JP4831664B2 - Cutinase variant with excellent thermostability - Google Patents

Description

The present invention relates to a parent cutinase mutant, particularly a cutinase mutant having improved thermostability, a DNA sequence encoding the mutant, a vector containing the DNA sequence, a transformant having the DNA sequence or vector, and a mutation It relates to a method for producing a body.

Cutinase [EC 3.1.1.74] is an esterolytic enzyme that can hydrolyze cutin. Cutinase has been found in many microorganisms (http://www.brenda.uni-koeln.de/php4, http://www.chem.qmw.ac.uk/iubmb/enzyme/).

Cutinase is also found in Aspergillus oryzae, and the amino acid sequence deduced from the base sequence encoded by the base sequence and the amino acid sequence has been reported (Genome structure and nucleotide sequence of a lipolytic enzyme gene of Aspergillus oryzae. K. Ohnishi, J. Toida, H. Nakazawa, J. Sekiguchi, FEMS Microbiol Lett. 126, 126145-126150 (1995), WO 2004038016).

Fungal cutinases are used in the textile industry for refining polyester fabrics or polyester fibers. In Patent Document 1, a method for degrading a biodegradable plastic using a filamentous fungus cutinase has been developed by the present inventors. Patent Document 2 and Patent Document 3 describe inventions related to cutinase specific substances that are more heat-stable. Actually, there is a specific disclosure of Humicola or Fusarium spp. A cutinase variant from a specific strain of Insolans.
WO 2004/038016 A1 Special table 2003-520016 gazette Special table 2003-534797 gazette

However, in order to efficiently decompose polyester containing biodegradable plastics, higher temperatures are more efficient due to its enzyme reaction mechanism, and the degradation rate of polyester molecules increases due to improved catalyst efficiency. Facilitates disassembly. For this reason, a cutinase having high thermostability is required.

As a result of studies to solve the above-mentioned problems, the present inventors have found a specific mutant of cutinase having improved thermostability and completed the present invention.

That is, the present invention relates to the following aspects.
[1] a) A variant of cutinase in which an additional peptide consisting of 11 or less amino acids is bound to the amino terminus of the parent cutinase, and / or
b) At least one amino acid residue corresponding to the following amino acid residues in the amino acid sequence of the cutinase of Aspergillus oryzae strain RIB-40: L2, G4, L8, P27, G28, A51, G54, K90, or A177 A variant of a parent cutinase comprising a substitution or deletion of the above, wherein the variant has better thermal stability than the parent cutinase.
[2] The mutant according to 1 above, wherein the parent cutinase is derived from a fungus belonging to the genus Aspergillus, Magnapolsa, Penicillium, Trichoderma, Rhizopus, metalithium, Monascus, Acremonium, or Mucor.
[3] The strain belonging to the genus Aspergillus is selected from the group consisting of Aspergillus oryzae, Aspergillus niger, Aspergillus soyae, and Aspergillus nidulans, or the strain belonging to the genus Magnapolsa is Magnapolsa grisea, 2. The mutant according to 2.
[4] The mutant according to the above 3, wherein the strain belonging to the genus Aspergillus is Aspergillus oryzae strain RIB40.
[5] The variant according to 4 above, wherein the amino acid sequence of the parent cutinase is represented by any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
[6] The mutant according to 1 above, wherein the amino acid sequence of the parent cutinase has at least 50% homology with the amino acid sequence of the cutinase of Aspergillus oryzae strain RIB40.
[7] The mutant according to the above item 6, wherein the amino acid sequence of the parent cutinase has at least 70% homology with the amino acid sequence of cutinase of Aspergillus oryzae strain RIB40.
[8] The mutant according to claim 7, wherein the amino acid sequence of the parent cutinase has at least 80% homology with the amino acid sequence of cutinase of Aspergillus oryzae strain RIB40.
[9] Substitution of amino acid residues is L2K / R, G4D / E / S / T / V, L8D, P27D, G28D, A51D / E / N / Q / R / S / T, G54C / D / N / The mutant according to any one of 1 to 8 above, comprising at least one selected from the group consisting of Q / S, K90E, and T177A.
[10] The mutant according to 9 above, wherein the amino acid residue substitution is G4D, G54S, or K90E.
[11] The variant according to any one of 1 to 10 above, wherein the additional peptide has ASTDPVDLQDT or STDPVDLQDR (amino acid single letter code), or a partial amino acid sequence thereof.
[12] The residual activity measured after treatment at 40 ° C. for 30 minutes is increased 1.1 times or more relative to the parent cutinase, and / or the residual activity measured after treatment at 50 ° C. for 30 minutes is the parent cutinase The mutant according to any one of the above 1 to 11, which has thermal stability such that it is increased 1.1 times or more relative to the above.
[13] The residual activity measured after treatment at 40 ° C. for 30 minutes is increased 1.15 times or more relative to the parent cutinase, and / or the residual activity measured after treatment at 50 ° C. for 30 minutes is the parent cutinase 13. The mutant according to 12 above, which has a thermal stability that is increased 1.18 times or more relative to the above.
[14] The mutant according to any one of 1 to 13 above, which has a hydrolytic activity on a biodegradable plastic.
[15] The variant according to the above 14, wherein the biodegradable plastic is selected from the group consisting of polylactic acid, polybutylene succinate, polybutylene succinate-co-adipate, polyhydroxybutyrate, and polycaprolactone.
[16] A DNA encoding a mutant of the parent cutinase according to any one of 1 to 15 above.
[17] The DNA of the above-mentioned 165, which hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA encoding the parent cutinase.
[18] An expression vector comprising the DNA sequence according to 16 or 17 above.
[19] A transformed cell having the DNA sequence described in 16 or 17 above or the vector described in 18 above.
[20] A method for producing the mutant according to any one of 1 to 15 above, wherein the transformed cell according to 19 is cultured, the mutant is expressed, and the mutant is recovered. Said method.
[21] The method according to 20 above, wherein the expressed mutant is secreted extracellularly and the mutant is recovered from the medium.

The present invention provides cutinase variants with improved thermal stability. The mutant can improve the thermal stability. Therefore, by using the cutinase mutant of the present invention, it is possible to improve the plastic decomposition efficiency per unit time by increasing the treatment temperature of the plastic decomposition process.

  In the mutant of the parent cutinase of the present invention, “corresponds to the following amino acid residues in the amino acid sequence of cutinase of Aspergillus oryzae strain RIB-40: L2, G4, L8, P27, G28, A51, G54, K90, or A177. The term “amino acid residue” refers to the above-mentioned amino acid sequence of Aspergillus oryzae strain RIB-40 when the Aspergillus oryzae strain RIB-40 and the other parent cutinase are aligned (aligned) using GENETYX. It means an amino acid residue on the amino acid sequence of the other parent cutinase corresponding to or corresponding to each position of the amino acid residue. Therefore, the corresponding amino acid residue numbers do not necessarily match between the amino acid sequences of the respective parent cutinases.

In the present specification, the amino acid number indicating the position of the mutation in the amino acid sequence is the first in the mature cutinase derived from Aspergillus oryzae strain RIB-40 shown in SEQ ID NO: 1 in each mutant. The amino acid corresponding to the amino acid is given as “1”.

  In the mutant of the parent cutinase of the present invention, the thermostability superior to that of the parent cutinase is compared to the heat stability of the parent cutinase, that is, the original cutinase without each amino acid mutation in the cutinase mutant of the present invention. Means significantly or substantially increased heat stability, and more specifically, in the following measurement method, the residual activity measured after treatment at 40 ° C. for 30 minutes is 1.1 times that of the parent cutinase. Or more, preferably increased by 1.15 times or more, and / or residual activity measured after treatment at 50 ° C. for 30 minutes is 1.1 times or more, preferably 1.18 times or more with respect to the parent cutinase, More preferably, it means that it has increased by 2 times or more.

The thermostability of the mutant of the parent cutinase of the present invention is specifically measured as follows.
That is, 100 μl of 0.1 M Tris-HCl buffer (pH 8.0) was added to 5 μl of purified enzyme, treated at 30 ° C., 40 ° C., 50 ° C., 60 ° C., 70 ° C. for 30 minutes, cooled to room temperature, Add 100 μl of 0.1 M Tris-HCl buffer (pH 8.0) containing 0.5 mM p-nitrophenyl-butyrate (pNP-butyrate) and measure the residual activity.

  The activity was determined by measuring the increase in absorbance at 410 nm at room temperature, and measuring the amount of p-nitrophnol (pNP) released to determine the esterase activity.

  The parent cutinase of the present invention is classified as [EC 3.1.1.74] according to the enzyme nomenclature. The parent cutinase may be derived from any organism, but for example, the fungus Aspergillus or the strain belonging to the genus Magnapolsa, Penicillium, Trichoderma, Rhizopus, Metalithium, Monascus, Acremonium, Mucor, preferably Aspergillus oryzae, Aspergillus niger, Aspergillus soyae, Aspergillus nidulans, or a strain belonging to Magnapolsa grisea, more preferably derived from Aspergillus oryzae strain RIB40.

Examples of the amino acid sequence of the parent cutinase derived from Aspergillus oryzae strain RIB40 include those shown in any of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

The cutinase shown in SEQ ID NO: 1 is an amino acid sequence of a mature cutinase derived from Aspergillus oryzae strain RIB40 (FERM P-18273), which is a preferred example of a bacterium belonging to the genus Aspergillus. The RIB40 strain is stored under the same number in the independent alcoholic beverage research institute: 3-7-1, Kagamiyama, Higashihiroshima City, and can be sold. It was deposited at the center (1-1, Higashi 1-chome, Tsukuba City, Ibaraki Prefecture) on March 28, 2001, and has been given a deposit number (FERM P-18273).

SEQ ID NO: 2 is the amino acid sequence of a cutinase mature product encoded by the gene described as the PBS-degrading enzyme analog gene “Homologue1” (SEQ ID NO: 4) in Table 8 of Patent Document 1. SEQ ID NO: 3 is the amino acid sequence of the mature cutinase encoded by the gene described as the PBS-degrading enzyme analog gene “Homologue2” (SEQ ID NO: 5) in Table 8 of Patent Document 1. In addition, the identity (homology) in the amino acid sequence between these cutinases is 54% between SEQ ID NO: 1 and SEQ ID NO: 2, and 78% between SEQ ID NO: 1 and SEQ ID NO: 3.

Alternatively, in the mutant of the present invention, the amino acid sequence of the parent cutinase has at least 50%, preferably 70%, more preferably 80% homology with the amino acid sequence of the cutinase of Aspergillus oryzae strain RIB40. is there. In particular, those capable of alignment with the Aspergillus oryzae strain RIB-40 cutinase are preferred.

  The cutinase mutant of the present invention includes a) a cutinase mutant in which an additional peptide consisting of 11 or less amino acids is bound to the amino terminus of the parent cutinase, and / or b) a cutinase of Aspergillus oryzae strain RIB-40. A variant of cutinase comprising a substitution or deletion of at least one amino acid residue corresponding to the following amino acid sequence numbers: L2, G4, L8, P27, G28, A51, G54, K90, or A177.

As an example of an additional peptide consisting of amino acids of 11 residues or less, a peptide having an amino acid sequence having ASDTPVDLQDT or STDPVDLQDR (amino acid single letter code) or a partial amino acid sequence thereof is preferable. Specific examples of amino acid residue substitution include L2K / R, G4D / E / S / T / V, L8D, P27D, G28D, A51D / E / N / Q / R / S / T, G54C / D / Mention may be made of N / Q / S, K90E, and T177A. In particular, substitution of G4D, G54S, or K90E is preferred. In addition to the above-described substitutions, one to several other amino acid substitutions, deletions, and / or as necessary, as long as the thermal stability of the cutinase mutant of the present invention is not substantially adversely affected. Alternatively, an insertion can be included.

  In the present invention, the nomenclature used to define the substitution of amino acid residues is identified by the amino acid before substitution and its position, and the amino acid after substitution. That is, “G4D” indicates that the 4th glycine has been substituted with asparagine, and the amino acid after substitution separated by a slash indicates that any of the amino acids has been substituted. That is, “G4D / S” indicates a mutation in which the fourth glycine is replaced with aspartic acid or serine.

  The cutinase mutant of the present invention has hydrolytic activity on various plastics such as polyester and polyamide, for example, biodegradable plastics, and can be used for enzymatic hydrolysis thereof. Here, “biodegradable plastic” means “a simple molecular level by maintaining the sufficient functions required for the purpose of use in the state of use, and by the action of microorganisms in soil or water when discarded. It is a substance that should be called “plastic that can be decomposed to the extent of” Based on the degree of degradation, it can be divided into “completely degradable biodegradable plastics” and “partially degradable (collapsed) biodegradable plastics”. It can be broadly divided into “natural polymer system” and “chemical synthesis system”. Any of these types of biodegradable plastics can be used in the method of the present invention.

  In particular, by treating biodegradable plastics comprising polylactic acid, polybutylene succinate, polybutylene succinate-co-adipate, polyhydroxybutyrate, and / or polycaprolactone with the cutinase variants of the present invention The biodegradable plastic can be efficiently decomposed.

  More specifically, by treating a biodegradable plastic comprising polylactic acid, polybutylene succinate, polybutylene succinate-co-adipate, polyhydroxybutyrate, and / or polycaprolactone with a cutinase variant, These polyesters can be hydrolyzed and solubilized constituent monomers, oligomers in which a plurality of them are polymerized, or salts thereof can be recovered. Here, the constituent monomer is synthesized by polycondensation, copolymerization, ring-opening polymerization or the like of polylactic acid, polybutylene succinate, polybutylene succinate-co-adipate, polyhydroxybutyrate, and / or polycaprolactone. It is a monomer used as a raw material in the process, and includes aliphatic carboxylic acid, aliphatic hydroxycarboxylic acid, aliphatic diol, aliphatic dicarboxylic acid, cyclic ester and the like.

  Furthermore, in all methods using the cutinase variant of the present invention, the plastic to be decomposed can take any form depending on the respective reaction system, such as an emulsified liquid and a solid pellet. Alternatively, it may be included as a constituent element of a so-called “composite material”. Here, the “composite material” is a material generally composed of two or more different substances, for example, a material composed of plastic, various metals, and other inorganic substances. Such composite materials are used for various purposes in various fields as various industrial materials.

  Therefore, by the above method, the plastic part is selectively decomposed from the composite material containing plastic, and the plastic is recovered as a monomer or oligomer, while other parts such as metal substantially free of plastic are removed. It can be recovered.

In the above method, the plastic degradation reaction using the cutinase mutant of the present invention may be carried out according to any reaction system known to those skilled in the art (for example, an aqueous system and a solid phase system) and reaction conditions depending on the purpose and scale. Can be selected and implemented. For example, examples of the solvent include water, a buffer solution, an organic solvent and water, or a mixture of an organic solvent and a buffer solution. A surfactant, an organic substance, an inorganic substance, or the like can be added to these solvents as necessary. By adding the cutinase mutant of the present invention to these decomposition reaction systems, the plastic can be decomposed. Furthermore, as disclosed in Patent Document 1, an appropriate biosurfactant known to those skilled in the art can be appropriately added to the reaction system, and the plastic can be decomposed in the presence of the biosurfactant. Those skilled in the art can appropriately select various conditions such as the amount and ratio of each substance added and the timing of addition.

  In order to stabilize the pH and improve the hydrolysis efficiency, it is preferable to add a buffer to the solvent. The pH of the solvent is preferably 5-12, more preferably 5-9, still more preferably 5-8.

  Alternatively, in the decomposition method described above, the plastic to be decomposed can coexist in the culture system of the transformant (cell) of the present invention, and the plastic can be decomposed by bringing the transformant into contact with the substance. This method can be performed in any culture system known to those skilled in the art, such as a liquid culture system containing a plastic emulsion and a solid culture system using a plastic solid pellet or plastic powder.

  In the above culture system, the solubilized constituent monomer obtained by decomposing the plastic or the substance itself such as an oligomer obtained by polymerizing a plurality of them is consumed as the sole nutrient source (carbon source / nitrogen source) of the transformant. Sometimes it is done. Alternatively, other carbon sources and nitrogen sources can be added separately to the culture system. For example, it contains various saccharides such as glucose as a carbon source, and an organic nitrogen source as a nitrogen source, such as peptone, meat extract, yeast extract, corn steep liquor, etc., inorganic nitrogen source such as ammonium sulfate, ammonium chloride, etc. Can do. Further, if desired, the medium may contain inorganic salts composed of cations such as sodium ion, potassium ion, calcium ion and magnesium ion and anions such as sulfate ion, chlorine ion and phosphate ion. . Furthermore, trace elements such as vitamins and nucleic acids can be contained.

  The culture conditions (medium, temperature, pH, etc.) of the transformant in the above culture system can be appropriately selected from conditions known to those skilled in the art according to the type of host. Further, any surfactant or biosurfactant known to those skilled in the art, such as disclosed in the above-mentioned International Publication, is allowed to coexist in the reaction system, for example, by adding it from the outside, It is also possible to further promote the decomposition of. These substances are not necessarily added at the same time, and can be added sequentially at each stage of the method.

  In addition, the cutinase variants of the present invention may be used in other known applications of lipases and cutinases, such as fibers containing polyester, fabric refining (WO 97/27237), bakery industry (WO 94/04035, and EP 585988). In the paper industry (EP 374700) and in the leather and wool industry, it can be used for removing grease and fat. Furthermore, it can also be used as a catalyst for organic synthesis.

  The cutinase mutant of the present invention can be produced by any method known in the art (for example, the method described in Patent Document 1). Hereinafter, a method for preparing (cloning) a DNA encoding the cutinase mutant of the present invention, a method for obtaining a cutinase mutant, and the like will be described.

Obtaining DNA sequence encoding cutinase As a representative example of DNA (gene) encoding parent cutinase, SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 2, encoding the amino acid sequence of cutinase derived from Aspergillus oryzae strain RIB40 A DNA having the base sequence shown in any of No. 3 can be mentioned. These DNAs can be easily isolated and cloned from any cell or microorganism that produces the cutinase using any method known in the art such as the method described in WO 2004/038016 A1, for example. I can do it. Alternatively, based on the information on the nucleotide sequence or amino acid production sequence of the DNA of the present invention described in the present specification, it should be prepared by chemical synthesis well known to those skilled in the art or by PCR using the primers of the present invention. You can also. Therefore, the DNA of the present invention can be any kind known to those skilled in the art, such as genomic DNA, cDNA, and synthetic DNA.

Construction of a cutinase mutant gene A cutinase mutant is a region-specific mutagenesis, region-specific amino acid insertion, site-specific mutagenesis, site-specific amino acid insertion, or localized random mutagenesis at a selected position, That is, it can be prepared by any method known to those skilled in the art, such as introducing amino acid substitutions randomly into selected positions or regions of the parent cutinase. For example, it can be performed using QuikChange II Site-Directed Mutagenesis Kits manufactured by Stratagene.

  Therefore, the DNA encoding the cutinase variant of the present invention thus prepared can hybridize under stringent conditions with a DNA comprising a base sequence complementary to the DNA encoding the parent cutinase.

  In the present specification, “under stringent conditions” means that the degree of homology between each base sequence is, for example, about 80% or more, preferably about 90% or more, more preferably about 95%, on the average on the whole. It means a condition in which a hybrid is specifically formed only between base sequences having high homology as described above. Specifically, for example, the conditions include a sodium concentration of 150 to 900 mM, preferably 600 to 900 mM, and pH 6 to 8 at a temperature of 60 to 68 ° C.

  Hybridization may be performed by a method known in the art, such as, for example, the method described in Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). It can carry out according to the method according to it. Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.

  The resulting DNA encoding the cutinase mutant can be inserted into an appropriate vector such as a plasmid, pET12b, pET15b, and pNGA142 to construct an expression vector.

  A gene encoding a cutinase mutant can be produced as a mature protein or a precursor protein (prepro form) using a so-called expression vector. The recombinant expression vector having a DNA sequence encoding the cutinase mutant of the present invention is any vector that can be handled by a recombinant DNA technique, such as a plasmid vector, a phage vector, and various hybrid vectors. . Various cells can be transformed with the expression vector thus obtained. This recombination DNA is any vector that can be handled by recombinant DNA techniques. These vectors can be appropriately selected depending on the host cell to be introduced. When the vector is introduced into a host cell, the whole or a part of the vector can be integrated into one or more places in the genome of the host cell. Examples of such vectors include pET-12Bb and pAUR101 for E. coli hosts.

  The expression vector of the present invention typically contains various regulatory sequences such as various promoters, ribosome binding sites, signal sequences, translation initiation sequences, and other exogenous or endogenous known to those skilled in the art. A gene encoding a protein, various drug resistance genes, a gene that complements auxotrophy, and the like can be optionally included.

  The promoter required to express the cutinase mutant of the present invention depends on the host cell obtained by transformation, but has transcription activity in the selected host cell and is homologous or heterologous to the host cell. It can be any DNA sequence that can be derived from the gene encoding the protein of interest.

  For example, examples of suitable promoters that regulate transcription of the gene encoding the cutinase mutant of the present invention hosted by a prokaryotic microorganism include the lac promoter of Escherichia coli Escherichia coli, the α-amylase gene (amyL) of Bacillus licheniformis Examples include promoters and promoters of the Bacillus amyloliquefaciens α-amylase gene (amyQ).

  Examples of promoters that can be used when a eukaryotic microorganism is used as a host include Saccharomyces cerevisiae galactosidase gene, Aspergillus oryzae takaamylase gene, enolase gene, xylanase gene, phosphoglucokinase gene, glucoamylase gene, Examples include promoters such as Rhizomucor Miehai aspartic protease gene, Aspergillus niger glucoamylase gene, Rhizomucor Miehai lipase gene.

  The obtained expression vector containing the DNA sequence encoding the cutinase mutant gene may be any known vector such as calcium chloride method, protoplast-PEG method, electroporation method, Ti plasmid method, particle gun method, baculovirus method, etc. This method can be introduced into a host cell to produce a transformant. Furthermore, the cotransfection method using a plurality of types of recombinant DNA is also possible.

  In the present invention, a prokaryotic microorganism, a eukaryotic microorganism, a plant cell, an insect cell, an avian cell containing an egg, a mammalian cell, or the like may be used as a host cell transformed with an expression vector containing a DNA sequence encoding a cutinase mutant. it can. For example, as an example of a prokaryotic microorganism, Escherichia, Bacillus, or Streptomyces genus Streptomyces griseus or Streptococcus sericolor can be used as a host. Examples of eukaryotes include yeasts such as Saccharomyces and Pichia, filamentous fungi such as Aspergillus, Penicillium, Rhizopus, Metalithium, Monascus, Acremonium, Mucor, and Basidiomycetes such as Trichoderma You can choose from. As insect cells, cells such as Drosophila melanogaster and silkworm can be used.

  The transformant according to the present invention may be transformed with one or more other DNAs for recombination containing a gene encoding any exogenous or endogenous protein in addition to the above vector. it can.

  Instead of the expression vector, it is also possible to obtain the transformed cell of the present invention using an appropriate DNA fragment itself containing a gene encoding a cutinase mutant obtained by PCR amplification or the like. In such a case, in addition to the DNA fragment, it can be used for transformation as a composition such as a solution optionally containing an appropriate buffer and other auxiliary agents.

Production of a cutinase mutant The present invention relates to a method relating to the production of a cutinase mutant in the present invention, and cultivates a transformed host cell containing a mutant gene under favorable conditions for production of the mutant, and expresses the mutant. Preferably, the method comprises secreting the expressed mutant extracellularly and recovering the mutant from the host cell and / or medium. The medium used for culturing the host cells can be any medium known to those skilled in the art for growing transformed host cells and expressing the cutinase variants of the invention.

  Cutinase variants secreted from host cells can be obtained by any suitable combination of means known to those skilled in the art, for example, separation of the medium and cells by centrifugation or filtration, and precipitation of the protein component of the medium by a salt such as ammonium sulfate, And subsequent use of hydrophobic chromatography, ion exchange chromatography, affinity chromatography, or other chromatography can be recovered from the medium.

  The present invention will be specifically described below with reference to examples, but the technical scope of the present invention is not limited to these descriptions. Various genetic manipulations in the examples were performed according to the methods described in Current Protocols in Molecular Biology (edited by Frederic M. Ausbel, et al., 1987).

Suddenly creating a cutinase mutant library To obtain a cDNA encoding cutinase, divide Aspergillus oryzae RIB-40 into a 100 ml solution in a 500 ml Erlenmeyer flask at 0.5 × 10 ⁶ cells / ml. Czapek-Dox medium (34 μg / ml chloramphenicol) (Nakajima, K., et al (2000) Curr) containing 1 (v / v)% PBS emulsion (Showa Polymer) as the sole carbon source Genet., 37, 322-327). After the addition, the cells were cultured at 30 ° C., 125 rpm for 5 days. The mycelium was filtered and collected with MIRACLOTH (CALBIOCHEM (registered trademark)). After measuring the wet weight of the obtained mycelium, it was ground in a mortar until it became powdery while pouring liquid nitrogen. The cells on the powder were transferred to a 50 ml tube containing 4 times the wet weight of Sepasol-RNA I Super, vigorously stirred, and then allowed to stand at room temperature for 5 minutes. After adding 1/5 volume of chloroform of Sepasol-RNA I Super and stirring well, the mixture was allowed to stand at room temperature for 3 minutes. After centrifuging at 10,000 xg, 4 ° C for 15 minutes, transfer the aqueous layer to a 15 ml tube, add the same amount of water-saturated acidic phenol / chloroform (phenol / chloroform = 1/1), mix, and then 12,000 xg, 4 The mixture was centrifuged at 10 ° C. for 10 minutes, and the supernatant was discarded and rinsed with 10 ml of 70% ethanol. After air drying, it was dissolved in an appropriate amount of DEPC (diethylpirocarbonate) -treated water to obtain a total RNA solution.

  Next, mRNA was purified from total RNA using Message Maker (Gibco BRL). The refining operation followed the manual attached to Message Maker. The procedure when the total RNA amount is 1 mg is shown below. Fill 1 mg of total RNA to 1.8 ml in a 15 ml tube with DEPC-treated water (final concentration 0.55 mg / ml), incubate at 65 degrees for 5 minutes, then rapidly cool on ice, 200 1 μl of oligo (dT) Cellulose Suspension was added after mixing well with 5 μl of 5 M NaCl, mixed well, and then incubated at 37 ° C. for 10 minutes. The sample was placed in a syringe and pushed down with a plunger, and 3 ml of Wash buffer 1 placed in a disposable cup was sucked with the syringe. The liquid in the syringe was well suspended, and the liquid was pushed out with a plunger. The same operation was performed with 3 ml of Wash buffer 2. Next, 1 ml of DEPC-treated water preheated to 65 degrees was sucked up with a syringe, suspended well, and then pushed out into a 15 ml tube. The same operation was performed again with DEPC-treated water, and the eluates obtained by the second operation were combined and centrifuged at 12,000 × g for 3 minutes at 4 ° C. to remove oligo (dT) Cellulose suspension. About 2 ml of this supernatant, add 20 μl of 5 mg / ml glycogen solution and 200 ml of 7.5 M ammonium acetate (pH 5.2), mix, dispense into 5 1.5 ml tubes and double the volume. Of ice-cold ethanol was added and left overnight at -20 ° C. After centrifugation at 12,000 × g and 4 ° for 30 minutes, the supernatant was discarded and rinsed with 75% ethanol. After air drying, it was dissolved in an appropriate amount of DEPC-treated water, and this was used as an mRNA solution.

Next, reverse transcription reaction was performed using the obtained mRNA as a template. In a 1.5 ml tube, add 5 μl of mRNA (92 ng / μl), 1 μl of oligo (dT) primer (0.5 μg / μl), 4 μl of dNTP mix (2.5 mM for each dNTP), and 2 μl of DEPC-treated water. In addition, after incubation at 70 ° C. for 10 minutes, the mixture was allowed to stand on ice for 1 minute or longer to anneal the oligo (dT) primer to the mRNA. Next, 4 μl of 5 × First Strand Buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl ₂ ) 2 ml of DTT was added to the reaction mixture, incubated at 42 degrees for 5 minutes, Add μl of reverse transcriptase SuperScript II RT (200 U / μl), stir by pipetting, incubate for 50 min at 42 ° C, add 1 ml of SuperScript II RT (200 U / μl), and stir by pipetting Then, it incubated at 42 degreeC for 50 minute (s), and reverse transcription reaction was performed. This solution was incubated at 70 ° C. for 15 minutes to inactivate reverse transcriptase. Further, 1 μl of RNaseH (10 U / μl) was added and incubated at 37 ° C. for 20 minutes to decompose unreacted mRNA. The solution obtained by this reverse transcription reaction was used as a cDNA library solution.

  Next, oligos based on the nucleotide sequence encoding the already known Aspergillus oryzae cutinase (Ohnishi, K., et al. (1995) FEMS Microbiol. Lett., 126 (2), 145-150). Nucleotides 5′-GCGTCGACGGATCCGGTGGACC-3 ′ (SEQ ID NO: 4) and 5′-GTTAGCAGCCGGATCCCGAAAATTTATCCCAGC-3 ′ (SEQ ID NO: 5) were synthesized. Using this primer set, PCR was performed using the cDNA library as a template to amplify cDNA encoding cutinase. The PCR apparatus used was PCR Thermal Cycler Personal (Takara Shuzo). The cDNA library used was 1 ml of the above cDNA library solution, and Ex Taq DNA polymerase (Takara Bio) was used as the DNA polymerase. Amplification reaction was performed by denaturing the template DNA at 95 ° C for 3 minutes, followed by 30 cycles of holding at 95 ° C for 1 minute, 60 ° C, 1 minute, 72 ° C for 1 minute, and then complete at 72 ° C for 12 minutes Stretched and held at 4 ° C. When the obtained PCR amplified fragment was subjected to agarose electrophoresis, amplification of a fragment consisting of about 626 base pairs was confirmed.

  The obtained PCR amplified fragment was digested with the restriction enzyme BamHI, subjected to agarose electrophoresis, stained with ethidium bromide, and then a DNA fragment consisting of about 608 base pairs was excised under UV irradiation. DNA was extracted from the gel using SUPREC-01 (Takara Bio) and used as an inserted DNA fragment. Next, 5 μg of pET12b plasmid DNA having the T7 promoter sequence and signal sequence (OmpT-leader sequence) in the base sequence is digested with BamHI (Takara Bio) at the position of the restriction enzyme BamHI recognition site immediately after the signal sequence. After performing phenol extraction and ethanol precipitation treatment, the phosphate group at the 5 ′ end was removed by alkaline phosphatase (Takara Bio). This reaction solution was subjected to phenol extraction and ethanol precipitation by a conventional method, and then dissolved in TE as vector DNA. Next, 1 μg of vector DNA and 1.5 μg of insert DNA were ligated with T4 DNA ligase (Takara Bio) to obtain ligated DNA.

Mix 2 μl of this ligated DNA solution with 10 μl of 10 × KCM (1 M KCl, 0.3 M CaCl ₂ , 0.5 M MgCl ₂ ), 7 μl of 30% (w / v) PEG # 6000, 81 μl of sterile water. 100 μl of transformable E. coli DH5α (Takara Bio) was added and allowed to stand in ice for 20 minutes and at room temperature for 10 minutes to obtain a transformed E. coli suspension. Next, the transformed Escherichia coli suspension was plated on an LB agar medium containing 50 μg / ml ampicillin and cultured at 37 ° C. for 16 hours. A single colony on the medium was picked up, transferred to an LB liquid medium containing 50 μg / ml ampicillin, and cultured at 37 ° C. for 16 hours. 1.5 ml of the culture solution was transferred to a 1.5 ml tube and centrifuged at 15,000 × g for 1 minute to precipitate the cells. After removing the medium, the cells are suspended in 100 μl of TGE (25 mM Tris-HCl buffer (pH 8.0), 10 mM EDTA, 50 mM glucose), and 200 μl of 1% (w / v) SDS is added thereto. 0.2 N NaOH containing was added and mixed gently, and left on ice for 5 minutes. Next, 150 μl of 3M sodium acetate pH 5.2 was added and mixed gently, centrifuged at 15,000 × g, 4 ° C. for 10 minutes, and the supernatant was transferred to a new 1.5 ml microtube. To this, 600 μl of 2-propanol was added and mixed well, followed by centrifugation at 15,000 × g, 4 ° C. for 10 minutes, and the supernatant was removed. The precipitate was washed with 70% (v / v) ethanol, centrifuged at 15,000 × g, 4 ° C. for 10 minutes, and the supernatant was completely removed to obtain a precipitate. The precipitate was dissolved in 0.1 ml of TE, and this was used as a plasmid DNA solution to obtain plasmid DNA for transformation of E. coli (pETCUTL1), which is one of the expression vectors of the present invention. The inserted cDNA fragment in this plasmid DNA and the base sequence including the T7 promoter sequence signal sequence were analyzed with the ABI PRISMTM 377 DNA sequencing system (PE BIOsystem) according to the protocol of ABI PRISMTM 377 DNA sequencer Long Read (PE Bioxystem) (sequence) Number 6).

  In this SEQ ID NO: 6, since the base sequence encoding the signal sequence is removed from the base sequence encoding cutinase derived from Aspergillus oryzae and inserted downstream of the OmpT signal sequence of pET12b, Aspergillus is translated into protein. OmpT signal sequence-addition peptide-mature cutinase sequence instead of the signal sequence encoded by the oryzae cutinase gene (SEQ ID NO: 6) Therefore, in this sequence, the amino terminal amino acid sequence of the mature enzyme after cleavage of the signal sequence is STDPVD.

  Using 5'-GATAAATTTCTCGAGCCGGCTGCTAAC-3 '(SEQ ID NO: 8) and 5'-GTTAGCAGCCGGCTCGAGAAAATTTATC-3' (SEQ ID NO: 9) using the plasmid DNA (pETCUTL1) having the gene encoding cutinase obtained by the method described above as a template Using the QuiageChangeTM Site-directed Mutagenesis kit manufactured by Staragene, a plasmid DNA was prepared by converting the BamHI cleavage site on the 3 'side of the cutinase gene into an XhoI cleavage site (pETCUTL1-X). 10 μg of an approximately 600 bp fragment excised from the newly prepared plasmid DNA (pETCUTL1-X) using the restriction enzymes BamHI and XhoI, 0.1 μM phosphate buffer containing 50 μl of 0.8 M hydroxylamine and 1 mM EDTA ( It was dissolved in pH 6.0) and incubated at 65 ° C. for 2 hours. After completion of the incubation, ethanol precipitation was performed by a conventional method and dissolved in 50 μl of TE to obtain a cutinase mutant gene library. The cutinase mutant gene library was prepared by subjecting plasmid DNA (pETCUTL1-X) cleaved with restriction enzymes BamHI and XhoI and treated with alkaline phosphatase to agarose electrophoresis, and collecting the collected vector DNA of about 4,657 bp with T4 DNA ligase (Takara). And ligated DNA was obtained. Mix 2 μl of this ligated DNA solution with 10 μl of 10 × KCM (1 M KCl, 0.3 M CaCl2, 0.5 M MgCl2), 7 Mμl of 30% (w / v) PEG # 6000, 81 μl of sterile water, Add μl of transformable Escherichia coli DH5α (Takara Bio), leave it in ice for 20 minutes and at room temperature for 10 minutes, add 1 ml of LB liquid medium, and incubate at 37 ° C for 1 hour. 100 μl of the solution was added to 3 ml of LB w / o NaCl liquid medium containing 50 μg / ml of ampicillin sodium and cultured with shaking at 37 ° C. for 16 hours. 1.5 ml of the culture solution was transferred to a 1.5 ml tube and centrifuged at 15,000 × g for 1 minute to precipitate the cells. After removing the medium, the cells are suspended in 100 μl of TGE (25 mM Tris-HCl buffer (pH 8.0), 10 mM EDTA, 50 mM glucose), and 200 μl of 1% (w / v) 0.2 N NaOH containing SDS was added, mixed gently, and left on ice for 5 minutes. Next, 150 μl of 3M sodium acetate pH 5.2 was added and mixed gently, centrifuged at 15,000 × g, 4 ° C. for 10 minutes, and the supernatant was transferred to a new 1.5 ml microtube. To this, 600 μl of 2-propanol was added and mixed well, followed by centrifugation at 15,000 × g, 4 ° C. for 10 minutes, and the supernatant was removed. The precipitate was washed with 70% (v / v) ethanol, centrifuged at 15,000 × g, 4 ° C. for 10 minutes, and the supernatant was completely removed to obtain a precipitate. The precipitate was dissolved in 0.1 ml of TE to obtain an amplified cutinase mutant gene library solution. Escherichia coli BL21-SI strain (Invitrogen) was transformed with this amplified cutinase mutant gene library to obtain a large number of single colonies on LB w / o NaCl agar medium.

Using a toothpick to select cutinase mutants, these single colonies were inoculated into LB w / o NaCl solid medium containing 50 μg / ml ampicillin sodium and 200 μl LB liquid medium containing 50 μg / ml ampicillin sodium. Each was cultured with shaking at 37 ° C. for 36 hours. After completion of the liquid culture, the culture supernatant was obtained by centrifugation at 3,000 × g for 15 minutes. To 10 μl of the obtained culture supernatant, add 100 μl 0.1 M Tris-HCl buffer pH 8.0, treat at 70 ° C. for 30 minutes, cool to room temperature, and then add 0.1 M Tris-HCl buffer containing 0.50 mM pNP-butyrate ( 100 μl of pH 8.0) was added and the remaining activity was measured. As a control, a culture supernatant of transformed Escherichia coli having a cutinase gene without mutation was used.

  As a result, a strain having a gene encoding a cutinase having heat resistance was obtained from about 9000 colonies by the above method. Table 1 shows the heat resistance of the enzymes in the culture supernatant of these strains.

DNA analysis LB w / o NaCl liquid medium containing 50 μg / ml ampicillin sodium according to a standard method to determine the nucleotide sequence of the gene encoding cutinase mutant AC obtained by the above method And incubate at 37 ° C. for 16 hours with shaking. After completion of the culture, plasmid DNA was recovered from the E. coli culture solution by the method described above. The base sequence of the recovered plasmid DNA was determined by the method described above. The gene encoding cutinase mutant AC contains DNAs of the base sequences shown in SEQ ID NOs: 10, 11, and 12, respectively. A G is mutated to A, and G4D is replaced by G, which is the 250th base, and A is replaced by A. G54S, A, which is the 358th base, is replaced by G. It was found that substitution of K90E occurred (these base sequence numbers depend on the base sequence of cutinase in the E. coli expression system shown in SEQ ID NO: 6).

Purification of cutinase mutant Colonies of E. coli transformants transformed with the plasmid DNA inserted with the above cutinase mutant gene were picked up with gauze, and 3 ml LB w / containing 50 μg / ml ampicillin sodium was collected. o Pre-cultured solution was obtained by culturing in NaCl liquid medium at 37 ° C for 16 hours. The obtained preculture was added to 1000 ml of LB liquid medium containing 50 μg / ml ampicillin sodium in a 5000 ml Erlenmeyer flask. After the addition, the mixture was cultured with shaking at 30 ° C. for 24-48 hours to induce and express the target enzyme protein in the culture supernatant. The culture solution was dispensed into a 500 ml centrifuge tube and centrifuged at 8,000 × g, 4 ° C. for 30 minutes to obtain a culture supernatant, which was used as a crude enzyme solution. Adsorbed protein was added to Octyl-Cellulofine (Seikagaku Corporation), which was equilibrated with 10 mM Trsi-HCl pH 8.0 containing 20% saturated ammonium sulfate. Was eluted with a linear gradient with 20-0% ammonium sulfate. The collected active fraction was sufficiently dialyzed against 10 mM Tris-HCl pH 8.0 and then subjected to DEAE-Toyopearl 650 M equilibrated with 10 mM Tris-HCl pH 8.0. Adsorbed protein was eluted with a 0-0.5 M NaCl linear gradient. The obtained active fraction was dialyzed again against 10 mM Tris-HCl pH 8.0, and then subjected to Hitrap Q (Amersham-Pharmacia) equilibrated with 10 mM Tris-HCl pH 8.0. Elute with a 0.5 M NaCl linear gradient.

Properties of Cutinase Variants The thermal stability of each cutinase variant obtained by the above method and the control cutinase without amino acid substitution was examined using the above measurement method. The obtained results are shown in FIG. As a result, it was confirmed that each cutinase mutant having an amino acid substitution has excellent thermal stability as compared with the control. Specifically, the residual activity measured after treatment at 40 ° C. for 30 minutes was increased by 1.1 times or more with respect to the control (parent cutinase). Further, the residual activity measured after treatment at 50 ° C. for 30 minutes was increased by 1.1 times or more with respect to the control (parent cutinase).

Construction of an expression system using the cutinase mutant Aspergillus oryzae as a host All of the above-mentioned mutant enzymes in Escherichia coli have 10 amino acid sequences: STDPVDLQDR added to the amino terminus of the enzyme native to Aspergillus. ing. Therefore, a cutinase mutant in which the above amino acid mutation was introduced into a native enzyme of Aspergillus oryzae that did not contain such an addition was prepared.

5'-CAATTGACTGATGGGGATGAGCTCCGGGATGGC-3 '(SEQ ID NO: 13) and Aspergillus oryzae expression plasmid DNA pNG-cut in an expression system using Aspergillus oryzae-derived cutinase described in Patent Document 1 as a host Primer set consisting of 5'-GCCATCCCGGAGCTCATCCCCATCAGTCAATTG-3 '(SEQ ID NO: 14) as a set, primer consisting of 5'-GGCGATGTTGCATGCAGAGTGTCGGACCC-3' (SEQ ID NO: 15) and 5'-GGGTCCGACACTCTGCATGCAACATCGCC-3 (SEQ ID NO: 16) A set of 5'-CAAGCTGTTTCCGAGTGTCCGGATACGCAGATCG-3 '(SEQ ID NO: 17) and 5'-GGGTCCGACACTCTGCATGCAACATCGCC-3 (SEQ ID NO: 18) is used as a set to make the above Escherichia coli It corresponds to the amino acid mutation position obtained from the mutation library prepared using G4D by introducing mutations into position, G54S, creates the K90E mutant was the Aspergillus expression plasmid with the cutinase variant gene with respective mutation each pNG-PcutG4D, pNG-PcutG54S, and pNG-PcutK90E.

In addition, in the E. coli expression system, the base sequence encoding the amino acid sequence excluding the signal sequence expected in Neisseria gonorrhoeae was connected downstream of the base sequence encoding the signal peptide of OmpT protein, so the SPVDL sequence that is an additional peptide Had been inserted. Even when Aspergillus oryzae was used as a host, 5'-CTGCAGTGGCAGCAGCAGCTAGCACGGATCCGGTGGACC-3 '(SEQ ID NO: 19) and 5'-GGTCCACCGGATCCGTGCTAGCTGCTGCTGCCACTGCAG-3' (SEQ ID NO: 20) were prepared to bind the additional peptide to the amino terminus. The ATD was added to the amino terminus of the cutinase expressed when used as a host, and the mutation of the ASTDPVDLQDT sequence in which Arg at the end of the added peptide was replaced with Thr was introduced using the QuikChangeTM Site-directed Mutagenesis kit from Staragene. A plasmid for gonococcal expression having a cutinase mutant gene having this mutation was designated as pNG-PcutNT.

The prepared plasmid DNA was confirmed for the base sequence by the conventional method in the manner described above, and used for transformation of Aspergillus oryzae. The nucleotide sequences of the cutinase mutants included in the plasmids for gonococcal expression of pNG-PcutG4D, pNG-PcutG54S, pNG-PcutK90E and pNG-PcutNT at this time are shown in SEQ ID NOs: 21, 22, 23, and 24, respectively. . For the transformation of Aspergillus oryzae, the method described in Patent Document 1 was followed. At the same time, Aspergillus oryzae transformed with pNG-Pcut without any mutation was used as a control.

  In addition, the high-expressing gonorrhea of the biodegradable plastic degrading enzyme cutinase gene transformed with plasmid DNA (pNG-cut) is the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1-1, Higashi 1-chome, Tsukuba, Ibaraki) ) On October 4, 2002, and is given the deposit number: FERM P-19054.

That is, a spore suspension of Aspergillus oryzae niaD300 strain was added to a YPD liquid medium and cultured with shaking at 30 ° C. for 12 hours. Mycelium was filtered and collected from the culture with MIRACLOTH (CALBIOCHEM (registered trademark)). The collected mycelium was transferred to a 50 ml centrifuge tube, then 10 ml protoplasting solution (0.6 M KCl, 5 mg / ml Lysing enzyme (Sigma Chemical), 10 mg / ml Cellulase Onozuka R-10 (Yakult) The suspension was added with 0.2 M phosphate buffer (pH 6.5) containing 10 mg / ml Yatalase (Takara Bio). The suspension was reacted with shaking at 30 ° C. and 90 rpm / min for 3 hours. After completion of the reaction, the mixture was filtered through MIRACLOTH, and centrifuged at 4 ° C., 5000 × g for 5 minutes to collect protoplasts. In a 50 ml plastic tube, add 0.1 ml of protoplast suspension prepared with 10 mM Tris-HCl buffer (pH 7.5) containing 1.2 M sorbitol and 50 mM CaCl2 to pH 1 × 10 ⁹ / ml with restriction enzyme MluI. 1 μl of cut plasmid DNA with 1 mg / ml cutinase mutant gene (pNG-PcutG4D, pNG-PcutG54S, pNG-PcutK90E, or pNG-PcutNT) and 12.5 μl of 50% (w / v) PEG A 10 mM Tris-HCl buffer solution (pH 7.5) containing # 4000 and 50 mM CaCl ₂ was added, and the mixture was left on ice for 30 minutes. Thereafter, 1 ml of 10 mM Tris-HCl buffer (pH 7.5) containing 50% (w / v) PEG # 4000 and 50 mM CaCl ₂ was added and mixed well. Further, 2 ml of 10 M Tris-HCl buffer (pH 7.5) containing 1.2 M sorbitol and 50 mM CaCl 2 was added and mixed well. 10 ml of Czapek-Dox soft agar medium preheated to 55 ° C. was added, quickly overlaid on the transformation agar medium, and statically cultured at 30 ° C. until conidia were formed.

  After conidia formation, scrape the conidia with ase, suspend in 0.01% (v / v) Tween80, dilute this suspension, spread on Czapek-Dox agar, and incubate at 30 ° C. Single spore separation was repeated. Single spore separation was confirmed by modifying the Hondl method (spore PCR method). A conidia was added to a 1.5 ml microtube containing 200 μl of YPD medium by ase and cultured at 30 ° C. for 40 hours. Transfer the cultured cells to a new 1.5 ml microtube and add 50 μl protoplasting solution (0.8 M KCl and 2.5 mg / ml Lysing enzyme (Sigma Chemical Co.), 10 mM containing 2.5 mg / ml Yatalase (Takara Bio). A citrate buffer solution (pH 6.5) was added and suspended, and the mixture was allowed to stand at 37 ° C. for 1 hour, and then left on ice for 5 minutes or more to precipitate the cells. 5 μl of this supernatant was used as a template DNA solution in the PCR reaction system. Two oligonucleotides (5'-TGCAGTGGCGGATCCGGTGGAC-3 'and 5'-GTAGAATCACGAATGGAGCCTTTGACGACC-3') were synthesized as primers for spore PCR. PCR Thermal Cycler PERSONL (Takara Shuzo) was used as the PCR device. As a positive control, the same procedure was performed using the plasmid DNA used for the transformation of Aspergillus oryzae as a template. The amplification reaction using Ex Taq polymerase (Takara Bio) is a cycle in which template DNA is denatured at 94 ° C for 3 minutes and kept at 94 ° C, 1 minute, 55 ° C, 1 minute, 72 ° C, 1 minute 30 seconds. After repeating 30 times, the film was fully extended at 72 ° C. for 95 seconds and then kept at 4 ° C. The obtained PCR amplified DNA fragment was subjected to agarose gel electrophoresis. As a result, a PCR-amplified DNA fragment appeared at the same size as the positive control, and a sequence linked to the cutinase mutant gene was inserted downstream of the glucoamylase promoter sequence on the chromosomal DNA, as well as the chimeric gene. Acquisition of an Aspergillus oryzae transformant was confirmed.

0.5 × 10 ⁶ spores / ml Transformed Aspergillus oryzae spores into YPM medium (1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v ) Maltose) and cultured with shaking at 30 ° C., 125 rpm for 16 hours. The culture was filtered through MIRACLOTH (CALBIOCHEM (registered trademark)), and the culture supernatant was collected as a filtrate. The supernatant fraction obtained by centrifuging this filtrate at 8000 × g and 4 ° C. for 20 minutes was used as a crude enzyme solution. Adsorbed protein was added to Octyl-cCellulofine (Seikagaku Corporation), which was equilibrated with 10 mM Trsi-HCl pH 8.0 containing 20% saturated ammonium sulfate. Was eluted with a linear gradient with 20-0% ammonium sulfate. The collected active fraction was sufficiently dialyzed against 10 mM Tris-HCl pH 8.0 and then subjected to DEAE-Toyopearl 650 M equilibrated with 10 mM Tris-HCl pH 8.0. Adsorbed protein was eluted with a 0-0.5 M NaCl linear gradient. The obtained active fraction was dialyzed against 10 mM MES buffer pH 5.5 and applied to a HiTrap SP column (Amersham Pharmacia Biotech) equilibrated with the same buffer solution. The adsorbed active fraction was 0-0.3 M NaCl. The purified enzyme was obtained by elution with a linear concentration gradient.

Amino terminal sequence of cutinase variant The amino terminal amino sequence of each cutinase variant obtained by the above method was determined. Specifically, 1 μg of the enzyme purified from the culture supernatant of the Aspergillus oryzae transformed strain transformed with pNG-PcutNT was subjected to SDS-polyacrylamide electrophoresis according to a conventional method, and then the gel was washed with distilled water. Transfer was performed on a PVDF membrane (Bio-Rad) equilibrated with 10 mM MES buffer (pH 11) containing 10% methanol using a Trans-Blot SD semi-dry transfer cell (Bio-Rad). The transcribed purified enzyme is stained with Komaji Brilliant Blue R-250, decolorized with 50% methanol solution, the stained band is cut out, and the amino acid sequence at the amino terminus is extracted by the Peptide Sequencing System 490 Procise (Applied Biosystem). It was determined. This revealed that the enzyme purified from the culture supernatant of Aspergillus oryzae transformed strain transformed with pNG-PcutNT has the ASPDV--sequence. On the other hand, 1 μg of the enzyme purified from the culture supernatant of Aspergillus oryzae transformed strain transformed with either pNG-PcutG4D, pNG-PcutG54S, or pNG-PcutK90E and 1 μg of the enzyme native to the koji mold as a control were After transferring to PVDF membrane, cut out the transferred portion of protein, wash the membrane with 60% methanol and 90% methanol, and incubate for 30 minutes at 37 ° C in 0.1 M acetate buffer containing 0.5% polyvinylpyrrolidone-K30. After that, the membrane was washed 10 times or more with distilled water. After washing, soak the membrane in methanol and add 5 ml Pfu pyroglutamate aminopeptidase (2 mU), 100 ml 0.1 M phosphate buffer (pH 7.0), 20 ml 0.1 M DTT, 75 ml distilled water, and treat at 50 ° C for 5 hours. did. After the treatment, the membrane was washed three times with distilled water, and the amino-terminal amino acid sequence was determined using a peptide sequencing system 490 Procise (Applied Biosystem). As a result, both enzymes had a QLTGG--sequence, indicating that glutamine at the first residue was pyroglutamylated. Thereby, it was shown that the cutinase mutant expressed using Aspergillus oryzae as a host has the amino acid sequences of SEQ ID NOs: 25, 26, 27, and 28. The thus confirmed cutinase variant shown in SEQ ID NO: 21 is G4D according to its amino acid sequence number, cutinase variant shown in SEQ ID NO: 22 is G54S, cutinase variant shown in SEQ ID NO: 23 is K90E, sequence Let the cutinase variant shown by the number 24 be a peptide addition cutinase.

Properties of Cutinase Mutants Expressed with Aspergillus oryzae as Hosts Using a cutinase mutant expressed with Aspergillus oryzae as a host and cutinase without amino acid substitution as a control, heat resistance was examined according to the activity measurement method. The obtained results are shown in FIG. As a result, it was confirmed that each cutinase mutant having an amino acid substitution has excellent thermal stability as compared with the control. Specifically, the residual activity measured after treatment at 40 ° C. for 30 minutes was increased by 1.1 times or more with respect to the control (parent cutinase). Further, the residual activity measured after treatment at 50 ° C. for 30 minutes was increased by 1.1 times or more with respect to the control (parent cutinase).

Degradation of PBSA using a cutinase mutant Using 0.1 M Tris-HCl buffer so that 5 μg / ml of cutinase mutant and cutinase native to the control Aspergillus oryzae were purified as described above. Prepared. PBSA emulsion (Bionore # 3001, Showa High Polymer) was diluted to 2% with the same buffer. 100 μl of the same buffer and 100 μl of diluted PBSA solution were added to 5 μl of the enzyme solution, and reacted at 50 ° C. for 6 hours. Thereafter, the decrease in turbidity was measured at 630 nm. As a result, the activity of the mutant enzyme was more than 10% higher than the native enzyme in the control Aspergillus oryzae, so this mutant can efficiently decompose PBSA at higher temperatures. Became clear.

PBSA hydrolysis method using Aspergillus oryzae having a cutinase mutant PBSA was decomposed using an Aspergillus oryzae transformed strain having a cutinase mutant gene transformed by the method described above.
That is, the spores of the cutinase mutant highly expressing strain were inoculated in a YPM medium (1% yeast extract, 2% peptone, 4% maltose) containing 2% PBSA emulsion so as to be 1 × 10 ⁶ cells / ml. After culturing at 30 ° C. for 24 hours, 1/10 volume of 1 M Tris buffer (pH 6.0) was added to the culture and incubated at 60 ° C. for 48 hours with stirring. At the same time, a high expression strain of cutinase native to Aspergillus oryzae was subjected to the same operation for comparison. The reaction solution was separated with miraculose, and the turbidity of the reaction solution was measured as absorbance at a wavelength of 630 nm to calculate the decomposition rate of PBSA. As a result, it was confirmed that the degradation rate increased by about 10% or more when digested with Aspergillus oryzae in which the cutinase mutant was highly expressed, compared to the strain in which the native enzyme was highly expressed.

  According to the present invention, the thermal stability of a cutinase capable of hydrolyzing an ester bond can be improved. Therefore, such cutinase mutants are useful in various industrial fields already described, particularly in various biological processes at high temperatures, medicine, cosmetics, food industry, recycling industry, and the like.

It is a graph which shows the thermostability of the mutant of the cutinase which has the heat resistance prepared in the colon_bacillus | E._coli expression system of this invention. Note that G14D, G64S, and K100E shown in FIG. 1 correspond to G4D, G54S, and K90E, which are mutants of the present invention, respectively. It is a graph which shows the more excellent thermal stability which the cutinase mutant prepared by the expression system which used Aspergillus oryzae of this invention as the host has.

Claims (9)

A cutinase variant comprising the amino acid sequence represented by any of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28 . The cutinase variant according to claim 1, which has a hydrolytic activity on a biodegradable plastic. The cutinase variant according to claim 2, wherein the biodegradable plastic is selected from the group consisting of polylactic acid, polybutylene succinate, polybutylene succinate-co-adipate, polyhydroxybutyrate, and polycaprolactone.   DNA encoding the cutinase variant according to claim 1. The DNA according to claim 4, comprising the base sequence represented by any of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24 . An expression vector comprising the DNA according to claim 4 or 5. A transformed cell comprising the DNA according to claim 4 or 5 or the vector according to claim 6. A method for producing the cutinase mutant according to any one of claims 1 to 3, wherein the transformed cell according to claim 7 is cultured, the mutant is expressed, and the mutant is recovered. Said method. The method according to claim 8, wherein the expressed cutinase mutant is secreted extracellularly and the mutant is recovered from the medium.

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