Patent Grant
9631189
The present invention relates to a technique for immobilizing microorganisms, and specifically to a method for attaching and releasing a microorganism having adhesiveness (a method including adhesion to a carrier and subsequent release). The present application claims priority based on Japanese Patent Application No. 2013-063695 filed on Mar. 26, 2013, and the content of the patent application is hereby incorporated by reference herein in its entirety.
Biocatalysts such as enzymes and microbial cells are useful for the production of fine chemicals, general-purpose chemicals, medical intermediates, and biofuels. Biocatalysts catalyze efficient and highly selective reaction under mild conditions such as normal temperature, normal pressure, and neutral conditions. However, bioprocess using biocatalysts requires high production costs, and this problem hinders its commercialization.
Immobilization of biocatalysts has been considered as an important strategy for the cost reduction of bioprocess, because it allows use of the catalyst in repeated and/or continuous reactions, simplifies recovery and isolation of the catalyst and product from the reactor, facilitates the regeneration of the catalyst, and allows the increase in the catalyst concentration per volume. In addition, the use of the whole cell catalyst using the whole of microbial cells markedly contributes to the cost reduction of bioprocess, because, for example, it dispenses with the isolation and purification of the enzyme, it has higher stability than a separated enzyme, it can be proliferated and reactivated, and it does not require the feeding of reducing power such as expensive NADH from the outside. Recently, problems such as restriction of the substance transportation speed and disorders in the cell surface layer, which are the major problems of the use of the whole cell, are being resolved by the development of the technique for surface display localizing the enzyme on the surface of microbial cells.
Prior art techniques for immobilizing microbial cells include entrapment in gel matrix, crosslinking, covalent bonding to a solid surface, and physical adsorption. The used gel, which has been most frequently used, has problems such as the restriction of the substance transportation speed in the gel, cell leakage from the gel, and fragility of the gel. The crosslinking and covalent bonding have problems such as inhibition by the crosslinking agent, and inactivation of cells by the bonding itself. The physical adsorption does not have sufficient adhesiveness for effectively immobilizing general microbial cells, and is only effective for some filamentous fungi. Recently, methodologies for using biofilm as a natural immobilization method are reported (Non-Patent Documents 1 to 8), but there is no method other than screening of the microorganism having the biofilm forming capacity and desired reaction activity, and versatility of the type of microorganisms and reactions is low. In addition, the method is not efficient because it depends on naturally formed biofilm, and is not on the level applicable to actual substance production. Accordingly, prior art immobilization methods are not truly effective, and have many problems, so that the development of a general-purpose and effective immobilization method has been desired.
Acinetobacter sp. Tol 5 (Acinetobacter sp. Bacterium, Tol 5 strain), which was isolated from a biofilter by the inventors, is a nonpathogenic gram negative bacterium which has high autoagglutinating properties of cells, and shows high adhesiveness to various material surfaces such as various hydrophobic plastic carriers and hydrophilic glass and metal surfaces. As a factor giving such adhesive properties which is not reported for other microorganisms, novel bacterionanofiber existing on the bacterial cell surface layer was discovered, and a new protein composing the nanofiber was identified. This protein belongs to the trimeric autotransporter adhesin (TAA) family, and the present inventors named it AtaA (Non-Patent Document 9). TAA is known as a pathogenic factor included in various gram-negative pathogenic bacteria for specifically adhering to the host cells and extracellular matrix such as collagen, fibronectin, and laminin, thereby infecting the host (Non-Patent Document 10). The protein belonging to the TAA family forms a homotrimer, and builds a common whole structure of head-stoke-membrane anchor domain from the amino terminal toward the carboxyl terminal. However, there are small to large single peptide chains having about 300 to over 3000 amino acid residues, and the amino acid sequences are diversified. The peptide chain of AtaA found by the inventors is composed of 3630 amino acids, and is one of the largest TAAs. It has a unique primary structure composed of a plurality of long repeated sequences arranged in a mosaic pattern on a long stoke. Only the AtaA exhibits nonspecific and high adhesiveness to various surfaces. In addition, the study of TAA focuses on pathogenic bacteria, and there is no study on TAA regarding nonpathogenic bacteria such as Tol 5. On the basis of the results of these studies, the inventors reported the method for imparting or enhancing non-specific adhesiveness and/or autoagglutination to the target microorganism through the introduction of the gene encoding AtaA (Patent Document 1). In Patent Document 1, AtaA and the gene encoding AtaA (ataA gene) were referred to as AadA and aadA gene, respectively.
As described above, the inventors have invented a novel method for immobilizing a microorganism using AtaA which shows unique adhesive properties and has a relatively simple structure (Patent Document 1). More specifically, they have succeeded in the addition of adhesiveness and autoagglutination through the introduction of the ataA gene into a microorganism having no adhesiveness or autoagglutination. This method is one of the physical adsorption methods, but the adhesiveness is based on the high adhesiveness of AtaA, so that high immobilizing power far higher than the prior art method is exhibited. In addition, the introduction and expression of the ataA gene allows adding adhesiveness to various microorganisms, so that the versatility is high. Furthermore, different from the biofilm through the matrix such as an extracellular polysaccharide and gel-entrapment method, the cells are directly immobilized on the surface through the cell surface protein, so that there is no problem of limitation of the substance transportation speed in the matrix. The material and shape of the carrier may be freely designed. It is a unique, effective, and versatile method for immobilizing a microorganism.
The method reported by the inventors (Patent Document 1) is a highly useful technique which allows immobilization of industrially useful microorganisms, and AtaA used therein has the potential of solving various problems with prior art immobilization techniques. Accordingly, the present invention is intended to provide a more useful immobilization technique, thereby promoting the utilization and application of AtaA which had been found and identified by the present inventors.
As described above, previous studies have revealed that AtaA imparts adhesiveness and/or autoagglutination to the target microorganism. However, details about the adhesion mechanism of AtaA have not been revealed. Therefore, the present inventors studied the adhesive properties of AtaA in detail. In the course of the study, an amazing phenomenon was observed; the AtaA nanofiber cut out and separated from the Tol 5 cell surface adheres to the material surface in the presence of a salt, but cannot adhere in pure water. On the basis of the discussion of this phenomenon, it was assumed that the adhesiveness of AtaA to microbial cells is dependent on the ionic strength, and various validation experiments were carried out. As a result of this, it was revealed that the adhesiveness of the target microorganism, to which AtaA was introduced and expressed, is dependent on the ionic strength. It was also revealed that the change of the ionic strength allows the control of adhesiveness of the target microorganism, and repeated attaching and releasing (adhesion and release) of the target microorganism for the carrier (release while keeping adhesion capacity). Furthermore, it was shown that the catalyst function of the microorganism, to which adhesion capacity is imparted, will not decrease even after repeated attaching and releasing.
The invention described below is mainly based on the above-described findings and results.
[1] A method for attaching and releasing a microorganism including the steps (1) and (2):
(1) a step of contacting a microorganism, into which DNA encoding autotransporter adhesin derived from a microorganism belonging to the genus Acinetobacter has been introduced to impart or enhance non-specific adhesiveness, with a carrier under a high ionic strength and thus attaching the microorganism to the carrier; and
(2) a step of releasing the microorganism from the carrier by washing under a low ionic strength.
[2] The method of [1], wherein the DNA is an ataA gene.
[3] The method of [1], wherein the DNA is any of the DNA of (a), (b) or (c):
(a) DNA composed of the base sequence of SEQ ID NO. 1;
(b) DNA composed of the base sequence having 90% or more homology with the base sequence of SEQ ID NO. 1, and encoding the protein having activity of imparting or enhancing non-specific adhesiveness to a microorganism;
(c) DNA composed of a part of the base sequence of SEQ ID NO. 1, and encoding the protein having activity of imparting or enhancing non-specific adhesiveness to a microorganism.
[4] The method of [3], wherein the DNA of the following (a) or (b) is introduced into the microorganism together with the DNA encoding an autotransporter adhesin:
(a) DNA composed of the base sequence of SEQ ID NO. 3;
(b) DNA composed of the base sequence having 90% or more homology with the base sequence of SEQ ID NO. 3.
[5] The method of [1], wherein the DNA of the following (a) or (b) including the above-described DNA has been introduced into the microorganism:
(a) DNA composed of the base sequence of SEQ ID NO. 5;
(b) DNA composed of the base sequence having 90% or more homology with the base sequence of SEQ ID NO. 5., and having activity of imparting or enhancing non-specific adhesiveness to a microorganism;
[6] The method of any one of [1] to [5], wherein the boundary ionic strength between the high and low ionic strengths is from 5 mM to 20 mM.
[7] The method of any one of [1] to [5], wherein the boundary ionic strength between the high and low ionic strengths is about 10 mM.
[8] The method of [7], wherein the high ionic strength is from 10 mM to 500 mM, and the low ionic strength is less than 10 mM.
[9] The method of [8], wherein the high ionic strength is from 20 mM to 200 mM.
[10] The method of any one of [1] to [9], wherein the following step (3-1) is carried out after the step (2):
(3-1) a step of recovering the released microorganism.
[11] The method of any one of [1] to [9], wherein the following step (3-2) is carried out after the step (2):
(3-2) a step of recovering the carrier.
[12] The method of any one of [1] to [11], wherein the following step (i) is carried out between the steps (1) and (2):
(i) a step of contacting the microorganism attached to the carrier with a liquid to be treated, and keeping the contact state.
[13] The method of [12], wherein the microorganism has capacity of producing a specific enzyme, and the liquid to be treated comprises the substrate of the enzyme.
[14] The method of any one of [1] to [13], wherein the microorganism is a microorganism belonging to the genus Escherichia.
The present invention relates to the method for attaching and releasing a microorganism using an autotransporter adhesin derived from a microorganism belonging to the genus Acinetobacter. In the present invention, “the attaching and releasing method” means the method including a step (operation) of attaching a microorganism to a carrier, and a step (operation) of releasing the microorganism from the carrier.
Autotransporter adhesins are proteins reported as adhesive nanofibers expressed by gram-negative bacteria, and are known to specifically interact with the tissues, cell surface molecules, and extracellular matrix of the host. Autotransporter adhesins are said to have functions such as adhesion, invasion, cytotoxicity, blood serum resistance, and intercellular propagation. Autotransporter adhesins have a common architecture including N-terminal signal peptide, internal passenger domain, and C-terminal translocator domain. Among them, the C-terminal translocator domain defines this protein family. Secretion of an autotransporter adhesin is initiated by a signal peptide, and starts from the passage through the inner membrane by the Sec system. Subsequently, the translocator domain is inserted into the outer membrane, and forms a β barrel structure. Finally, the passenger domain passes through the tunnel formed with the barrel, and appears on the surface of the bacterial cells. Autotransporter adhesins are classified into monomeric autotransporter adhesins and trimeric autotransporter adhesins (Shane E. Cotter, Neeraj K. Surana and Joseph W. St Geme III 2005. Trimeric autotransporters: a distinct subfamily of Autotransporter proteins. TRENDS in Microbiology. 13: 199-205). The translocator domain of a monomeric autotransporter adhesin is considered to form a β barrel structure composed of 12 transmembrane antiparallel β sheets by one subunit. However the translocator domain of a trimeric autotransporter adhesin is known to form a trimer which is stable to heat and resistant to SDS in the outer membrane, and the subunit having four β sheets is oligomerized to form a 12-strand β barrel structure by three subunits. Furthermore, actually the passenger domain of all the monomeric autotransporter adhesins is linked to the bacteria surface in the translocator domain through a noncovalent bond, or released extracellularly. On the other hand, for all the trimeric autotransporter adhesin proteins, the passenger domain likely remains to be linked to the translocator domain through a covalent bond.
The trimeric autotransporter adhesin is abbreviated as TAA (trimeric autotransporter adhesin), and also referred to as Oca family (Oligomeric Coiled-coil Adhesin Family) which is a new class forming a coiled coil having a common oligomer structure (Andreas Roggenkamp, Nikolaus Ackermann, Christoph A. Jacobi, Konrad Truelzsch, Harald Hoffmann, and Jurgen Heesemann 2003. Molecular analysis of transport and oligomerization of the Yersinia enterocolitica adhesin YadA. J Bacteriol. 185: 3735-3744).
As shown by the below-described examples, the attaching and releasing method of the present invention is based on the surprising finding that the adhesiveness of the trimeric autotransporter adhesin AtaA derived from the Acinetobacter sp. Tol5 strain depends on the ionic strength, and includes the following steps (1) and (2):
(1) a step of contacting a microorganism, into which DNA encoding autotransporter adhesin derived from a microorganism belonging to the genus Acinetobacter has been introduced to impart or enhance non-specific adhesiveness, with a carrier under a high ionic strength and thus attaching the microorganism to the carrier; and
(2) a step of releasing the microorganism from the carrier by washing under a low ionic strength.
In the step (1), more specifically the adhesion step, firstly, a microorganism, into which DNA encoding TAA derived from a microorganism belonging to the genus Acinetobacter (adhesiveness-imparting DNA) has been introduced to impart or enhance non-specific adhesiveness (hereinafter referred to as “adhesiveness-imparted microorganism”) is provided.
The adhesiveness-imparting DNA is preferably the ataA gene isolated and identified from the Acinetobacter sp. Tol5 strain. The ataA gene is composed of the base sequence represented by SEQ ID NO. 1, and encodes the protein AtaA represented by SEQ ID NO. 2. The Acinetobacter sp. Tol5 strain has capacity of degrading toluene and was isolated from the exhaust gas treating reactor, and deposited to International Patent Organism Depositary, National Institute of Technology and Evaluation (NITE IPOD) under the accession number FERM P-17188 (Tsukuba Center Chuo No. 6, 1-1-1 Higashi Tsukuba-shi, Ibaraki, Japan).
In one preferred embodiment, the DNA composed of the base sequence represented by SEQ ID NO. 1 is used as the adhesiveness-imparting DNA, or its equivalent in terms of function may be used. Examples of the DNA which is functionally equivalent to the DNA composed of the base sequence represented by SEQ ID NO. 1 include the DNA which is composed of the base sequence having 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, most preferably 98% or more homology (or identity) with the base sequence represented by SEQ ID NO. 1, and encodes the protein having activity of imparting or enhancing non-specific adhesiveness to a microorganism. Another example is the DNA which hybridizes with the DNA composed of the base sequence complementary to the base sequence represented by SEQ ID NO. 1 under stringent conditions, and encodes the protein having activity of imparting or enhancing non-specific adhesiveness to a microorganism. Yet another example is the DNA which is composed of a part of the base sequence represented by SEQ ID NO. 1, and encodes the protein having activity of imparting or enhancing non-specific adhesiveness to a microorganism, in other words, the DNA of the deleted gene composed of the base sequence prepared by deleting several tens to several thousands of continuous base sequences from the base sequence represented by SEQ ID NO. 1 at one or more points, and the protein translated by it will not lose its activity of imparting or enhancing non-specific adhesiveness.
Stringent conditions mean the conditions which form a specific hybrid and no nonspecific hybrid, and examples thereof include low and high stringent conditions, but are preferably high stringent conditions. The low stringent conditions mean, for example, the conditions including washing after hybridization at 42° C., 5×SSC, 0.1% SDS, preferably at 50° C., 5×SSC, 0.1% SDS. The high stringent conditions mean, for example, the conditions including washing after hybridization at 65° C., 0.1×SSC and 0.1% SDS.
The mutation in the base sequence preferably keeps the domain structure composed of a signal peptide, a head domain, a neck domain, a stoke domain, and a membrane anchor domain. In the base sequence represented by SEQ ID NO. 1, the signal peptide corresponds to the bases of the positions 1 to 171, the head domain corresponds to the bases of the positions 322 to 807 and 8989 to 9444, the neck domain corresponds to the bases of the positions 886 to 957 and 9445 to 9516, the stoke domain corresponds to the bases of the positions 1216 to 8898 and 9517 to 10611, and the membrane anchor domain corresponds to the bases of the positions 10612 to 10890.
In the DNA composed of a part of the base sequence represented by SEQ ID NO. 1, the several tens to several thousands of continuous base sequences which can be deleted are preferably the sequence region encoding one or both stoke domains, one head domain, and one neck domain, and one or a plurality of continuous sequences may be deleted. It is more preferred that the region (the positions 322 to 8898) encoding the whole region from the head domain close to the amino terminal to the stoke domain, or the region (the position 8989 to 10611) encoding the whole region from the head domain close to the carboxy terminal to the stoke domain be deleted from the above-described each domains. It is even more preferred that a plurality of repeated regions found in the coding region of the stoke domain be deleted in such a manner that they each appear once without repetition. It is most preferred that any one of the plurality of repeated regions which is found in the coding region of the stoke domain.
The non-specific adhesiveness of the target microorganism is further improved by introducing the DNA composed of the base sequence represented by SEQ ID NO. 3 into the target microorganism, together with the DNA encoding the autotransporter adhesin. A gene functionally equivalent to the DNA composed of the base sequence represented by SEQ ID NO. 3 may be introduced. Examples of the DNA functionally equivalent to the DNA composed of the base sequence represented by SEQ ID NO. 3 include the DNA composed of the base sequence having 90% or more, preferably 95% or more, and more preferably 98% or more homology with the DNA composed of the base sequence represented by SEQ ID NO. 3. Other examples include the DNA which hybridizes with the DNA having a base sequence complementary to the base sequence represented by SEQ ID NO. 3 under stringent conditions. The base sequence represented by SEQ ID NO. 3 is a sequence found immediately below the AtaA gene of the Tol5 strain, and encodes ORF of the protein showing homology with the outer membrane protein ompA gene, BamE gene, and omlA gene included in gram-negative bacteria. Tol5-OmlT (referred to as Tol5-OmpA in Patent Document 1) as the ORF is composed of 264 amino acids (SEQ ID NO. 4) encoded by the 795-bp gene (SEQ ID NO. 3).
The operon containing the DNA encoding the autotransporter adhesin may be introduced to the target microorganism. For example, the introduction of the DNA composed of the base sequence represented by SEQ ID NO. 5 into the target microorganism allows the introduction of the DNA encoding the above-described outer membrane protein to the target microorganism together with the DNA encoding the autotransporter adhesin. Other operon functionally equivalent to the above-described operon may be introduced. Examples of the functionally equivalent operon include the operon composed of the DNA which is composed of a base sequence having 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, most preferably 98% or more homology with the base sequence represented by SEQ ID NO. 5, and has activity of imparting or enhancing non-specific adhesiveness and/or autoagglutination to the host microorganism. The base sequence represented by SEQ ID NO. 5 is the operon (ataA-omLT operon) isolated and identified from the Tol5 strain, and contains the promoter/ribosome bound site (the positions 1 to 106), ataA gene (the positions 107 to 10999) and Tol5-omlT gene (the positions 11064 to 11858). The E. coli DH5α transformed by the vector including the operon is deposited as “DH5α-XLTOPO::aadA-ompA to NITE Patent Microorganisms Depositary (NPMD) (2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan) under the accession number NITE BP-490 (date of accession Feb. 19, 2008).
The microorganism (target microorganism) subjected to the introduction of the adhesiveness-imparting DNA may be selected from various microorganisms. The target microorganism is not particularly limited, and examples include the microorganisms having no or weak non-specific adhesiveness. The target microorganism may be wild strain, mutant, or gene recombinant strain. An appropriate microorganism is selected according to the intended use of the present invention. Examples of the target microorganism include a bacterium belonging to the genus Escherichia such as Escherichia coli, a bacterium belonging to the genus Acinetobacter such as Acinetobacter calcoaceticus, a bacterium belonging to the genus Ralstonia such as Ralstonia eutropha, a bacterium belonging to the genus Pseudomonas such as Pseudomonas putida and Pseudomonas fluorescens, a bacterium belonging to the genus Aeromonas such as Aeromonas caviae, a bacterium belonging to the genus Alcaligenes such as Alcaligenes latus, a bacterium belonging to the genus Xanthomonas such as Xanthomonas campestris, a bacterium belonging to the genus Desulfomonile such as Desulfomonile tiedjei, a bacterium belonging to the genus Desulfuromonas such as Desulfuromonas chloroethenica, a bacterium belonging to the genus Chromobacterium such as Chromobacterium chocolatum, a bacterium belonging to the genus Burkholderia such as Burkholderia arboris, a bacterium belonging to the genus Rhodobacter, a bacterium belonging to the genus Acidovorax such as Acidovorax facilis, and a bacterium belonging to the genus Zymomonas such as Zymomonas mobilis.
Through the introduction of the adhesiveness-imparting DNA to the target microorganism for transformation, a microorganism having imparted or enhanced non-specific adhesiveness is obtained. Typically, the adhesiveness-imparting DNA is linked to an appropriate vector, and the target microorganism (host microorganism) is transformed by the vector, thereby obtaining a microorganism having imparted or enhanced non-specific adhesiveness. Specifically, the DNA is introduced to the host microorganism by multicopy, or the DNA is linked under control of the promoter which is expressed constitutively, or the DNA is linked under control of an enzyme-inducible promoter, thereby obtaining a microorganism having imparted or enhanced non-specific adhesiveness.
Firstly, the intended DNA is linked to the vector, thereby making a recombinant vector. Phages, cosmids, artificial chromosomes, or plasmids which can autonomously replicate in the host cell are used as the vector. Furthermore, plasmids which are integrated into the chromosome can be used as the expression cassette. In this case, the vector must have autonomous replication capability in the host (for example, Escherichia coli) in which the expression cassette is constructed, but the autonomous replication capability is not necessarily required in the host into which the expression cassette is introduced (for example, Acinetobacter sp. bacteria). The recombinant vector may be, for example, a shuttle vector designed so as to be useful for both of Escherichia coli and the bacterium belonging to the genus Acinetobacter.
Examples of the plasmid include the plasmid derived from Escherichia coli (for example, pET21a(+), pET32a(+), pET39b(+), pET40b(+), pET43.1a(+), pET44a(+), pKK223-3, pGEX4T, pUC118, pUC119, pUC18, and pUC19), and Escherichia coli-Acinetobacter shuttle vector plasmid pARP3 (Non-Patent Document 9), and examples of the phage DNA include λ, phage (λ, gt11, λ, ZAP, and the like). Alternatively, a commercially available cloning vector such as pCR4-TOPO (registered trademark) may be used for cloning and sequence confirmation.
Insertion of DNA into a vector is carried out by, for example, cleaving purified DNA with an appropriate restrictive enzyme, inserting it into the restriction site or multicloning site of an appropriate vector DNA, and then linking it to a vector. For example, the intended DNA may be synthesized by a commonly known method, and incorporated into a vector. The DNA may be amplified by the PCR method using a primer so as to contain the sites cleaved by an appropriate restrictive enzyme at each end. The conditions of PCR reaction may be appropriately established by those skilled in the art.
The recombinant vector may be linked to, in addition to the promoter and DNA of the present invention, as necessary, a cis element such as an enhancer, a selected marker, and a ribosome-binding sequence (SD sequence). Examples of the selection marker include, but not limited to, drug-resistant markers such as kanamycin, ampicillin, tetracycline, and chloramphenicol, and auxotrophic markers such as leucine, histidine, lysine, methionine, arginine, tryptophan, and uracil.
The promoter is not particularly limited, and may be appropriately selected by those skilled in the art according to the host microorganism. For example, when the host is Escherichia coli, T7 promoter, lac promoter, trp promoter, or λ-PL promoter may be used. A promoter composed of the base sequence represented by SEQ ID NO. 6, and those functionally equivalent to the promoter, such as a promoter composed of the base sequence having 90% or more, preferably 95% or more, more preferably 98% or more homology with the base sequence represented by SEQ ID NO. 6 are also preferred.
Linking of the DNA fragment and vector fragment may use a known DNA ligase. The DNA fragment and vector fragment are linked together after annealing, thereby making a recombinant vector. The recombinant vector can be obtained by ligation reaction under normal conditions preferably using a commercially available ligation kit, such as Ligation High (manufactured by Toyobo Co., Ltd.).
The recombinant DNA technique including cloning, ligation, and PCR may use, for example, those described in Sambrook, J et al., Molecular Cloning 2nd ed., 9. 47-9. 58, Cold Spring Harbor Lab. Press (1989) and Short Protocols In Molecular Biology, Third Edition, A Compendium of Methods from Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
The vector thus obtained may be as necessary purified by, for example, the boiling method, alkali SDS method, magnetic beads method, or a commercially available kit using any of these principles, and followed by, for example, concentration by any concentration means such as the ethanol precipitation method or polyethylene glycol precipitation method.
The method for introducing a recombinant vector into the target microorganism is not particularly limited, and examples thereof include the heat shock method using calcium ions, the electroporation method, and the lipofection method.
The transformed microorganism containing the intended DNA may be screened by forming a colony on an LB medium agar plate containing antibiotics such as ampicillin and kanamycin using the marker gene included in the recombinant vector. In order to check whether the cloned host microorganism is transformed by the recombinant vector, a part of the microorganism may be subjected to the amplification confirmation of insert by the PCR method, or the sequence analysis by the dideoxy method using a sequencer. Other than the method of introducing an autonomously replicable plasmid, an introduction method of chromosome incorporation type may be used, the method including placing a region homologous to the chromosomal gene in the vector, and causing homologous recombination to introduce the target gene.
The method for culturing the transformed microorganism thus obtained in a medium is carried out according to an ordinary method used for culturing the target microorganism. The medium for culturing the transformed microorganism obtained by using a microorganism such as Escherichia coli as the host may be a natural or synthetic medium as long as it contains, for example, a carbon source, a nitrogen source, inorganic salts which can be assimilated by the microorganism, and allows efficient culturing of the transformed microorganism. Specific examples include the M9 medium, M9G medium, BS medium, LB medium, Nutrient Broth medium, meat extract medium, SOB medium, and SOC medium.
The carbon source may be a carbon compound which can be assimilated, and examples thereof include saccharides such as glucose, polyols such as glycerin, alcohols such as methanol and ethanol, or organic acids such as pyruvic acid, succinic acid, citric acid, and lactic acid, fatty acids, and oil and fat. The nitrogen source may be a nitrogen compound which can be assimilated, and examples thereof include peptone, meat extract, yeast extract, casein hydrolysate, soybean cake alkali extract, alkylamines such as methylamine, and ammonia or salts thereof. In addition, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and an anti-foaming agent may be used as necessary. Furthermore, a protein expression inducer such as isopropyl-β-D-thiogalactopyranoside may be added to the medium as necessary.
The culturing is usually carried out under aerobic conditions such as shake culture or aeration stirred culture, preferably at 0 to 40° C., more preferably 10 to 37° C., particularly preferably 15 to 37° C. During the culturing period, the medium pH may be changed as appropriate within the range which allows the growth of the host and will not impair activity of the autotransporter adhesin, and is preferably about pH4 to 8. The pH is adjusted using, for example, an inorganic or organic acid, or an alkaline solution. During culturing, as necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium.
As described above, a microorganism having imparted or enhanced non-specific adhesiveness is obtained. The non-specific adhesiveness of the microorganism thus obtained may be evaluated using the adhesion test (CV adhesion test) by crystal violet staining. The specific method is, for example, as follows: the culture broth is subjected to centrifugation, the culture supernatant is removed, an inorganic salt medium containing neither carbon source nor nitrogen source or a salt solution is added to the bacterial cell pellet, a bacterial cell suspension is obtained by ultrasonication, the turbidity OD660 of the bacterial cell suspension is adjusted to a constant value (in the vicinity of 0.5) with the medium or the salt solution, the suspension is added to each well of a 96-well polystyrene plate in portions of 200 μl and incubated for 2 hours at a temperature optimum for the microorganism, the suspension in the well is thoroughly removed by a pipette, the well is washed with 200 μl of the inorganic salt medium or the salt solution twice and air-dried, 1% crystal violet aqueous solution is added to the wells and incubated for 15 minutes at room temperature, the crystal violet is removed by a pipette, the wells are washed three times with 200 μl of the inorganic salt medium, the crystal violet is eluted from the attached bacterial cells stained with 70% aqueous ethanol solution, and then the absorbance A590 is measured. When the absorbance A590 is 0.7 or more, preferably 1.0 or more, more preferably 1.5 or more, the microorganism is regarded as a microorganism having non-specific adhesiveness. The adhesion test may be carried out by any modified method of the above-described method, as long as the method is based on a principle that microbial cells are attached to the inner wall of an appropriate container under non-growth conditions, the attached cells are stained with an appropriate staining agent, and the amount of the attached cells are quantified by quantifying the staining agent or the number of stained cells. For example, the material and volume of the plate, the number of wells, the type and amount of the staining agent and cleaning liquid, the staining time and temperature, the concentration of suspended cells, the apparatus used for quantifying the staining agent and the number of stained cells (for example, spectrophotometer, plate reader, and microscope) may be appropriately selected. The staining agent may be safranine or a fluorescent dye. The adhesiveness may be evaluated by the comparison with a wild strain (negative control) having no adhesiveness-imparting DNA.
In the step (1) of the present invention, the prepared adhesiveness-imparted microorganism is contacted with a carrier under conditions where the adhesive protein, which is an expression product of the adhesiveness-imparting DNA, exhibits adhesiveness, more specifically, under a high ionic strength, thereby attaching the adhesiveness-imparted microorganism to the carrier. The border between the high ionic strength and low ionic strength (the conditions of the below-described step (2)) can be varied by the DNA used for imparting adhesiveness, and may be established by those skilled in the art based on a preliminary experiment with reference to the disclosure of the present description. It is preferred that the ionic strength at which the adhesiveness drastically changes be used as the “border” herein. For example, the border between the high ionic strength and the low ionic strength may be from 5 mM to 20 mM. In this case, the high ionic strength is, for example, from 10 mM to 500 mM, preferably from 20 mM to 200 mM. If a too high ionic strength is used, activity and existence of the adhesiveness-imparted microorganism can be adversely affected. Specific examples of the border include 5 mM, 7 mM, 10 mM, and 15 mM.
The type and constitution of the solution used in the step (1) (solution of high ionic strength) are not specifically limited. For example, various buffer solutions, various salt solutions, and media may be used.
The microorganism used in the present invention shows non-specific adhesiveness, so that various carriers (immobilizing carriers) may be used. The surface properties (for example, hydrophilicity and hydrophobicity), material, and form of the carrier are not particularly limited. Examples of the material include polyethylene, polystyrene, polycarbonate, silicon, nylon, polypropylene, polyvinyl alcohol, urethane, chitosan, cellulose derivatives, glass, ceramic, and metal, and examples of the form include plate, sphere, granules, nonwoven fabric, fiber, film, and sponge (foam).
Two or more adhesiveness-imparted microorganisms may be used in combination. This embodiment is useful when, for example, treatment or reaction is continuously carried out in two or more stages using the adhesiveness-imparted microorganism attached to a carrier, which is obtained by the step (1).
A contact state of the adhesiveness-imparted microorganism and carrier may be formed by dropping or adding a suspension of the adhesiveness-imparted microorganism to the carrier, or put the carrier into a solution containing the adhesiveness-imparted microorganism. This operation is preferably followed by incubation for 1 minute to 3 hours, thereby increasing the rate of adhesion. Alternatively, the adhesiveness-imparted microorganism may be grown in a culture broth containing salts in the presence of the carrier, thereby attaching the microorganism to the carrier simultaneously with growth.
The adhesiveness-imparted microorganism attached to the carrier by the step (1) is usually subjected to one or more treatment or reaction (details about this treatment or reaction will be described below). In the present invention, thereafter, the step (2), more specifically, the release step is carried out. In the step (2), the adhesiveness-imparted microorganism is released from the carrier by washing under a low ionic strength. In the present invention, the release of the adhesiveness-imparted microorganism is carried out by a simple operation of washing under a low ionic strength. This point is one of the greatest features of the present invention, and is advantageous in practicality and versatility.
In the present invention, the border between the high and low ionic strengths is as described above, and the low ionic strength is preferably less than 10 mM (0 mM or more and below 10 mM). The low ionic strength is more preferably 0 mM to 5 mM.
The buffer solution, medium, or the like satisfying the above-described conditions may be used as washing water. In order to achieve more reliable releasing and simple operation, the washing operation preferably uses water substantially free of ions or containing very few ions, more specifically, deionized water, distilled water, pure water, or ultrapure water. For example, the washing operation is carried out by keeping the carrier to which the adhesiveness-imparted microorganism is attached is kept in washing water for a predetermined time (for example, one minute to three hours) (during this time, the washing water may be stirred, and the carrier may be shaken), or washing water is continuously or intermittently sprayed over the carrier to which the adhesiveness-imparted microorganism is attached. The washing water may be replaced by a new one during the washing operation. In addition, the washing operation may be repeated twice or more.
The steps (1) and (2), more specifically the adhesion to the carrier and release may be repeated. In this case, the adhesiveness-imparted microorganism and/or carrier may be replaced with a new one (for example, the same type, similar one, or different one) in midstream.
In one embodiment, after the step (2), the released adhesiveness-imparted microorganism is recovered (step (3-1)). The recovered adhesiveness-imparted microorganism may be reused for the method of the present invention or other purpose. For example, a new carrier is put into the suspension of the adhesiveness-imparted microorganism recovered with the washing water, that is, the suspension of the released adhesiveness-imparted microorganism, and salts are dissolved, thereby attaching (immobilizing) the adhesiveness-imparted microorganism to the carrier. The recovered adhesiveness-imparted microorganism may be reused in such a manner after regeneration.
In another embodiment, after the step (2), the carrier is recovered (step (3-2)). The recovered carrier may be reused for the method of the present invention or other purpose. The recovered carrier may be reused after regeneration or activation. This embodiment is particularly useful when durability of the carrier is high, or the carrier is expensive.
The method of the present invention is applicable to various uses using an immobilized microorganism, such as the production of medicines, medical intermediates, and medical raw materials, the production of pesticides, the production of bioethanol, the production of biodiesel, the synthesis of chemicals, the production of foods (for example, isomerized sugars, maltodextrins, oligosaccharides, synthetic sweeteners, amino acids, peptides, and vitamins), and treatment of sewage, drains, industrial waste liquids, and industrial waste water. Therefore, in one embodiment of the present invention, the following step, more specifically, a step of contacting the adhesiveness-imparted microorganism attached to the carrier with the liquid to be treated, and keeping the contact state (step (i)) is carried out between the steps (1) and (2). The liquid to be treated is selected according to the intended use. For example, when the production or synthesis of a substance accompanied by enzyme reaction (for example, the above-described medicines, bioethanol, biodiesel, chemicals, and food) is carried out, a solution containing the substrate of the enzyme is used as the liquid to be treated. In this case, an adhesiveness-imparted microorganism having capacity of producing the specific enzyme causing the enzyme reaction is used. Examples of the specific enzyme include lipase, protease, peptidase, esterase, cellulase, hemicellulase, α-amylase, β-amylase, β-glucanase, glutaminase, isomerase, dehydrogenase, reductase, peroxidase, kinase, phosphatase, glycosyl transferase, and dechlorination enzymes. The enzyme produced by the adhesiveness-imparted microorganism may be an intracellular enzyme, extracellular enzyme, enzyme localized in the bacterial surface layer, or enzyme displayed at the cell surface.
The step (i) may be carried out using, for example, a continuous reaction vessel/reaction chamber, or batch type reaction vessel/reaction chamber.
In order to advance the utilization and application of AtaA as the trimeric autotransporter adhesin (TAA) derived from Acinetobacter sp. Tol 5, the adhesive properties of AtaA were studied in detail.
1. Method and Material
(1) Strain and Culture Conditions
The Tol 5 strain and its mutant were cultured at 28° C. in an inorganic salt medium or LB medium containing 0.05% toluene. The Acinetobacter sp. bacterium ADP1 strain and its mutant were cultured at 30° C. in an LB medium. The antibiotics were added as necessary at the following concentrations. Ampicillin: 500 μg/mL, gentamycin: 10 μg/mL for Tol 5-derived mutant. Ampicillin: 100 μg/mL, gentamycin 10 μg/mL for ADP1-derived mutant. For inducing the ataA gene, 0.5% arabinose was added.
(2) Construction of p3CAtaA
Using overlap PCR, the whole length of the DNA sequence encoding the HRV3C protease recognition site was inserted into the ataA gene. The first PCR was carried out using the primer set composed of the Bgl II ataA S primer (5′-GGTTTGAGCAATAAAGATCTAAATTCAAC-3′: SEQ ID NO. 7) and 3C protease ataA AS primer (5′-GGGTCCCTGAAAGAGGACTTCAAGCCCACCACCAAGATAATTGACTAC-3′: SEQ ID NO. 8), or the set composed of the XbaI ataA AS primer (5′-TGGGTCTAGAGAATTAGTCAATCAC-3′: SEQ ID NO. 9) and 3C protease ataA S primer (5′-CTTGAAGTCCTCTTTCAGGGACCCGGTGGTGGGGCAGGTTATGACAAC-3′: SEQ ID NO. 10). PrimeSTAR Max DNA polymerase (manufactured by Takara Bio Inc.) was used. Amplification of the DNA fragment of interest from the template plasmid (pTA2-ataA) was confirmed by agarose electrophoresis. Using the amplification product, Bgl II ataA S primer, and XbaI ataA AS primer, the second PCR was carried out. The amplified 3CataA fragment was subcloned into the pTA2 vector (manufactured by Toyobo Co., Ltd.). The vector construct pTA2-3C thus obtained was treated with Bgl II and Xba I, and the generated DNA fragment (containing the C-terminal side head, HRV 3C recognition site, and membrane binding anchor region) was ligated with the pTA2-ataA plasmid, and used as pTA2-3CataA. Finally, the 3CataA gene was subcloned into the pARP3 vector (Non-Patent Document 9), thereby obtained the plasmid p3CataA. The 4140 strain, which is the ataA gene-deletion mutant of Tol 5, was transformed by conjugation with the donor strain E. coli S17-1 (Simon, R.; Priefer, U.; Puhler, A., Bio-Technol 1983, 1, (9), 784-791) that had p3CAtaA.
(3) SDS-PAGE and Immunoblotting
The bacteria cell suspension or protein solution was dissolved in a 2×SDS-PAGE loading solution, and heated at 97° C. for 5 minutes. These samples were isolated by electrophoresis on 7.5% polyacrylamide using a tris-glycine SDS buffer solution, and subjected to CBB staining. In the immunoblotting, the protein isolated by SDS-PAGE was transferred to a PVDF membrane by the semi-dry method. The membrane was subjected to blocking treatment by incubation for one hour in a PBS buffer solution containing 5% skim milk, and reacted with the anti-AtaA antiserum against AtaA699-1014 for 1 hour. The membrane was washed with a PBST buffer solution for 10 minutes, and allowed to react with a peroxidase-bound anti-rabbit IgG secondary antibody at room temperature for one hour. The membrane was washed with a PBST buffer solution three times, and the bound antibody was detected with ECL Prime Detection Reagent (GE Healthcare).
(4) Flow Cytometry
The bacteria cell suspension was fixed sequentially in 2% and 4% paraformaldehyde for 10 minutes, and the cells were recovered by centrifugation. The cells were washed with deionized water twice, and then allowed to react with the anti-AtaA antiserum in PBS for 30 minutes. After washing with PBS once, the cells were allowed to react with Alexa Flour 488-bound anti-rabbit IgG secondary antibody in an NET buffer solution. The stained cells were washed with PBS twice, resuspended in deionized water, and analyzed with flow cytometry (FACSCant, BD).
(5) Adhesion Test
The bacterial strain cultured in an LB medium overnight was inoculated in an LB medium in an amount of 1/100, and cultured under shaking at 115 rpm. Tol 5 and its mutant were cultured at 28° C. for 8 hours, and ADP1 and its mutant were cultured at 30° C. for 12 hours. When the ataA gene was induced, 0.5% arabinose was added upon inoculation. After culturing, the bacterial cells were collected by centrifugation, washed three times with deionized water, and suspended again in KCl aqueous solutions at different concentrations. Finally, the cell concentration OD660 was adjusted to 0.5, and 200 μl of the suspension was transferred to a 96-well polystyrene (PS) plate or glass plate. The cells were incubated at 28° C. for 2 hours for attaching them thereto, the wells were washed twice with a KCl solution having the same concentration, and the attached cells were stained with 1% crystal violet for 15 minutes. After staining, the wells were washed another three times with a KCl solution having the same concentration, the staining agent was eluted from the cells with 70% ethanol, and quantified by measuring the absorbance at 590 nm.
(6) Release Test
In the same manner as in the adhesion test, a 100 mM KCl suspension of ADP1(pAtaA) cells was prepared. This suspension was added to a 96-well PS plate or glass plate, and incubated at 28° C. for 2 hours thereby attaching the cells thereto. Thereafter, the plate was washed three times with a KCl solution having the same concentration or deionized water, and the remaining cells were stained with 1% crystal violet. Thereafter, the cells were washed with KCl having the same concentration or deionized water, and then the staining agent was quantified in the same manner as in the adhesion test.
(7) Re-Adhesion Test
In the same manner as in the adhesion test, a 100 mM KCl suspension of bacterial cells was prepared. The bacterial cells in the bacterial suspension were attached to a 96-well PS plate in the same manner, and three wells were used for quantification of attached cells. This procedure is the same as in the adhesion test. Other wells were washed three times with deionized water, the bacterial cells released in the cleaning liquid were collected by centrifugation and suspended again in 100 mM KCl, and the OD660 was readjusted to 0.5. This cell suspension was used for the re-adhesion test. Thereafter, the same operation was repeated.
(8) Measurement of Esterase Activity
The ADP1(pAtaA) cells were subjected to re-adhesion test by the above-described method. The 96-well plate to which the cells are attached to was washed with 100 mM KCl, and then 200 μl of 4-NBP reaction solution (1.9 mM p-nitrophenyl butyrate, 1.1% triton-X 100, 50 mM 3,3-dimethylglutaric acid, 50 mM tris, 50 mM 2-amino-2-methyl-1,3-propanediol) was added, and incubated for 30 minutes at room temperature. Thereafter, the absorbance at 405 nm, which is the absorption wavelength of paranitrophenol as the product, was measured using a microplate reader, thereby determining esterase activity.
2. Result and Discussion
(1) Separation and Purification of AtaA Protein and Analysis of Adhesive Properties
In order to cut out AtaA from the cell surface of Tol 5, the recognized sequence of HRV 3C protease (LEVLFQGP: SEQ ID NO. 11) was inserted into the FGG region, which is supposed to form a loop near the outer membrane binding anchor domain (MAD) of AtaA, together with the linker composed of three glycine residues (GGG) (
Subsequently, 3CAtaA was cleaved from the cell surface by HRV 3C protease, and precipitated and isolated by 30% ammonium sulfate. The isolate was subjected to SDS-PAGE and Western blotting, and confirmed to be a monomer of 3CAtaA (the result not shown). Furthermore, the isolate was subjected to Native PAGE, and the protein having a molecular weight of 720 kDa or more was detected (the result not shown). AtaA belonging to the TAA family is supposed to have a homotrimer structure, and the 3CAtaA, which had been actually obtained by cleaving from the vicinity of the outer membrane binding region, followed by isolation and purification, was shown to maintain a polymer structure.
The adhesive properties of the separated and purified 3CAtaA itself to the PS surface and glass surface were studied by ELISA. The influence on adhesion was analyzed with the ionic strength changed by potassium chloride (KCl). 3CAtaA adhered to both the surfaces in the presence of a small amount of ions. However, the adhesiveness drastically decreased at 10 mM or lower, and it hardly adhered to the PS surface or glass surface in pure water containing no ion (
(2) Ionic Strength Dependency of Microbial Cell Adhesion by AtaA
On the basis of the above-described discovery, whether the adhesion of microbial cells by AtaA is influenced by the ionic strength was studied using a Tol 5 wild strain. As a result, it was found that the adhesion of the Tol 5 cells itself is dependent on the ionic strength (
In order to examine whether the ionic strength dependency of the microbial cell adhesion directly reflects the adhesive properties of AtaA, ADP1 belonging to the genus Acinetobacter, which is a different bacterium belonging to the same genus, and ADP1(pAtaA) including the ataA gene were studied for their adhesive properties. It has already reported that ADP1 originally has no adhesiveness or autoagglutination, while ADP1(pAtaA) shows high adhesiveness and autoagglutination (Non-Patent Document 9). This was confirmed also this time. The ADP1 wild strain hardly adhered to the PS surface or glass surface irrespective of the ionic strength (
The adhesiveness of general microbial cells is far lower than the adhesiveness by AtaA, and is not at the level useful for immobilizing the microorganism, but is known to show similar ionic strength dependency, and is explained by the DLVO theory. According to this theory, the energy necessary for the approach and attachment of microbial cells to the surface is the total of the energy applied to both of Van der Waals attraction and electrostatic repulsive force, so that the decrease of the ionic strength increases the electrostatic repulsive force, and thus hinders attachment of microbial cells. When the ionic strength is below a certain level, the energy of microorganisms to approach the surface cannot be exceeded by the motile energy or the energy of Brownian motion of microbial cells themselves. Because of this energy barrier, microbial cells cannot directly reach the surface, so they attach to the surface via the extracellular polymer or nanofibers on the cell surface. The radiuses of curvature of the extracellular polymer and nanofiber are far smaller than cells themselves, so that there is no energy barrier. There should be no energy barrier in the adhesion of AtaA as nanofiber, and the loss of adhesion ability in pure water cannot be explained by this theory. However, the ionic strength dependency of the cell adhesion itself is similar to the general microorganism adhesive properties reflecting the DLVO theory. Therefore, in order to confirm whether the surface potentials of ADP1 and ADP1(pAtaA) are different or not, the ionic strength dependencies of the electrophoresis of the cells were compared. As a result, their profiles of the electrophoresis speed completely identical to each other, and the ionic strength dependencies were the same regardless of the presence or absence of AtaA (the result not shown). As described above, for the ADP1 wild strain, the adhesiveness is not improved by the increase of the ionic strength. Therefore, the high adhesiveness of the ADP1(pAtaA) cells in the presence of ions is brought about by AtaA, and the loss of adhesiveness under a low ionic strength and in pure water reflects the adhesive properties of AtaA, so that the difference from the DLVO theory is strongly suggested. In any case, this proves that the microbial cells, to which adhesiveness was imparted by AtaA, hardly adhere in pure water or under an extremely low ionic strength.
(2) Technique for Attaching and Releasing Microorganism Using AtaA
Using the above-described adhesive properties of AtaA and adhesive properties of microorganism imparted by AtaA, it was attempted to release the microbial cells, which had been once immobilized by AtaA, by washing with pure water. The ADP1(pAtaA) cells, which had been immobilized on a PS plate or glass plate in 100 mM KCl, was washed with pure water or 100 mM KCl. As a result, it was shown that the microbial cells were effectively removed from the both plates by washing with pure water (
Finally, with the aim of actually applying the technique of attaching and releasing the microorganism immobilized by AtaA, which has been developed this time, to bioprocess, the change in the enzymatic activity of the immobilized cells was studied by repeating the attaching and releasing. The ADP1 cells originally have esterase on the cell surface. Therefore, using the esterase activity as the index, the influence of attaching and releasing on the catalyst function of a whole cell catalyst was evaluated. The immobilized ADP1(pAtaA) cells were released with pure water, suspended again in a 100 mM KCl solution, and immobilized again, and then the immobilized microorganism cells were subjected to the quantification of the esterase activity. The attaching and releasing operation and esterase activity measurement were repeated. The result indicates that the esterase activity of the immobilized microorganism cells did not decrease even after repetition of the attaching and releasing (
As described above, the method for directly immobilizing a microorganism using AtaA is applicable to various microorganisms and carriers, and is a unique and versatile technique for immobilizing a whole cell catalyst without the problems of prior art immobilization methods. This novel method allows repeating attaching and releasing immobilized microbial cells with keeping their catalyst function, and contributes to the design of a more flexible and practical bioprocess.
The ADP1(pAtaA) cells were suspended in pure water in a conical flask, and then a black sponge (polyurethane foam) was put in. Firstly, the liquid was turbid because of the bacterial cells dispersed in the liquid (
The method of the present invention allows releasing and recovering a microorganism which has been immobilized (attached) on a carrier, immobilizing the recovered microorganism on the carrier again, and further repeating the immobilizing and releasing. The present invention realizing such use of a microorganism by simple operations will bring about innovative effect in the bioprocess using an immobilized microorganism. Of special note is that the immobilized microorganism can be released by an extremely simple operation. The use of the method of the present invention allows free reuse of a microorganism and a carrier, such as the use of a once used immobilized microorganism by immobilizing it again on another carrier, replacement of a microorganism having weakened activity on a carrier with a fresh cells having high activity, or re-activation and re-immobilization of released cells having decreased activity. The present invention is widely useful, and is useful for, for example, conventional fermentation industry and waste disposal, and is markedly effective for the production of biomass energy and green biotechnology using microbial cells. The application of the present invention will allow cost reduction, improvement of efficiency of the production process, and the like.
The present invention will not be limited to the description of the embodiments and examples of the present invention. Various modifications readily made by those skilled in the art are also included in the present invention, without departing from the scope of claims. The entire contents of the articles, unexamined patent publications, and patent applications specified herein are hereby incorporated herein by reference.
SEQ ID NO. 7: Explanation of artificial sequence: primer
SEQ ID NO. 8: Explanation of artificial sequence: primer
SEQ ID NO. 9: Explanation of artificial sequence: primer
SEQ ID NO. 10: Explanation of artificial sequence: primer
SEQ ID NO. 11: Explanation of artificial sequence: recognition sequence of HRV 3C protease
| Number | Date | Country | Kind |
|---|---|---|---|
| 2013-063695 | Mar 2013 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2014/056966 | 3/14/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/156736 | 10/2/2014 | WO | A |
| Number | Date | Country |
|---|---|---|
| WO-2009104281 | Aug 2009 | WO |
| Entry |
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| Number | Date | Country | |
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| 20160046923 A1 | Feb 2016 | US |
