JP6610868B2 - Method for attaching and detaching microorganisms - Google Patents

Description

  The present invention relates to a microorganism immobilization technique. Specifically, the present invention relates to a method for attaching and detaching microorganisms imparted with adhesion (a method involving attachment to a carrier and subsequent desorption) and a desorption method.

  Biocatalysts such as enzymes and microbial cells are useful for producing fine chemicals, general-purpose chemicals, pharmaceutical intermediates, biofuels and the like. Biocatalysts catalyze efficient and highly selective reactions under mild conditions such as normal temperature, normal pressure, and neutrality. However, a bioprocess using a biocatalyst has a high production cost, which hinders practical use.

  Immobilization of biocatalysts enables repeated use and continuous reaction of the catalyst, facilitates recovery and separation of the catalyst and product from the reactor, facilitates catalyst regeneration, It has been considered as an important strategy for reducing the cost of bioprocesses because it enables the catalyst concentration to be increased. In addition, the use of whole cell catalysts that use whole microbial cells eliminates the need for enzyme separation and purification, is more stable than the separated enzyme, is capable of growth and reactivation, is expensive This greatly contributes to the cost reduction of bioprocesses because it is not necessary to supply reducing power such as NADH from the outside. In particular, the problem of mass transport rate limiting and obstacles in the cell surface, which has been a major problem in the use of all cells, has recently been opened up by the surface display technology that localizes enzymes on the surface of microbial cells. .

  Conventional methods for immobilizing microbial cells include gel entrapment, cross-linking, covalent bonding, and physical adsorption. The most commonly used gel entrapment methods have problems such as the rate of mass transport within the gel, cell leakage from the gel, and fragility of the gel. In the cross-linking method and the covalent bond method, there are problems such as inhibition by a cross-linking agent and cell inactivation due to the binding itself. In the physical adsorption method, it is not possible to expect an adsorption force for effectively fixing ordinary microbial cells, and it is effective only for some filamentous fungi. Recently, methodologies using biofilms as natural immobilization methods have also been reported (Non-Patent Documents 1 to 8), but only a method of screening microorganisms having both biofilm-forming ability and target reaction activity is available. No, the versatility of microorganisms and reaction types is low. In addition, it is not an efficient method because it relies on a naturally formed biofilm, and it is not at a level applicable to actual substance production. Therefore, it cannot be said that the conventional immobilization method is truly effective, and there are many problems. Therefore, the development of a general-purpose and effective immobilization method has been desired.

  Acinetobacter sp. Tol 5 (Acinetobacter genus Tol 5 strain) previously isolated from the biofilter by the present inventor has high cell self-aggregation property, and hydrophilic glass and metal surfaces from various hydrophobic plastic carriers. Until now, it is a non-pathogenic Gram-negative bacterium with high adhesion to various material surfaces. We discovered a novel bacterio nanofiber that exists on the surface of bacterial cells as a factor that brings about such adhesion characteristics that have not been reported in other microorganisms, and also identified a new protein that constitutes the nanofiber. This protein belongs to the trimeric autotransporter adhesin (TAA) family, and the inventor named it AtaA (Non-patent Document 9). TAA is known as a virulence factor that various gram-negative pathogenic bacteria specifically adhere to extracellular cells such as host cells and collagen, fibronectin, and laminin to infect the host (Non-patent Document 10). ). Proteins belonging to the TAA family form homotrimers and have a common overall structure of head-stalk-outer membrane binding site from the amino terminus to the carboxyl terminus. However, the number of amino acid residues in a single peptide chain ranges from as small as 300 to as large as more than 3000, and the amino acid sequence is diverse. The peptide chain of AtaA discovered by the present inventor consists of 3630 amino acids, and is the largest in TAA. It has a unique primary structure in which multiple long repeating arrays are arranged in a mosaic pattern on a long stalk. Only AtaA exhibits nonspecific and high adhesion to various surfaces. TAA research is focused on pathogenic bacteria, and there are no examples of TAA studies on non-pathogenic bacteria such as Tol 5. Based on the above research results, the present inventor has reported a method for imparting or enhancing nonspecific adhesion and / or aggregation to a target microorganism by introducing a gene encoding AtaA (Patent Document 1). In Patent Document 1, AtaA and a gene (ataA gene) encoding the same are referred to as AadA and aadA gene, respectively.

International Publication No. 2009/104281 Pamphlet International Publication No. 2014/156736 Pamphlet

Junter, G. A .; Jouenne, T., Immobilized viable microbial cells: from the process to the proteome... Or the cart before the horse. Biotechnol. Adv. 2004, 22, (8), 633-658. Qureshi, N .; Annous, B. A .; Ezeji, T. C .; Karcher, P .; Maddox, I. S., Biofilm reactors for industrial bioconversion processes: using potential of enhanced reaction rates.Microb. Cell. Fact. 2005, 4, 24. Li, XZ; Webb, JS; Kjelleberg, S .; Rosche, B., Enhanced benzaldehyde tolerance in Zymomonas mobilis biofilms and the potential of biofilm applications in fine-chemical production. Applied and Environmental Microbiology 2006, 72, (2), 1639 -1644. Gross, R .; Hauer, B .; Otto, K .; Schmid, A., Microbial biofilms: new catalysts for maximizing productivity of long-term biotransformations. Biotechnology and Bioengineering 2007, 98, (6), 1123-1134. Li, X. Z .; Hauer, B .; Rosche, B., Single-species microbial biofilm screening for industrial applications.Appl.Microbiol. Biotechnol. 2007, 76, (6), 1255-1262. Rosche, B .; Li, X. Z .; Hauer, B .; Schmid, A .; Buehler, K., Microbial biofilms: a concept for industrial catalysis? Trends Biotechnol 2009, 27, (11), 636-43. Halan, B .; Buehler, K .; Schmid, A., Biofilms as living catalysts in continuous chemical syntheses.Trends Biotechnol 2012, 30, (9), 453-65. Cheng, K. C .; Demirci, A .; Catchmark, J. M., Advances in biofilm reactors for production of value-added products.Appl Microbiol Biotechnol 2010, 87, (2), 445-56. Ishikawa, M .; Nakatani, H .; Hori, K., AtaA, a new member of the trimeric autotransporter adhesins from Acinetobacter sp. Tol 5 mediating high adhesiveness to various abiotic surfaces.PLoS One 2012, 7, (11), e48830 . Linke, D .; Riess, T .; Autenrieth, I. B .; Lupas, A .; Kempf, V. A., Trimeric autotransporter adhesins: variable structure, common function.Trends Microbiol. 2006, 14, (6), 264-270.

  As described above, the present inventor has previously invented a novel method for immobilizing microorganisms using AtaA that exhibits unique adhesive properties and has a relatively simple structure (Patent Document 1). That is, by introducing the ataA gene into a microorganism that does not have adhesion or aggregation, the inventors succeeded in imparting adhesion and aggregation. Although this method can be said to be one of the physical adsorption methods, the adsorption force is based on the high adhesion force of AtaA, and thus exhibits a high immobilization force that cannot be compared with the conventional method. Moreover, if the ataA gene can be introduced and expressed, adhesion can be imparted to various microorganisms, and thus versatility is high. In addition, unlike biofilm and gel entrapment methods attached by a matrix such as extracellular polysaccharide, cells are directly fixed to the surface by surface proteins, so there is no problem of mass transport rate limiting in the matrix. . The material and shape of the carrier can be designed freely. This is a unique and effective method for immobilizing microorganisms.

  The method (Patent Document 1) reported by the present inventor is a highly useful technique that enables immobilization of industrially useful microorganisms, and AtaA used therein solves various problems in the conventional immobilization technique. It has the potential to be overcome. The present inventor examined the attachment characteristics of AtaA in detail in order to advance the use and application of AtaA. As a result, a surprising fact that the adhesion of the target microorganism can be manipulated by changing the ionic strength was found, and a patent application was filed (Patent Document 2). The ability to immobilize microorganisms on a carrier and desorb from the carrier by a simple operation of changing the ionic strength is extremely useful in practice. However, it may be difficult to change the ionic strength depending on the form of use. For example, when implemented on a large scale, a large amount of solution needs to be exchanged at the time of attachment / detachment (or removal), and the operation becomes complicated in some cases. In view of the usefulness and versatility of AtaA, its use is considered to expand more and more in the future, and it is desired to provide a new means for manipulating adhesion in order to cope with various usage forms. The object of the present invention is to respond to such a demand and to further use and apply AtaA.

Under the above problems, the present inventor decided to further study the adhesion characteristics of AtaA. As a result, it was found that a specific substance / component inhibits adhesion of AtaA in a concentration-dependent manner and also inhibits aggregation of bacterial cells by AtaA (in other words, promotes dispersion). It should be noted with respect to the latter fact that the effect of dispersing the formed aggregates and the effect of peeling the microbial aggregates fixed on the surface from the surface were also recognized.
Based on the above results, the following inventions are provided. In addition, according to this invention, several choices are provided as attachment / detachment or removal conditions. Therefore, it becomes possible to select the optimum conditions in consideration of the influence on the microorganisms to be used, and the versatility and utility value of the immobilization technique using AtaA is enhanced.
[1] A method for attaching and detaching microorganisms, including the following steps (1) and (2):
(1) A step of bringing a microorganism to which nonspecific adhesion is imparted or enhanced by introducing DNA encoding an autotransporter adhesin derived from an Acinetobacter genus microorganism into contact with the carrier, and attaching the microorganism to the carrier ;
(2) The microorganism attached to the carrier is subjected to the following conditions (A) to (F), that is,
(A) containing 0.01% (w / v) or more of casamino acid;
(B) containing 0.01% (w / v) or more of lactic acid;
(C) containing 0.01% (w / v) or more of yeast extract;
(D) containing 0.01% (w / v) or more of tryptone;
(E) containing skim milk of 0.1% (w / v) or more;
(F) containing 1 mM or more of glutamic acid;
Treatment with a solution satisfying at least one of the conditions to desorb the microorganism from the carrier.
[2] The method according to [1], wherein the following step (i) is performed between step (1) and step (2):
(I) A step of bringing the microorganism attached to the carrier into contact with a liquid to be treated and maintaining the contact state.
[3] The method according to [2], wherein the microorganism has an ability to produce a specific enzyme, and the liquid to be treated contains a substrate of the specific enzyme.
[4] The method according to [1], wherein the following step (3-1) and / or step (3-2) is performed after step (2):
(3-1) recovering the detached microorganisms;
(3-2) A step of recovering the carrier.
[5] A method for detaching microorganisms, comprising the following step (I):
(I) A microorganism having nonspecific adhesion imparted or enhanced by introduction of DNA encoding an autotransporter adhesin derived from an Acinetobacter microorganism, which adheres to a carrier under the following conditions ( A) to (F), that is,
(A) containing 0.01% (w / v) or more of casamino acid;
(B) containing 0.01% (w / v) or more of lactic acid;
(C) containing 0.01% (w / v) or more of yeast extract;
(D) containing 0.01% (w / v) or more of tryptone;
(E) containing skim milk of 0.1% (w / v) or more;
(F) containing 1 mM or more of glutamic acid;
Treatment with a solution satisfying at least one of the conditions.
[6] The method according to claim 5, wherein the following step (3-1) and / or step (3-2) is performed after step (I):
(3-1) recovering the detached microorganisms;
(3-2) A step of recovering the carrier.
[7] The concentration of casamino acid in the condition (A) is 0.1% (w / v) or more,
The concentration of lactic acid in the condition (B) is 0.1% (w / v) or more,
The concentration of the yeast extract in the condition (C) is 0.1% (w / v) or more,
The tryptone concentration in the condition (D) is 0.1% (w / v) or more,
The concentration of glutamic acid in the condition (F) is 10 mM or more,
[1] The method according to any one of [6].
[8] The method according to any one of [1] to [7], wherein the DNA is ataA gene.
[9] The method according to any one of [1] to [7], wherein the DNA is the following DNA (a), (b), or (c):
(A) DNA consisting of the base sequence represented by SEQ ID NO: 1;
(B) a DNA encoding a protein consisting of a base sequence represented by SEQ ID NO: 1 and having a homology of 90% or more and having an activity of imparting or enhancing nonspecific adhesion to microorganisms;
(C) DNA encoding a protein consisting of a part of the base sequence represented by SEQ ID NO: 1 and having an activity of imparting or enhancing nonspecific adhesion to microorganisms.
[10] The method according to [9], wherein the DNA of the following (a) or (b) is introduced into the microorganism together with DNA encoding an autotransporter adhesin:
(A) DNA consisting of the base sequence represented by SEQ ID NO: 3;
(B) DNA comprising a nucleotide sequence having 90% or more homology with the nucleotide sequence represented by SEQ ID NO: 3.
[11] The method according to any one of [1] to [7], wherein the DNA of the following (a) or (b) including the DNA is introduced into the microorganism:
(A) DNA consisting of the base sequence represented by SEQ ID NO: 5;
(B) A DNA comprising a nucleotide sequence having 90% or more homology with the nucleotide sequence represented by SEQ ID NO: 5, and having an activity of imparting or enhancing nonspecific adhesion to microorganisms.
[12] The method according to any one of [1] to [11], wherein the microorganism is an Escherichia bacterium.

Results of adhesion inhibition test. The inhibitory ability of various additives (casamino acid, lactic acid, yeast extract, tryptone, skim milk, glutamic acid) on the adhesion of Acinetobacter sp. Tol 5 wild strain to the polystyrene plate was evaluated. Glucose and cellobiose were used as comparative controls. Continuation of FIG. Results of cell self-aggregation inhibition test. The inhibitory ability of various additives (casamino acid, lactic acid, glutamic acid, yeast extract, tryptone) on the aggregation of Acinetobacter sp. Tol 5 wild strain was evaluated. Glucose and glycerol served as comparative controls. The addition amount was 1% (w / v) or 100 mM. Results of adhesion inhibition test. The inhibitory ability of various additives (casamino acid, lactic acid) on the adhesion of Acinetobacter sp. Tol 5 wild strain to the glass plate was evaluated. The processed slide glass was observed with a confocal laser microscope. Upper row is observed from above, lower row is observed from the side. Left: treated with additive-free inorganic salt medium, center: treated with casamino acid (2.5% (w / v)), right: treated with sodium lactate (2.5% (w / v)). The result of the peeling test of the microbial cells adhering to the glass plate. The peeling effect of Acinetobacter sp. Tol 5 wild strain adhering to the glass plate was examined for casamino acid and lactic acid. After a series of treatments, the cells attached to the slide glass were fluorescently stained and observed with a confocal laser microscope. Upper row is observed from above, lower row is observed from the side. Left: treated with additive-free inorganic salt medium, center: treated with casamino acid (2.5% (w / v)), right: treated with sodium lactate (2.5% (w / v)). The result of the adhesion inhibition test using microorganisms to which adhesion was imparted by introduction of the ataA gene. We evaluated the ability of casamino acid and lactic acid to inhibit the adhesion of ADP1 strain ADP1 introduced with ataA gene to polystyrene plates. Glucose was used as a comparative control.

  The present invention relates to a method for attaching and detaching a microorganism using an autotransporter adhesin derived from an Acinetobacter microorganism. In the present invention, the “detaching method” refers to a method including a step (operation) for attaching a microorganism to a carrier and a step (operation) for removing the microorganism attached to the carrier.

  Autotransporter adhesin is a protein reported as an adhesive nanofiber of gram-negative bacteria, and is known to interact specifically with host tissues, cell surface molecules, and extracellular matrix. . The autotransporter adhesin is said to have functions such as adhesion, invasion, cytotoxicity, serum resistance, and cell-to-cell transmission. Autotransporter adhesins have a common region organization: N-terminal signal peptide, internal passenger domain, and C-terminal translocator domain. Among them, the C-terminal translocator domain is a domain that defines this genus. Secretion of the autotransporter adhesin is initiated by the signal peptide and begins with passage through the inner membrane by the Sec system. Subsequently, the translocator domain is inserted into the outer membrane, forming a β-barrel structure. Finally, the passenger domain passes through the tunnel formed by the barrel and appears on the surface of the cells. Autotransporter adhesins are classified into monomeric autotransporter adhesins and trimeric autotransporter adhesins (Shane E. Cotter, Neeraj K. Surana and Joseph W. St GemeIII 2005. Trimeric autotransporters: a distinct subfamily of Autotransporter proteins.TRENDS in Microbiology.13: 199-205). The translocator domain of the monomeric autotransporter adhesin is thought to form a β-barrel structure consisting of 12 transmembrane antiparallel β-sheets from one subunit. However, the translocator domain of the trimer autotransporter adhesin forms a trimer that is heat-stable and strong in SDS in the outer membrane, and the subunits with four β sheets are oligomerized into three subunits. It is known that a β-barrel structure of 12 strands is formed. Furthermore, virtually all monomeric autotransporter adhesin passenger domains are either non-covalently linked to the bacterial surface with the translocator domain or released extracellularly, whereas all trimeric autotrans In the porter adhesin protein, the passenger domain is thought to remain covalently linked to the translocator domain.

  Trimeric autotransporter adhesin is abbreviated as TAA (trimeric autotransporter adhesin) and is also called the Oca family (Oligomeric Coiled-coil Adhesin Family) as a new class that creates coiled coils with 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).

The attachment / detachment method of the present invention is based on the surprising finding that a specific substance can control the adhesion of AtaA and also exhibits an aggregation inhibition effect, as shown in the Examples below. The following steps ( Includes 1) and (2).
(1) A step of bringing a microorganism to which nonspecific adhesion is imparted or enhanced by introducing DNA encoding an autotransporter adhesin derived from an Acinetobacter genus microorganism into contact with the carrier, and attaching the microorganism to the carrier (2) The microorganism attached to the carrier is subjected to the following conditions (A) to (F), that is,
(A) containing 0.01% (w / v) or more of casamino acid;
(B) containing 0.01% (w / v) or more of lactic acid;
(C) containing 0.01% (w / v) or more of yeast extract;
(D) containing 0.01% (w / v) or more of tryptone;
(E) containing skim milk of 0.1% (w / v) or more;
(F) containing 1 mM or more of glutamic acid;
Treating with a solution satisfying at least one of the conditions and desorbing the microorganism from the carrier

  In the step (1), that is, the attachment step, first, DNA encoding TAA derived from an Acinetobacter genus microorganism (hereinafter referred to as “adhesion-imparting DNA”) is introduced to impart or enhance nonspecific adhesion. Prepared microorganisms (hereinafter referred to as “adhesion-imparting microorganisms”).

  As the adhesion-imparting DNA, ataA gene isolated and identified from Acinetobacter sp. Tol5 strain is preferably used. The ataA gene consists of the base sequence represented by SEQ ID NO: 1 and encodes the protein AtaA represented by SEQ ID NO: 2. Acinetobacter sp. Tol5 strain is a strain having a toluene resolution isolated from an exhaust gas treatment reactor, and under the accession number FERM P-17188, National Institute of Technology and Evaluation Biotechnology Center Patent Biodeposition Center (NITE IPOD) ( 2-10, Kazusa Kamashiga, Kisarazu City, Chiba Prefecture, Japan, Room 120).

  In a preferred embodiment, DNA consisting of the base sequence represented by SEQ ID NO: 1 is employed as the adhesion-imparting DNA, but DNA functionally equivalent to the DNA may be used. The DNA functionally equivalent to the DNA consisting of the base sequence represented by SEQ ID NO: 1 is 70% or more, preferably 80% or more, more preferably 90% or more, more than the base sequence represented by SEQ ID NO: 1. Preferably, a DNA encoding a protein consisting of a base sequence having a homology (or identity) of 95% or more, most preferably 98% or more, and having an activity of imparting or enhancing nonspecific adhesion to a microorganism Can be mentioned. Alternatively, a protein that hybridizes with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1 under stringent conditions and has an activity of imparting or enhancing nonspecific adhesion to a microorganism. For example, the encoding DNA. Furthermore, it consists of a part of the base sequence represented by SEQ ID NO: 1 and encodes a protein having an activity of imparting or enhancing nonspecific adhesion to microorganisms, in other words, represented by SEQ ID NO: 1. Deletion that does not lose the activity that imparts or enhances nonspecific adhesion to the translated protein. Examples include genetic DNA.

  Stringent conditions refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed, and include low stringency conditions and high stringency conditions. High stringency conditions are preferred. . Low stringent conditions are conditions for washing with, for example, 42 ° C., 5 × SSC, 0.1% SDS, preferably 50 ° C., 5 × SSC, 0.1% SDS. is there. Highly stringent conditions are conditions in which, for example, washing at 65 ° C., 0.1 × SSC and 0.1% SDS is performed after washing.

  It is preferable that the mutation in the base sequence also maintains the domain structure composed of a signal peptide, a head domain, a neck domain, a stalk domain, and a membrane anchor domain. In the base sequence represented by SEQ ID NO: 1, the signal peptide corresponds to bases at positions 1 to 171, the head domain corresponds to bases at positions 322 to 807 and 8989 to 9444, the neck domain corresponds to positions 886 to 957, and The stalk domain corresponds to the bases at positions 9445 to 9516, the stalk domain corresponds to the bases at positions 1216 to 8898 and 9517 to 10611, and the membrane anchor corresponds to the bases at positions 10612 to 10890.

  In DNA consisting of a part of the base sequence represented by SEQ ID NO: 1, several tens to several hundreds of continuous base sequences that can be trimmed are preferably one or both stalk domains, one head domain, one In the sequence region encoding the neck domain, one continuous sequence or a plurality of continuous sequences may be deleted. More preferably, it encodes the entire region from the head domain close to the amino terminus to the stalk domain (positions 322 to 8898) or the entire region from the head domain close to the carboxy terminus to the stalk domain. It is preferable to cut the region (8989 to 10611). More preferably, it is preferable that a plurality of repetitive regions appearing in the code region of the stalk domain are removed so that the repetitive region disappears once each time. Most preferably, any one of a plurality of repetitive regions found in the code region of the stalk domain is removed.

  By introducing a DNA consisting of the base sequence represented by SEQ ID NO: 3 into the target microorganism together with the DNA encoding the autotransporter adhesin, the nonspecific adhesion of the target microorganism can be further improved. You may introduce | transduce a gene functionally equivalent to DNA which consists of a base sequence represented by sequence number 3. The DNA functionally equivalent to the DNA consisting of the base sequence represented by SEQ ID NO: 3 is 90% or more, preferably 95% or more, more preferably 98% or more homologous to the base sequence represented by SEQ ID NO: 3. DNA consisting of a nucleotide sequence having a property. Alternatively, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 3 can be mentioned. The base sequence represented by SEQ ID NO: 3 is a sequence found immediately downstream of the Tol5 strain's AtaA gene, and is homologous to the outer membrane protein ompA gene, BamE gene, omlA gene and the like possessed by Gram-negative bacteria. It encodes the ORF of a protein that indicates The ORF, Tol5-tpgA (referred to as Tol5-OmpA in Patent Document 1) consists of 264 amino acids (SEQ ID NO: 4) encoded by a 795 bp gene (SEQ ID NO: 3).

  An operon containing DNA encoding the autotransporter adhesin may be introduced into the target microorganism. For example, by introducing a DNA consisting of the base sequence represented by SEQ ID NO: 5 into the target microorganism, the DNA encoding the outer membrane protein can be introduced into the target microorganism together with the DNA encoding the autotransporter adhesin. . An operon functionally equivalent to the operon may be introduced. The functionally equivalent operon is 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 98% or more with the nucleotide sequence represented by SEQ ID NO: 5. Examples thereof include an operon consisting of a DNA having a homologous base sequence and having an activity of imparting or enhancing nonspecific adhesion and / or aggregation to a host microorganism. The base sequence represented by SEQ ID NO: 5 is an operon isolated from Tol5 strain (ataA-tpgA operon), promoter / ribosome binding site (positions 1 to 106), ataA gene (positions 107 to 10999) and Contains the Topg5 tpgA gene (positions 11064-11858). E. coli DH5α transformed with a vector incorporating the operon is referred to as “DH5α-XLTOPO :: aadA-ompA” as an independent administrative agency, National Institute of Technology and Evaluation Microbiology (NPMD) (Chiba, Japan). 2-5-8, Kazusa Kamashitsu, Kisarazu City, Prefecture No. 122) is deposited with a deposit number of NITE BP-490 (date of deposit: February 19, 2008).

  Various microorganisms can be used as the microorganism (target microorganism) into which the adhesion-imparting DNA is introduced. Although it does not restrict | limit especially as a target microorganism, For example, the microorganisms which do not have nonspecific adhesion or are weak are mentioned. The target microorganism may be a wild strain, a mutant strain, or a genetically modified strain. An appropriate microorganism is selected according to the application of the present invention. Examples of microorganisms that can be used as target microorganisms include bacteria belonging to the genus Escherichia, such as Escherichia coli, bacteria belonging to the genus Acinetobacter, such as Acinetobacter calcoaceticus, bacteria such as Ralstonia Ralstonia eutropha, Pseudomonas bacteria such as Pseudomonas putida, Pseudomonas fluorescens, Aeromonas bacteria such as Aeromonas caviae Genus bacteria such as Alcaligenes latus, Xanthomonas bacteria such as Xanthomonas campestris Desulfomonile bacteria such as Desulfomonile tiedjei, Desulfuromonas bacteria such as Desulfuromonas chloroethenica, Chromobacterium, For example, Chromobacterium chocolatum, Burkholderia genus bacteria, eg Burkholderia arboris, Rhodobacter genus, Acidvoraxax genus bacteria, eg Acidborax facilis (Acidovorax facilis), bacteria of the genus Zymomonas, such as Zymomonas mobilis.

  By introducing the adhesion-imparting DNA into the target microorganism and transforming it, a microorganism with nonspecific adhesion imparted or enhanced can be obtained. Typically, a microorganism having nonspecific adhesion imparted or enhanced can be obtained by ligating the adhesion-imparting DNA to an appropriate vector and transforming the target microorganism (host microorganism) with the vector. . Specifically, the DNA is introduced into a host microorganism in multiple copies, the DNA is linked under the control of a constitutively expressed promoter, or the DNA is linked under the control of an inducible enzyme promoter. Thus, a microorganism having nonspecific adhesion imparted or enhanced can be obtained.

  First, a target DNA is ligated into a vector to produce a recombinant vector. For the above vector, a phage, cosmid, artificial chromosome or plasmid that can autonomously grow in a host cell is used. For example, when a plasmid is introduced into a chromosome as an expression cassette, the expression cassette is constructed. However, the autonomous replication ability in a host (for example, Acinetobacter genus bacteria) into which the expression cassette is introduced is not necessarily required. As these recombinant vectors, for example, a shuttle vector designed so as to be usable in both E. coli and Acinetobacter bacteria can be used.

  Examples of the plasmid include plasmids derived from E. coli (for example, pET21a (+), pET32a (+), pET39b (+), pET40b (+), pET43.1a (+), pET44a (+), pKK223-3, pGEX4T, pUC118 , PUC119, pUC18, pUC19, etc.), E. coli-Acinetobacter shuttle vector plasmid pARP3 (Non-patent Document 9), and the like, and phage DNA include λ phage (λgt11, λZAP, etc.). A commercially available cloning vector such as pCR4-TOPO (registered trademark) may be used for cloning and sequence confirmation.

  In order to insert DNA into a vector, first, a method in which the purified DNA is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or a multicloning site of an appropriate vector DNA, and linked to the vector is employed. . For example, the target DNA can be synthesized by a generally known method, and in order to incorporate it into a vector, it is amplified by a PCR method using primers so as to contain an appropriate restriction enzyme cleavage site at both ends. Also good. The conditions for the PCR reaction can be appropriately determined by those skilled in the art.

  In addition, in addition to the promoter and the DNA of the present invention, a cis element such as an enhancer, a selection marker, a ribosome binding sequence (SD sequence) and the like may be linked to the recombinant vector as necessary. Examples of selectable markers include, but are not limited to, drug resistance markers such as kanamycin, ampicillin, tetracycline, 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 depending on the host microorganism. For example, when the host is Escherichia coli, T7 promoter, lac promoter, trp promoter, λ-PL promoter, arabinose promoter and the like can be used.

  In order to link the DNA fragment and the vector fragment, a known DNA ligase may be used. Then, the DNA fragment and the vector fragment are annealed and then ligated to prepare a recombinant vector. Preferably, a recombinant vector can be obtained by performing a ligation reaction under defined conditions using a commercially available ligation kit such as ligation high (manufactured by Toyobo Co., Ltd.).

  Recombinant DNA techniques including cloning, ligation, PCR, etc. are described in, for example, Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press (1989) and Short Protocols In Molecular Biology, Those described in Third Edition, A Compendium of Methods from Current Protocols in Molecular Biology, John Wiley & Sons, Inc. can be used.

  If necessary, the obtained vector is purified by a boil method, an alkaline SDS method, a magnetic bead method and a commercially available kit using these principles, and further concentrated by, for example, an ethanol precipitation method or a polyethylene glycol precipitation method. It can be concentrated by means.

  The method for introducing the recombinant vector into the target microorganism is not particularly limited, and examples thereof include a heat shock method using calcium ions, an electroporation method, and a lipofection method.

  The transformed microorganism containing the target DNA can be selected by forming a colony on an LB medium agar plate containing antibiotics such as ampicillin and kanamycin, for example, with the marker gene of the recombinant vector, In order to confirm whether the cloned host microorganism has been transformed with a recombinant vector, using a part, confirm the amplification of the insert by the PCR method, or perform the sequence analysis by the dideoxy method using a sequencer. Also good. In addition to the form of introducing autonomously replicable plasmids, it is also possible to use a chromosomal integration method in which a region homologous to a chromosomal gene is placed in a vector and homologous recombination occurs to introduce the target gene. Good.

  The method of culturing the obtained transformed microorganism in a medium is performed according to a usual method used for culturing a target microorganism. As a medium for culturing transformed microorganisms obtained using microorganisms such as Escherichia coli as a host, it contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms, and can efficiently culture transformed microorganisms. As long as the medium can be used, either a natural medium or a synthetic medium may be used. Specific examples include M9 medium, M9G medium, BS medium, LB medium, Nutrient Broth medium, meat extract medium, SOB medium, SOC medium, and the like.

  The carbon source may be any assimitable carbon compound, for example, sugars such as glucose, polyols such as glycerin, alcohols such as methanol and ethanol, or organic substances such as pyruvic acid, succinic acid, citric acid or lactic acid. In addition to acids, fatty acids and oils can be used. The nitrogen source may be any available nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolyzate, soybean extract alkaline extract, alkylamines such as methylamine, or ammonia or a salt thereof. Etc. can be used. In addition, salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, zinc, a specific amino acid, a specific vitamin, an antifoaming agent, and the like may be used as necessary. Moreover, you may add protein expression inducers, such as isopropyl- (beta) -D-thiogalactopyranoside, to a culture medium as needed.

  The culture is usually carried out under aerobic conditions such as shaking culture or aeration and agitation culture, preferably at 0 to 40 ° C, more preferably at 10 to 37 ° C, particularly preferably at 15 to 37 ° C. During the culture period, the pH of the medium can be appropriately changed within the range in which the host can grow and the activity of the autotransporter adhesin is not impaired, but is preferably in the range of about pH 4-8. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.

As described above, a microorganism having nonspecific adhesion imparted or enhanced can be obtained. The non-specific adhesion of the obtained microorganism can be evaluated using an adhesion test (CV adhesion test) by crystal violet staining. As an example of a specific procedure, the cell culture solution is centrifuged, the culture supernatant is removed, an inorganic salt medium or salt solution containing neither a carbon source nor a nitrogen source is added to the cell pellet, and the cells are sonicated. Obtain a body suspension, adjust the turbidity OD _{660 of the} cell suspension to a constant (near 0.5) with medium or salt solution, and add 200 μl of suspension to each well of a 96-well polystyrene plate. Add each solution, incubate for 2 hours at the optimal temperature of the microorganism, remove all suspension in the well with a pipette, wash the well twice with 200 μl of mineral salt medium or salt solution, air dry, and 1% crystal Add violet aqueous solution to the well, incubate for 15 minutes at room temperature, remove crystal violet with a pipette, wash the well 3 times with 200 μl inorganic salt medium, and then crystal violet from the adherent cells stained with 70% ethanol aqueous solution And the absorbance A ₅₉₀ is measured. A microorganism having an absorbance A ₅₉₀ of 0.6 or more, preferably 0.8 or more, and more preferably 1.0 or more can be evaluated as a microorganism having nonspecific adhesion. Principle of quantifying the amount of adherent cells by attaching microbial cells to the inner wall of an appropriate container under non-proliferation conditions, staining the attached cells with an appropriate stain, and quantifying the number of stains or stained cells Based on the above, the adhesion test may be performed by a modified method of the above method. For example, the material and volume of the plate, the number of wells, the type and amount of stain and washing solution, the staining time and temperature, the concentration of suspended cells, and the device used for quantification of the number of stains and stained cells (absorptiometer, plate reader, A microscope or the like) can be selected as appropriate. Safranin or fluorescent dyes can also be used as the staining agent. Adhesiveness can be evaluated by comparison with a wild strain (negative control) into which no adhesion-imparting DNA has been introduced.

  In the step (1) of the present invention, the prepared adhesion-imparting microorganism is brought into contact with a carrier. The contact here is performed under conditions that allow the adhesion-imparting microorganism to adhere to the carrier. The adherence-imparting microorganism has a high adhesion and exhibits adhesion to various carriers under normal culture conditions. Therefore, in special cases (typically, when the desorption treatment liquid employed in step (2) described later is used as a medium or under low ionic strength (see the above-mentioned International Publication No. 2014/156736 pamphlet)) It adheres to a support | carrier by contact except. One specific example of the conditions under which adhesion is possible is under high ionic strength (see the above-mentioned pamphlet of International Publication No. 2014/156736). Under high ionic strength, the adhesion protein, which is the expression product of the adhesion-imparting DNA, exhibits adhesion. The standard that defines high ionic strength (ie, the boundary between high ionic strength and low ionic strength) may vary depending on the DNA used to impart adhesion, but those skilled in the art will be able to understand the disclosure of the present specification and international publication. It can be set by a preliminary experiment while referring to the description in the pamphlet of No. 2014/156736. The ionic strength at which the adhesiveness changes rapidly may be adopted as the “boundary” here. For example, the boundary between high ionic strength and low ionic strength can be set between ionic strengths 5 mM and 20 mM. In this case, as the high ionic strength, for example, an ionic strength of 10 mM to 500 mM, preferably 20 mM to 200 mM is employed. Adopting an ionic strength higher than necessary is undesirable because it affects the activity and survival of the adhesion-imparting microorganism. Specific examples of the boundary include 5 mM, 7 mM, 10 mM, and 15 mM.

  There are no particular restrictions on the type and composition of the solution used in step (1). For example, various buffers, various salt solutions, culture solutions, and the like can be used.

  Since the microorganisms used in the present invention exhibit nonspecific adhesion, various carriers (immobilization carriers) can be used. The surface characteristics (for example, hydrophilicity and hydrophobicity), material, shape and the like of the carrier are not particularly limited. Examples of materials are polyethylene, polystyrene, polycarbonate, silicon, nylon, polypropylene, polyvinyl alcohol, polyurethane, chitosan, cellulose derivatives, glass, ceramic, and metal. Examples of shapes are plate, spherical, granular, non-woven, and fibrous. , Film-like, sponge-like (foam).

  Two or more types of adhesion-imparting microorganisms may be used in combination. Such an embodiment is useful, for example, when two or more stages of treatments or reactions are continuously performed using the adhesion-imparting microorganism attached to the carrier obtained in step (1).

  The contact state between the adhesion-imparting microorganism and the carrier can be formed by dropping or adding a suspension of the adhesion-imparting microorganism to the carrier, or by introducing the carrier into a solution containing the adhesion-giving microorganism. it can. After such an operation, in order to increase the adhesion rate, for example, it may be incubated for about 1 minute to 3 hours. Alternatively, the adhesion-imparting microorganism can be allowed to adhere to the carrier simultaneously with the growth by growing it in a culture medium containing a salt in the presence of the carrier.

The adhesion-imparting microorganism attached to the carrier in the step (1) is usually subjected to one or more treatments or reactions (details of the treatment or reaction will be described later). In the present invention, thereafter, the step (2), that is, the desorption step is performed. In the step (2), the adhesion-imparting microorganism attached to the carrier is treated with a solution that satisfies a predetermined condition, and the adhesion-imparting microorganism is desorbed from the carrier. Specifically, a solution satisfying at least one of the following conditions (A) to (F) is used as the desorption treatment liquid.
(A) Contains 0.01% (w / v) or more of casamino acid (B) Contains 0.01% (w / v) or more of lactic acid (C) Contains 0.01% (w / v) or more of yeast extract (D ) Containing tryptone 0.01% (w / v) or more (E) Containing skim milk 0.1% (w / v) or more (F) Containing 1 mM or more glutamic acid

  The solution used in this step (hereinafter referred to as “desorption treatment liquid” for convenience of explanation) is one or two or more substances that have been confirmed to have an adhesion inhibitory effect and an aggregation inhibitory effect by the inventors' investigation. Contains by concentration. In step (2), treatment with a separately prepared detachment treatment liquid (mode 1), as well as a solution containing an adhesion-imparting microorganism attached to the carrier (immediately before carrying out step (2)) It can be carried out by adding the necessary components (that is, at least one of casamino acid, lactic acid, yeast extract, tryptone, skim milk, and glutamic acid) (Aspect 2). In the case of the aspect 1, for example, the solution containing the adhesion-imparting microorganism attached to the carrier is replaced with a desorption treatment solution and maintained for a predetermined time (for example, 1 minute to 5 hours). Alternatively, the adhesion-imparting microorganism attached to the carrier is collected, transferred into a desorption treatment solution, and maintained for a predetermined time (for example, 1 minute to 5 hours). A method of spraying a desorption treatment liquid continuously or intermittently on the carrier to which the adhesion-imparting microorganism is attached can also be employed. Aspect 2 has various advantages such as being able to eliminate the problem of disposal associated with the replacement of the solution, no need to prepare a desorption treatment solution, and simpler operation, and is more practical. It can be said.

  As described above, in the desorption treatment solution, casamino acid, lactic acid, yeast extract, tryptone, skim milk, and glutamic acid are used as active ingredients. Although not particularly important in view of the fact that each active ingredient has a concentration-dependent effect, if the upper limit of the content of each active ingredient in the conditions (A) to (F) is exemplified, for casamino acid, 20% (w / v), 20% (w / v) for lactic acid, 20% (w / v) for yeast extract, 20% (w / v) for tryptone ), 20% (w / v) for skim milk and 2M for glutamic acid.

  Since the effect increases in a concentration-dependent manner, it is advantageous in principle to employ a desorption treatment liquid having a high concentration of active ingredient in order to increase or improve the effect. Therefore, in the case of casamino acid, lactic acid, yeast extract, or tryptone, the concentration of the active ingredient in the desorption treatment solution is preferably 0.1% (w / v) or more (as a specific example, 0.2% (w / v), 0.3% (w / v), 0.4% (w / v), 0.5% (w / v), 0.6% (w / v), 0.7% (w / v), 0.8% (w / v), 0.9% (W / v)), more preferably 1% (w / v) or more (specific examples are 1% (w / v), 1.5% (w / v), 2% (w / v), 2.5% ( w / v), 3% (w / v), 3.5% (w / v), 4% (w / v), 4.5% (w / v)), more preferably 5% (w / v) or more (Specific examples are 5% (w / v), 5.5% (w / v), 6% (w / v), 6.5% (w / v), 7% (w / v), 7.5% (w / v v), 8% (w / v), 8.5% (w / v), 9% (w / v), 9.5% (w / v), 10% (w / v)). In the case of skim milk, 0.1% (w / v) is sufficient, but 1% (w / v) or more (specific examples are 1% (w / v), 1.5% (w / v)) , 2% (w / v), 2.5% (w / v), 3% (w / v), 3.5% (w / v), 4% (w / v), 4.5% (w / v)) Or 5% (w / v) or more (specific examples are 5% (w / v), 5.5% (w / v), 6% (w / v), 6.5% (w / v) ), 7% (w / v), 7.5% (w / v), 8% (w / v), 8.5% (w / v), 9% (w / v), 9.5% (w / v), The effect can be increased by 10% (w / v)). In the case of glutamic acid, preferably 10 mM or more (specific examples are 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM), more preferably 100 mM or more (specific examples are 100 mM, 200 mM, 300 mM, 400 mM) More preferably, it is 500 mM or more (specific examples are (500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM).

  The casamino acid used in the condition (A) is a protein acid degradation product and has a high amino acid content. For example, casamino acid (product name: High Daisamino acid “Digo” or casamino acid “Digo”) provided by Nippon Pharmaceutical Co., Ltd., or casamino acid (product name: casamino acid) provided by Nacalai Tesque can be used.

  In order to prepare a desorption treatment solution that satisfies the condition (B), a lactate salt (for example, sodium lactate, potassium lactate, calcium lactate) can be used. Numerous lactic acids and lactates are commercially available (for example, provided by Wako Pure Chemicals, Nacalai Tesque) and are readily available.

  Yeast extract (yeast extract) is obtained by extracting useful components of yeast by treatment with autolysis, enzymes, hot water and the like. For example, yeast extract (product name Bacto Yeast Extract) provided by Becton Dickinson & Company, yeast extract provided by Nippon Pharmaceutical Co., Ltd. (product name powdered yeast extract SH), and the like can be used.

  Tryptone is a tryptic digest of proteins. For example, tryptone (product name Bacto Tryptone) provided by Becton Dickinson and Company, tryptone (product name High Polypeptone) provided by Nippon Pharmaceutical Co., Ltd., and the like can be used.

  Skimmed milk (skim milk powder) is obtained by concentrating and drying skim milk into a powder form. For example, skim milk provided by Wako Pure Chemical Industries, Ltd. and Nacalai Tesque can be used.

  Glutamic acid is an amino acid. For example, L-glutamic acid provided by Wako Pure Chemical Industries, Ltd. and Nacalai Tesque can be used.

  The desorption treatment solution may contain other components (for example, medium components such as LB medium, inorganic salts, etc.) as long as the effect of desorption by the active ingredients is not affected.

  It is also possible to repeat the steps (1) and (2), that is, the attachment and detachment to the carrier. In this case, the adhesion-imparting microorganism and / or carrier may be changed to a new one (same type, similar type, different type, etc.).

  In one embodiment, after the step (2), the detached adhesion-imparting microorganism is recovered (step (3-1)). The collected adhesion-imparting microorganism can be reused in the method of the present invention or in another application. For example, the adhesion-imparting microorganism collected together with the desorption treatment liquid is contacted with a new carrier after the desorption treatment liquid is removed by washing or the like, and the adhesion-imparting microorganism is again attached (immobilized) to the carrier. Is possible. After the collected adhesion-imparting microorganism is regenerated, it may be used for such reuse.

  In another embodiment, the carrier is recovered after step (2) (step (3-2)). The recovered carrier can be reused in the method of the present invention or in another application. The recovered carrier may be used for such reuse after being regenerated or activated. This aspect is particularly useful when the durability of the carrier is high or when the carrier is expensive.

  The method of the present invention can be used in various applications using immobilized microorganisms, for example, production of pharmaceuticals, pharmaceutical intermediates and pharmaceutical raw materials, agricultural chemicals production, bioethanol production, biodiesel production, chemical synthesis, food products (isomerism) It is applicable to the production of saccharified sugars, maltodextrins, oligosaccharides, synthetic sweeteners, amino acids, peptides, vitamins, etc.) and treatment of sewage / drainage / industrial waste / industrial wastewater. Therefore, in one aspect of the present invention, the following steps between step (1) and step (2), that is, the step of bringing the adhesion-imparting microorganism attached to the carrier into contact with the liquid to be treated and maintaining the contact state (step) (I)) is performed. A liquid to be treated is used according to the application. For example, in the case of producing or synthesizing a substance accompanying an enzyme reaction (for example, the above-mentioned pharmaceutical, bioethanol, biodiesel, chemical, food), a solution containing an enzyme substrate is used as a liquid to be treated. In this case, an adhesion-imparting microorganism having the ability to produce a specific enzyme responsible for the enzyme reaction is used. Examples of specific enzymes include lipase, protease, peptidase, esterase, cellulase, hemicellulase, α-amylase, β-amylase, β-glucanase, glutaminase, isomerase, dehydrogenase, reductase, peroxidase, kinase, phosphatase, glycosyltransferase, deoxidation Mention may be made of chlorinases. The enzyme produced by the adhesion-imparting microorganism may be an intracellular enzyme, an extracellular enzyme, a bacterial cell surface localized enzyme, or a surface layer-displayed enzyme.

  For example, a continuous reaction vessel / reaction vessel or a batch-type (batch type) reaction vessel / reaction vessel can be used for carrying out the step (i).

  As one aspect, the present invention also provides a method for detaching (peeling) an adhesion-imparting microorganism. The detachment method of the present invention can be used, for example, for removing the immobilized adhesion-imparting microorganism. For example, when an adhesion-imparting microorganism immobilized on a carrier forms an agglomerate and the agglomerate develops into a large biofilm, resulting in a material transport rate-determining effect, the biofilm is removed. Useful for scraping.

  The carrier can be reused after the adhesion-imparting microorganism is desorbed. In other words, the carrier is recovered and reused as necessary. The detached adhesion-imparting microorganism should be reused when it is alive and maintains the desired properties (for example, it expresses an enzyme required for a specific reaction). Is possible. Thus, also in the said aspect, you may decide to perform said process (3-1) or process (3-2), or both of these processes.

  The following experiment was conducted in order to find a substance capable of controlling non-specific adhesion and aggregation of the protein AtaA derived from Acinetobacter genus Tol 5 strain.

1. Adhesion inhibition test on 96-well polystyrene plate Acinetobacter sp. Tol 5 wild strain expressing adhesion protein AtaA was inoculated in 1/100 volume in LB medium and cultured with shaking at 115 rpm and 28 ° C for 8 hours. . After incubation, collect cells by centrifugation, wash 3 times with deionized water, and place additives (tryptone, yeast extract, casamino acid, lactic acid (using DL sodium lactate), glucose, cellobiose, skim milk, glutamic acid) Quantitative (tryptone, yeast extract, casamino acid, lactic acid, glucose, cellobiose and skim milk 0.01% (w / v), 0.1% (w / v), 1% (w / v) or 5% (w / v) Glutamic acid was resuspended in mineral salt medium with or without (0%) 1 mM, 10 mM, 100 mM or 500 mM). A cell suspension was prepared so that OD660 indicating the cell concentration was 0.5, and 200 μL of the suspension was transferred into a 96-well polystyrene plate. The microorganism cells were allowed to adhere to the inner wall of the well by allowing to stand at 4 ° C. for 2 hours. The wells were washed twice with an inorganic salt medium, and the attached microbial cells were stained with crystal violet. After staining, the cells were washed three times with an inorganic salt medium, crystal violet was eluted in 70% ethanol, and the amount of attached cells was quantified by measuring the absorbance at 590 nm. The results are shown in FIGS. Casamino acid, lactic acid, yeast extract, tryptone, skim milk, and glutamic acid inhibited AtaA adhesion in a concentration-dependent manner.

2. Cell self-aggregation inhibition test A wild strain of Acinetobacter sp. Tol 5 expressing the adhesion protein AtaA was inoculated in an amount of 1/100 in LB medium and cultured with shaking at 115 rpm and 28 ° C. for 8 hours. After incubation, the cells are collected by centrifugation, washed 3 times with deionized water, and 1% of additives (tryptone, yeast extract, casamino acid, lactic acid (using DL sodium lactate), glutamic acid, glucose, glycerose) However, in the case of lactic acid, it was precisely resuspended in an inorganic salt medium containing 1.12% = 0.1 M) or containing no additive (all containing kanamycin at a final concentration of 50 μg / ml). 8 mL of the cell suspension was prepared so that OD660 indicating the cell concentration was 0.5, and transferred to a test tube. After allowing to stand at 28 ° C. for 3 hours to settle the cell aggregate of microbial cells, the supernatant was collected and the turbidity of the cell suspension was measured with OD660. The aggregation rate was calculated from the decrease rate of turbidity from 0.5 before standing. The results are shown in FIG. Lactic acid, glutamic acid, yeast extract, tryptone, and casamino acid also exhibited an aggregation inhibitory effect. The aggregation inhibitory effect of casamino acid is particularly high.

3. Test for inhibition of adhesion to glass plate 1/100 of the wild strain of Acinetobacter sp. Tol 5 expressing the adhesion protein AtaA was inoculated into LB medium and cultured with shaking at 115 rpm and 28 ° C. for 8 hours. After cultivation, the cells are collected by centrifugation, washed 3 times with deionized water, and contain 2.5% (w / v) of casamino acid or sodium lactate (-). What was resuspended in the mineral salt medium was placed on a glass slide. After allowing microbial cells to adhere to the surface of the slide glass by allowing it to stand for 1 hour, the slide glass was gently moved up and down 5 times in the same solution in which the microbial cells were suspended to remove excess cells. . The remaining adherent cells were immersed in an inorganic salt medium containing 1/1000 of SYTO 9, and allowed to stand for 30 minutes for fluorescence staining. After removing the staining solution and washing with an inorganic salt medium, the stained adherent cells were observed with a confocal laser microscope (oscillation 473 nm, detection 520 nm). The results are shown in FIG. Casamino acid and sodium lactate exhibited an excellent adhesion inhibitory effect.

4). Peeling test of cells attached to glass plate Inoculate 1/100 amount of wild strain of Acinetobacter sp. Tol 5 expressing adhesion protein AtaA in LB medium and shake at 115 rpm, 28 ° C for 8 hours Cultured. After incubation, the cells were collected by centrifugation, washed 3 times with deionized water, and resuspended in an inorganic salt medium, and placed on a slide glass. After allowing microbial cells to adhere to the surface of the slide glass by allowing to stand for 1 hour, the slide glass is contained in (−) inorganic salt medium containing 2.5% (w / v) casamino acid or sodium lactate. Washed by gently raising and lowering 5 times. The remaining adherent cells were immersed in an inorganic salt medium containing 1/1000 of SYTO 9, and allowed to stand for 30 minutes for fluorescence staining. After removing the staining solution and washing with an inorganic salt medium, the stained adherent cells were observed with a confocal laser microscope (oscillation 473 nm, detection 520 nm). The results are shown in FIG. Casamino acid and sodium lactate also exhibited the effect of peeling the attached cells. In particular, the effect of casamino acid is high.

5. Adhesion-imparting microorganism adhesion inhibition test In accordance with a previously reported method (Appl Microbiol Biotechnol. 2015 Jun; 99 (12): 5025-32 .; PLoS One. 2012; 7 (11): e48830.) The ataA gene was introduced and expressed. The ADP1 strain originally does not have ataA gene and does not exhibit adhesion, but by introducing the ataA gene of Tol 5 strain, it will show the same level of adhesion as Tol 5 strain (additive of FIG. 6) (Refer to the value of 0% concentration). Using the ADP1 strain after gene transfer, the above 1. The adhesion inhibition test was conducted in the same manner as above. The results are shown in FIG. Similar to the case of Tol 5 strain (1. above), adhesion was inhibited by casamino acid and lactic acid in a concentration-dependent manner. However, glucose did not have an inhibitory effect (comparison test)

<Discussion>
We succeeded in identifying a substance that can control AtaA adhesion. In addition to being able to inhibit the attachment of AtaA, it is worthy of note that it was also possible to peel off the adherent cells once attached. Casamino acid exhibits a particularly excellent effect and can be evaluated as highly useful.

  According to the method of the present invention, it is possible to desorb and recover microorganisms immobilized (attached) to the carrier, to fix the collected microorganisms to the carrier again, and to repeat the immobilization and desorption. is there. The present invention that realizes such a utilization form of microorganisms by a simple operation has an innovative effect on a bioprocess using immobilized microorganisms. It should be noted that the immobilized microorganism can be detached by a very simple operation. For example, the target microorganism can be desorbed from the carrier by an operation of adding a specific substance to a solution containing the target microorganism attached to or immobilized on the carrier. If the method of the present invention is used, the immobilized microorganism once used can be re-immobilized on another carrier and used, or the microorganism having weak activity on the carrier can be replaced with fresh cells having high activity, or Reusing microorganisms and carriers, such as reactivating and re-immobilizing cells with reduced activity that have been detached. The application of the present invention is wide, for example, not only for use in the traditional fermentation industry and waste treatment, but also extremely effective for the production of biomass energy and green biotechnology using microbial cells. By applying the present invention, it is possible to realize cost reduction, production process efficiency, and the like.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (11)

( 1) A step of bringing a microorganism to which nonspecific adhesion is imparted or enhanced by introducing DNA encoding an autotransporter adhesin derived from an Acinetobacter microorganism into contact with the carrier, and attaching the microorganism to the carrier ;
(2) The microorganism attached to the carrier is subjected to the following conditions (A) to (F), that is,
(A) containing 0.01% (w / v) or more of casamino acid;
(B) containing 0.01% (w / v) or more of lactic acid;
(C) containing 0.01% (w / v) or more of yeast extract;
(D) containing 0.01% (w / v) or more of tryptone;
(E) containing skim milk of 0.1% (w / v) or more;
(F) containing 1 mM or more of glutamic acid;
Treating with a solution that satisfies at least one of the conditions, and desorbing the microorganism from the carrier, comprising :
The DNA is
(A) DNA consisting of the base sequence represented by SEQ ID NO: 1;
(B) a DNA comprising a base sequence having 90% or more homology with the base sequence represented by SEQ ID NO: 1 and encoding a protein having an activity of imparting or enhancing nonspecific adhesion to microorganisms; or
(C) From the nucleotide sequence represented by SEQ ID NO: 1 to (i) the stalk domain at positions 9517 to 10611, (ii) the head domain at positions 8989 to 9444, or (iii) from the head domain close to the carboxy terminus to the stalk domain The above-mentioned method, which is a DNA encoding a protein comprising a sequence obtained by cutting the region encoding the entire region of, and having an activity of imparting or enhancing nonspecific adhesion to microorganisms . The method according to claim 1, wherein the following step (i) is performed between step (1) and step (2):
(I) A step of bringing the microorganism attached to the carrier into contact with a liquid to be treated and maintaining the contact state.   The method according to claim 2, wherein the microorganism has an ability to produce a specific enzyme, and the liquid to be treated contains a substrate of the specific enzyme. The method according to claim 1, wherein the following step (3-1) and / or step (3-2) is performed after step (2):
(3-1) recovering the detached microorganisms;
(3-2) A step of recovering the carrier. ( I) A microorganism having nonspecific adhesion imparted or enhanced by introduction of DNA encoding an autotransporter adhesin derived from an Acinetobacter microorganism, which adheres to a carrier under the following conditions ( A) to (F), that is,
(A) containing 0.01% (w / v) or more of casamino acid;
(B) containing 0.01% (w / v) or more of lactic acid;
(C) containing 0.01% (w / v) or more of yeast extract;
(D) containing 0.01% (w / v) or more of tryptone;
(E) containing skim milk of 0.1% (w / v) or more;
(F) containing 1 mM or more of glutamic acid;
Treating with a solution that satisfies at least one of the following conditions :
The DNA is
(A) DNA consisting of the base sequence represented by SEQ ID NO: 1;
(B) a DNA comprising a base sequence having 90% or more homology with the base sequence represented by SEQ ID NO: 1 and encoding a protein having an activity of imparting or enhancing nonspecific adhesion to microorganisms; or
(C) From the nucleotide sequence represented by SEQ ID NO: 1 to (i) the stalk domain at positions 9517 to 10611, (ii) the head domain at positions 8989 to 9444, or (iii) from the head domain close to the carboxy terminus to the stalk domain The above-mentioned method, which is a DNA encoding a protein comprising a sequence obtained by cutting the region encoding the entire region of, and having an activity of imparting or enhancing nonspecific adhesion to microorganisms . The method according to claim 5, wherein the following step (3-1) and / or step (3-2) is performed after step (I):
(3-1) recovering the detached microorganisms;
(3-2) A step of recovering the carrier. The concentration of casamino acid in the condition (A) is 0.1% (w / v) or more,
The concentration of lactic acid in the condition (B) is 0.1% (w / v) or more,
The concentration of the yeast extract in the condition (C) is 0.1% (w / v) or more,
The tryptone concentration in the condition (D) is 0.1% (w / v) or more,
The concentration of glutamic acid in the condition (F) is 10 mM or more,
The method according to any one of claims 1 to 6.   The method according to any one of claims 1 to 7, wherein the DNA is an ataA gene. The method according to any one of claims 1 to 8 , wherein the DNA of the following (a) or (b) is introduced into the microorganism together with DNA encoding an autotransporter adhesin:
(A) DNA consisting of the base sequence represented by SEQ ID NO: 3;
(B) DNA comprising a nucleotide sequence having 90% or more homology with the nucleotide sequence represented by SEQ ID NO: 3. The method according to any one of claims 1 to 7, wherein the following DNA (a) or (b) including the DNA is introduced into the microorganism:
(A) DNA consisting of the base sequence represented by SEQ ID NO: 5;
(B) A DNA comprising a nucleotide sequence having 90% or more homology with the nucleotide sequence represented by SEQ ID NO: 5, and having an activity of imparting or enhancing nonspecific adhesion to microorganisms. The method according to any one of claims 1 to 10 , wherein the microorganism is an Escherichia bacterium.

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