JP7080122B2 - How to break down extracts, soluble fractions, proteins, biofilms - Google Patents

Description

The present invention relates to methods for degrading extracts, soluble fractions, proteins and biofilms.

Membranes are used in the parts responsible for the filtration process in various equipment. Occasionally, fouling of the membrane produces a biofilm, which can interfere with the operation of the equipment. Previous studies have revealed that foulants contain a type of uronic acid-containing polysaccharide and that there are bacteria with the ability to break down such polysaccharides. In recent years, attempts to decompose foulants by enzymes expressed in bacteria have also been reported (see Patent Document 1).

Japanese Unexamined Patent Publication No. 2007-268379

Therefore, an object of the present invention is to effectively decompose a biofilm such as foulant generated in a membrane used for filtration.

The gist of the present invention is as follows.

[1]
A method for degrading a biofilm using a protein, which is encoded by the base sequence of SEQ ID NO: 1 (No. 2567) .
[2]
The biofilms are starch, glycogen, pectin, guar gum, gum arabic, xanthan gum, gellan gum, tamarind gum, locust bean gum, carrageenan, carboxymethyl cellulose (CMC), gelatin, agar, glucomannan, heparin, hyaluronic acid, alginic acid, propylene alginate. The method according to [1] , which comprises at least one selected from the group consisting of glycol, purulan, psyllium, carrageen and propylene glycol.

According to the present invention, a biofilm such as foulant formed on a membrane used for filtration can be effectively decomposed.

It is a figure which shows the result when the experiment which decomposed xanthan etc. was performed using the culture supernatant of the mixed bacterial solution obtained from activated sludge. It is a figure which shows the result when SDS-PAGE was performed about the soluble fraction of 50 kDa or more of the culture medium of a mixed bacterium. It is a figure which shows the fraction profile when the soluble fraction of 50 kDa or more of the culture solution of a mixed bacterium was purified by anion exchange column chromatography. It is a figure which shows the result when SDS-PAGE was performed for each fraction when the soluble fraction of 50 kDa or more of the culture solution of a mixed bacterium was purified by anion exchange column chromatography. In the figure, arrow A indicates the position of the band of 70 kDa, and arrow B indicates the position of the band of 50 kDa. It is a figure which shows the result when SDS-PAGE about the purified protein A solution was performed. It is a figure which shows the result when SDS-PAGE was performed on the culture medium which culture | cultivated the mixed bacterium which has a foulant decomposing activity in various BS media (glucose-containing BS medium, glucose and mannose-containing BS medium, xantan-containing BS medium). In the figure, M indicates a marker, lane 1 indicates a culture medium in a BS medium containing 20 mM glucose, lane 2 indicates a culture medium in a BS medium containing 10 mM glucose and 10 mM mannose, and lane 3 indicates a 0.05% xanthan. The culture medium in the containing BS medium is shown. In the figure, the arrow of lane 1 and the arrow of lane 2 both indicate the position of the band of 50 kDa, and the arrow of lane 3 indicates the position of the band of 50 kDa and the position of the band of 70 kDa. (A) shows a GPC profile for a control. The vertical axis shows the intensity (mV), and the horizontal axis shows the passage of time (minutes). The upper right figure of (A) shows an enlarged profile of the portion after 4 minutes to 7 minutes in particular. (B) shows the GPC profile for the sample. The vertical axis shows the intensity (mV), and the horizontal axis shows the passage of time (minutes). The upper right figure of (A) shows an enlarged profile of the portion after 4 minutes to 7 minutes in particular. (A) is a figure which shows the growth state of 6 kinds of fungi (No. B1 to No. B6) isolated from the mixed bacterial solution which has a foulant decomposing activity. (B) is No. 1 among 6 types of bacteria (No. B1 to No. B6) isolated from the mixed bacterial solution having foulant-degrading activity. It is a figure which shows the growth state of B2. No. It is a figure which shows the result when the experiment which decomposes xanthan was performed using the B2 culture supernatant. No. It is a figure which shows the result when SDS-PAGE was performed on the B2 culture supernatant. In the figure, M indicates a marker, and lane 1 is No. B2 culture broth is shown, lane 2 shows the purified protein A solution, and lane 3 is No. The B2 culture supernatant is shown. It is a figure which shows the result of SDS-PAGE about the protein (unpurified) encoded by the mass-expressed SEQ ID NO: 1 (No. 2567). In the figure, M indicates a marker, C indicates a control, lane 1 indicates a pellet, and lane 2 indicates a culture medium supernatant. Further, super indicates a supernatant and ppt indicates a precipitate. It is a figure which shows the result of SDS-PAGE about the protein (purified by Ni-Sepharose) encoded by the mass-expressed SEQ ID NO: 1 (No. 2567). In the figure, M indicates a marker, C indicates a control, FT indicates a washing solution, lane 1 indicates a first eluate, lane 2 indicates a second eluate, and lane 3 indicates a third eluate. , Lane 4 shows the 4th eluate, and lane 5 shows the 5th eluate. Further, super indicates a supernatant and ppt indicates a precipitate.

Hereinafter, embodiments for carrying out the present invention (hereinafter, also referred to as “the present embodiment”) will be described in detail. The present invention is not limited to the following embodiments, and can be variously modified and implemented within the scope of the gist thereof.

(Extract, soluble fraction)
The extract and soluble fraction of this embodiment are derived from a culture of a bacterium belonging to the genus Paenartrobacter.

Examples of the species of the bacterium belonging to the genus Paenarthrobacter include Paenarthrobacter nicotinovorans, Paenarthrobacter histidinovorans and the like.
These species may be used alone or in combination of two or more.

As the species, nicotinovorans, which makes it possible to obtain high biofilm-degrading activity, is particularly preferable.
The genus Paenartrobacter nicotinovorans can be theoretically referred to as the genus Arthrobacter nicotinovalans (Arthrobacter nicotinovorans).

Among those belonging to the genus Paenarthrobacter nicotinovorans, more specifically, 18140-B2 is preferable.
18140-B2 was deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center on July 7, 2017 under the accession number: NITE P-02506.

In particular, the extract of the present embodiment may be an extract from a culture of strain 18140-B2 (accession number: NITE P-02506) of the genus Paenartrobacter.

In particular, the soluble fraction of the present embodiment may be a fraction of 50 kDa or more of the culture of the genus Paenarthrobacter.
Here, the soluble fraction refers to a water-soluble fraction of the extract of the culture.

According to the extract and the soluble fraction of the present embodiment, biofilms such as foulants formed on the membrane used for filtration can be effectively decomposed.

[Culture method]
The culture method is not particularly limited, and a method usually used in the art may be used, and examples thereof include batch culture (batch culture), fed-batch culture (half-batch culture), and continuous culture (perfusion culture). Be done.

[Culture conditions]
The culture conditions are not particularly limited, and standard cell culture conditions can be used.
Depending on the nature of the host cell, the culture can also be performed under aerobic, anaerobic, or anaerobic conditions.
The culture temperature is preferably 20 ° C. to 37 ° C., the culture atmosphere is preferably a CO ₂ concentration of about 6% to about 84%, and the pH is preferably about 5 to about 9.

[Culture medium]
The medium used is not particularly limited, and examples thereof include a natural medium and a synthetic medium, which are media containing a carbon source which is a nutrient source for bacteria, and optionally include a nitrogen source, an organic micronutrient source, and an inorganic substance ( It may be used as a medium containing (minerals) and the like.
Carbohydrates such as monosaccharides, disaccharides, oligosaccharides, polysaccharides, and converted sugars; compounds having 1 carbon atom (hereinafter referred to as C1 compounds); lipids (oils and the like); fatty acids; glycerin fatty acid esters; Polypeptides; renewable carbon sources; yeast extracts; phospholipids; glycerolipids; glycerol; acetates and the like.
These carbon sources may be used alone or in combination of two or more.

Details about the carbon source are given below.
Among carbohydrates, monosaccharides include triose such as ketotriose (dihydroxyacetone) and aldotriose (glyceraldehyde); tetrose such as ketotetroth (elittlerose) and aldotethrose (erythrose, treose); ketopentose (librose, xylrose). , Pentose such as aldopentose (ribose, arabinose, xylose, liquisource), deoxy sugar (deoxyribose); ketohexose (psicose, fructose, sorbose, tagatose), ald hexose (allose, altrose, glucose, mannose, growth, idose) , Galactose, talose), hexoses such as deoxy sugars (fucose, fukuros, ramnorth); heptoses such as sedhepturose; C6 sugars such as fructose, mannose, galactose, glucose; ..
Examples of the disaccharide include sucrose, lactose, maltose, trehalose, turanose, cellobiose and the like, and sucrose and lactose are preferable.
Examples of oligosaccharides include trisaccharides such as raffinose, meregitos, and maltotriose; tetrasaccharides such as acarbose and stachiose; and other oligosaccharides such as fructooligosaccharide (FOS), galactooligosaccharide (GOS), and mannan oligosaccharide (MOS). Can be mentioned.
Specific examples of the polysaccharides include starch, glycogen, pectin, guar gum, gum arabic, xanthan gum, gellan gum, tamarind gum, locust bean gum, carrageenan, carboxymethyl cellulose (CMC), gelatin, agar, glucomannan, heparin, and hyalurone. Examples thereof include acid, alginic acid, propylene glycol alginate, purulan, psyllium, carrageenan, and propylene glycol, preferably containing β-1,4 bonds, and preferably xanthan gum and gellan gum.
Examples of invert sugar include those obtained by hydrolyzing sucrose.
Examples of the compound having 1 carbon atom (C ₁ compound) include methanol, formaldehyde, formate, carbon monoxide, carbon dioxide and the like.
Examples of the lipid include substances containing 1 or more saturated fatty acids and / or unsaturated fatty acids of C ₄ or higher, and oils that are liquid at room temperature are particularly preferable.
Here, as the oil, vegetable oils such as soybean, corn, canola, jatrofa, palm, peanut, sunflower, coconut, mustard, cottonseed, palm kernel oil, olive, red flower, sesame, flaxseed, oily microbial cells, and Nankinhaze. , And animal oils.
Examples of the fatty acid include compounds represented by the formula RCOOH (“R” represents a hydrocarbon group), and examples thereof include unsaturated fatty acids and saturated fatty acids. Here, the unsaturated fatty acid is a compound having at least one carbon-carbon double bond in "R", and examples thereof include oleic acid, vaccenic acid, linoleic acid, palmiterazic acid, and arachidonic acid. The saturated fatty acid is a compound in which "R" is a saturated aliphatic group, and examples thereof include docosanoic acid, icosanoic acid, octadecanoic acid, hexadecanoic acid, tetradecanoic acid, and dodecanoic acid. As the fatty acid, C ₂ to C ₂₂ fatty acids are preferable, and C ₁₂ fatty acid, C ₁₄ fatty acid, C ₁₆ fatty acid, C ₁₈ fatty acid, C ₂₀ fatty acid, and C ₂₂ fatty acid are more preferable.
Salts of these fatty acids, derivatives, and salts of derivatives are also mentioned. Examples of the salt include lithium salt, potassium salt, sodium salt and the like.
Examples of the glycerin fatty acid ester include monoglyceride, diglyceride, triglyceride and the like.
Examples of the polypeptide include a microbial polypeptide, a vegetable polypeptide and the like, where the microbial polypeptide includes a yeast- or bacterial-derived polypeptide and the like, and examples of the vegetable polypeptide include soybeans. Examples thereof include corn, canola, jatrofa, palm, peanut, sunflower, coconut, mustard, cottonseed, palm kernel oil, olive, red flower, sesame, and polypeptide derived from flaxseed.
Examples of the renewable carbon source include biomass carbon sources, and in particular, biomass carbon sources obtained by hydrolyzing all or part of them are preferable. Here, examples of the biomass carbon source include cellulosic substrates such as waste wood, paper, and pulp, leafy plants, and fruit meat; and parts of plants such as stalks, grains, roots, and stalks. Examples of plants used as a biomass carbon source include legumes such as corn, wheat, rye, sorghum, triticate, rice, awa, barley, cassava, and pea, potatoes, sweet potatoes, bananas, sugar cane, and tapioca. When a renewable carbon source such as biomass is used in the medium, pretreatment is preferable, and examples of the pretreatment include enzymatic pretreatment, chemical pretreatment, and a combination thereof.

For example, examples of the carbon source include a combination of lipids, oils, fats and oils, fatty acids, glycerin fatty acid esters and carbohydrates such as glucose.
Examples of the carbon source include yeast extract or a combination of yeast extract and another carbon source such as glucose, and a combination of yeast extract and a C ₁ compound such as carbon dioxide or methanol is preferable.

As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolyzate, ammonia gas, aqueous ammonia and the like can be used.
As the organic micronutrient source, it is desirable to contain an appropriate amount of a required substance such as vitamin B1 or L-homoserin or yeast extract.
As the inorganic substance (mineral), in addition to these, potassium phosphate, magnesium sulfate, iron ion, manganese ion and the like are added in a small amount, if necessary.

As the medium to be used, a commercially available standard medium is preferable. A medium suitable for culturing a specific host can be appropriately selected and used.
The standard medium is not particularly limited, and examples thereof include ready-made media such as standard agar medium, SCD agar medium, and R2A agar medium.

The extraction method is not particularly limited, and a method usually used in the art may be used, and centrifugation, filter filtration, or the like may be used.

(protein)
The protein of the present embodiment is contained in the extract of the present embodiment and the soluble fraction of the present embodiment.

Specifically, the protein of the present embodiment includes the bases of SEQ ID NO: 1 (No. 2567), SEQ ID NO: 2 (No. 753), SEQ ID NO: 3 (No. 246), and SEQ ID NO: 4 (No. 2584). It may contain a protein encoded by a base sequence having at least one base sequence selected from the group consisting of sequences, and may be a protein encoded by these base sequences.

The amino acid sequence of the protein of the present embodiment is not limited to the amino acid sequence encoded by the above-mentioned four types of base sequences, and variants of the above-mentioned protein can also be used. Examples of the variants include those having amino acid substitutions, deletions, insertions, additions, etc. to all amino acids.
The ratio of substitutions, deletions, insertions, additions, etc. to all amino acids varies depending on the type of amino acid residue and the position of the amino acid residue in the protein conformation, but may be 10% or less, preferably 5%. It is less than or equal to, more preferably 2% or less, still more preferably 1% or less, and particularly preferably 0.5%.
The identity of the amino acid sequence of the protein of the present embodiment with respect to the amino acid sequence encoded by the above-mentioned four base sequences may be 90% or more, preferably 95% or more, and more preferably 98% or more. Yes, more preferably 99% or more, and particularly preferably 99.5% or more.

Those having a substitution include, in particular, those having a conservative substitution.
"Conservative substitution" refers to substitution between amino acids having similar properties, specifically, in the case of aromatic amino acids, substitution between Ph, Trp, Tyr, and in the case of hydrophobic amino acids. Is a substitution between Leu, Ile and Val, a substitution between Gln and Asn in the case of polar amino acids, a substitution between Lys, Arg and His in the case of basic amino acids, and a substitution between Lys, Arg and His in the case of acidic amino acids. Refers to the substitution between Asp and Glu, and in the case of an amino acid having a hydroxyl group, the substitution between Ser and Thr.
Substitutions that are considered conservative substitutions include, specifically, Ala to Ser or Thr, Arg to Gln, His or Lys, Asn to Glu, Gln, Lys, His or Asp. Substitution of Asp to Asn, Glu or Gln, Substitution of Cys to Ser or Ala, Substitution of Gln to Asn, Glu, Lys, His, Asp or Arg, Substitution of Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Replacement of Lys with Asn, Glu, Gln, His or Arg, replacement of Met with Ile, Leu, Val or Phe, replacement of Phe with Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitutions include Thr to Ser or Ala, Trp to Ph or Tyr, Tyr to His, Phe or Trp, and Val to Met, Ile or Leu.

According to the protein of the present embodiment, a biofilm such as foulant formed on a membrane used for filtration can be effectively decomposed.

(gene)
The gene of this embodiment encodes the protein of this embodiment.

The gene of this embodiment consists of a group consisting of the base sequences of SEQ ID NO: 1 (No. 2567), SEQ ID NO: 2 (No. 753), SEQ ID NO: 3 (No. 246), and SEQ ID NO: 4 (No. 2584). It may contain a base sequence having 90% or more identity with at least one selected base sequence, and may be a base sequence having 90% or more identity with these base sequences.

This base sequence is not limited to the above base sequence itself as long as it has the function of the protein encoded by the base sequence, and a variant of the above base sequence can also be used.

The mutant may be obtained by a naturally occurring mutation or by an artificial mutation (eg, site-specific mutagenesis).

As described above, the identity of the variant of the base sequence with respect to the original base sequence may be 90% or more, preferably 95% or more, further preferably 98% or more, still more preferably 99% or more, and particularly preferably 99%. It is 5% or more.

The codon encoding the amino acid of the above protein can be changed according to the frequency of use of the codon in the host expressing the protein, whereby the base sequence of the gene encoding the above protein can be changed. can.

According to the gene of this embodiment, a biofilm such as foulant generated in a membrane used for filtration can be effectively decomposed.

(Recombinant vector)
The recombinant vector of the present embodiment contains the gene of the present embodiment.

The vector can be constructed by using genetic engineering methods such as PCR method, crossover PCR method, in-fusion method, and λ-Red method by appropriately using enzymes such as restriction enzymes, DNA polymerases, and ligases. ..

The vector used for preparing the recombinant vector of the present embodiment is not particularly limited, and examples thereof include a plasmid vector and a viral vector. The vector may be a DNA vector or an RNA vector, but a DNA vector is preferable. Examples of the vector include a monocistronic vector, a bicistronic vector, a polycistronic vector and the like.
The vector may be appropriately selected depending on the host used. For example, in the case of E. coli, a ColE-based plasmid represented by a pBR322 derivative, a pACYC-based plasmid having a p15A origin, a pSC-based plasmid, etc. Examples thereof include a mini F plasmid derived from F factor such as Bac system.

According to the recombinant vector of the present embodiment, a biofilm such as foulant formed on a membrane used for filtration can be effectively decomposed.

(Transformant)
The transformant of the present embodiment contains the vector of the present embodiment.

Examples of the method for gene transfer used in the preparation of the transformant of the present embodiment include methods known in the art.

As such a method, for example, a plasmid vector having a gene encoding a target protein such as an enzyme is subjected to a host cell by a method using a cell (competent cell) treated with calcium chloride, an electroporation method, or the like. Examples thereof include a method of introducing into the chromosome of the bacterium by infecting the bacterium with a phage vector into which the gene encoding the target protein has been introduced.

The host is not particularly limited, and examples thereof include any cell, but a bacterium or a fungus is preferable.

The bacterium may be a gram-positive bacterium or a gram-negative bacterium.
The gram-positive bacteria include Bacillus bacterium, Listeria bacterium, Staphylococcus bacterium, Streptococcus bacterium, Enterococcus bacterium, Clostridium Clo. Examples thereof include bacteria of the genus Corynebacterium, bacteria of the genus Streptomyces, and bacteria of the genus Bacillus and bacteria of the genus Corynebacterium are particularly preferable.
Examples of the gram-negative bacterium include Escherichia bacterium, Pantoea bacterium, Salmonella bacterium, Vivrio bacterium, Serratia bacterium, Enterobacter bacterium and the like. In particular, bacteria of the genus Escherichia, bacteria of the genus Pantoea, and bacteria of the genus Enterobacter are preferred.
In particular, as the bacterium belonging to the genus Escherichia, Escherichia coli is particularly preferable. Examples of the strain of E. coli include those known in the art, and examples thereof include DH5 and JM109.

As fungi, the genus Saccharomyces, the genus Schizoccharomyces, the genus Yarrowia, the genus Trichoderma, the genus Aspergillus, the genus Aspergillus, the genus Fuzarium, etc. However, microorganisms of the genus Saccharomyces, the genus Schizoccharomyces, the genus Yarrowia, or the genus Trichoderma are preferred.

Examples of the host include insect cells, animal cells, plant cells and the like, in addition to the above-mentioned bacteria and fungi.

The host may be used alone or in combination of two or more.

In gene transfer, it is preferable to enhance the activity of the target protein by increasing the number of copies of the target gene on the chromosome. When introducing a gene of an enzyme that is not originally possessed by the host microorganism, it is preferable that the microorganism has at least one copy of the gene on the chromosome, and that the enzyme possessed by the host microorganism is present. When introducing a gene, it is preferable that the microorganism has a plurality of copies of the gene on the chromosome, preferably 2 copies or more, and more preferably 3 copies or more.
In the gene transfer in the present invention, the transfer of the target gene can also be achieved by introducing a plasmid carrying the gene into a host cell.
Here, the increase in the number of copies can also be achieved by transferring the target gene onto the genome of the host using a transposon, Mu phage, or the like.

The system used for the above-mentioned gene transfer is preferably constructed so that the expression of the target gene can be induced in the following culture by a conventional method. Examples of such induction include IPTG induction, arabinose induction and the like. Here, examples of the promoter used in the vector include tryptophan promoters such as trc and tac, lac promoter, T7 promoter, T5 promoter, T3 promoter, SP6 promoter, arabinose-inducing promoter, cold shock promoter, tetracycline-inducible promoter and the like. Be done.

The details of culturing the transformant will be described below.

[Culture method]
The culture method is not particularly limited, and a method usually used in the art may be used, and examples thereof include batch culture (batch culture), fed-batch culture (half-batch culture), and continuous culture (perfusion culture). Be done.

[Culture conditions]
The culture conditions are not particularly limited, and standard cell culture conditions can be used.
Depending on the nature of the host cell, the culture can also be performed under aerobic, anaerobic, or anaerobic conditions.
The culture temperature is preferably 20 ° C. to 37 ° C., the culture atmosphere is preferably a CO ₂ concentration of about 6% to about 84%, and the pH is preferably about 5 to about 9.

[Culture medium]
The medium used is not particularly limited, and examples thereof include a natural medium and a synthetic medium, which are media containing a carbon source which is a nutrient source for bacteria, and optionally include a nitrogen source, an organic micronutrient source, and an inorganic substance ( It may be used as a medium containing (minerals) and the like.
Carbohydrates such as monosaccharides, disaccharides, oligosaccharides, polysaccharides, and converted sugars; compounds having 1 carbon atom (hereinafter referred to as C1 compounds); lipids (oils and the like); fatty acids; glycerin fatty acid esters; Polypeptides; renewable carbon sources; yeast extracts; phospholipids; glycerolipids; glycerol; acetates and the like.
These carbon sources may be used alone or in combination of two or more.

The details of the carbon source may be as described above for the preparation of the extract for the present embodiment and the culture for preparing the soluble fraction of the present embodiment.
In particular, as polysaccharides, specifically, starch, glycogen, pectin, guar gum, gum arabic, xanthan gum, gellan gum, tamarind gum, locust bean gum, carrageenan, carboxymethyl cellulose (CMC), gelatin, agar, glucomannan, heparin. , Hyaluronic acid, alginic acid, propylene glycol alginate, purulan, psyllium, carrageenan, propylene glycol, and xanthan gum and gellan gum can be used from the viewpoint of efficiently obtaining a transformant having an activity of cleaving β-1,4 bond. preferable.

According to the transformant of the present embodiment, a biofilm such as foulant formed on a membrane used for filtration can be effectively decomposed.

(How to decompose biofilm)
The method for decomposing the biofilm of the present embodiment uses the extract of the present embodiment, the soluble fraction of the present embodiment, or the protein of the present embodiment.

In the method for decomposing the biofilm of the present embodiment, it is preferable to perform mixing under aerobic conditions of 20 ° C. or higher.

Hereinafter, embodiments of the present invention, such as a method for decomposing an extract, a soluble fraction, a protein, and a biofilm, have been exemplified with reference to the drawings. The invention is not limited to the above-exemplified embodiments.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to the following examples.

<Discovery of activated sludge>
Fowling occurred in the filtration membrane used for filtration in the membrane separation activated sludge method.
Analysis of the foulant revealed that it contained a type of polysaccharide containing uronic acid and the like.
On the other hand, when sludge derived from wastewater treatment activated sludge from a sugar factory was added to the above foulant, it was found that the foulant was decomposed.

Add 2 mL of the culture supernatant of the culture obtained by culturing about 1 g of the above sludge in a xanthan medium at 28 ° C. for 72 hours on a 20 mL scale to 500 mg of xanthan gum, 500 mg of polygalactucuronic acid, 500 mg of glucuronic acid, 500 mg of galacturonic acid, and 500 mg of glucose, respectively. , Prepare the sample solution,
Each of these solutions was cultured at 28 ° C., and the concentration (ppm) of uronic acid produced by the decomposition of the biofilm in the solution was measured over time.
A control with only the culture supernatant was also prepared.
It was found that the culture supernatant of the mixed bacterial solution obtained from sludge having foulant decomposing activity (activated sludge) has an activity of decomposing a biofilm containing xanthan, which is a cell-derived polysaccharide.
FIG. 1 shows the results of an experiment in which xanthan and the like were decomposed using a culture supernatant of a mixed bacterial solution obtained from activated sludge.

<Identification of biofilm-degrading active protein>
An experiment was conducted to identify a protein having an activity of degrading a biofilm containing xanthan and the like.

The above mixed bacteria were cultured in BS-xanthan medium (final xanthan concentration 0.5%) for 2 weeks, and then cultured in fermenter for 2 days.
Centrifugation was performed on the culture solution, the supernatant was collected, and the supernatant was fractionated by an ultrafiltration membrane (AHP-0013 manufactured by Asahi Kasei Corporation). I got a fraction.
Soluble fractions of 50 kDa or more were filter sterilized, protein components were precipitated with 10% trichloroacetic acid (TCA) and SDS-PAGE was performed. The gel after electrophoresis was stained with CBB and silver.
FIG. 2 shows the results when SDS-PAGE was performed on a soluble fraction of 50 kDa or more of the culture solution of the mixed bacterium.

The soluble fraction of 50 kDa or more was purified by anion exchange column chromatography to obtain a fraction according to the molecular weight.
Purification by anion exchange column chromatography was performed according to the following.
Column: Q Sepharose High Performance (manufactured by GE Healthcare) 5 mL
Conditions and operations:
The column was equilibrated using 50 mL (about 10 times the column) of 20 mM Tris-HCl buffer (pH 7.4).
-Stepwise elution was performed while increasing the NaCl concentration using 4 mL each of 20 mM Tris-HCl buffer (pH 7.4) containing NaCl at 0.1 M, 0.25 M, 0.4 M, and 0.5 M, respectively, and 1 mL each. Sampling was performed to obtain each fraction.
FIG. 3 shows a fraction profile when a soluble fraction of 50 kDa or more of the culture solution of the mixed bacterium was purified by anion exchange column chromatography.

In addition, each fraction was subjected to SDS-PAGE. The gel after electrophoresis was silver-stained.
FIG. 4 shows the results when SDS-PAGE was performed on each fraction when the soluble fraction of 50 kDa or more of the culture solution of the mixed bacterium was purified by anion exchange column chromatography. In the figure, arrow A indicates the position of the band of 70 kDa, and arrow B indicates the position of the band of 50 kDa.

As shown in FIG. 4, a protein having a molecular weight of about 50 kDa (hereinafter, also referred to as “protein A”), a protein having a molecular weight of about 70 kDa (hereinafter, also referred to as “protein B”), and the like were found by the above purification. ..

For the fraction containing mainly protein A (fraction 7 etc. in FIG. 4) obtained by the purification shown in FIG. 4 above, a 50 kDa spin column (manufactured by Takara Bio Inc.) was used to purify the buffer. SDS-PAGE was performed on the prepared purified protein A solution after desalting by substituting with. The gel after electrophoresis was stained with CBB.
FIG. 5 shows the results when SDS-PAGE was performed on the purified protein A solution.

If the characteristic proteins such as protein A and protein B found by purification as described above are xanthan-dependent, it can be said that these proteins are characteristically possessed by bacteria having xanthan as a carbon source.
Therefore, as a comparative experiment, mixed bacteria having foulant-degrading activity were cultured in BS-xanthan medium and also in BS medium containing a carbon source other than xanthan.
The medium used was a BS medium containing 20 mM glucose, a BS medium containing 10 mM glucose and 10 mM mannose, and a BS medium containing 0.05% xanthan.
SDS-PAGE was performed on the culture medium. The gel after electrophoresis was stained with silver.
FIG. 6 shows the results when SDS-PAGE was performed on a culture medium obtained by culturing a mixed bacterium having foulant-degrading activity in various BS media (glucose-containing BS medium, glucose and mannose-containing BS medium, xantan-containing BS medium). In the figure, M indicates a marker, lane 1 indicates a culture medium in a BS medium containing 20 mM glucose, lane 2 indicates a culture medium in a BS medium containing 10 mM glucose and 10 mM mannose, and lane 3 indicates a 0.05% xanthan. The culture medium in the containing BS medium is shown. In the figure, the arrow of lane 1 and the arrow of lane 2 both indicate the position of the band of 50 kDa, and the arrow of lane 3 indicates the position of the band of 50 kDa and the position of the band of 70 kDa.

As shown in FIG. 6, a band having a molecular weight of about 50 kDa was observed in all three samples, but the band in the culture medium (lane 3) in the BS medium containing 0.05% xanthan was the above-mentioned protein A in terms of molecular weight. It was concluded that it is a band equivalent to. In lane 3, a band corresponding to protein B having a molecular weight of about 70 kDa was also confirmed.
From this result, it was suggested that protein A and protein B are peculiar to bacteria having xanthan as a carbon source.

Next, it was confirmed by the following experiment whether or not the extract containing a soluble fraction of 50 kDa or more has biofilm decomposition activity.
The xanthan solution was prepared by dissolving 0.5 g of xanthan (manufactured by Sigma-Aldrich) in 1000 mL of 20 mM phosphate buffer.
A control in which 1 mL of a 50 mM phosphate buffer was added to 100 mL of the xanthane solution and a sample in which 1 mL of the purified protein A solution was added to 100 mL of the xanthane solution and incubated for 24 hours were analyzed by gel permeation chromatography (GPC).

Analysis by gel permeation chromatography (GPC) was performed according to the following.
Equipment: Hitachi Chromaster series Sorting column: Shodex OHpak SB-806 HQ
Guard column: OHpak SB-G manufactured by Shodex
Measuring solvent: 50 mM KH2PO4 (pH 4)
Sample concentration: The liquid after incubation was filtered through a 0.45 μm filter and used as it was. Injection amount: 20 μL
Measurement temperature: 40 ° C
Flow velocity: 1 mL / min Eight types of pullulan with different molecular weights (molecular weight: 1660000, 380000, 186000, 100,000, 48000, 23700, 12200, 5800) were used to prepare the calibration curve. A calibration curve was created using an approximate expression of a cubic straight line.
FIG. 7A shows a GPC profile for the control. The vertical axis shows the intensity (mV), and the horizontal axis shows the passage of time (minutes). The upper right figure shows an enlarged profile of the part after 4 minutes to 7 minutes in particular.
FIG. 7B shows the GPC profile for the sample. The vertical axis shows the intensity (mV), and the horizontal axis shows the passage of time (minutes). The upper right figure shows an enlarged profile of the part after 4 minutes to 7 minutes in particular.

As shown in FIG. 7 (A), in the case of control, the peak of xanthan (molecular weight about 10 ⁶ to ¹⁰⁷ ) is observed during the retention time of about 5 to about 6 minutes, and the retention time of the peak top is 5. It was .3 minutes.
On the other hand, as shown in FIG. 7B, in the case of the sample to which the purified protein A solution was added, a peak was observed during the retention time of about 5 minutes to about 6 minutes, but the retention time was 5.4 minutes. Along with a peak top, a peak top with a retention time of 5.8 minutes was also observed.
From this result, it was found that the peak of xanthan was shifted to the low molecular weight side, and it was found that xanthan was decomposed by protein A, and it was suggested that about half of the xanthan was decomposed from the analysis of the peak area (data not shown). Was done.

From the above, it was found that an extract containing a soluble fraction of 50 kDa or more containing protein A having a molecular weight of about 50 kDa has an activity of degrading a biofilm.

<Identification of strains with foulant-degrading activity>
When the above-mentioned mixed bacterial solution having foulant-degrading activity was cultured in an SCD medium (BBL®, manufactured by Becton Dickinson) at 30 ° C. for 3 to 5 days under aerobic conditions, 6 types of bacteria were obtained. (No. B1 to No. B6) were isolated. Mold and yeast were excluded.
FIG. 8A shows the growth states of 6 types of bacteria (No. B1 to No. B6) isolated from the mixed bacterial solution having foulant-degrading activity.

No. B1 to No. When B6 was cultured in BS-xanthan medium at 30 ° C. for 3 to 5 days under aerobic conditions, No. Only B2 grew.
FIG. 8B shows No. 1 among 6 types of bacteria (No. B1 to No. B6) isolated from the mixed bacterial solution having foulant-degrading activity. Shows the growth state of B2.

This No. B2 was deposited as 18140-B2 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center on July 7, 2017, with the accession number: NITE P-02506.

No. It was analyzed whether or not the B2 culture supernatant had xanthan degrading activity.
No. 1 in 100 mL of 1 g / L xanthan aqueous solution. 10 mL of B2 culture supernatant was added, and the mixture was incubated for 20 days and analyzed by gel permeation chromatography (GPC).
In FIG. 9, No. The results of the experiment of decomposing xanthan using the B2 culture supernatant are shown.

As shown in FIG. 9, the xanthan concentration became almost 0 after the culture time was 7 days.

No. The proteins in the B2 culture supernatant were analyzed.
No. SDS-PAGE was performed on the B2 culture supernatant. The gel after electrophoresis was stained with silver. At this time, as a control, No. A B2 culture solution (left figure) and a purified protein A solution (middle figure) were prepared and compared and examined.
In FIG. 10, No. The results when SDS-PAGE was performed on the B2 culture supernatant are shown. In the figure, M indicates a marker, and lane 1 is No. B2 culture broth is shown, lane 2 shows the purified protein A solution, and lane 3 is No. The B2 culture supernatant is shown.
As shown in FIG. 10, a band having a molecular weight of about 50 kDa was detected, and No. It was suggested that the above-mentioned protein A was present in the B2 culture supernatant.

In addition, No. The strain of B2 was identified.
No. Using the UltraClean Microbial DNA kit manufactured by MO BIO from the B2 cells according to the manufacturer's manual, No. Genomic DNA of B2 fungus was extracted.
Sequence analysis was performed on genomic DNA using a next-generation sequencer.
Sequence analysis was performed according to the following.
Equipment: Illumin-HiSeq
Analysis method: Paired End
Read base length: 100 bases / 1 read Number of acquired reads: 10 million read pairs (20 million reads) / sample Acquisition data amount: 2 Gb / sample

No. A 16S rDNA nucleotide sequence analysis was performed on the genomic DNA sequence of the B2 fungus.
The 16S rDNA nucleotide sequence analysis was performed according to the following.
DNA extraction: achromopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.)
PCR amplification: PrimeSTAR HS DNA Polymerase (manufactured by Takara Bio Inc.)
Cycle Sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (manufactured by Applied Biosystems)
Sequence: ABI PRISM 3130 x1 Genetic Analyzer System (manufactured by Applied Biosystems)
BLAST homology search;
Software: Technosuruga Lab Microbial Identification System (Technosuruga Lab)
Database: DB-BA11.0 (Technosuruga Lab), International Base Sequence Database (DDBJ / ENA (EMBL) / GeneBank)

As a result, it was found that the genomic DNA has a base sequence having 99.8% identity with the Paenarthrobacter genus nicotinovorans (or Arthrobacter genus nicotinovorans). No. B2 was identified as Paenarthrobacter genus nicotinovorans.

In addition, No. The sequence of the genomic DNA of the B2 fungus was annotated by RAST (http://last.nmpdr.org/).
As a result, four base sequences having high similarity to the base sequence encoding glucanase were found.
The four base sequences were SEQ ID NO: 1 (No. 2567), SEQ ID NO: 2 (No. 753), SEQ ID NO: 3 (No. 246), and SEQ ID NO: 4 (No. 2584).
The nucleotide sequence of SEQ ID NO: 1 was similar to the sequence encoding the endoglucanase A precursor. The nucleotide sequence of SEQ ID NO: 2 was similar to the sequence encoding endo-1,4-β-glucanase. The nucleotide sequence of SEQ ID NO: 3 was similar to the sequence encoding the endoglucanase, family 26, S-layer domain protein. The nucleotide sequence of SEQ ID NO: 4 was similar to the sequence encoding the endoglucanase, family 26, S-layer domain protein.
The attached sequence listing shows the base sequences of SEQ ID NO: 1 to SEQ ID NO: 4.

In particular, SEQ ID NO: 1 (No. 2567) was expressed in large quantities using Escherichia coli by genetic recombination.

A vector pBIC1 containing the nucleotide sequence of SEQ ID NO: 1 (No. 2567) was prepared by a conventional method.

The prepared vector pBIC1 was introduced into Escherichia coli by a conventional method and transformed.

The transformed Escherichia coli was cultured by a conventional method, expression was induced, and a large amount of the protein encoded by SEQ ID NO: 1 (No. 2567) was expressed. The obtained protein was purified using a column.

The resulting protein was analyzed.
SDS-PAGE was performed on the obtained protein solution. The gel after electrophoresis was stained with an antibody.
FIG. 11 shows the results of SDS-PAGE for the protein (unpurified) encoded by SEQ ID NO: 1 (No. 2567) expressed in large quantities. In the figure, M indicates a marker, C indicates a control, lane 1 indicates 150 ng, and lane 2 indicates 75 ng. Further, super indicates a supernatant and ppt indicates a precipitate.
FIG. 12 shows the results of SDS-PAGE for the protein encoded by SEQ ID NO: 1 (No. 2567) expressed in large quantities (purified by Ni-Sepharose). In the figure, M indicates a marker, C indicates a control, FT indicates a washing solution, lane 1 indicates a first eluate, lane 2 indicates a second eluate, and lane 3 indicates a third eluate. , Lane 4 shows the 4th eluate, and lane 5 shows the 5th eluate. Further, super indicates a supernatant and ppt indicates a precipitate.
As shown in FIGS. 11 and 12, a band having a molecular weight of about 50 kDa was detected, and No. It was suggested that the above-mentioned protein A was present in the B2 culture supernatant.

When the xanthan degrading activity was confirmed for the protein encoded by the above-mentioned mass-expressed SEQ ID NO: 1 (No. 2567), xanthan degrading activity was found.
It was analyzed whether the protein solution had xanthan degrading activity.
1 mL of the above-mentioned protein solution was added to 100 mL of a 1 g / L xanthan aqueous solution, incubated for 24 hours, and analyzed by gel permeation chromatography (GPC).

According to the present invention, a biofilm such as foulant formed on a membrane used for filtration can be effectively decomposed.

Biomaterial Depositary Organization: National Institute of Technology and Evaluation Patent Microorganisms Depositary Center Address: Asahi Kasei Co., Ltd. Microza Technology Development Department Contract number: NITE P-02506
Date of deposit: July 7, 2017

Claims (2)

A method for degrading a biofilm using a protein, which is encoded by the base sequence of SEQ ID NO: 1 (No. 2567) . The biofilms are starch, glycogen, pectin, guar gum, gum arabic, xanthan gum, gellan gum, tamarind gum, locust bean gum, carrageenan, carboxymethyl cellulose (CMC), gelatin, agar, glucomannan, heparin, hyaluronic acid, alginic acid, propylene alginate. The method of claim 1 , comprising at least one selected from the group consisting of glycols, purulans, psylliums, carrageenans, propylene glycols.

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