JP2006238801A - New polyhydroxyalkanoic acid-degrading microorganism and method for producing degradation enzyme - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for promoting the degradation of a polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid, especially 3-hydroxybutyric acid/3-hydroxyhexanoic acid copolyester. <P>SOLUTION: A microorganism for degrading the polyhydroxyalkanoic acid composed of the 3-hydroxyhexanoic acid, especially the 3-hydroxybutyric acid/3-hydroxyhexanoic acid copolyester, is separated from environments. A depolymerase is highly efficiently produced by isolating a depolymerase gene from the strain and then expressing the gene in a host cell. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to a novel polyhydroxyalkanoic acid (hereinafter abbreviated as PHA) -degrading microorganism. The present invention also relates to an enzyme that degrades the PHA, the enzyme gene, and a method for producing the enzyme. More specifically, Bacillus bacteria and Cellulomonas bacteria that efficiently decompose PHA composed of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, enzymes that degrade PHA isolated from both microorganisms, both The present invention relates to a method for using microorganisms and a method for producing an enzyme that degrades PHA by culturing both microorganisms. Furthermore, the present invention relates to a method for producing an enzyme that degrades PHA by culturing the enzyme gene isolated from the Bacillus bacterium and the transformant transformed with the enzyme gene expression vector.

In recent years, composting has been promoted as a method for reusing waste from school lunches, restaurants and the like as products effective for agriculture. Compost used as a soil amendment has the desirable effect of reducing the amount of chemical fertilizer input and controlling plant diseases, because it increases the organic matter content, moisture and nutrient retention in the soil. By using compost for agriculture in this way, it becomes possible to convert the problem of waste disposal into sustainable agriculture with low input.

However, in order to compost the waste and use it for agricultural purposes, it is necessary to separate and remove the plastic waste contained therein, and such separation and removal require a great deal of labor. It has become a big issue.

To date, it has been known that polyesters accumulate in cells as an energy storage substance in many microorganisms. These polyesters (PHA), which are homopolymers or copolymers of 3-hydroxyalkanoic acid, are thermoplastic polymers and have been attracting attention as environmentally friendly plastics because they are decomposed by microorganisms in composting and natural environments. . For this reason, such biodegradable plastics are being developed for a wide range of applications such as agricultural materials, food containers, packaging materials, sanitary goods, garbage bags, etc. for which one way is required. In particular, food containers and packaging materials are highly expected because they do not need to be separated and removed for composting.

However, taking into account the current amount of plastic used, it is expected that it will not be possible to treat it simply by waiting for decomposition by microorganisms present in the natural world and composting facilities. Therefore, a method of blending a microbial medium component with a biodegradable material (polylactic acid) to promote degradation by microorganisms (Patent Document 1), lipase, amylase, cellulase, lactic acid dehydrogenation into a biodegradable plastic material (polylactic acid) Method of adding hydrolase such as enzyme (Patent Document 2), blending amount and average volume of inorganic and / or organic filler blended when producing biodegradable plastic material (aliphatic polyester) molded product in specific range A method for suppressing biodegradation and collapse during use of a molded product (Patent Document 3) has been proposed.

Moreover, the search of the microorganism which decomposes | disassembles polyhydroxybutyric acid (henceforth PHB) which is a homopolymer of 3-hydroxybutyric acid (henceforth 3HB) was also performed (patent document 4). A microorganism belonging to the genus Pseudomonas isolated from soil was isolated, and it was found that this strain produces depolymerase, an enzyme that degrades PHB. By using this microorganism and the same enzyme, PHB can be efficiently decomposed. In addition, cloning of a depolymerase gene and production of a highly efficient depolymerase by applying a gene recombination technique have been carried out (Patent Document 5). Furthermore, a microorganism and a degrading enzyme that effectively degrade a homopolyester of ω-hydroxyalkanoic acid such as 4-hydroxyalkanoic acid or a copolyester containing the same have been reported (Patent Document 6).

PHA is classified into short-chain-length PHA (hereinafter abbreviated as PHA _SCL ; 3 to 5C-atoms) and medium-chain-length PHA (hereinafter abbreviated as PHA _MCL ; 6 to 14 C-atoms). Although strains that degrade PHA are widely distributed in bacteria and molds, many microorganisms that degrade PHA _SCL and PHA _MCL have been isolated (Non-patent Document 1). Most bacteria specifically degrade PHA _SCL or PHA _MCL . Moreover, although Streptomyces exforiatus has been found as a microorganism capable of degrading both types of PHA, it has been found that it has two or more types of degrading enzymes corresponding to each type of PHA. (Non-patent document 2, Non-patent document 9).

Degradation of PHA in the environment is mainly performed by a depolymerase produced by the strain. The depolymerase is classified into a type that is secreted outside the cell (Extracellular Depolymerase) and a type that exists inside the cell (Intracellular Depolymerase). The Of these, the former mainly contributes to PHA decomposition in the environment. Various depolymerases have been purified from these strains and their enzymatic properties have been elucidated. As a result of examining PHB, polyhydroxyvaleric acid (hereinafter abbreviated as PHV), polyhydroxyoctanoic acid (hereinafter abbreviated as PHO), etc., the substrate specificity of these _{depolymerases} is specific to either PHA _SCL or PHA _MCL. (Non-Patent Document 1, Non-Patent Document 2).
Extracellular Depolymerase that degrades PHA _SCL has been well studied and the primary structure has been elucidated. The structure consists of a catalytic domain containing an active residue at the amino terminus, a substrate binding domain for adsorbing to a hydrophobic polymer material at the carboxyl terminus, and a linker domain between the two domains. In the catalytic domain, there are serine, histidine and aspartic acid as active residues, among which serine residues are present in the lipase box-like pentapeptide sequence (Gly-Xaa ₁ -Ser-Xaa ₂ -Gly) Patent Document 1, Non-Patent Document 10).
There are few studies on Extracellular Depolymerase that degrades PHA _MCL, and only one example of Pseudomonas fluorescens depolymerase has been studied at the molecular level. In the primary structure of this depolymerase, there is a catalytic domain possessed by Extracellular Depolymerase that degrades PHA _SCL, and there are also similar active residues. However, the catalytic domain is at the carboxyl terminus, and has a structure different from Extracellular Depolymerase that degrades PHA _SCL (Non-patent Document 11).

On the other hand, application development of PHA _SCL such as PHBV which is a copolyester of PHB, 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) has been advanced. However, since these PHAs have high crystallinity and are hard and brittle, their practical application range is limited. For this reason, research aimed at improving this property has been conducted.

PHA _MCL is known to have lower crystallinity and higher elasticity than PHB and PHBV (Non-patent Document 3), and is expected to be widely applied. However, PHA _MCL was produced by introducing a PHA synthase gene derived from the genus Pseudomonas into various hosts, but none of these were low in productivity and suitable for industrial production (non-patented). Document 4, Non-Patent Document 5, Non-Patent Document 6).

In recent years, research has been conducted on a copolymerized polyester (hereinafter abbreviated as PHBH) of 3HB and 3-hydroxyhexanoic acid (hereinafter abbreviated as 3HH) and a method for producing the same (Patent Document 7 and Patent Document 8). This PHBH gradually shows a soft property from a hard and brittle property as the 3HH composition increases, and it becomes clear that changing the 3HH composition has a wide range of physical properties applicable from hard polymers to soft polymers. (Non-Patent Document 7). In addition, research on production of PHBH has been carried out, and efforts toward commercialization have been accelerated (Patent Document 9, Non-Patent Document 8).

However, little research has been conducted on PHBH degrading enzymes, which are hybrids of PHA _SCL and PHA _MCL . Depolymerases found to date specifically degrade PHA _SCL or PHA _MCL , but with regard to PHBH, there is little knowledge that PHB-degrading enzymes produced by Alcaligenes faecalis cannot hydrolyze the bond between 3HB and 3HH. (Non-Patent Document 1). Therefore, a microorganism that degrades PHBH, which is expected to be applied to a wide range of uses, has been desired. Furthermore, a method for producing the same degrading enzyme at low cost has been demanded.
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An object of the present invention is to provide a microorganism that degrades hybrid PHBH in view of the above situation. Another object of the present invention is to provide a highly efficient method for producing the depolymerase by isolating a depolymerase gene from the microorganism and expressing the gene in a host cell. According to the present invention, it becomes possible to decompose PHBH discarded in large quantities efficiently and inexpensively.

As a result of intensive studies to solve the above problems, the present inventors have performed primary and secondary fermentation in Niigata City, Niigata Prefecture, which is a cold rice cultivation region, and decomposed PHBH from the produced compost extract. Bacillus megaterium and Cellulomonas cellulans were isolated. In addition, the present invention was completed by cloning the depolymerase gene from Bacillus megaterium and successfully producing the depolymerase by expressing the gene in a host cell.

That is, the present invention provides the following (1) to (12).
(1) A microorganism that degrades polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid
(2) The microorganism according to (1), which decomposes polyhydroxyalkanoic acid composed of 3-hydroxybutyric acid (3) The polyhydroxyalkanoic acid is a copolyester of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid (1 ) Or the microorganism described in (2) (4) the microorganism is Bacillus megaterium N18-25-9 (FERM AP-20422), the microorganism described in any one of (1) to (3) (5) the microorganism is Cellulomonas The microorganism (6) or the following (a), (b) or (c) polypeptide according to any one of (1) to (3) which is cellulans N18-25-10 (FERM AP-20423):
(A) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 (b) by a DNA that hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 A polypeptide that is encoded and has an activity of degrading polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid (c), wherein one or several amino acids are deleted in the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, A polypeptide comprising an amino acid sequence substituted and / or added and having an activity of decomposing polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid (7) The following (d), (e), (f) Or (g) polynucleotide:
(D) a polynucleotide comprising the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 (e) a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 A polynucleotide encoding a polypeptide having the activity of degrading polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid (f) 80% or more of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 (G) a polynucleotide encoding a polypeptide having an activity of degrading polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid and comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 Amino in which one or several amino acids have been deleted, substituted and / or added A promoter sequence is linked upstream of the polynucleotide described in the polynucleotide (8) (7), which encodes a polypeptide comprising the sequence and having an activity of decomposing polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid, And the transformant (11) (1)-() transformed with the vector (10) containing the expression cassette described in the expression cassette (9) (8) with the terminator sequence linked downstream (10) (9) A method for producing a polypeptide having the activity of decomposing polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid, comprising culturing the microorganism according to 5) or the transformant according to (10) (12) poly Hydroxyalkanoic acid copolyester of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid Manufacturing method of a (11), wherein the polypeptide

Hereinafter, the present invention will be described in detail.
PHA in the present invention is represented by the following general formula.

(In the formula, R represents an alkyl group having 1 to 13 carbon atoms, and m represents an integer of 2 or more. The m Rs may be the same or different.)
The microorganism of the present invention decomposes PHA composed of 3HH, and preferably decomposes PHA composed of 3HB. More preferably, it is represented by the following general formula:

(M and n in the formula represent an integer of 1 or more) A microorganism decomposing a copolyester composed of 3HB and 3HH.

(Separation of PHA-degrading microorganisms)
The microorganism of the present invention can be separated from the environment such as soil, compost, activated sludge, river, seawater and the like. Various methods are known for the separation of microorganisms (Agricultural Chemistry Experiments, Vol. 2). Any separation method can be used as long as it can separate PHA composed of 3HH, particularly microorganisms that degrade PHBH. Good. As an example, various samples obtained from the environment are suspended in sterilized water or physiological saline to prepare a cell suspension. The suspension is diluted to an appropriate bacterial concentration, applied to a solid medium containing PHA composed of 3HH, particularly PHBH, as the only or main carbon source, and cultured at a temperature at which the strain can grow. The culture temperature is not particularly limited as long as the microorganism can grow. The PHA added to the medium can be used in any shape, but a fine powder is preferable. The concentration to be added is not particularly limited, but 0.05% to 5%, which is clouded by PHA added to the solid medium, is preferable, and 0.1% to 0.5% is more preferable. Microorganisms that decompose PHA composed of 3HH can be detected and separated by forming a halo (dissolution zone) indicating that the polymer has been decomposed around the growing bacterial colonies.

In addition, the microorganism suspension is concentrated by adding and culturing a cell suspension separated from various samples in a medium containing PHA composed of 3HH, particularly PHBH as the sole or main carbon source. You can also. Thereafter, the microorganism can be separated by the method.

A preferred example of separation of a PHA composed of 3HH according to the present invention, particularly a microorganism that degrades PHBH, is as follows.

A compost extract obtained from compost subjected to primary and secondary fermentation in Niigata City, Niigata Prefecture is applied to a general bacterial medium (NB medium) to form bacterial cell colonies. These colonies are applied to NB medium containing 0.3% of powdered PHBH. After the cultivation, two types of microorganisms that have formed a PHBH dissolution zone around the bacterial colonies are separated. Identification of these strains can be performed by mycological properties and sequence comparison of 16S ribosomal DNA. As a result of identification of this strain by the latter, it was revealed that it was Bacillus megaterium and Cellulomonas cellulans, and Bacillus megaterium N18-25-9 (reception date: February 22, 2005, receipt number: FERM AP-20422), respectively. Cellulomonas cellulans N18-25-10 (reception date: February 22, 2005, reception number: FERM AP-20423). These two strains are deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, located at 1st, 1st, 1st East, Tsukuba, Ibaraki, Japan.

Physiological tests were performed according to general methods (Takeharu Hasegawa, Classification and Identification of Microorganisms, The University of Tokyo Press, 155245 (1975), Sneath et al., Bergey's Manual of Systematic Bacteriology, Vol2, Williams & Wilkins, 1988 )). Bacillus megaterium N18-25-9 (FERM AP-20422) is a Gram-positive bacterium, is an aerobic bacilli, forms spores, has a growth temperature of 20-45 ° C., and other physiological properties are as follows: It was as Table 1. Cellulomonas cellulans N18-25-10 (FERM AP-20423) is a Gram-positive bacterium, an aerobic short bacillus, and other physiological properties are as shown in Table 1 below.

Bacillus megaterium N18-25-9 (FERM AP-20422) was able to oxidize or assimilate glycerol, glucose, fructose, mannitol, cerbose, maltose, lactose, melibiose, sucrose, trehalose, raffinose and glycogen. It was also possible to hydrolyze esculin and starch.
Cellulomonas cellulans N18-25-10 (FERM AP-20423) can oxidize or assimilate L-arabinose, D-xylose, galactose, glucose, fructose, mannose, cellobiose, maltose, sucrose and glycogen. And the starch could be hydrolyzed.

(Cloning of depolymerase gene)
1. Construction of Genomic Library Various host / vector systems can be used to construct a genomic library of microorganisms that degrade PHA composed of 3HH, particularly PHBH. Although not particularly limited by the host, a PHA composed of 3HH, particularly a host that does not degrade PHBH, is preferable, and Escherichia coli is more preferable. The vector is not particularly limited as long as it is replicated and maintained in the host. For details, it is possible to refer to an experimental document such as Molecular Cloning (Sambrook et al., CSH Laboratory Press).

Various known methods can be used for the preparation of PHA composed of 3HH, particularly microorganism DNA that degrades PHBH. As an example, microbial cells are collected from the culture solution of the microorganism by centrifugation or the like, and a lytic enzyme treatment such as lysozyme is performed. Next, after freeze-thawing treatment, lysis is performed by overlaying the SDS solution and mixing the interface. A phenol / chloroform solution is added to the lysate, and after denaturation treatment of proteins and the like, ethanol is added to the aqueous layer. Genomic DNA can be prepared by winding the precipitated DNA with a glass rod or the like.

The prepared genomic DNA can be cleaved using physical cleavage or various restriction enzymes. Although the cutting method is not particularly limited, for example, a DNA fragment of 8 to 10 Kb can be purified by partial cleavage with the restriction enzyme Sau3AI. Plasmids, phages and the like can be used as vectors, but are not particularly limited. It can be used for the construction of a genomic library by treating with a restriction enzyme according to the DNA fragment to be inserted. For example, a DNA fragment of 8 to 10 Kb partially cleaved with the restriction enzyme Sau3AI can be inserted into a vector such as pUC19 cleaved with the restriction enzyme BamHI, and a genomic library can be constructed by transformation into E. coli. .

2. Cloning of the depolymerase gene A genomic library is spread on a solid medium containing PHA composed of 3HH, particularly PHBH as the sole or main carbon source, and cultured at a temperature at which the strain can grow. When the host used to construct the genomic library secretes depolymerase, the colony forming the same polymer lysis zone larger than the host alone is a clone containing the depolymerase gene, and the host is a depolymerase such as E. coli. When the polymerase is not produced, the colony forming the polymer dissolution zone is a clone containing the depolymerase gene. The cloned gene can be identified by analyzing the nucleotide sequence or the like.

Preferable examples of the depolymerase gene according to the present invention include a depolymerase gene derived from Bacillus megaterium N18-25-9 (FERM AP-20422) represented by SEQ ID NO: 1 or SEQ ID NO: 2. However, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA shown in SEQ ID NO: 1 and SEQ ID NO: 2 is also a PHA whose polypeptide translated from the DNA is composed of 3HH. As long as it has an activity of degrading, it is included in the depolymerase gene. Here, stringent conditions refer to, for example, conditions where the salt concentration is 6 × SSC and the temperature is 68 ° C. (Molecular Cloning (Sambrook et al., CSH Laboratory Press)).
A DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA represented by SEQ ID NO: 1 or SEQ ID NO: 2 is a base sequence complementary to the DNA represented by SEQ ID NO: 1 or SEQ ID NO: 2. Means DNA obtained by using colony hybridization method, plaque hybridization method, Southern hybridization method, etc., with DNA consisting of as a probe. Specifically, colony or plaque-derived DNA is immobilized. After the hybridization was performed at a salt concentration of 6 × SSC and 68 ° C., the SSC solution having a concentration of 0.1 to 2 times (the composition of the SSC solution having a concentration of 1 was 150 mM sodium chloride and 15 mM sodium citrate). And filter under conditions of 65 ° C Can include DNA can be identified by washing the.
Hybridization can be performed according to the method described in Molecular Cloning, A laboratory manual, second edition (Cold Spring Harbor Laboratory Press, 1989) and the like.

A polypeptide comprising a base sequence having 80% or more homology with the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and having an activity of decomposing polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid The encoding polynucleotide is also included in the polymerase gene. More preferred is a nucleotide sequence having 90% or more homology, more preferably 95% or more homology, particularly preferably 99% or more homology.
Here, “homology (%)” means that the two polynucleotides to be compared are optimally aligned and the nucleobases (eg, A, T, C, G, U, or I) match in both sequences. The number of positions is divided by the total number of comparison bases, and the result is expressed by a value obtained by multiplying by 100.
Sequence identity can be calculated using, for example, the following tools for sequence analysis: GCG Wisconsin Package (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group 37, 575 ScienceDenM Rice, P. (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Center, Hinxton Hall, Cambridge, CB10 1RQ, England, and the ExPASyWr ty Hospital and University of Geneva, Geneva, Switzerland).

Furthermore, one or several amino acids are deleted, substituted and / or added in the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, and has an activity of decomposing polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid A polynucleotide encoding a polypeptide is also included in the polymerase gene.
“Deletion, substitution and / or addition of one or several amino acids” means that a sufficient number of amino acids have been deleted by a known method such as site-directed mutagenesis. , Substituted and / or added, usually 1 to 3.
A polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 can be obtained by using the Current Protocols in Molecular Biology (John Wiley and Sons, Inc.). As long as it has an activity of decomposing PHA composed of 3HH, it is included in the polynucleotide of the present invention.

As will be described later, the depolymerase gene derived from Bacillus megaterium N18-25-9 (FERM AP-20422) has a portion predicted to function as a secretory sequence. SEQ ID NO: 2 is SEQ ID NO: 1 This is obtained by removing a portion predicted to function as a secretory sequence from the base sequence of.

(Method for producing depolymerase)
Production of Depolymerase Using PHA Comprising 1.3HH, In particular, Microorganism that Decomposes PHBH The method for producing a polypeptide having activity of degrading PHA comprised of 3HH of the present invention comprises culturing the microorganism. It consists of things.
Various carbon sources, nitrogen sources, inorganic salts, and other organic nutrient sources can be arbitrarily used for culturing this strain.
As the carbon source, for example, glucose, sucrose, fats and oils can be used.
Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, peptone, meat extract, yeast extract, and the like.
Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride.
Examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, and proline; vitamins such as vitamin B1, vitamin B12, and vitamin C. In addition, rice bran obtained in large quantities during rice milling, molasses produced as a by-product during sugar production, and the like can also be used as the medium.

The culture temperature should just be the temperature which the microbe can grow. The culture time is not particularly limited as long as the cells can be produced efficiently. The culture solution obtained by such culture can be used as a depolymerase solution or a bacterial cell preparation. In addition, the bacterial cell culture solution can be separated into a culture solution and a bacterial cell by centrifugation or the like. The former can be used as a depolymerase solution, and the latter as a cell preparation. In addition, depolymerase purified from the culture solution by various methods can also be used.

2. Production of depolymerase using depolymerase gene By expressing the cloned depolymerase gene in a host, depolymerase can be produced from the transformant. That is, the method for producing a polypeptide having activity of decomposing PHA composed of 3HH according to the present invention comprises culturing the transformant. Since the transformant introduced with the gene obtained by the depolymerase gene cloning expresses the depolymerase, this transformant is used as a cell preparation for degrading PHA composed of 3HH, particularly PHBH. Can be used. The culture solution can be used as a depolymerase solution.

Various host / vector systems can be used for more efficient expression of the gene. Bacteria, yeast, animal cells, etc. can be used as the host / vector system, but there is no particular limitation as long as the gene can be expressed and produced efficiently. If the cloned depolymerase gene has a promoter and terminator necessary for expression of the gene and can be expressed in the host, it can be used as it is. Moreover, the expression vector can be constructed by preparing an expression cassette in which various promoters and terminators are connected to DNA encoding the depolymerase gene, and incorporating this expression cassette into the vector. An expression cassette in which a promoter sequence is linked upstream of a polynucleotide that is a DNA encoding the depolymerase gene and a terminator sequence is linked downstream is also one aspect of the present invention.

As an example, when using E. coli, various expression vectors can be constructed for efficient gene expression. A depolymerase expression cassette can be constructed in which the gene is connected downstream of the lactose operon promoter, the tryptophan operon promoter, the previous two fusion promoters, the promoter of λ phage, etc., and a terminator is connected downstream of the gene. . The terminator may be an rrn terminator, but is not particularly limited. In the production of these expression cassettes, depolymerase can be secreted outside the cells by connecting a DNA sequence that functions as a secretion signal in the host. As an example, the depolymerase (SEQ ID NO: 3) of Bacillus megaterium N18-25-9 (FERM AP-20422) is predicted to function as a secretory sequence from the first methionine to the 24th alanine of the same sequence. Thus, the DNA encoding this sequence can be used for secretion of various depolymerases. The amino acid sequence of SEQ ID NO: 4 is obtained by removing the portion predicted to function as a secretory sequence from the sequence of SEQ ID NO: 3.

The following polypeptides (a), (b) or (c) are also one aspect of the present invention.
(A) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 (b) by a DNA that hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 A polypeptide that is encoded and has an activity of degrading polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid (c), wherein one or several amino acids are deleted in the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 In the “stringent conditions” and (c) in the polypeptide (b) having the activity of degrading polyhydroxyalkanoic acid composed of a substituted and / or added amino acid sequence and composed of 3-hydroxyhexanoic acid “Amino with one or several amino acids deleted, substituted and / or added The preparation of "are as described above.

A depolymerase expression vector can be constructed by introducing the expression cassette thus prepared into a vector that is replicated and maintained in the host. The produced expression vector can be transformed into a host to produce a depolymerized transformant. This transformant can secrete depolymerase outside the cell when the expression cassette contains a secretion signal DNA, and can accumulate the depolymerase inside the cell when it does not have the same sequence. .
A vector containing the above expression cassette and a transformant transformed with the vector are also one aspect of the present invention.

Various carbon sources, nitrogen sources, inorganic salts, and other organic nutrient sources can be arbitrarily used for culturing the transformed strain.
Examples of carbon sources that can be used include glucose, sucrose, molasses, and fats.
Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, peptone, meat extract, yeast extract, and the like.
Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride.
Examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, and proline; vitamins such as vitamin B1, vitamin B12, and vitamin C.

The culture temperature may be any temperature at which the transformed strain can grow. The culture time is not particularly limited as long as it can efficiently produce depolymerase. In this way, depolymerase can be produced efficiently.

According to the present invention, it is possible to efficiently treat a waste composed of a biodegradable polymer (PHA composed of 3HH, particularly PHBH), which is expected to be widely used in the future.

EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention does not limit the technical scope by these Examples.
Example 1 Isolation of Biodegradable Plastic Degrading Bacteria Copolyester PHBH (powder, film, molecular weight 1.2 million, 3HH composition 6 mol%) of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid provided by Kaneka Corporation It was done.
In Niigata City, Niigata Prefecture, which is a cold rice-growing region, using an extract obtained from the primary and secondary fertilized compost, the microorganisms that formed colonies on the NB medium (Table 2) were treated with 0.3% PHBH in the NB medium. Cultures on the mixed agar medium were searched for microorganisms forming a lysis zone. As a result, two strains N-18-25-9 and N-18-25-10 were obtained. The N-18-25-9 strain also formed a polymer dissolution zone in an agar medium supplemented with PHB as a carbon source.

(Example 2) Identification of degrading bacteria N-18-25-9 and N-18-25-10 strains were identified by a microorganism identification method using 16S rDNA sequences.
Chromosomal DNA was extracted from the two strains (see Example 3 for the method), and 16S rDNA of each strain was amplified by PCR using universal primer SEQ ID NO: 5 and SEQ ID NO: 6. After the amplified DNA was cloned into T-vector (manufactured by Promega), the sequence of about 500 bases on the 5 ′ side of the 16S rDNA was determined. The respective base sequences are registered in DDBJ as N-18-25-9 (registration number AB190040) and N-18-25-10 (registration number AB190041).
NCBI Nucleotide-nucleotide BLAST (blastn) (http://www.ncbi.nlm.nih.gov/BLAST/), RDPII Sequence match (http: // cmtp / cmp) / .Msu.edu / seqmatch / seqmatch-intro.jsp) sequence comparison was performed. As a result, N-18-25-9 strain showed Bacillus megaterium AC46b1 (Aj7171781) and 99% similarity from the analysis results using both programs, so Bacillus megaterium N18-25-9 (Received date: 2005) On February 22, it was named with the receipt number: FERM AP-20422). The N18-25-10 strain showed Cellulomonic cellulans DSM43879 (X79455.1) and 98% similarity from the analysis results using both programs, so Cellulomonas celluans N18-25-10 (Received date: February 22, 2005) Day, receipt number: FERM AP-20423). These two strains are deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, located at 1st, 1st, 1st East, Tsukuba, Ibaraki, Japan.

(Example 3) Preparation of chromosomal DNA N-18-25-9 strain cultured overnight in 50 ml of NB medium (Table 2) was collected and 7.5 ml of saline-EDTA (0.15 M NaCl, 0. It was suspended in 1M ethylenediaminetetraacetic acid (pH 8.0). In a 50 ml centrifuge tube, lysozyme was added to the cell suspension to a final concentration of 1 mg / ml, dissolved well, and incubated at 37 ° C. for 15 minutes with occasional stirring. As soon as the viscosity started to increase, it was frozen in a -80 ° C freezer. If no increase in viscosity was seen after 30 minutes, more lysozyme was added. Next, after putting the frozen cells in a 60 ° C. water bath, 7.5 ml of Tris-SDS (0.1 M Tris-HCL (pH 9.0), 1% sodium dodecyl sulfate) is added, and a sterilized glass rod is added. The cells were lysed while mixing the interface between the frozen cells and the SDS solution. While confirming the increase in viscosity, the sample was incubated for 10 minutes when it reached approximately 60 ° C., and then ice-cooled. After adding 15 ml of phenol / chloroform solution, the mixture was vigorously shaken for 15 minutes with ice age. After centrifugation at 7500 rpm for 20 minutes at 4 ° C., the upper aqueous layer was transferred to a new 50 ml centrifuge tube, and phenol / chloroform extraction was performed again. 30 ml of ethanol cooled at −20 ° C. was added, and the precipitated DNA was wound on a glass rod while stirring with a glass rod. The obtained DNA was washed with 10 ml of 70% ethanol and dissolved in an appropriate amount of TE (10 mM Tris-HCL (pH 8.0), 1 mM EDTA) to obtain a chromosomal DNA solution.

(Example 4) Preparation of genomic library Total DNA of N-18-25-9 strain was partially treated with restriction enzyme Sau3AI, and then fractionated by the following method.
Using 10% sucrose TES (10 mM Tris-HCl (pH 8.0), 1 mM EDTE, 10 mM NaCl) solution and 40% sucrose TES solution, 10%, 11.25%, 12.5%, 13.75%, 15%, 16.25%, 17.5%, 18.75%, 20%, 21.25%, 22.5%, 23.75%, 25%, 26.25%, 27.5%, 28 Create sucrose TES solutions with concentrations of .75%, 30%, 31.25%, 32.5%, 33.75%, 35%, 36.25%, 37.5%, 38.75%, 40% did. These solutions were attached to a Beckman Coulter tube (14 × 89 mm) so that the tip of a micropipette for 50 μl was placed at the bottom in 500 μl, and gently stacked in the order of thin solution to thick solution using a peristaltic pump. The tube containing the sucrose concentration gradient solution was allowed to stand overnight at 4 ° C., and then the DNA solution was overlaid, and centrifuged with SW41 Ti rotor (Beckman Coulter) at 26000 rpm for 18 hours at 15 ° C. After centrifugation, 500 μl was fractionated from the bottom using a peristaltic pump, and the size of the DNA fragment was confirmed by agarose electrophoresis. Fractions containing the desired 8-10 kbp DNA fragment were collected, extracted with phenol / chloroform and precipitated with ethanol, and then dissolved in an appropriate amount of TE.
The DNA fragment prepared as described above was ligated with pUC19 that had been digested with the restriction enzyme BamHI and then BAP-treated. It was transformed into E. coli DH5α, and a part was plated on an agar medium to examine the size of the library and the insertion efficiency. The rest was cultured overnight in LB medium containing ampicillin, the plasmid was extracted, and the cells were stored at −80 ° C. as a 15% glycerol solution.

Example 5 Cloning of Depolymerase Gene A genomic library consisting of about 2,500 clones was prepared from Bacillus megaterium N18-25-9 (FERM AP-20422) by the construction of the genomic library. As a result of plating this library on NB medium containing 0.3% PHBH, one clone which formed a clear zone (lysis zone) was found. When the plasmid was recovered from this clone and the recovered plasmid (named as pUCBM-A) was transformed again into E. coli, the transformed strain formed a polymer lysis zone.
Next, the base sequence of the inserted fragment was determined in order to determine the gene site imparting the polymer dissolution zone forming ability (PHBH resolution). A restriction enzyme map of the DNA is shown in FIG.
As a result of base sequence analysis, a conserved sequence of PHB depolymerase known as an enzyme that degrades biodegradable plastics in other microorganisms was found in the DNA. Therefore, after treating pUCBM-A with restriction enzymes SacI and HincII, a DNA fragment of about 2.7 kbp containing only the ORF portion having the same conserved sequence was excised and cloned into pUC19 (named pUCBM-ASH). When pUCBM-ASH was transformed into E. coli DH5α, a polymer dissolution zone was formed on NB medium containing PHBH or LB medium containing PHBH. From the above results, this ORF portion was determined to be a depolymerase gene, and this gene was named PhaZ _Bm .

(Example 6) Analysis of depolymerase gene The depolymerase gene of megaterium N18-25-9 (FERM AP-20422) consisted of 1573 bases (GC content 40.3%), 590 amino acids, and the expected molecular weight was 62.3 KDa. A Shine-Dalgarno sequence (TAAGGAGGA) was present 10 bases upstream of the ATG start codon. In addition, expected promoter sequences -35 (TTGATT) and -10 (CATCTT) were observed (FIG. 2, SEQ ID NO: 7).
The base sequence and amino acid sequence of the depolymerase gene are shown in SEQ ID NOs: 1 and 3, respectively, and the base sequence and amino acid sequence excluding the part that is predicted to function as a secretory sequence from these sequences, respectively, Shown in SEQ ID NOs: 2 and 4.
In many cases, a signal peptide is added to the N-terminus of a depolymerase known to date. Therefore, the presence or absence of a signal peptide in PhaZ _Bm was analyzed using SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/). Analysis using SignalP-NN (neural networks) and integration of C score, Y score, which predicts the cleavage site, and S score, which predicts the signal peptide, resulted in the 24th alanine of the polypeptide shown in SEQ ID NO: 3. And the 25th alanine (SSA-AG) were expected to cleave. Moreover, the same result was obtained also by the analysis result using SignalP-HMN (hidden Markov model).

It is a restriction enzyme map of the DNA fragment containing the depolymerase gene of Bacillus megaterium N18-25-9. It is the base sequence and amino acid sequence of a DNA fragment containing the depolymerase gene of Bacillus megaterium N18-25-9. It is the base sequence and amino acid sequence of a DNA fragment containing the depolymerase gene of Bacillus megaterium N18-25-9. It is the base sequence and amino acid sequence of a DNA fragment containing the depolymerase gene of Bacillus megaterium N18-25-9. It is the base sequence and amino acid sequence of a DNA fragment containing the depolymerase gene of Bacillus megaterium N18-25-9.

Claims (12)

A microorganism that degrades polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid. The microorganism according to claim 1, which decomposes polyhydroxyalkanoic acid composed of 3-hydroxybutyric acid. The microorganism according to claim 1 or 2, wherein the polyhydroxyalkanoic acid is a copolyester of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid. The microorganism according to any one of claims 1 to 3, wherein the microorganism is Bacillus megaterium N18-25-9 (FERM AP-20422). The microorganism according to any one of claims 1 to 3, wherein the microorganism is Cellulomonas cellulans N18-25-10 (FERM AP-20423). The following polypeptide (a), (b) or (c):
(A) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 (b) by a DNA that hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 A polypeptide that is encoded and has an activity of degrading polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid (c), wherein one or several amino acids are deleted in the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, A polypeptide comprising an amino acid sequence substituted and / or added and having an activity of decomposing polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid. The following polynucleotide (d), (e), (f) or (g):
(D) a polynucleotide comprising the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 (e) a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 A polynucleotide encoding a polypeptide having the activity of degrading polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid (f) 80% or more of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 (G) a polynucleotide encoding a polypeptide having an activity of degrading polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid and comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 Amino in which one or several amino acids have been deleted, substituted and / or added Polynucleotide encoding a polypeptide having polyhydroxyalkanoate decomposing activity consists of it, and 3-hydroxy hexanoic acid sequence. An expression cassette comprising a promoter sequence linked upstream of the polynucleotide of claim 7 and a terminator sequence linked downstream. A vector containing the expression cassette according to claim 8. A transformant transformed with the vector according to claim 9. A method for producing a polypeptide having an activity of degrading polyhydroxyalkanoic acid composed of 3-hydroxyhexanoic acid, comprising culturing the microorganism according to claim 1 or 5 or the transformant according to claim 10. The method for producing a polypeptide according to claim 11, wherein the polyhydroxyalkanoic acid is a copolyester of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

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