was converted to BL21 (DE3) ( Novagen).

Example 6 : Expression and refolding of algicidal active protease

The transformant obtained in Example 5 was inoculated in an LB medium (Difco) to which kanamycin (50 µg/ml) was added and cultured at 37° C. for 12 hours, and the culture was then in the same medium as above. After inoculating at 1% (v/v) and incubating for 3 hours, IPTG (Sigma) was added at a final concentration of 1 mM to overexpress the protease. In order to analyze the expression level and timing of each protease, the samples were sampled every 1 hour and analyzed by SDS-PAGE, and the expression level of the protease after 3 hours of incubation was shown in FIG. 5.

As shown in FIG. 5, it was confirmed that the protease was expressed in all except the plasmid into which the PCR product 10476_3 was inserted.

In addition, as a result of confirming whether the expressed protease was produced in any of the soluble fraction, the insoluble fraction, and the periplasmic region according to the protocol of the pET system, it was confirmed that most of the proteases were insoluble proteins.

In addition, the insoluble fractions of 10476_1, 10476_6, 10476_7 (SEQ ID NO: 27), 10476_8 (SEQ ID NO: 26) and 10476_9 obtained above were treated with 8M urea to solubilize the protease, and then buffered again. A solution (50 mM Tris (pH 8.0), 1 mM CaCl ₂ ) was substituted to perform refolding to obtain a protease ( see FIG. 6 ).

As a result, it was confirmed that all proteases were refolded.

Example 7 : Degradation activity measurement of algicidal protease

The proteolytic enzymes obtained in Example 6 were each developed on a gelatin substrate gel to measure proteolytic activity (see [Park, HI, J. Biol. Chem., 275:20540-20544, 2000]). ), and the results are shown in FIG. 7.

As shown in FIG. 7 , proteins 10476_1 and 10476_6 to 10476_8 except for the 10476_9 protein exhibited proteolytic activity.

In addition, the band indicating the activity of the 10476_8 protein (SEQ ID NO: 26) containing the processing peptide gradually descends over time, and then exhibits a size similar to that of the 10476_7 protein (SEQ ID NO: 27) expressing the mature region. I got it.

Therefore, it can be expected that the processing peptide of the 10476_8 protein is converted into a mature protein through autocleavage over time.

In addition, from the result of the 10476_1 protein, the presence of the N-terminal His tag interferes with the proteolytic activity, and from the result of the 10476_9 protein, it can be expected that the C-terminal deletion mutation does not show activity because it is not processed. .

Example 8 : Observation of the processing phenomenon of algicidal protease

In order to observe the processing phenomenon of the algicidal protease of the present invention, the protein degradation activity analysis was performed in the same manner as in Example 7, except that 10476_8 protein (SEQ ID NO: 26) was reacted at 4° C. for 1 week. After performing, it was confirmed by SDS-PAGE, and the results are shown in FIG. 8.

As shown in FIG. 8 , it was confirmed that the 10476_8 protein was transformed into a mature protease by decomposing the processing peptide 3 days after the reaction, and appeared as two main bands after 7 days. In addition, when considering the amount of major protein and the amount of low-molecular peptide increased with time, it was confirmed that self-cleavage was accompanied during the maturation process.

In addition, as a result of analyzing the 10476_9 protein in the same manner as above, the 10476_9 protein, a mutant containing the processing peptide and in which 5 amino acids were deleted at the C-terminus, was well isolated, but the phenomenon that the size of the band decreases like the 10476_8 protein was observed Didn't. Therefore, since the 10476_9 protein does not show activity by not being processed, it can be seen that the C-terminal amino acid plays an important role in the processing.

Test Example 1 : Identification of biochemical properties of algicidal active protease

10476_7 protein (SEQ ID NO: 27) containing only mature protein was combined with the resistance to detergent of protease and stability using EDTA to dissolve the proteins using SDS. After diluting and refolding with a buffer solution containing HCl, 1 mM EDTA, and 1% Triton X-100, proteolytic activity according to temperature and pH was measured according to the method described in [Park, HI, 2000]. , The results are shown in Figs. 9 and 10, respectively.

9 and 10 , the 10476_7 protein exhibited optimal activity at pH 7-8 and about 20°C.

In addition, 5 ㎍ of the refolded protein was added to a buffer solution containing 1 mM zinc or calcium ions in 0.1% gelatin, 50 mM Tris-HCl (pH 8.0), and 100 mM KCl, and reacted at 20° C. for 2 hours. Thereafter, 5% TCA solution (w/v) was added and left on ice for 10 minutes, centrifuged at 10,000 rpm for 5 minutes, and then protein was quantified using the supernatant. The inhibition of activity by metal ions was measured compared to the control group containing no metal, and the results are shown in FIGS. 11 and 12.

As shown in FIG. 11 , the inhibitory activity of zinc ions against 10476_7 protein was confirmed.

In addition, as shown in FIG. 12 , calcium ions are essential for the activity of the protease, and the maximum activity of the protease was exhibited by the calcium ions of 80 to 100 mM.

Test Example 2 : Analysis of algicidal activity of algicidal protease

In order to confirm the algicidal activity of 10476_7 protease (SEQ ID NO: 27), Cochlodinium polykrikoides prepared in Example 2, Thalassiosira sp., heterosigma acacio prepared in Example 2 ( Heterosigma akashiwo ), Skeletonema costatum , Alexandrium ( Alexandrium sp), Catoceros cuvisetus (Chaetoceros cuvisetus ), Gymnodinium (Gymnodniium sp) 50 μl of the above protein (100 μg/ml) in a culture solution Added. After 24 hours, the culture solution was stained with Lugol's iodine solution (1%), and then the number of cells was measured, and the control group to which protease was not added and the experimental group to which protease was added. The number of cells and the degree of inhibition were compared in (Experimental Groups 1 and 2), and the results are shown in Table 3 below.

Assurance of killing against Cocrodinium polyclicoides Control Experimental group 1 Experimental group 2 Cell count
(Cell/ml) 1,360(130) 960(30) 1040(90) (Cell/ml) 1,330(120) 940(60) 900(110) Degree of inhibition (%) - 40.7 30.0 Inhibition degree (%) = ((control-experimental group)/experimental group)×100
( ): Standard Deviation

As shown in Table 3, it was confirmed that the growth of the strain was effectively inhibited when the protease of SEQ ID NO: 1 was treated on the Cocrodinium polyclicoides strain. In addition, US dollars, Mr come la species (Thalassiosira sp.), Heterocyclic Sigma Oh Casio (Heterosigma akashiwo), skeletal Loreto nematic Costa lithium (Skeletonema costatum), Alexandria Solarium (Alexandrium sp), caviar Tosh Ross (Chaetoceros cuvisetus), Jim nodi uranium ( Gymnodinium sp) was also confirmed to be effective.

Example 9 : Search for genes with homology

As a result of analyzing the amino acid sequence of various marine microorganisms using NCBI Blast based on the amino acid sequence (SEQ ID NO: 1) of the protease of the present invention in which the algicidal effect was confirmed, as shown in FIG . 13, the Flavobacterium zone Soniae ( Flavobacterium johnsoniae ) UW101 (ZP_01247095, SEQ ID NO: 4), Croceibacter atlanticus HTCC2559 (ZP_00951344, SEQ ID NO: 5) and Leeuwenhoekiella blandensis (Leuwenhoekiella blandensis) MED217 (ZP_01247095, SEQ ID NO: 4) , SEQ ID NO: 6) showed 40% or more identity in a gene coverage of 80% or more, and in particular, the mature protease portion after the processing peptide showed a high homology of 50% or more. In addition, as shown in Fig. 14 , it also showed high homology with the KAOT1_11562 gene (SEQ ID NO: 7) of Cordy aljishida.

Example 10 : Expression of homologous algicidal protein

In order to express the algicidal protein showing homology with the algicidal protease of SEQ ID NO: 1, the primer combinations in Table 4 below, and the gene encoding the protein having the amino acid sequence of SEQ ID NO: 4 to 7, respectively as a template PCR was performed in the same manner as in Example 5, except that (SEQ ID NOs: 8 to 11) was used, and the results are shown in FIG. 15.

PCR product primer Sequence number ZP_00951344 HTCC2559m_t 18 HTCC2559m_bc 19 11562 OT11562m_t 20 OT11562m_bc 21 ZP_01059780 MED217m_t 22 MED217m_bc 23 ZP_01247095 fjohnm_t 24 fjohnm_bc 25

Thereafter, the PCR product was cloned into the pET24 expression vector in the same manner as in Example 5, except that each PCR product was used, and the protein was overexpressed in the same manner as in Example 6 and analyzed by SDS-PAGE. All proteins were well expressed, but all were detected in the insoluble fraction. Thus, to measure the activity, after dissolving the proteins in the same manner as in Test Example 1 (before), this was used as a buffer solution containing 50mM Tris-HCl, 1 mM EDTA, and 1% Triton X-100 not containing SDS. Diluted and refolded (after) was attempted, and the results are shown in FIG. 16.

As shown in FIG. 16, in the case of other proteins except for the protein of Crosseybacter atlanticus HTCC2559 (SEQ ID NO: 5), a large amount of mature protein could be obtained.

Test Example 11 : Analysis of algicidal activity of homologous algicidal protein

Flavobacterium johnsoniae UW101 (ZP_01247095, SEQ ID NO: 4), Croceibacter atlanticus HTCC2559 (ZP_00951344, SEQ ID NO: 5), Leeuwenhoekiella blendensis blandensis ) MED217 (ZP_01059780, SEQ ID NO: 6), In order to confirm the algicidal activity of the protein prepared in the KAOT1_11562 gene (SEQ ID NO: 7) of Cordy aljishida, Cocrodinium polyclicoides prepared in Example 2 ( Cochlodinium polykrikoides), US dollars, Mr come la species (Thalassiosira sp.), heterocyclic Sigma Oh Casio (Heterosigma akashiwo), skeletal Loreto nematic Costa lithium (Skeletonema costatum), Alexandria Solarium (Alexandrium sp), caviar Tosh Ross (Chaetoceros cuvisetus), Jim 50 μl of the protein (100 μg/ml) was added to the culture medium of Gymnodium sp. After 24 hours, the culture solution was stained with Lugol's iodine solution (1%), and then the number of cells was measured, and the control group to which protease was not added and the experimental group to which protease was added. The number of cells and the degree of inhibition of (Experimental Groups 1 and 2) were compared, and as a result , Cocrodinium polyclicoides , Thalassiosira sp., Heterosigma akashiwo, and Skeletonema Costa It was confirmed that there is also an effect on lithium ( Skeletonema costatum ), alexandrium ( Alexanderium sp), cateoceros cuvisetus , and gymnodinium (Gymnodinium sp).

Killing verification of red tide killing protein FJ
(ZP_01247095)
(SEQ ID NO: 4) CRO
(ZP_00951344)
(SEQ ID NO: 5) MED
(ZP_01059780)
(SEQ ID NO: 6) KAOT1_10476
(SEQ ID NO: 1) KAOT1_11562
(SEQ ID NO: 7) Cochlodinium polykrikoides ++++ ++++ ++++ ++++ ++++ Thalassiosira sp. ++++ ++++ ++++ ++++ ++++ Heterosigma akashiwo ++++ ++++ ++++ ++++ ++++ Skeletonema costatum ++ +++ ++++ + + Alexandrium sp. ++++ ++++ ++++ ++++ ++++ Chaetoceros cuvisetus ++++ ++++ ++++ ++++ ++++ Gymnodinium sp. ++++ ++++ ++++ ++++ ++++ Inhibition degree = ((control group-experimental group)/experimental group)×100
+ means <25%
++ means 25-50%
++ means 50-75%
++++ means >75% inhibition

Korea Research Institute of Bioscience and Biotechnology KCTC11320BP 20080408

<110> KOREA OCEAN RESEARCH AND DEVELOPMENT INSTITUTE <120> PROTEASE HAVING ALGICIDAL ACTIVITY, GENE ENCODING THE SAME AND ALGICIDAL FORMULATION COMPRISING THE SAME <130> F <160> 29 <170> KopatentIn 1.71 <210> 1 <211> 278 <212> PRT <213> KAOT1_10476 of Kordia algicida OT-1 <400> 1 Met Lys Lys Ile Lys Asn Leu Thr Phe Ala Leu Ala Leu Gly Leu Gly 1 5 10 15 Ile Thr Met Val Ser Cys Ser Lys Asp Thr Ala Val Val Asp Glu Glu 20 25 30 Gln Asp Thr Ala Ile Gly Ile Pro Gln Asp Val Leu Gln Lys Ala Gln 35 40 45 Ser Leu His Phe Asn Thr Phe Asp Met Gln Glu Ala Ser Phe Glu Lys 50 55 60 Pro Asn Gly Lys Ile Glu Glu Gly Tyr Met Met Glu Gly Asp Ile Phe 65 70 75 80 Phe Thr Arg Asp Gln Leu Met Asn Met Glu Leu Gly Gly Asp Ile Thr 85 90 95 Ser Lys Gln Tyr Arg Thr Asn Asn Leu Val Ser Pro Gly Val Ile Thr 100 105 110 Ile Ile Gly Tyr Thr Gly Asn Asn Ser Asn Gly Leu Thr Thr Lys Met 115 120 125 Gln Thr Gly Leu Arg Trp Ala Val Asp Asn Tyr Asn Ala Leu Asn Leu 130 135 140 Ser Ile Ser Phe Gln Leu Thr Phe Gly Thr Asp Tyr Gln Asn Lys Asp 145 150 155 160 Met Val Val Tyr Gln Val Gln Gly Gly Ala Gly Gly Ser Ala Gly Phe 165 170 175 Pro Ser Gly Gly Asn Pro Tyr Lys Trp Val Lys Ile Asn Ser Gly Met 180 185 190 Ala Pro Tyr Ser Asn Asn Val His Glu His Val Ile Gly His Glu Ile 195 200 205 Gly His Ser Ile Gly Phe Arg His Ser Asp Tyr Phe Ser Arg Gln Ser 210 215 220 Cys Gly Gln Asn Ser Asn Glu Gly Ser Ala Gly Val Gly Ala Ile His 225 230 235 240 Ile Pro Gly Thr Pro Thr Gly Trp Asp Pro Thr Ser Leu Met Asn Ala 245 250 255 Cys Phe Ser Ser Ser Glu Asp Gly Glu Phe Asn Gly Asn Asp Ile Thr 260 265 270 Ala Leu Asn Phe Leu Tyr 275 <210> 2 <211> 834 <212> DNA <213> KAOT1_10476 of Kordia algicida OT-1 <400> 2 atgaaaaaaa ttaaaaactt aacctttgcg ttggcattag ggttagggat cactatggta 60 tcttgtagta aagacactgc cgtagtagac gaggagcaag acacagctat cggaatccca 120 caagacgtac ttcaaaaagc acaatcactt cacttcaaca cattcgatat gcaagaagca 180 tctttcgaaa agccaaatgg taaaatcgaa gaaggatata tgatggaagg tgatatcttc 240 tttacacgtg accaattaat gaacatggaa ctcggtggag atattacaag caaacaatat 300 cgtacaaaca atttagtttc tccaggagta attacaatca ttggatatac aggaaataac 360 tctaatggat taacaactaa aatgcaaact ggacttagat gggcagtaga taactataat 420 gcattaaacc taagtattag tttccaatta acatttggta ctgactatca aaataaagat 480 atggtagtat atcaagtaca aggtggagca ggtggatctg caggtttccc ttcaggagga 540 aatccataca agtgggttaa aattaactca ggtatggctc cttacagtaa caatgtacat 600 gaacatgtaa ttggacatga aataggacac tcgattggat tccgtcattc agactacttc 660 agcagacaaa gttgtggaca aaattcaaat gaaggtagtg caggagttgg agcaatccat 720 attccaggaa cacctacagg ttgggatcca acatctttaa tgaatgcttg tttcagttct 780 tcagaagatg gagaatttaa cggaaacgac attactgctt taaacttctt atac 834 <210> 3 <211> 20 <212> PRT <213> N-terminus of KAOT_1 10476 <400> 3 Gln Tyr Val Thr Asn Asn Leu Val Ser Pro Gly Val Ile Thr Ile Ile 1 5 10 15 Gly Tyr Thr Phe 20 <210> 4 <211> 276 <212> PRT <213> Flavobacterium johnsoniae UW101 (ZP_01247095) <400> 4 Met Lys Lys Ile Lys Ser Ile Leu Ile Leu Ser Phe Thr Ala Leu Val 1 5 10 15 Leu Leu Ser Cys Asn Lys Glu Asp Glu Thr Val Ser Ser Gly Gln Glu 20 25 30 Ser Leu Lys Val Thr Pro Glu Val Leu Glu Lys Leu Lys Ser Leu Ser 35 40 45 Leu Asn Thr Ser Asp Val Gln Val Ile Gln Asn Thr Ser Leu Glu Gly 50 55 60 Ala Val Glu Asp Ala Phe Leu Val Glu Gly Asp Ile Ile Ile Thr Gln 65 70 75 80 Ala Gln Leu Asn Lys Met Asp Leu His Gly Gly Ile Thr Thr Glu Gln 85 90 95 Tyr Arg Thr Thr Asn Leu Val Ser Ala Pro Arg Thr Ile Lys Val Val 100 105 110 Gly Leu Ser Gly Thr Gly Thr Thr Ala Leu Thr Thr Asn Met Arg Asn 115 120 125 Gly Leu Gln Ala Ala Ile Asn Arg Tyr Asn Asn Leu Gly Leu Ser Ile 130 135 140 Asn Phe Thr Leu Thr Phe Ser Ser Ser Thr Ser Gly Ala Asn Ile Val 145 150 155 160 Val Arg Arg Gln Thr Gly Ser Ala Gly Gly Val Ala Gly Phe Pro Ser 165 170 175 Gly Gly Asn Pro Tyr Asn Ser Val Thr Leu Tyr Ser Gly Leu Asp Ser 180 185 190 Tyr Ser Thr Asn Val Asn Ala His Val Ala Ala His Glu Ile Gly His 195 200 205 Cys Ile Gly Leu Arg His Thr Asp Trp Phe Ser Arg Gln Ser Cys Gly 210 215 220 Gln Asn Ser Asn Glu Gly Thr Ala Gly Val Gly Ala Ile Leu Ile Pro 225 230 235 240 Gly Thr Pro Ser Gly Tyr Asp Ala Thr Ser Tyr Met Arg Ala Cys Phe 245 250 255 Gly Ser Asn Glu Thr Gly Ala Phe Asn Ala Asn Asp Ile Thr Ala Leu 260 265 270 Asn Tyr Leu Tyr 275 <210> 5 <211> 284 <212> PRT <213> Croceibacter atlanticus HTCC2559 (ZP_00951344) <400> 5 Met Val Phe Val Ser Cys Glu Lys Asp Asn Asp Thr Asn Asn Ala Glu 1 5 10 15 Val Ala Asp Thr Asn Glu Val Thr Thr Gly Ser Leu Glu Thr Leu Gly 20 25 30 Val Asn Thr Asp Leu Thr Pro Thr Asn Leu Asp Gln Ala Thr Leu Asp 35 40 45 Leu Ile Ala Ser Lys His Leu Ser Pro Ile Gly Ala Gln Glu Glu Leu 50 55 60 Arg Tyr Leu Pro Asp Gly Thr Ser Glu Lys Ala Ile Arg Ile Glu Gly 65 70 75 80 Asp Ile Val Met Thr Lys Ala Glu Leu Glu Glu Leu Glu Phe Asn Gly 85 90 95 Tyr Ser Asn Glu Asn Ala Gln Tyr Ser Thr Asn Ala Leu Val Ser Pro 100 105 110 Gln Thr Ile Thr Ile Ile Gly Tyr Thr Gly Gly Ser Gln Ala Leu Thr 115 120 125 Ser Ser Glu Gln Thr Ala Leu Gln Trp Ala Val Ala Asn Tyr Asn Arg 130 135 140 Leu Asn Leu Asn Ile Asn Phe Ser Leu Thr Phe Gly Thr Asn Tyr Gln 145 150 155 160 Asn Lys Asp Met Val Val Tyr Asn Asn Thr Val Asn Asn Pro Ser Gly 165 170 175 Ala Gly Gly Ser Ala Gly Phe Pro Ser Gly Gly Asn Pro His Lys Phe 180 185 190 Val Gln Ile Tyr Gly Leu Ser Asn Tyr Asn Thr Asn Val Ile Glu His 195 200 205 Val Ile Thr His Glu Ile Gly His Ser Val Gly Phe Arg His Thr Asp 210 215 220 Tyr Phe Ser Arg Gln Ser Cys Gly Gln Asn Thr Asn Glu Gly Thr Ala 225 230 235 240 Gly Val Gly Ala Asn His Ile Pro Gly Thr Pro Thr Gly Tyr Asp Ser 245 250 255 Thr Ser Ile Met Leu Ala Cys Phe Ser Ser Gly Glu Asp Gly Glu Phe 260 265 270 Asn Ser Asn Asp Ile Thr Ala Leu Asn Tyr Leu Tyr 275 280 <210> 6 <211> 270 <212> PRT <213> Leeuwenhoekiella blandensis MED217 (ZP_01059780) <400> 6 Met Thr Ser Cys Ser Glu Asp Ala Glu Gln Thr Thr Leu Pro Glu Ala 1 5 10 15 Gln Pro Thr Ser Asn Leu Glu Val Ser Ser Glu Ile Ile Gln Gln Val 20 25 30 Glu Asp Leu Gly Met Asn Ala Asn Tyr Val Arg Trp Asp Asp Phe Tyr 35 40 45 Phe Pro Asp Gly Ser Ser Glu Pro Arg Leu Phe Leu Glu Glu Asp Val 50 55 60 Val Val Thr Pro Glu Gln Leu Ser Ser Met Ser Ala Asn Val Glu Glu 65 70 75 80 Ser Lys Ser Gly Lys Gly Asp Ser Lys Gln Tyr Arg Thr Ser Ala Leu 85 90 95 Val Ser Gln Gly Arg Thr Ile Ser Ile Ile Gly Tyr Thr Gly Gly Ser 100 105 110 Gln Ala Leu Ser Gln Lys Glu Arg Thr Ala Leu Gln Trp Ala Val Ala 115 120 125 Asn Tyr Asn Arg Leu Ser Gly Val Ser Ile Ser Phe Asn Leu Thr Phe 130 135 140 Gly Thr Asp Tyr Gln Asn Lys Asp Met Val Val Tyr Asn Asn Thr Val 145 150 155 160 Asn Asn Pro Ser Gly Ala Gly Gly Ser Ala Gly Phe Pro Ser Asn Gly 165 170 175 Leu Pro Tyr Lys Phe Val Gln Ile Tyr Gly Leu Ala Asn Tyr Asp Thr 180 185 190 Asn Val Val Glu His Val Met Thr His Glu Ile Gly His Ser Val Gly 195 200 205 Phe Arg His Thr Asp Trp Phe Ser Arg Gln Ser Cys Gly Gln Asn Val 210 215 220 Tyr Glu Gly Gly Asp Ala Asn His Val Ser Gly Thr Pro Thr Gly Tyr 225 230 235 240 Asp Ser Thr Ser Ile Met Leu Ala Cys Phe Ser Ala Asn Glu Asp Gly 245 250 255 Glu Phe Asn Ala Asn Asp Ile Thr Ala Leu Asn Asn Met Tyr 260 265 270 <210> 7 <211> 378 <212> PRT <213> Kordia algicida KAOT1_11562 <400> 7 Met Lys Arg Asn Phe Lys Ile Gln Met Leu Tyr Val Leu Leu Thr Met 1 5 10 15 Leu Leu Leu Ala Gly Cys Gln Asn Asp Thr Glu Glu Glu Leu Val Glu 20 25 30 Glu Ala Thr Thr Ala Val Glu Arg Val Asp Val Lys Trp Leu Ala Pro 35 40 45 Asp Asp His Pro Val Val Gln Leu Leu Tyr Ser Arg Gly Tyr Glu Arg 50 55 60 Gly Thr Ile Tyr Glu Thr Asp Glu His Phe Leu Ala Pro Pro Asp Leu 65 70 75 80 Leu Tyr Ser Lys Asp Ile Asn Asp Tyr Asp Leu Ser Asp Asn Gly Ser 85 90 95 Asn Ala Glu Gln Ala Tyr Asn Thr Gly Lys Leu Val Ser Leu Asn Arg 100 105 110 Met Arg Ile Asn Val Phe Leu Asp Asn Ser Ile Gly Thr Asp Leu Gln 115 120 125 Thr Gln Ser Val Asn Ala Met Asn Glu Leu Asn Gly Ile Asn Asn Cys 130 135 140 Ala Leu Phe Phe Val Arg Val Phe Asn Ala Asn Gln Ala Gln Ile Thr 145 150 155 160 Ile Arg Ser Asp Phe Gly Ala Glu Ser Asn Asn Val Leu Gly Arg Ala 165 170 175 Gly Phe Pro Ser Asn Gly Arg Pro Phe Asp Thr Val Thr Leu Asn Val 180 185 190 Asp Arg Leu Asp Asp Phe Gly Ala Asp Ile Arg Arg Asn Thr Ile Ile 195 200 205 His Glu Leu Gly His Cys Val Gly Leu Arg His Thr Asp Trp Gln Ala 210 215 220 Asn Arg Glu Arg Ser Ala Val Asn Ile Pro Gly Thr Ser Ala Asn Asp 225 230 235 240 Thr Gly Ser Ile Met Trp His Thr Ile Asn Gly Gly Thr Pro Phe Thr 245 250 255 Asn Gly Asp Leu Thr Ala Phe Arg Ala Leu Phe Pro Arg Ala Leu Arg 260 265 270 Ile Asp Val Val Asn Glu Ile Asp Asp Tyr Asp Tyr Ser Gly Glu Ile 275 280 285 Tyr Val Leu Asp Asn Val Phe Val Asp Val Phe Thr Asp Gly Ser Tyr 290 295 300 Ser Thr Gln Thr Thr Leu Asn Arg Asn Val Asn Val Ser Tyr Arg Ile 305 310 315 320 Asn Val Gln Glu Tyr Asn His Thr Ser Gly Thr Tyr Tyr Tyr Ser Arg 325 330 335 Asn Arg Thr Leu Thr Ala Gly Asn Asn Arg Tyr Tyr Ile Asp Asp Glu 340 345 350 Glu Glu Glu Cys Ser Pro Tyr Gln Gly Glu Thr Cys Thr Arg Gln Asp 355 360 365 Leu Glu Ile Arg Leu Ala Thr Ser Ile Leu 370 375 <210> 8 <211> 828 <212> DNA <213> Flavobacterium johnsoniae UW101 (ZP_01247095) <400> 8 atgaaaaaaa ttaaatcaat cctgattctg tcatttacag cattagtact attgtcttgt 60 aacaaagaag atgagactgt atcatctggt caagagtctt taaaagtaac accagaggtg 120 ttggaaaaac ttaaatcact ttctttgaac acctcagatg tgcaggtgat ccaaaatact 180 agtttagaag gtgctgttga agacgcgttc cttgtagaag gggatattat catcacgcag 240 gcacaattaa acaaaatgga tcttcacgga ggtattacaa cagaacaata ccgtactact 300 aatttagtat ctgctccaag aacgatcaaa gttgttggtt tatcaggaac tggtacaaca 360 gccctgacta ccaatatgcg taacggactg caggctgcta taaacagata caataattta 420 ggattatcta taaactttac tttgactttt agttcaagta cttcaggcgc aaacattgta 480 gtgcggagac aaaccggatc tgccggcgga gtagctggtt tcccttcagg aggaaatcca 540 tataattcag ttaccttata ttcaggatta gattcttatt caacaaatgt aaacgcacac 600 gttgcggcac atgaaatagg acattgtatt ggtctgcgtc atacagactg gttcagccgt 660 caaagctgcg ggcagaattc aaatgaagga actgctggtg taggagcaat tcttattccg 720 ggaacacctt ctggatatga tgctacttct tacatgagag cgtgtttcgg ttcaaacgaa 780 accggtgctt tcaatgctaa tgatattaca gcattgaact atttatat 828 <210> 9 <211> 852 <212> DNA <213> Croceibacter atlanticus HTCC2559 (ZP_00951344) <400> 9 atggtattcg tttcttgtga gaaagataat gatacaaaca acgcagaggt tgcagacact 60 aatgaagtta caacaggttc tcttgaaact ttaggtgtta atacagacct taccccaaca 120 aatttagatc aagctacttt agatttaatt gcttctaaac atcttagtcc tatcggagct 180 caagaagagt taagatattt accagacggt acatcagaaa aagctatacg tatagaaggt 240 gatatcgtaa tgactaaagc tgaacttgaa gagttggagt ttaatggtta ttcaaatgaa 300 aatgctcaat atagtactaa cgcattagtt agtcctcaaa caattacaat tattggttat 360 actggtggaa gccaggcgtt aactagtagc gagcaaactg cattacaatg ggcagttgca 420 aactataata gattaaacct taacatcaat ttctctttaa cttttggtac aaactaccaa 480 aacaaagata tggttgttta caacaatact gtaaataacc caagtggtgc aggaggatct 540 gcaggtttcc caagtggagg aaacccacat aaatttgtac agatttatgg attaagtaac 600 tataacacaa atgtaattga gcacgttatt actcacgaaa taggacactc tgtaggattc 660 cgtcacacag attactttag ccgtcagagt tgtggtcaaa atacaaacga aggaactgca 720 ggagttggtg ctaaccacat cccaggaact ccaacaggat atgattctac gtctattatg 780 ttagcttgtt ttagcagtgg tgaagatgga gagtttaaca gtaatgatat tactgcttta 840 aactaccttt at 852 <210> 10 <211> 810 <212> DNA <213> Leeuwenhoekiella blandensis MED217 (ZP_01059780) <400> 10 atgacctctt gttcagaaga tgctgagcaa actacgttgc cagaagcaca acctacttcc 60 aaccttgagg tttcttcaga aattatccag caagttgaag atttaggcat gaacgctaat 120 tatgtgcgtt gggatgattt ttattttcct gatggttctt ctgaacctcg tctattttta 180 gaagaagatg ttgtcgtaac tcctgaacaa ttatcctcaa tgtccgctaa cgttgaagaa 240 tcaaaatcag gaaaaggtga ttctaaacaa tatcgcactt ctgctttggt aagtcaggga 300 agaacgattt ctatcatcgg ttataccggc ggtagtcagg cgctaagcca aaaagagcgt 360 accgccttac aatgggctgt tgcaaattac aatcggttaa gcggtgtaag catcagcttc 420 aatttgacat ttggtaccga ctatcaaaac aaagacatgg tagtttacaa caacacggta 480 aataacccaa gtggcgcagg tggtagtgcc ggtttcccga gtaatggact tccttataaa 540 tttgttcaga tctatgggct cgctaattac gacaccaatg ttgttgaaca cgtaatgacg 600 cacgagatag gtcattcggt tggatttaga cacacagatt ggtttagtag acaaagctgt 660 ggacagaacg tttatgaagg tggtgacgca aatcacgttt cgggaacccc aacaggttac 720 gactcgactt caattatgct agcttgcttt agtgccaatg aagatggcga atttaatgcc 780 aatgatatta ccgcattaaa caatatgtac 810 <210> 11 <211> 1134 <212> DNA <213> Kordia algicida KAOT1_11562 <400> 11 atgaaaagaa attttaaaat ccaaatgctg tatgttttgt tgacaatgct cctgcttgca 60 ggttgtcaaa acgatacgga agaagaatta gtagaagaag caacaactgc tgtagaacgt 120 gttgatgtaa aatggcttgc accagacgat catcctgtgg tacaattatt atacagcaga 180 ggttatgaac gaggaacaat ctatgaaact gatgagcatt ttttagcgcc accagattta 240 ttgtacagta aagatatcaa cgattatgac ctatctgaca atggtagcaa tgccgaacaa 300 gcctacaaca cagggaaatt agtatctcta aacagaatgc gtatcaatgt gtttttagac 360 aactctattg ggacagattt gcaaacacaa tccgtaaatg caatgaatga acttaacgga 420 atcaacaatt gtgctttatt ctttgtacgt gttttcaatg ccaatcaggc acaaattacg 480 atacgaagtg actttggagc agaatccaat aatgttttag gtcgtgcagg ttttccatct 540 aacggacgtc catttgatac agtgacccta aacgttgatc gtttggatga cttcggagct 600 gacattcgta gaaacaccat tattcatgaa cttggtcatt gtgtcggatt gcgccataca 660 gattggcaag ccaacagaga acgtagtgct gtaaacattc ctggtacaag tgccaatgat 720 acaggatcaa tcatgtggca taccatcaat ggaggaacac cttttacaaa cggagattta 780 actgctttta gagccttatt tccacgagct ttgcgcatag atgttgtcaa tgaaattgac 840 gattatgatt acagtggtga aatttatgtt ttagacaatg tatttgtcga tgtctttacc 900 gatggaagct acagtacgca aacaacgctt aaccgaaatg taaacgtaag ttaccgtatt 960 aacgtacaag aatacaatca cacctcagga acttattatt acagtcgaaa tcgaacttta 1020 acggctggta ataatcgtta ttatattgat gatgaggaag aagaatgttc gccataccaa 1080 ggagaaacct gtacacgaca agaccttgaa atcagattgg caacttcaat acta 1134 <210> 12 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> OT1m_t <400> 12 cgacccggca tatgcaatat cgtacaaaca atttagtttc 40 <210> 13 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> OT1m_bc <400> 13 ctccacatct cgagctagta taagaagttt aaagcag 37 <210> 14 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> OT1m_bH <400> 14 ctccacatct cgaggtataa gaagtttaaa gcagtaatg 39 <210> 15 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> OT1delta5_bc <400> 15 ctccacatct cgagctaagc agtaatgtcg tttccgttaa a 41 <210> 16 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> OT1delta26_bc <400> 16 ctccacatct cgagctaaga tgttggatcc caacctg 37 <210> 17 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> OT1p_t <400> 17 cgacccggca tatgagtaaa gacactgccg tagtagac 38 <210> 18 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> HTCC2559m_t <400> 18 cgacccggca tatgcaatat agtactaacg cattagttag 40 <210> 19 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> HTCC2559m_bc <400> 19 ctccacatct cgagttaata aaggtagttt aaagcag 37 <210> 20 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> OT11562m_t <400> 20 cgacccggca tatgcaagcc tacaacacag ggaaattag 39 <210> 21 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> OT11562m_bc <400> 21 ctccacatct cgagttatag tattgaagtt gccaatc 37 <210> 22 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> MED217m_t <400> 22 cgacccggca tatgcaatat cgcacttctg ctttggtaag 40 <210> 23 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> MED217m_bc <400> 23 ctccacatct cgagctagta catattgttt aatgcgg 37 <210> 24 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> fjohnm_t <400> 24 cgacccggca tatgcaatac cgtactacta atttagtatc 40 <210> 25 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> fjohnm_bc <400> 25 ctccacatct cgagttaata taaatagttc aatgctg 37 <210> 26 <211> 251 <212> PRT <213> KAOT1_10476 of Kordia algicida OT-1 lacking singal peptide <400> 26 Val Val Asp Glu Glu Gln Asp Thr Ala Ile Gly Ile Pro Gln Asp Val 1 5 10 15 Leu Gln Lys Ala Gln Ser Leu His Phe Asn Thr Phe Asp Met Gln Glu 20 25 30 Ala Ser Phe Glu Lys Pro Asn Gly Lys Ile Glu Glu Gly Tyr Met Met 35 40 45 Glu Gly Asp Ile Phe Phe Thr Arg Asp Gln Leu Met Asn Met Glu Leu 50 55 60 Gly Gly Asp Ile Thr Ser Lys Gln Tyr Arg Thr Asn Asn Leu Val Ser 65 70 75 80 Pro Gly Val Ile Thr Ile Ile Gly Tyr Thr Gly Asn Asn Ser Asn Gly 85 90 95 Leu Thr Thr Lys Met Gln Thr Gly Leu Arg Trp Ala Val Asp Asn Tyr 100 105 110 Asn Ala Leu Asn Leu Ser Ile Ser Phe Gln Leu Thr Phe Gly Thr Asp 115 120 125 Tyr Gln Asn Lys Asp Met Val Val Tyr Gln Val Gln Gly Gly Ala Gly 130 135 140 Gly Ser Ala Gly Phe Pro Ser Gly Gly Asn Pro Tyr Lys Trp Val Lys 145 150 155 160 Ile Asn Ser Gly Met Ala Pro Tyr Ser Asn Asn Val His Glu His Val 165 170 175 Ile Gly His Glu Ile Gly His Ser Ile Gly Phe Arg His Ser Asp Tyr 180 185 190 Phe Ser Arg Gln Ser Cys Gly Gln Asn Ser Asn Glu Gly Ser Ala Gly 195 200 205 Val Gly Ala Ile His Ile Pro Gly Thr Pro Thr Gly Trp Asp Pro Thr 210 215 220 Ser Leu Met Asn Ala Cys Phe Ser Ser Ser Glu Asp Gly Glu Phe Asn 225 230 235 240 Gly Asn Asp Ile Thr Ala Leu Asn Phe Leu Tyr 245 250 <210> 27 <211> 180 <212> PRT <213> KAOT1_10476 of Kordia algicida OT-1 lacking signal and processing peptide <400> 27 Gln Tyr Arg Thr Asn Asn Leu Val Ser Pro Gly Val Ile Thr Ile Ile 1 5 10 15 Gly Tyr Thr Gly Asn Asn Ser Asn Gly Leu Thr Thr Lys Met Gln Thr 20 25 30 Gly Leu Arg Trp Ala Val Asp Asn Tyr Asn Ala Leu Asn Leu Ser Ile 35 40 45 Ser Phe Gln Leu Thr Phe Gly Thr Asp Tyr Gln Asn Lys Asp Met Val 50 55 60 Val Tyr Gln Val Gln Gly Gly Ala Gly Gly Ser Ala Gly Phe Pro Ser 65 70 75 80 Gly Gly Asn Pro Tyr Lys Trp Val Lys Ile Asn Ser Gly Met Ala Pro 85 90 95 Tyr Ser Asn Asn Val His Glu His Val Ile Gly His Glu Ile Gly His 100 105 110 Ser Ile Gly Phe Arg His Ser Asp Tyr Phe Ser Arg Gln Ser Cys Gly 115 120 125 Gln Asn Ser Asn Glu Gly Ser Ala Gly Val Gly Ala Ile His Ile Pro 130 135 140 Gly Thr Pro Thr Gly Trp Asp Pro Thr Ser Leu Met Asn Ala Cys Phe 145 150 155 160 Ser Ser Ser Glu Asp Gly Glu Phe Asn Gly Asn Asp Ile Thr Ala Leu 165 170 175 Asn Phe Leu Tyr 180 <210> 28 <211> 753 <212> DNA <213> KAOT1_10476 of Kordia algicida OT-1 lacking signal peptide <400> 28 gtagtagacg aggagcaaga cacagctatc ggaatcccac aagacgtact tcaaaaagca 60 caatcacttc acttcaacac attcgatatg caagaagcat ctttcgaaaa gccaaatggt 120 aaaatcgaag aaggatatat gatggaaggt gatatcttct ttacacgtga ccaattaatg 180 aacatggaac tcggtggaga tattacaagc aaacaatatc gtacaaacaa tttagtttct 240 ccaggagtaa ttacaatcat tggatataca ggaaataact ctaatggatt aacaactaaa 300 atgcaaactg gacttagatg ggcagtagat aactataatg cattaaacct aagtattagt 360 ttccaattaa catttggtac tgactatcaa aataaagata tggtagtata tcaagtacaa 420 ggtggagcag gtggatctgc aggtttccct tcaggaggaa atccatacaa gtgggttaaa 480 attaactcag gtatggctcc ttacagtaac aatgtacatg aacatgtaat tggacatgaa 540 ataggacact cgattggatt ccgtcattca gactacttca gcagacaaag ttgtggacaa 600 aattcaaatg aaggtagtgc aggagttgga gcaatccata ttccaggaac acctacaggt 660 tgggatccaa catctttaat gaatgcttgt ttcagttctt cagaagatgg agaatttaac 720 ggaaacgaca ttactgcttt aaacttctta tac 753 <210> 29 <211> 540 <212> DNA <213> KAOT1_10476 of Kordia algicida OT-1 lacking signal and processing peptide <400> 29 caatatcgta caaacaattt agtttctcca ggagtaatta caatcattgg atatacagga 60 aataactcta atggattaac aactaaaatg caaactggac ttagatgggc agtagataac 120 tataatgcat taaacctaag tattagtttc caattaacat ttggtactga ctatcaaaat 180 aaagatatgg tagtatatca agtacaaggt ggagcaggtg gatctgcagg tttcccttca 240 ggaggaaatc catacaagtg ggttaaaatt aactcaggta tggctcctta cagtaacaat 300 gtacatgaac atgtaattgg acatgaaata ggacactcga ttggattccg tcattcagac 360 tacttcagca gacaaagttg tggacaaaat tcaaatgaag gtagtgcagg agttggagca 420 atccatattc caggaacacc tacaggttgg gatccaacat ctttaatgaa tgcttgtttc 480 agttcttcag aagatggaga atttaacgga aacgacatta ctgctttaaa cttcttatac 540 540

Claims (5)

A tortoise active protease consisting of the amino acid sequence of SEQ ID NO: 4. A polynucleotide encoding the protease of claim 1. 3. The polynucleotide according to claim 2, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8. A pharmaceutical preparation containing the protease of claim 1 as an active ingredient. A method for controlling red tide, comprising treating the fungicide of claim 4 in an area where red tide has occurred.

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