KR101059873B1 - Novel Peroxidase Variants with Increased Radical Stability - Google Patents

Abstract

The present invention relates to a peroxidase (CiP) protein variant derived from Coprinus cinereus , in which the amino acid at position 229 of the CiP protein represented by the amino acid sequence of SEQ ID NO: 1 is substituted. CiP protein variants according to the present invention selected from the group consisting of proteins represented by the amino acid sequence of SEQ ID NO: 2 to 4 has an increased radical stability than the existing CiP protein can be usefully used in the industry using peroxidase .

Peroxidase, mutation, radical stability

Description

Novel peroxidase variants having improved radical stability

The present invention relates to a peroxidase (CiP) protein variant derived from Coprinus cinereus , which has increased radical stability, and more particularly CiP protein variant having higher radical stability than conventional CiP protein, The present invention relates to a gene encoding the same, a recombinant vector including the gene, and a host cell including the recombinant vector.

Peroxidase is an enzyme that functions to oxidize a substrate in the presence of hydrogen peroxide and is widely known in all living organisms. It is a commercially important enzyme used in polymer synthesis and environmental pollutant treatment processes. In recent years, research has been conducted to utilize peroxidase as an enzyme catalyst for enzymatic polymerization to polymerize phenolic polymers.

Peroxidase is an oxidizing agent that, upon encountering hydrogen peroxide, forms two active oxygen complexes, which desorb the hydrogen atoms from the substrate, resulting in oxidation of the substrate. In the case of phenol, phenoxy radicals are produced, and polymer polymerization proceeds by coupling of these radicals to produce phenolic polymers. Such radical coupling reactions are used to remove phenolic or aromatic contaminants from wastewater or to produce industrially useful polymers.

However, peroxidase has a problem in that industrial utilization is limited because it is rapidly inactivated during the polymerization reaction. In other words, when hydrogen peroxide is present at high concentrations, the reducing substrate (eg phenol) generates a radical called compound III (eg, phenoxy radical), which causes the destruction of heme or protein by generating hydroxyl free radicals. Oxidation causes peroxidase to lose activity. Even in the presence of a reducing agent, inactivation of the peroxidase occurs due to the action of peroxidase and compound III radicals such as phenoxy radicals during the oxidation reaction.

In order to overcome this problem, there has been proposed a method of maintaining a low concentration of radicals by adding stepwise diluted hydrogen peroxide or by supplying a sufficient reducing substrate, but the low activity of phenoxidase has never been improved.

Accordingly, there is a demand for providing and securing a lipase suitable for a polymer polymerization system due to increased radical stability for a wide range of applications of phenoxidase.

The present invention has been made in view of the above requirements, and an object of the present invention is to provide a CiP protein variant with increased radical stability.

Another object of the present invention is to provide a gene encoding the CiP protein variant.

It is another object of the present invention to provide a recombinant vector comprising the gene.

It is another object of the present invention to provide a host cell comprising the recombinant vector.

In order to achieve the above object, the present invention is selected from the group consisting of a protein represented by the amino acid sequence of SEQ ID NO: 2 to 4, the amino acid at position 229 of the CiP protein represented by the amino acid sequence of SEQ ID NO: 1 CiP protein variants are provided.

In order to achieve the above another object, the present invention provides a gene encoding the CiP protein variant sequence.

In order to achieve the above another object, the present invention provides a recombinant vector comprising the gene.

In order to achieve the above another object, the present invention provides a host cell comprising the recombinant vector.

CiP protein variants with increased radical stability according to the present invention exhibit increased radical stability compared to native CiP proteins to maintain the activity of peroxidase and thus include phenolic polymers from the essential components of biosensors and immunoassays. From biological enzymes for the synthesis of fine compounds, it can be usefully used in a variety of industries, including analytical diagnostics, fine chemicals.

The present invention is a group consisting of a protein represented by the amino acid sequence of SEQ ID NO: 2 to 4 of the amino acid at position 229 of the Coprius cinereus-derived peroxidase (CiP) protein represented by the amino acid sequence of SEQ ID NO: 1 CiP protein variants, characterized in that selected from.

In addition, CiP protein variants of the invention include within the scope of the invention functional equivalents or functional derivatives having substantially equivalent functional activity. As an example of such a functional equivalent, amino acid residues in the amino acid sequences represented by SEQ ID NOs: 2 to 4 may include variants in which they are deleted, inserted, non-conservative or conservatively substituted or mutated by a combination thereof.

In some cases, CiP protein variants of the invention may have modifications that increase or decrease their physical and chemical properties such that phosphorylation, sulfation, acrylation, glycosylation, methylation ( It may be modified by methylation, farnesylation, acetylation, amylation, and the like, so long as the activity of CiP protein variants increased by this modification is substantially maintained. Derivatives are also included within the scope of the present invention.

The CiP protein variant represented by the amino acid sequence of SEQ ID NO: 2 is a substitution of alanine for phenylalanine at position 229 of the native CiP protein represented by the amino acid sequence of SEQ ID NO: 1, about 70-fold increase compared to the native CiP protein. Have enzymatic activity (see Table 1).

CiP protein variant represented by the amino acid sequence of SEQ ID NO: 3 is a phenylalanine at position 229 of the native CiP protein represented by the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine, about 30 times compared to the native CiP protein Have increased enzymatic activity (see Table 1).

The CiP protein variant represented by the amino acid sequence of SEQ ID NO: 4 is a phenylalanine at position 229 of the native CiP protein represented by the amino acid sequence of SEQ ID NO: 1 substituted with leucine, about 30-fold increase compared to the native CiP protein. Have enzymatic activity (see Table 1).

The present invention also provides a gene encoding the CiP protein variant.

The gene encoding the CiP protein variant does not change the amino acid sequence of the CiP protein variant due to the degeneracy of the codon or in consideration of the preferred codon in the organism to express the CiP protein variant. Various modifications may be made to the coding region within, and various modifications or modifications may be made within a range that does not affect the expression of the protein in portions other than the coding region, and such modified genes are also included in the scope of the present invention. Gene encoding the CiP protein variant in the present invention is preferably a gene represented by the nucleotide sequence of SEQ ID NO: 11 to 13. The gene encoding the CiP protein variant of the present invention may be provided by a vector expressing it.

The present invention provides a recombinant expression vector comprising a gene encoding the CiP protein variant.

In the present invention, "vector" refers to a means for expressing CiP protein variants by introducing DNA encoding CiP protein variants into a host cell, and includes all conventional ones, including plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Vector, and is preferably a plasmid vector.

Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, initiation codons, termination codons, polyadenylation signals and enhancers, and can be prepared in various ways depending on the purpose. For example, when the host is yeast, AOX1 promoter, PHO5 promoter, PGK promoter, GAP promoter and ADH promoter can be used. The start codon and the stop codon must be functional in the individual when the gene construct is administered and must be in frame with the coding sequence. In addition, the expression vector comprises a selectable marker for selecting a host cell containing the vector and, in the case of a replicable expression vector, a replication origin. Vectors can either autonomously replicate or integrate into host cell DNA.

Specifically, the recombinant expression vector according to the present invention may be prepared by inserting a gene encoding the CiP protein variant sequence into a pPICZαA vector comprising an AOX1 promoter.

The present invention provides a host cell transformed with the recombinant expression vector to produce CiP protein variants.

Since expression amounts and formulas of proteins vary depending on the host cell, the host cell that is most suitable for the purpose is selected and used. Host cells include Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis or Staphylococcus. Prokaryotic host cells such as, but are not limited to. In addition, fungi (e.g. Aspergillus), yeast (e.g. Pichia pastoris, Saccharomyces cerevisiae, Szozocaromyces ( Eukaryotic cells such as Schizosaccharomyces, Neurospora crassa, etc. can be used as host cells, among which Pichia Pastoris is preferred.

In order to transform the host cell with the recombinant expression vector according to the present invention, a method known in the art may be used, and such a method may include electroporation, plasma fusion, and calcium phosphate (CaPO ₄ ) precipitation. And calcium chloride (CaCl ₂ ) precipitation, and the like.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

<Reagents and Samples>

ABTS (2,2'-azinobis (ethylbenzthiazoline-6-sulfonate)), H ₂ O ₂ and trypsin, which are substrates for measuring peroxidase activity, were purchased from Sigma (USA), Tag DNA polymer. Aze was purchased from Roche Applied Science (USA). Pichia pastoris strain X-33 (natural) and plasmid pPICZαA were purchased from Invitrogen (USA) and the yeast transformation kit (MicroPulser ^™ ) was purchased from Bio-Rad (USA). Water and ethanol used for high performance liquid chromatography (HPLC) were provided by Burdick & Jackson Lab., USA. Microbial culture media such as yeast extract, peptone and tryptone were purchased from Becton Dichinson Co .; USA.

Example 1: Preparation of pPICZαA-rCiP

This amino acid sequence is based on the amino acid sequence of the peroxidase (CiP; GenBank Accession No. X70789) of Coprinus cinereus , which is published in GenBank, a gene sequence database of the National Institutes of Health (NCBI). RCiP was synthesized by considering the codon usage of Pichia Pastoris (GenScript, USA). The structural regions of the rCiP gene were amplified using the primers SEQ ID NOs: 19 and 20 by PCR. The PCR was performed using 100 μL of reaction solution (100 ng rCiP synthetic gene, 1 μL SEQ ID NO: 19 and primers of SEQ ID NO: 20, 50 pmol), 12 μL magnesium chloride (25 mM), 10 μL reaction buffer (10 ×), 2.5 μL Deoxynucleotide (2.5 mM), 0.5 μL of Tag DNA polymerase (5 units / μL) and water were reacted for 5 minutes at 95 ° C. using a My cycler ^™ (BioRad, USA), 95 The reaction was carried out for 30 seconds at 30 ° C., 30 seconds at 54 ° C., and 1 minute at 72 ° C. for 30 cycles, and then terminated at 70 ° C. for 10 minutes. PCR 2.1-TOPOp-rCiP was prepared by ligation of the PCR product with the T-vector pCR 2.1-TOPO vector (Invitrogen, USA), followed by sequencing (solgent, Korea) to mutated rCiP sequences. It was confirmed that none. After treatment with the restriction enzymes EcoRI and NotI to each of the pCR 2.1-TOPOp-rCiP and PICZaA vectors (Invitrogen, USA), the desired DNA fragments were purified using a gel extraction kit (Qiagen, Germany). . Then, the cleaved pPICZaA and rCiP DNA fragments were ligated using ligase (TAKARA, Japan).

Example 2 Selection of Amino Acids to Cause Mutation

As mentioned above, the action of phenoxy radicals and peroxidase can lead to inactivation of CiP. Thus, in order to investigate the modification of CiP by phenoxy radicals or phenol substrates during the phenol oxidation reaction, CiP separated after phenol oxidation reaction was analyzed by mass spectrometry. As described below, proteolytic digestion of the isolated CiP and the original CiP using trypsin was followed by LC MS / MS analysis.

18 mM phenol was added to 25 ml of phosphate buffer (0.1 M, pH 7.0) to prepare a reaction mixture. CiP was added to a final concentration of 25 U / mL and 18 mM hydrogen peroxide was added to initiate the reaction. The reaction mixture was incubated with gentle stirring for 40 minutes at room temperature and protein samples were precipitated by rapid addition of four times cold acetone. The mixture was incubated overnight at -20 ° C and centrifuged at 13,000 g, 4 ° C for 20 minutes. The supernatant was removed and the precipitate was washed by adding 1 mL of cold acetone. The precipitate sample was incubated on ice for 15 minutes and then centrifuged at 13,000 g, 4 ° C. for 20 minutes. The supernatant containing acetone was removed and the precipitate was lyophilized. The precipitate obtained from the acetone precipitation was analyzed by 10% SDS-PAGE (sodium dodecyl sulfate-polyacryl amide gel). Gels were stained with Colloidal Coomassie Brilliant Blue G-250 (CBB; Sigma, USA) for 30 minutes and the remaining staining was washed with 10% methanol containing 8% acetic acid. Appropriate protein bands containing CiP (46 kDa) were cut out and trypsin digested. Each gel slice in the microtube was destained 3 or 4 times for 10 minutes each until 200% of the gel slice was cleaned with 200 μL of a 1: 1 mixture of 25 mM ammonium bicarbonate (NH ₄ HCO ₃ , pH 7.8) and acetonitrile. Speed back (SpeedVac ^™ ; Savant Instruments, USA). The dried gel pieces were incubated with 100 mL of 10 mM dithiothreitol / 100 mM ammonium bicarbonate solution at 56 ° C. for 60 minutes. After removing the sample solution, 100 μL of a 55 mM iodoacetamide / 100 mM ammonium bicarbonate solution prepared immediately was added and incubated for 45 minutes at room temperature while blocking light. Gel slices were washed with 100 mM ammonium bicarbonate solution with acetonitrile and dried completely in a vacuum concentrator. Destained gel slices were incubated overnight at 37 ° C. with trypsin (0.02 μg / μl, w / v) dissolved in 25 mM ammonium bicarbonate solution and 30 μL of acetonitrile and 5% formic acid (50:50, v / v) Extracted twice. Acidified peptides were decontaminated using a microcolumn made of GelLoader tip (Eppendorf, Germany) and packed with 0.1 μL of POROS R2 resin (Perseptive Biosystems, USA). The loaded peptide was washed with 5% formic acid, diluted with 1 μL of deionized water, 50% acetonitrile in 0.15 formic acid, dried in a vacuum centrifuge and dissolved in 4 μL of 0.1% formic acid.

Trypsinized peptides were isolated and analyzed with an HP 1100 HPLC nano-flow system using a splitter (Agilent) connected on-line to LCQ DECA ion traps (Thermo Fisher Scientific, Calif.). Zorbax 300SB-C18 resin (particle size 5 μm; Agilent Technologies, USA) was packed in a home-built fused silica column (100 mm length × 75 μm I.D., tip diameter 10 μm). The bound peptide was diluted with a 50 minute gradient of 5 to 90% (v / v) of acetonitrile containing 0.1% (v / v) formic acid at a flow rate of 0.2 μL / min. A injection voltage of 1.7 kV was applied at the ESI source and the transfer capillary temperature was set to 180 ° C. MS scan in cation mode was controlled by Xcalibur 1.2 software. Precursor ions were selected in the range of m / z 350 to 2,000 for MS / MS fragmentation within a ± 3 m / z window for continuous MS / MS scan in data-dependent mode. Exclusion dynamic mode was applied to exclude the most concentrated ions selected from further selection over 2 minutes. MS / MS data were obtained using a 2 m / z unit ion isolation window in an automated acquisition control (AGC) mode (AGC values of 5.00e + 04 and 1.00e + 04 were set for total MS and MS / MS). . The standardized CID was set at 35.0.

The peak list of MS / MS spectra was exported as each file in data format using Bioworks 3.3 software (Thermo Fisher Scientific, USA) under the following settings: peptide mass range, 500 to 3500 Da; Minimum total ionic strength threshold, 10000; Minimum number of fragment ions, 20; Precursor mass tolerance, 1.4 amu; Group scan, 1; Minimum number of groups, 1. NCBI non-redundant database (2009.7.) Or certain variables (trypsin as the enzyme with one) by limiting the single text file generated by the automated combination of data files created from each analysis to the species. potential missed cleavage; monoisotope mass selected, a 2-2.5 Da peptide mass tolerance and a 1-Da MS / MS toloerance; carbamidomethylation of cysteines, oxidation of methionine, deamidation of asparagine / glutamine (formally 'deamidation' May have deisotoping artefacts), 1 to 4 phenylene (s) modification of phenylalanine as variable modifications; singly, doubly, or triply charged state; Mascot) was investigated in the peroxidase protein database. Only significant hits by the Massscott probability analysis (p <0.05) were considered.

MS / MS spectra of the fragment (m / z 838.880) and sequence information via fresh sequencing are shown in FIGS. 2A and 2B. The fragment sequence of inactivated CiP (m / z 838.880) was KGTTQPGPSLG F AEELSPFPGEFRM (residues 219-241) and was modified by the phenol molecule, whereas the original CiP fragment (m / z 1211.621) was not. The peptide modified with phenol was KGTTQPGPSLG F AEELSPFPGEFRM, with only one underlined phenylalanine residue (F229) covalently linked with one phenol or phenol oligotrimer. As a result, it was found that the site for inactivating CiP by covalently bonding with a phenol radical was a phenylalanine residue at the 229 position.

Example 3: Preparation of CiP Protein Variant F229A

CiP protein variant F229A was prepared by replacing phenylalanine at position 229 of CiP with alanine by a site-directed mutagenesis method using specific primers.

<3-1> Variation of position 229 of CiP

First, the DNA corresponding to the front of the CiP gene was amplified by PCR using a 5 'flanking primer (SEQ ID NO: 21) and a mutagenic primer (SEQ ID NO: 24) having a cleavage site of restriction enzyme EcoRI. The DNA corresponding to the back was amplified by PCR using a 3'- flanking primer (SEQ ID NO: 22) and a mutagenesis primer (SEQ ID NO: 23) having a cleavage site of the enzyme NotI. The PCR was performed using 100 μL of reaction solution (100 ng pPICZαA-rCiP, 1 μL mutagenic primer (SEQ ID NO: 23 or SEQ ID NO: 24; 50 pmol), 1 μL 5'- or 3'- flanking primer (SEQ ID NO: 21 or SEQ ID NO: 22, 50 pmol), 12 μL magnesium chloride (25 mM), 10 μL reaction buffer (10 ×), 2.5 μL deoxynucleotide (2.5 mM), 0.5 μL of Tag DNA polymerase (5 units / μL) and Water) was reacted for 5 minutes at 95 ° C using My cycler ^™ (BioRad, USA), 30 seconds at 95 ° C, 30 seconds at 54 ° C, and 1 minute at 72 ° C as 30 cycles. After cycling reaction, it terminated by making it react at 70 degreeC for 10 minutes. Both PCR products were purified using a PCR purification kit (Qiagen).

By PCR using the two amplified DNA fragments, the primer of SEQ ID NO: 21, and the primer of SEQ ID NO: 22 (adding the same amount of the two PCR products instead of pPICZαA-rCiP and using 1 μL of two flanking primers (50 μL) Except that the reaction conditions are the same as those of PCR).

<3-2> Plasmid Expression Expressing CiP Protein Variant F229A

The gene of this CiP protein variant was cloned into pCR 2.1-TOPO vector (Invitrogen, USA), and then sequenced (solgent, Korea) to sequence all PCR products and variant genes (Solgent, Korea) to confirm that the mutation occurred. It was. After treatment with the restriction enzymes EcoRI and NotI for each of the CiP variant genes and PICZaA vectors (Invitrogen, USA) cloned into pCR 2.1-TOPOp, the desired DNA fragments were purified using a gel extraction kit (Qiagen, Germany). Thereafter, the cleaved pPICZaA and CiP DNA fragments were ligated using ligase (TAKARA, Japan).

<3-3> Transformation of Pichia Pastoris

Pichia pastoris strain X-33 (Invitrogen, USA) was transfected with the CiP protein variant gene cloned in pPICZαA by electroshock gene transfer and YPDS medium containing 100 μg / mL Zeocin (1). Selection was made by incubating in% yeast extract, 2% peptone, 2% glucose and 1M sorbitol.

<3-4> Purification of CiP Protein Variant F229A

The selected positive clones were subjected to BMMY medium containing 100 μg / mL zeocin (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB (yeast nitrogen base), 400 mg / l biotin) biotin), 0.5% methanol) was incubated for 5 days at 30 rpm at 220 rpm, sterile methanol (0.5%) was added to the medium every 24 hours. The highest activity culture was centrifuged at 3880 g for 20 minutes, and the supernatant was concentrated by ultrafiltration (ultrafiltration; 10 kDa MWCo; Amicon) while desalting with 0.1 M Tris-HCl buffer (pH 7.0). Finally purified using a size exclusion column (SESE) column (Superose 6 10/300 GL; GE Healthcare, USA) using 0.1 M Tris-HCl buffer (pH 7.0) as eluent. Fractions showing the highest ABTS activity were combined and again concentrated by ultrafiltration (10 kDa MWCo; Amicon). Total protein concentration was determined using the Bicinchoninic acid (BCA) method (Pierce, USA).

Example 4: Preparation of CiP Protein Variant F229I

CiP protein variant F229I was prepared by substituting isoleucine for phenylalanine at position 229 in CiP, except that the primers of SEQ ID NO: 25 and 26 were used instead of SEQ ID NOs: 23 and 24, respectively. .

Example 5: Preparation of CiP Protein Variant F229L

CiP protein variant F229L was prepared by substituting leucine for phenylalanine at position 229 of CiP in the same manner as in Example 3 except that the primers of SEQ ID NOs: 27 and 28 were used instead of SEQ ID NOs: 23 and 24, respectively.

Comparative Example 1: Purification of Natural CiP Protein

pPICZαA-rCiP was purified by the method described in <3-3> and <3-4> of Example 3.

Comparative Example 2: Preparation of CiP Protein Variants F229G, F229V, F229H, F229Y, and F229W

CiP protein variant F229G was prepared by replacing glycine with phenylalanine at position 229 of CiP in the same manner as in Example 3, except that the primers of SEQ ID NOs: 29 and 30 were used instead of SEQ ID NOs: 23 and 24, respectively.

CiP protein variant F229V was prepared by substituting valine for phenylalanine at position 229 of CiP in the same manner as in Example 3 except for using the primers SEQ ID NOs: 31 and 32 instead of SEQ ID NOs: 23 and 24, respectively.

CiP protein variant F229H was prepared by substituting histidine for phenylalanine at position 229 in CiP, except that the primers of SEQ ID NOs: 33 and 34 were used instead of SEQ ID NOs: 23 and 24, respectively.

CiP protein variant F229Y was prepared by substituting tyrosine for phenylalanine at position 229 of CiP, except that the primers of SEQ ID NOs: 35 and 36 were used instead of SEQ ID NOs: 23 and 24, respectively.

CiP protein variant F229W was prepared by substituting tryptophan for phenylalanine at position 229 of CiP in the same manner as in Example 3, except that the primers of SEQ ID NOs: 37 and 38 were used instead of SEQ ID NOs: 23 and 24, respectively.

Experimental Example 1 Radical Stability of Natural CiP Protein and CiP Protein Variants

The CiP protein was stirred in phosphate buffer (100 mM, pH 6.0) containing 0.5 mM phenol and 0.5 mM hydrogen peroxide for 20 minutes at 25 ° C. to initiate the phenol oxidation reaction. Samples were taken every 5 minutes after initiating the phenol oxidation reaction and centrifuged at 4,000 rpm for 15 minutes to remove polymerized precipitate. The supernatant was collected and analyzed for concentration of phenol using liquid chromatography (Agilent model 1200 liqhid chromatography; USA). Analytical conditions indicated that the column maintained Zorbax XDB-C ₁₈ (150 × 0.3 mm, 3.5 μm; Agilent, USA) at 25 ° C., the mobile phase was 0.3% acetic acid (70%) and methanol (30%), flow rate Was measured at 280 nm using a diode-array detector at 1.0 ml / min. Phenol concentrations were quantified using a calibration curve. Turnover capacity is the ratio of phenol consumed per enzyme consumed for 20 minutes after the start of the reaction.

Peroxidase activity was measured using a method of oxidizing ABTS and measuring the amount of color development at 420 nm using an absorption spectrometer. 20 μL of the sample was prepared using 2 mL of ABTS-H ₂ O ₂ (0.18 mM ABTS and 2.9 mM). H ₂ O ₂ , pH 5.0) and a quartz cuvette were mixed and absorbance change at 420 nm was measured at 25 ° C. using a UV-Vis spectrometer (Shimadzu; Japan) (molar extinction coefficient of ABTS). : 34,799 M ⁻¹ cm ⁻¹ ).

Table 1 shows changes in phenol concentrations over time for the native CiP protein, and the CiP protein variants prepared through Examples 3 to 5 and Comparative Example 2. And shown in Figure 3a, the enzyme activity is shown in Table 1 and Figure 3b. In addition, the turnover capacity is shown in Table 2.

On the other hand, in order to explain that the amino acid substitution at position 229 affects the stability of the CiP protein against the phenol radical and the turnover capacity of the CiP protein, comparing the highest Occupied Molecular Orbital (HOMO) energy level of each amino acid, Table 3 shows.

HOMO energy levels of amino acids were calculated by molecular dynamics, Hyperchem 8.0.3. Semi-empirical calculations using molecular dynamics were performed to perform geometric optimization of amino acids using MM + force fields. After completing the geometric optimization of the amino acids, single point out calculations were performed, with semi-experimental calculations performed using Parametric Method No. 3 (PM3) based on the Restricted Hartree-Fock (RHF) method. HOMO energy of amino acids was obtained.

       Phenol Concentration and Enzyme Activity with Time after Oxidation SEQ ID NO:
Phenol Concentration [μM] Enzyme Activity [U / ml] Early final Spent phenol [%] Early final residual
activation[%]  1 (natural) 0.51 0.41 20 0.9 0.01 One 2 (F229A) 0.54 0.07 87 1.12 0.80 71 3 (F229I) 0.48 0.08 83 1.13 0.34 30 4 (F229L) 0.5 0.08 84 1.08 0.32 30 7 (F229H) 0.5 0.19 62 0.99 0.02 2 9 (F229W) 0.51 0.21 59 1.31 0.11 8

Turnover Capacity (Growth Rate) SEQ ID NO: Turnover Capacity [mM / U] (Growth Rate) 1 (natural) 0.11 (1.0) 2 (F229A) 1.47 (13.1) 3 (F229I) 0.51 (4.5) 4 (F229L) 0.55 (4.9) 7 (F229H) 0.32 (2.8) 9 (F229W) 0.25 (2.2)

HOMO energy levels of amino acids SEQ ID NO: E _HOMO (eV) 1 (natural) -9.63 2 (F229A) -10.32 3 (F229I) -10.24 4 (F229L) -10.28 7 (F229H) -9.2 9 (F229W) -8.76

As shown in Table 1, CiP protein variants of SEQ ID NOS: 2 to 4 according to the present invention are about 90% of the initial phenol is oxidized to form phenolic polymers, whereas CiP protein variants of SEQ ID NOs: 7 and 9 are about 50 %, Only about 20% of the native CiP protein of SEQ ID NO: 1 was oxidized. In addition, the CiP protein variants of SEQ ID NOS: 2-4 have about 70-fold, 30-fold, and 30-fold increased enzymatic activity, respectively, compared to the native CiP protein of SEQ ID NO: 1. In particular, the native CiP protein and CiP protein variants of SEQ ID NOs: 7 and 9 lost activity within 10 minutes, while the CiP protein variants of SEQ ID NO: 2 maintained 80% of their initial activity 20 minutes after the start of the reaction. On the other hand, CiP protein variants of SEQ ID NOs: 5, 6 and 8 showed no peroxidase activity when tested by ABTS as substrate.

In addition, as shown in Table 2, the CiP protein variants of SEQ ID NOS: 2 to 4 have increased turnover doses of about 14, 5, and 5 times, respectively, compared to the native CiP protein of SEQ ID NO: 1.

On the other hand, since the energy of HOMO is related to the oxidation potential, the higher the HOMO energy value, the more susceptible to the attack of oxidation and radicals. As shown in Table 3, it can be seen that HOMO energy levels of alanine, isoleucine and leucine are lower than the values of phenylalanine, histidine, tyrosine and tryptophan. The stability of native CiP protein and CiP protein variants correlated with HOMO energy levels. This supports that the factor that inactivates CiP during the oxidation reaction is the attack and binding of phenoxy radicals to the 229 position of CiP.

Experimental Example 2: Kinetics of Peroxidase

Catalyst for various concentrations of ABTS by reacting CiP protein with ABTS at various concentrations (0, 10, 20, 30, 40, 50, 60, 75 and 100 μM) and measuring absorbance at 420 nm By measuring the reaction rate, kinetic parameters were determined.

The reaction was initiated by adding 0.05 μM of CiP protein to the reaction mixture of 100 μM of H ₂ O ₂ and ABTS ranging from 0 to 100 μM. All the samples were dissolved in 50 mM phosphate-citrate buffer (pH 5.0). Michaelis-Menten of CiP protein variants, and CiP protein variants prepared through Examples 3 to 5 and Comparative Example 2 above, from a Michaelis-Menten equation based on a Hanes-Woolf plot graphically plotting the reaction rate against ABTS concentration Constant (K _m ), maximum turnover number (k _cat ) and catalytic efficiency (k _cat / K _m ) were derived (see Table 4).

Hanes-Woolf plots for the native CiP protein, and CiP protein variants prepared through Examples 3 to 5 and Comparative Example 2 are shown in FIG. 4.

Kinetic Properties SEQ ID NO: K _m [μM] k _cat [S ^-1 ] k _cat / K _m [μM · S ⁻¹ ] 1 (natural) 7.700 3.918 0.509 2 (F229A) 129.194 87.146 0.675 3 (F229I) 46.857 31.740 0.677 4 (F229L) 38.351 23.600 0.615 7 (F229H) 37.380 13.080 0.350 9 (F229W) 29.323 12.080 0.412

As shown in Table 4, the substrate binding affinity of the CiP protein variant of SEQ ID NO: 2 was decreased (K _m = 129.19 μM), K _cat value was increased to show high K _cat / K _m value.

Experimental Example 3: Molecular Docking Simulation

Molecular docking simulations of the CiP protein variants of SEQ ID NOS: 2, 3 and 9 and phenolic molecules were performed using Discovery Studio version 2.1 (Accelrys, USA). As the structure of CiP, the structure of ARP ( Arthromyces ramosus peroxidase; PDB ID: 1ARP) was used.

The structure of the CiP protein was modeled with the SCRWL 3.0 program based on a combination of rotamer libraries, and the structure of the phenol was made with Discovery Studio's Fragment Builder tools. CHARMm force fields were applied to the molecules and molecular docking simulations were performed using the CDOCKER module.

The phenol was docked to the CiP protein and produced a possible CiP protein-phenol complex. Residues within 8 μs of the CiP protein were used as binding sites and other residues were fixed. Molecular docking simulations were performed with default parameters of the CDOCKER module. Ten low docking scores were used to calculate the binding free energy of the complex. The binding free energy of the complex was calculated according to Discovery Studio's protocol. The complex of CiP protein and phenol with the lowest binding free energy was used for structural analysis. The binding distance of CiP protein was determined by calculating the distance between the δN atom of 56His and the active OH group using the modeled structure of the complex. The binding free energy and binding distance of the modeled composites are shown in Table 5.

Binding free energy and distance of the modeled composite SEQ ID NO: Binding free energy [kcal / mol] Distance [Å] 1 (natural) -15.1 11.3 2 (F229A) -7.6 13.2 3 (F229I) -10.3 14.1 9 (F229W) -12.0 9.6

Molecular docking simulations showed that the F229 residue of CiP was located at the inlet of the heme pocket of CiP, resulting in hydrophobic interaction with the aromatic ring of the substrate. This is an important property for the bonding of the substrate to the aromatic ring. In the modeled complex, the F229 residue of CiP had an aromatic-aromatic interaction with phenol. The phenol interacts in parallel with F229 and the active OH group was directed to the δN atom of 56His known as the catalytic moiety for electron transfer (see FIGS. 5A, 5B, 5C and 5D). As shown in Table 5, the CiP protein variant of SEQ ID NO: 9 had similar interactions, but was closer to the phenol compared to the native form (11.3 kV) (9.6 kV). This supports the consumption of more phenol (50%) than the native CiP protein (20%), although the CiP protein variant of SEQ ID NO: 9 has similar residue activity in the oxidation reaction of phenol. In addition, if the native CiP protein and CiP protein variant of SEQ ID NO: 9 have a similar aromatic environment for radical attack, the close bond distance will provide a favorable environment for the oxidation of phenol mediated by CiP.

In contrast to native CiP proteins having aromatic residues and CiP protein variants of SEQ ID NO: 9, CiP protein variants of SEQ ID NOS: 2 and 3 having hydrophobic residues showed different binding from phenols. CiP protein variants of SEQ ID NO: 2 and 3 have longer binding distances compared to native CiP protein (11.3 kPa, -15.1 kcal / mol) and CiP protein variant (9.6 kPa, -12.0 kcal / mol) 13.2 kV and 14.1 kV) and high binding free energy (-7.61 kcal / mol and -10.35 kcal / mol). Removal of the aromatic moiety at the inlet of the heme pocket reduces the specific binding affinity for the aromatic substrate because it disrupts specific aromatic-aromatic interactions. As shown in Table 3, the CiP protein variant of SEQ ID NO: 2 showed a reduced K _m value (129.1939 μM) compared to native CiP protein (7.7003 μM) for ABTS, an aromatic substrate. This binding affinity (K _m value) is consistent with the results of the same modeling of the high binding free energy of the CiP protein variant of SEQ ID NO: 2 while the native CiP protein has low binding free energy. The above results, in which the CiP protein variant of SEQ ID NO: 2 has a long binding distance and high binding free energy compared to the native CiP protein, suggest that the aromatic residue plays an important role in binding to the aromatic substrate. 3A and 3B, the CiP protein variants of SEQ ID NOS: 2 and 3 show higher enzymatic activity after phenol oxidation compared to the native CiP protein and CiP protein variants of SEQ ID NO: 9. That is, CiP protein variants with aliphatic residues, especially CiP protein variants of SEQ ID NO: 2, are more stable to radical attack compared to aromatic variants due to the removal of aromatic residues. Therefore, it was found that the peroxidase had high radical stability by removing the aromatic residue at position 229 of CiP.

The modeling results are in agreement with experimental results that the F229 residue of CiP is important for the binding and radical attack of aromatic substrates. Such molecular docking simulations suggest that variants with radical stability can be constructed by removing aromatic residues to minimize loss of binding affinity even for other peroxidases.

1 is a recombinant plasmid vector Gene map for pPICZαA-rCiP.

2A and 2B are MS / MS spectra of inactivated CiP fragment (m / z 838.880) (FIG. 2A) and sequence information via new sequencing (FIG. 2B), respectively.

3A and 3B are graphs showing changes in phenol concentration (FIG. 3A) and enzyme activity (FIG. 3B) with time when the natural type CiP protein or CiP protein variant is oxidized with phenol.

4 is a Hanes-Woolf plot for obtaining kinetic parameters when natural CiP protein or CiP protein variant is oxidized with ABTS.

5A, 5B, 5C and 5D show the native CiP protein of FIG. 1 (FIG. 5A), the CiP protein variant of FIG. 2 (FIG. 5B), the CiP protein variant of FIG. 3 (FIG. 5C), and Figure shows the molecular docking simulation results of the phenol and the CiP protein variant of SEQ ID NO.

Table 1 is a table showing the phenol concentration and enzyme activity over time when the natural type CiP protein or CiP protein variant and phenol oxidized.

Table 2 is a table showing the turnover capacity when the natural CiP protein or CiP protein variant is oxidized with phenol.

Table 3 is a table showing HOMO energy levels of amino acids of native CiP protein and CiP protein variants.

Table 4 is a table showing the kinetic parameters obtained based on the results of ABTS oxidation of the native CiP protein or CiP protein variants.

Table 5 is a table showing binding free energy and binding distance of the modeled complex as a result of molecular docking simulation.

<110> KOREA research institute of chemical technology <120> Peroxidase variants having improved radical stability <130> FPD / 200909-0140 <160> 38 <170> KopatentIn 1.71 <210> 1 <211> 343 <212> PRT <213> Artificial Sequence <220> <223> CiP protein derived from Coprinus cinereus <400> 1 Gln Gly Pro Gly Gly Gly Gly Ser Val Thr Cys Pro Gly Gly Gln Ser   1 5 10 15 Thr Ser Asn Ser Gln Cys Cys Val Trp Phe Asp Val Leu Asp Asp Leu              20 25 30 Gln Thr Asn Phe Tyr Gln Gly Ser Lys Cys Glu Ser Pro Val Arg Lys          35 40 45 Ile Leu Arg Ile Val Phe His Asp Ala Ile Gly Phe Ser Pro Ala Leu      50 55 60 Thr Ala Ala Gly Gln Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Ile  65 70 75 80 Ala His Ser Asn Ile Glu Leu Ala Phe Pro Ala Asn Gly Gly Leu Thr                  85 90 95 Asp Thr Ile Glu Ala Leu Arg Ala Val Gly Ile Asn His Gly Val Ser             100 105 110 Phe Gly Asp Leu Ile Gln Phe Ala Thr Ala Val Gly Met Ser Asn Cys         115 120 125 Pro Gly Ser Pro Arg Leu Glu Phe Leu Thr Gly Arg Ser Asn Ser Ser     130 135 140 Gln Pro Ser Pro Pro Ser Leu Ile Pro Gly Pro Gly Asn Thr Val Thr 145 150 155 160 Ala Ile Leu Asp Arg Met Gly Asp Ala Gly Phe Ser Pro Asp Glu Val                 165 170 175 Val Asp Leu Leu Ala Ala His Ser Leu Ala Ser Gln Glu Gly Leu Asn             180 185 190 Ser Ala Ile Phe Arg Ser Pro Leu Asp Ser Thr Pro Gln Val Phe Asp         195 200 205 Thr Gln Phe Tyr Ile Glu Thr Leu Leu Lys Gly Thr Thr Gln Pro Gly     210 215 220 Pro Ser Leu Gly Phe Ala Glu Glu Leu Ser Pro Phe Pro Gly Glu Phe 225 230 235 240 Arg Met Arg Ser Asp Ala Leu Leu Ala Arg Asp Ser Arg Thr Ala Cys                 245 250 255 Arg Trp Gln Ser Met Thr Ser Ser Asn Glu Val Met Gly Gln Arg Tyr             260 265 270 Arg Ala Ala Met Ala Lys Met Ser Val Leu Gly Phe Asp Arg Asn Ala         275 280 285 Leu Thr Asp Cys Ser Asp Val Ile Pro Ser Ala Val Ser Asn Asn Ala     290 295 300 Ala Pro Val Ile Pro Gly Gly Leu Thr Val Asp Asp Ile Glu Val Ser 305 310 315 320 Cys Pro Ser Glu Pro Phe Pro Glu Ile Ala Thr Ala Ser Gly Pro Leu                 325 330 335 Pro Ser Leu Ala Pro Ala Pro             340 <210> 2 <211> 343 <212> PRT <213> Artificial Sequence <220> <223> CiP protein variant (F229A) <400> 2 Gln Gly Pro Gly Gly Gly Gly Ser Val Thr Cys Pro Gly Gly Gln Ser   1 5 10 15 Thr Ser Asn Ser Gln Cys Cys Val Trp Phe Asp Val Leu Asp Asp Leu              20 25 30 Gln Thr Asn Phe Tyr Gln Gly Ser Lys Cys Glu Ser Pro Val Arg Lys          35 40 45 Ile Leu Arg Ile Val Phe His Asp Ala Ile Gly Phe Ser Pro Ala Leu      50 55 60 Thr Ala Ala Gly Gln Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Ile  65 70 75 80 Ala His Ser Asn Ile Glu Leu Ala Phe Pro Ala Asn Gly Gly Leu Thr                  85 90 95 Asp Thr Ile Glu Ala Leu Arg Ala Val Gly Ile Asn His Gly Val Ser             100 105 110 Phe Gly Asp Leu Ile Gln Phe Ala Thr Ala Val Gly Met Ser Asn Cys         115 120 125 Pro Gly Ser Pro Arg Leu Glu Phe Leu Thr Gly Arg Ser Asn Ser Ser     130 135 140 Gln Pro Ser Pro Pro Ser Leu Ile Pro Gly Pro Gly Asn Thr Val Thr 145 150 155 160 Ala Ile Leu Asp Arg Met Gly Asp Ala Gly Phe Ser Pro Asp Glu Val                 165 170 175 Val Asp Leu Leu Ala Ala His Ser Leu Ala Ser Gln Glu Gly Leu Asn             180 185 190 Ser Ala Ile Phe Arg Ser Pro Leu Asp Ser Thr Pro Gln Val Phe Asp         195 200 205 Thr Gln Phe Tyr Ile Glu Thr Leu Leu Lys Gly Thr Thr Gln Pro Gly     210 215 220 Pro Ser Leu Gly Ala Ala Glu Glu Leu Ser Pro Phe Pro Gly Glu Phe 225 230 235 240 Arg Met Arg Ser Asp Ala Leu Leu Ala Arg Asp Ser Arg Thr Ala Cys                 245 250 255 Arg Trp Gln Ser Met Thr Ser Ser Asn Glu Val Met Gly Gln Arg Tyr             260 265 270 Arg Ala Ala Met Ala Lys Met Ser Val Leu Gly Phe Asp Arg Asn Ala         275 280 285 Leu Thr Asp Cys Ser Asp Val Ile Pro Ser Ala Val Ser Asn Asn Ala     290 295 300 Ala Pro Val Ile Pro Gly Gly Leu Thr Val Asp Asp Ile Glu Val Ser 305 310 315 320 Cys Pro Ser Glu Pro Phe Pro Glu Ile Ala Thr Ala Ser Gly Pro Leu                 325 330 335 Pro Ser Leu Ala Pro Ala Pro             340 <210> 3 <211> 343 <212> PRT <213> Artificial Sequence <220> <223> CiP protein variant (F229I) <400> 3 Gln Gly Pro Gly Gly Gly Gly Ser Val Thr Cys Pro Gly Gly Gln Ser   1 5 10 15 Thr Ser Asn Ser Gln Cys Cys Val Trp Phe Asp Val Leu Asp Asp Leu              20 25 30 Gln Thr Asn Phe Tyr Gln Gly Ser Lys Cys Glu Ser Pro Val Arg Lys          35 40 45 Ile Leu Arg Ile Val Phe His Asp Ala Ile Gly Phe Ser Pro Ala Leu      50 55 60 Thr Ala Ala Gly Gln Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Ile  65 70 75 80 Ala His Ser Asn Ile Glu Leu Ala Phe Pro Ala Asn Gly Gly Leu Thr                  85 90 95 Asp Thr Ile Glu Ala Leu Arg Ala Val Gly Ile Asn His Gly Val Ser             100 105 110 Phe Gly Asp Leu Ile Gln Phe Ala Thr Ala Val Gly Met Ser Asn Cys         115 120 125 Pro Gly Ser Pro Arg Leu Glu Phe Leu Thr Gly Arg Ser Asn Ser Ser     130 135 140 Gln Pro Ser Pro Pro Ser Leu Ile Pro Gly Pro Gly Asn Thr Val Thr 145 150 155 160 Ala Ile Leu Asp Arg Met Gly Asp Ala Gly Phe Ser Pro Asp Glu Val                 165 170 175 Val Asp Leu Leu Ala Ala His Ser Leu Ala Ser Gln Glu Gly Leu Asn             180 185 190 Ser Ala Ile Phe Arg Ser Pro Leu Asp Ser Thr Pro Gln Val Phe Asp         195 200 205 Thr Gln Phe Tyr Ile Glu Thr Leu Leu Lys Gly Thr Thr Gln Pro Gly     210 215 220 Pro Ser Leu Gly Ile Ala Glu Glu Leu Ser Pro Phe Pro Gly Glu Phe 225 230 235 240 Arg Met Arg Ser Asp Ala Leu Leu Ala Arg Asp Ser Arg Thr Ala Cys                 245 250 255 Arg Trp Gln Ser Met Thr Ser Ser Asn Glu Val Met Gly Gln Arg Tyr             260 265 270 Arg Ala Ala Met Ala Lys Met Ser Val Leu Gly Phe Asp Arg Asn Ala         275 280 285 Leu Thr Asp Cys Ser Asp Val Ile Pro Ser Ala Val Ser Asn Asn Ala     290 295 300 Ala Pro Val Ile Pro Gly Gly Leu Thr Val Asp Asp Ile Glu Val Ser 305 310 315 320 Cys Pro Ser Glu Pro Phe Pro Glu Ile Ala Thr Ala Ser Gly Pro Leu                 325 330 335 Pro Ser Leu Ala Pro Ala Pro             340 <210> 4 <211> 343 <212> PRT <213> Artificial Sequence <220> <223> CiP protein variant (F229L) <400> 4 Gln Gly Pro Gly Gly Gly Gly Ser Val Thr Cys Pro Gly Gly Gln Ser   1 5 10 15 Thr Ser Asn Ser Gln Cys Cys Val Trp Phe Asp Val Leu Asp Asp Leu              20 25 30 Gln Thr Asn Phe Tyr Gln Gly Ser Lys Cys Glu Ser Pro Val Arg Lys          35 40 45 Ile Leu Arg Ile Val Phe His Asp Ala Ile Gly Phe Ser Pro Ala Leu      50 55 60 Thr Ala Ala Gly Gln Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Ile  65 70 75 80 Ala His Ser Asn Ile Glu Leu Ala Phe Pro Ala Asn Gly Gly Leu Thr                  85 90 95 Asp Thr Ile Glu Ala Leu Arg Ala Val Gly Ile Asn His Gly Val Ser             100 105 110 Phe Gly Asp Leu Ile Gln Phe Ala Thr Ala Val Gly Met Ser Asn Cys         115 120 125 Pro Gly Ser Pro Arg Leu Glu Phe Leu Thr Gly Arg Ser Asn Ser Ser     130 135 140 Gln Pro Ser Pro Pro Ser Leu Ile Pro Gly Pro Gly Asn Thr Val Thr 145 150 155 160 Ala Ile Leu Asp Arg Met Gly Asp Ala Gly Phe Ser Pro Asp Glu Val                 165 170 175 Val Asp Leu Leu Ala Ala His Ser Leu Ala Ser Gln Glu Gly Leu Asn             180 185 190 Ser Ala Ile Phe Arg Ser Pro Leu Asp Ser Thr Pro Gln Val Phe Asp         195 200 205 Thr Gln Phe Tyr Ile Glu Thr Leu Leu Lys Gly Thr Thr Gln Pro Gly     210 215 220 Pro Ser Leu Gly Leu Ala Glu Glu Leu Ser Pro Phe Pro Gly Glu Phe 225 230 235 240 Arg Met Arg Ser Asp Ala Leu Leu Ala Arg Asp Ser Arg Thr Ala Cys                 245 250 255 Arg Trp Gln Ser Met Thr Ser Ser Asn Glu Val Met Gly Gln Arg Tyr             260 265 270 Arg Ala Ala Met Ala Lys Met Ser Val Leu Gly Phe Asp Arg Asn Ala         275 280 285 Leu Thr Asp Cys Ser Asp Val Ile Pro Ser Ala Val Ser Asn Asn Ala     290 295 300 Ala Pro Val Ile Pro Gly Gly Leu Thr Val Asp Asp Ile Glu Val Ser 305 310 315 320 Cys Pro Ser Glu Pro Phe Pro Glu Ile Ala Thr Ala Ser Gly Pro Leu                 325 330 335 Pro Ser Leu Ala Pro Ala Pro             340 <210> 5 <211> 343 <212> PRT <213> Artificial Sequence <220> <223> CiP protein variant (F229G) <400> 5 Gln Gly Pro Gly Gly Gly Gly Ser Val Thr Cys Pro Gly Gly Gln Ser   1 5 10 15 Thr Ser Asn Ser Gln Cys Cys Val Trp Phe Asp Val Leu Asp Asp Leu              20 25 30 Gln Thr Asn Phe Tyr Gln Gly Ser Lys Cys Glu Ser Pro Val Arg Lys          35 40 45 Ile Leu Arg Ile Val Phe His Asp Ala Ile Gly Phe Ser Pro Ala Leu      50 55 60 Thr Ala Ala Gly Gln Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Ile  65 70 75 80 Ala His Ser Asn Ile Glu Leu Ala Phe Pro Ala Asn Gly Gly Leu Thr                  85 90 95 Asp Thr Ile Glu Ala Leu Arg Ala Val Gly Ile Asn His Gly Val Ser             100 105 110 Phe Gly Asp Leu Ile Gln Phe Ala Thr Ala Val Gly Met Ser Asn Cys         115 120 125 Pro Gly Ser Pro Arg Leu Glu Phe Leu Thr Gly Arg Ser Asn Ser Ser     130 135 140 Gln Pro Ser Pro Pro Ser Leu Ile Pro Gly Pro Gly Asn Thr Val Thr 145 150 155 160 Ala Ile Leu Asp Arg Met Gly Asp Ala Gly Phe Ser Pro Asp Glu Val                 165 170 175 Val Asp Leu Leu Ala Ala His Ser Leu Ala Ser Gln Glu Gly Leu Asn             180 185 190 Ser Ala Ile Phe Arg Ser Pro Leu Asp Ser Thr Pro Gln Val Phe Asp         195 200 205 Thr Gln Phe Tyr Ile Glu Thr Leu Leu Lys Gly Thr Thr Gln Pro Gly     210 215 220 Pro Ser Leu Gly Gly Ala Glu Glu Leu Ser Pro Phe Pro Gly Glu Phe 225 230 235 240 Arg Met Arg Ser Asp Ala Leu Leu Ala Arg Asp Ser Arg Thr Ala Cys                 245 250 255 Arg Trp Gln Ser Met Thr Ser Ser Asn Glu Val Met Gly Gln Arg Tyr             260 265 270 Arg Ala Ala Met Ala Lys Met Ser Val Leu Gly Phe Asp Arg Asn Ala         275 280 285 Leu Thr Asp Cys Ser Asp Val Ile Pro Ser Ala Val Ser Asn Asn Ala     290 295 300 Ala Pro Val Ile Pro Gly Gly Leu Thr Val Asp Asp Ile Glu Val Ser 305 310 315 320 Cys Pro Ser Glu Pro Phe Pro Glu Ile Ala Thr Ala Ser Gly Pro Leu                 325 330 335 Pro Ser Leu Ala Pro Ala Pro             340 <210> 6 <211> 343 <212> PRT <213> Artificial Sequence <220> <223> CiP protein variant (F229V) <400> 6 Gln Gly Pro Gly Gly Gly Gly Ser Val Thr Cys Pro Gly Gly Gln Ser   1 5 10 15 Thr Ser Asn Ser Gln Cys Cys Val Trp Phe Asp Val Leu Asp Asp Leu              20 25 30 Gln Thr Asn Phe Tyr Gln Gly Ser Lys Cys Glu Ser Pro Val Arg Lys          35 40 45 Ile Leu Arg Ile Val Phe His Asp Ala Ile Gly Phe Ser Pro Ala Leu      50 55 60 Thr Ala Ala Gly Gln Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Ile  65 70 75 80 Ala His Ser Asn Ile Glu Leu Ala Phe Pro Ala Asn Gly Gly Leu Thr                  85 90 95 Asp Thr Ile Glu Ala Leu Arg Ala Val Gly Ile Asn His Gly Val Ser             100 105 110 Phe Gly Asp Leu Ile Gln Phe Ala Thr Ala Val Gly Met Ser Asn Cys         115 120 125 Pro Gly Ser Pro Arg Leu Glu Phe Leu Thr Gly Arg Ser Asn Ser Ser     130 135 140 Gln Pro Ser Pro Pro Ser Leu Ile Pro Gly Pro Gly Asn Thr Val Thr 145 150 155 160 Ala Ile Leu Asp Arg Met Gly Asp Ala Gly Phe Ser Pro Asp Glu Val                 165 170 175 Val Asp Leu Leu Ala Ala His Ser Leu Ala Ser Gln Glu Gly Leu Asn             180 185 190 Ser Ala Ile Phe Arg Ser Pro Leu Asp Ser Thr Pro Gln Val Phe Asp         195 200 205 Thr Gln Phe Tyr Ile Glu Thr Leu Leu Lys Gly Thr Thr Gln Pro Gly     210 215 220 Pro Ser Leu Gly Val Ala Glu Glu Leu Ser Pro Phe Pro Gly Glu Phe 225 230 235 240 Arg Met Arg Ser Asp Ala Leu Leu Ala Arg Asp Ser Arg Thr Ala Cys                 245 250 255 Arg Trp Gln Ser Met Thr Ser Ser Asn Glu Val Met Gly Gln Arg Tyr             260 265 270 Arg Ala Ala Met Ala Lys Met Ser Val Leu Gly Phe Asp Arg Asn Ala         275 280 285 Leu Thr Asp Cys Ser Asp Val Ile Pro Ser Ala Val Ser Asn Asn Ala     290 295 300 Ala Pro Val Ile Pro Gly Gly Leu Thr Val Asp Asp Ile Glu Val Ser 305 310 315 320 Cys Pro Ser Glu Pro Phe Pro Glu Ile Ala Thr Ala Ser Gly Pro Leu                 325 330 335 Pro Ser Leu Ala Pro Ala Pro             340 <210> 7 <211> 343 <212> PRT <213> Artificial Sequence <220> <223> CiP protein variant (F229H) <400> 7 Gln Gly Pro Gly Gly Gly Gly Ser Val Thr Cys Pro Gly Gly Gln Ser   1 5 10 15 Thr Ser Asn Ser Gln Cys Cys Val Trp Phe Asp Val Leu Asp Asp Leu              20 25 30 Gln Thr Asn Phe Tyr Gln Gly Ser Lys Cys Glu Ser Pro Val Arg Lys          35 40 45 Ile Leu Arg Ile Val Phe His Asp Ala Ile Gly Phe Ser Pro Ala Leu      50 55 60 Thr Ala Ala Gly Gln Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Ile  65 70 75 80 Ala His Ser Asn Ile Glu Leu Ala Phe Pro Ala Asn Gly Gly Leu Thr                  85 90 95 Asp Thr Ile Glu Ala Leu Arg Ala Val Gly Ile Asn His Gly Val Ser             100 105 110 Phe Gly Asp Leu Ile Gln Phe Ala Thr Ala Val Gly Met Ser Asn Cys         115 120 125 Pro Gly Ser Pro Arg Leu Glu Phe Leu Thr Gly Arg Ser Asn Ser Ser     130 135 140 Gln Pro Ser Pro Pro Ser Leu Ile Pro Gly Pro Gly Asn Thr Val Thr 145 150 155 160 Ala Ile Leu Asp Arg Met Gly Asp Ala Gly Phe Ser Pro Asp Glu Val                 165 170 175 Val Asp Leu Leu Ala Ala His Ser Leu Ala Ser Gln Glu Gly Leu Asn             180 185 190 Ser Ala Ile Phe Arg Ser Pro Leu Asp Ser Thr Pro Gln Val Phe Asp         195 200 205 Thr Gln Phe Tyr Ile Glu Thr Leu Leu Lys Gly Thr Thr Gln Pro Gly     210 215 220 Pro Ser Leu Gly His Ala Glu Glu Leu Ser Pro Phe Pro Gly Glu Phe 225 230 235 240 Arg Met Arg Ser Asp Ala Leu Leu Ala Arg Asp Ser Arg Thr Ala Cys                 245 250 255 Arg Trp Gln Ser Met Thr Ser Ser Asn Glu Val Met Gly Gln Arg Tyr             260 265 270 Arg Ala Ala Met Ala Lys Met Ser Val Leu Gly Phe Asp Arg Asn Ala         275 280 285 Leu Thr Asp Cys Ser Asp Val Ile Pro Ser Ala Val Ser Asn Asn Ala     290 295 300 Ala Pro Val Ile Pro Gly Gly Leu Thr Val Asp Asp Ile Glu Val Ser 305 310 315 320 Cys Pro Ser Glu Pro Phe Pro Glu Ile Ala Thr Ala Ser Gly Pro Leu                 325 330 335 Pro Ser Leu Ala Pro Ala Pro             340 <210> 8 <211> 343 <212> PRT <213> Artificial Sequence <220> <223> CiP protein variant (F229Y) <400> 8 Gln Gly Pro Gly Gly Gly Gly Ser Val Thr Cys Pro Gly Gly Gln Ser   1 5 10 15 Thr Ser Asn Ser Gln Cys Cys Val Trp Phe Asp Val Leu Asp Asp Leu              20 25 30 Gln Thr Asn Phe Tyr Gln Gly Ser Lys Cys Glu Ser Pro Val Arg Lys          35 40 45 Ile Leu Arg Ile Val Phe His Asp Ala Ile Gly Phe Ser Pro Ala Leu      50 55 60 Thr Ala Ala Gly Gln Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Ile  65 70 75 80 Ala His Ser Asn Ile Glu Leu Ala Phe Pro Ala Asn Gly Gly Leu Thr                  85 90 95 Asp Thr Ile Glu Ala Leu Arg Ala Val Gly Ile Asn His Gly Val Ser             100 105 110 Phe Gly Asp Leu Ile Gln Phe Ala Thr Ala Val Gly Met Ser Asn Cys         115 120 125 Pro Gly Ser Pro Arg Leu Glu Phe Leu Thr Gly Arg Ser Asn Ser Ser     130 135 140 Gln Pro Ser Pro Pro Ser Leu Ile Pro Gly Pro Gly Asn Thr Val Thr 145 150 155 160 Ala Ile Leu Asp Arg Met Gly Asp Ala Gly Phe Ser Pro Asp Glu Val                 165 170 175 Val Asp Leu Leu Ala Ala His Ser Leu Ala Ser Gln Glu Gly Leu Asn             180 185 190 Ser Ala Ile Phe Arg Ser Pro Leu Asp Ser Thr Pro Gln Val Phe Asp         195 200 205 Thr Gln Phe Tyr Ile Glu Thr Leu Leu Lys Gly Thr Thr Gln Pro Gly     210 215 220 Pro Ser Leu Gly Tyr Ala Glu Glu Leu Ser Pro Phe Pro Gly Glu Phe 225 230 235 240 Arg Met Arg Ser Asp Ala Leu Leu Ala Arg Asp Ser Arg Thr Ala Cys                 245 250 255 Arg Trp Gln Ser Met Thr Ser Ser Asn Glu Val Met Gly Gln Arg Tyr             260 265 270 Arg Ala Ala Met Ala Lys Met Ser Val Leu Gly Phe Asp Arg Asn Ala         275 280 285 Leu Thr Asp Cys Ser Asp Val Ile Pro Ser Ala Val Ser Asn Asn Ala     290 295 300 Ala Pro Val Ile Pro Gly Gly Leu Thr Val Asp Asp Ile Glu Val Ser 305 310 315 320 Cys Pro Ser Glu Pro Phe Pro Glu Ile Ala Thr Ala Ser Gly Pro Leu                 325 330 335 Pro Ser Leu Ala Pro Ala Pro             340 <210> 9 <211> 343 <212> PRT <213> Artificial Sequence <220> <223> CiP protein variant (F229W) <400> 9 Gln Gly Pro Gly Gly Gly Gly Ser Val Thr Cys Pro Gly Gly Gln Ser   1 5 10 15 Thr Ser Asn Ser Gln Cys Cys Val Trp Phe Asp Val Leu Asp Asp Leu              20 25 30 Gln Thr Asn Phe Tyr Gln Gly Ser Lys Cys Glu Ser Pro Val Arg Lys          35 40 45 Ile Leu Arg Ile Val Phe His Asp Ala Ile Gly Phe Ser Pro Ala Leu      50 55 60 Thr Ala Ala Gly Gln Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Ile  65 70 75 80 Ala His Ser Asn Ile Glu Leu Ala Phe Pro Ala Asn Gly Gly Leu Thr                  85 90 95 Asp Thr Ile Glu Ala Leu Arg Ala Val Gly Ile Asn His Gly Val Ser             100 105 110 Phe Gly Asp Leu Ile Gln Phe Ala Thr Ala Val Gly Met Ser Asn Cys         115 120 125 Pro Gly Ser Pro Arg Leu Glu Phe Leu Thr Gly Arg Ser Asn Ser Ser     130 135 140 Gln Pro Ser Pro Pro Ser Leu Ile Pro Gly Pro Gly Asn Thr Val Thr 145 150 155 160 Ala Ile Leu Asp Arg Met Gly Asp Ala Gly Phe Ser Pro Asp Glu Val                 165 170 175 Val Asp Leu Leu Ala Ala His Ser Leu Ala Ser Gln Glu Gly Leu Asn             180 185 190 Ser Ala Ile Phe Arg Ser Pro Leu Asp Ser Thr Pro Gln Val Phe Asp         195 200 205 Thr Gln Phe Tyr Ile Glu Thr Leu Leu Lys Gly Thr Thr Gln Pro Gly     210 215 220 Pro Ser Leu Gly Trp Ala Glu Glu Leu Ser Pro Phe Pro Gly Glu Phe 225 230 235 240 Arg Met Arg Ser Asp Ala Leu Leu Ala Arg Asp Ser Arg Thr Ala Cys                 245 250 255 Arg Trp Gln Ser Met Thr Ser Ser Asn Glu Val Met Gly Gln Arg Tyr             260 265 270 Arg Ala Ala Met Ala Lys Met Ser Val Leu Gly Phe Asp Arg Asn Ala         275 280 285 Leu Thr Asp Cys Ser Asp Val Ile Pro Ser Ala Val Ser Asn Asn Ala     290 295 300 Ala Pro Val Ile Pro Gly Gly Leu Thr Val Asp Asp Ile Glu Val Ser 305 310 315 320 Cys Pro Ser Glu Pro Phe Pro Glu Ile Ala Thr Ala Ser Gly Pro Leu                 325 330 335 Pro Ser Leu Ala Pro Ala Pro             340 <210> 10 <211> 1032 <212> DNA <213> Artificial Sequence <220> <223> gene sequence encoding CiP protein of sequence No: 1 <400> 10 caaggtcctg gtggtggtgg atctgttact tgtccaggag gtcaaagcac atcaaattct 60 cagtgctgtg tttggtttga tgtactggac gatcttcaaa ccaacttcta ccaagggtca 120 aaatgcgaat cacctgtcag aaaaatcttg cgtatcgtct tccatgatgc catcggattt 180 agtccagctt taacggctgc cggtcaattc ggtggaggtg gtgcggatgg atctattatc 240 gcccattcta acatcgaact tgcattccct gcaaacggtg gattgaccga tacaattgaa 300 gcattacgtg cagtcggtat taatcacggt gtctctttcg gtgatttaat ccagttcgct 360 accgcggttg gtatgtccaa ttgtccgggt tcacccagat tagaatttct gaccggaaga 420 agtaactcgt cccaaccatc acctccgagt ctgatcccag gtcctggtaa tacggttact 480 gccattctag acaggatggg cgatgctgga ttttctcccg atgaggtcgt cgatttgcta 540 gcagcacatt ctttagcgtc tcaagagggt ttgaacagtg ccatatttag gtccccgttg 600 gatagtaccc cccaggtatt cgacacacag ttctatatcg aaaccctgtt gaagggtact 660 actcaacccg gtccttcatt gggttttgca gaagaattgt ctccgttccc tggagaattt 720 agaatgagat cggatgctct tttggcaaga gactccagaa ctgcgtgtag gtggcaatcc 780 atgacttcaa gtaacgaggt tatgggtcaa aggtatagag cagccatggc gaaaatgtca 840 gtgttgggtt tcgacagaaa tgcgttgaca gattgctccg acgttatccc ttcggccgtt 900 agtaataacg ctgctccagt cattccagga ggtttgactg tcgatgatat agaggtatct 960 tgcccgtctg aaccatttcc agaaatcgcc actgcatccg gtccattgcc atcacttgct 1020 cctgctccat aa 1032 <210> 11 <211> 1032 <212> DNA <213> Artificial Sequence <220> <223> gene sequence encoding CiP protein variant of sequence No: 2 <400> 11 caaggtcctg gtggtggtgg atctgttact tgtccaggag gtcaaagcac atcaaattct 60 cagtgctgtg tttggtttga tgtactggac gatcttcaaa ccaacttcta ccaagggtca 120 aaatgcgaat cacctgtcag aaaaatcttg cgtatcgtct tccatgatgc catcggattt 180 agtccagctt taacggctgc cggtcaattc ggtggaggtg gtgcggatgg atctattatc 240 gcccattcta acatcgaact tgcattccct gcaaacggtg gattgaccga tacaattgaa 300 gcattacgtg cagtcggtat taatcacggt gtctctttcg gtgatttaat ccagttcgct 360 accgcggttg gtatgtccaa ttgtccgggt tcacccagat tagaatttct gaccggaaga 420 agtaactcgt cccaaccatc acctccgagt ctgatcccag gtcctggtaa tacggttact 480 gccattctag acaggatggg cgatgctgga ttttctcccg atgaggtcgt cgatttgcta 540 gcagcacatt ctttagcgtc tcaagagggt ttgaacagtg ccatatttag gtccccgttg 600 gatagtaccc cccaggtatt cgacacacag ttctatatcg aaaccctgtt gaagggtact 660 actcaacccg gtccttcatt gggtgctgca gaagaattgt ctccgttccc tggagaattt 720 agaatgagat cggatgctct tttggcaaga gactccagaa ctgcgtgtag gtggcaatcc 780 atgacttcaa gtaacgaggt tatgggtcaa aggtatagag cagccatggc gaaaatgtca 840 gtgttgggtt tcgacagaaa tgcgttgaca gattgctccg acgttatccc ttcggccgtt 900 agtaataacg ctgctccagt cattccagga ggtttgactg tcgatgatat agaggtatct 960 tgcccgtctg aaccatttcc agaaatcgcc actgcatccg gtccattgcc atcacttgct 1020 cctgctccat aa 1032 <210> 12 <211> 1032 <212> DNA <213> Artificial Sequence <220> <223> gene sequence encoding CiP protein variant of sequence No: 3 <400> 12 caaggtcctg gtggtggtgg atctgttact tgtccaggag gtcaaagcac atcaaattct 60 cagtgctgtg tttggtttga tgtactggac gatcttcaaa ccaacttcta ccaagggtca 120 aaatgcgaat cacctgtcag aaaaatcttg cgtatcgtct tccatgatgc catcggattt 180 agtccagctt taacggctgc cggtcaattc ggtggaggtg gtgcggatgg atctattatc 240 gcccattcta acatcgaact tgcattccct gcaaacggtg gattgaccga tacaattgaa 300 gcattacgtg cagtcggtat taatcacggt gtctctttcg gtgatttaat ccagttcgct 360 accgcggttg gtatgtccaa ttgtccgggt tcacccagat tagaatttct gaccggaaga 420 agtaactcgt cccaaccatc acctccgagt ctgatcccag gtcctggtaa tacggttact 480 gccattctag acaggatggg cgatgctgga ttttctcccg atgaggtcgt cgatttgcta 540 gcagcacatt ctttagcgtc tcaagagggt ttgaacagtg ccatatttag gtccccgttg 600 gatagtaccc cccaggtatt cgacacacag ttctatatcg aaaccctgtt gaagggtact 660 actcaacccg gtccttcatt gggtattgca gaagaattgt ctccgttccc tggagaattt 720 agaatgagat cggatgctct tttggcaaga gactccagaa ctgcgtgtag gtggcaatcc 780 atgacttcaa gtaacgaggt tatgggtcaa aggtatagag cagccatggc gaaaatgtca 840 gtgttgggtt tcgacagaaa tgcgttgaca gattgctccg acgttatccc ttcggccgtt 900 agtaataacg ctgctccagt cattccagga ggtttgactg tcgatgatat agaggtatct 960 tgcccgtctg aaccatttcc agaaatcgcc actgcatccg gtccattgcc atcacttgct 1020 cctgctccat aa 1032 <210> 13 <211> 1032 <212> DNA <213> Artificial Sequence <220> <223> gene sequence encoding CiP protein variant of sequence No: 4 <400> 13 caaggtcctg gtggtggtgg atctgttact tgtccaggag gtcaaagcac atcaaattct 60 cagtgctgtg tttggtttga tgtactggac gatcttcaaa ccaacttcta ccaagggtca 120 aaatgcgaat cacctgtcag aaaaatcttg cgtatcgtct tccatgatgc catcggattt 180 agtccagctt taacggctgc cggtcaattc ggtggaggtg gtgcggatgg atctattatc 240 gcccattcta acatcgaact tgcattccct gcaaacggtg gattgaccga tacaattgaa 300 gcattacgtg cagtcggtat taatcacggt gtctctttcg gtgatttaat ccagttcgct 360 accgcggttg gtatgtccaa ttgtccgggt tcacccagat tagaatttct gaccggaaga 420 agtaactcgt cccaaccatc acctccgagt ctgatcccag gtcctggtaa tacggttact 480 gccattctag acaggatggg cgatgctgga ttttctcccg atgaggtcgt cgatttgcta 540 gcagcacatt ctttagcgtc tcaagagggt ttgaacagtg ccatatttag gtccccgttg 600 gatagtaccc cccaggtatt cgacacacag ttctatatcg aaaccctgtt gaagggtact 660 actcaacccg gtccttcatt gggtttggca gaagaattgt ctccgttccc tggagaattt 720 agaatgagat cggatgctct tttggcaaga gactccagaa ctgcgtgtag gtggcaatcc 780 atgacttcaa gtaacgaggt tatgggtcaa aggtatagag cagccatggc gaaaatgtca 840 gtgttgggtt tcgacagaaa tgcgttgaca gattgctccg acgttatccc ttcggccgtt 900 agtaataacg ctgctccagt cattccagga ggtttgactg tcgatgatat agaggtatct 960 tgcccgtctg aaccatttcc agaaatcgcc actgcatccg gtccattgcc atcacttgct 1020 cctgctccat aa 1032 <210> 14 <211> 1032 <212> DNA <213> Artificial Sequence <220> <223> gene sequence encoding CiP protein variant of sequence No: 5 <400> 14 caaggtcctg gtggtggtgg atctgttact tgtccaggag gtcaaagcac atcaaattct 60 cagtgctgtg tttggtttga tgtactggac gatcttcaaa ccaacttcta ccaagggtca 120 aaatgcgaat cacctgtcag aaaaatcttg cgtatcgtct tccatgatgc catcggattt 180 agtccagctt taacggctgc cggtcaattc ggtggaggtg gtgcggatgg atctattatc 240 gcccattcta acatcgaact tgcattccct gcaaacggtg gattgaccga tacaattgaa 300 gcattacgtg cagtcggtat taatcacggt gtctctttcg gtgatttaat ccagttcgct 360 accgcggttg gtatgtccaa ttgtccgggt tcacccagat tagaatttct gaccggaaga 420 agtaactcgt cccaaccatc acctccgagt ctgatcccag gtcctggtaa tacggttact 480 gccattctag acaggatggg cgatgctgga ttttctcccg atgaggtcgt cgatttgcta 540 gcagcacatt ctttagcgtc tcaagagggt ttgaacagtg ccatatttag gtccccgttg 600 gatagtaccc cccaggtatt cgacacacag ttctatatcg aaaccctgtt gaagggtact 660 actcaacccg gtccttcatt gggtggtgca gaagaattgt ctccgttccc tggagaattt 720 agaatgagat cggatgctct tttggcaaga gactccagaa ctgcgtgtag gtggcaatcc 780 atgacttcaa gtaacgaggt tatgggtcaa aggtatagag cagccatggc gaaaatgtca 840 gtgttgggtt tcgacagaaa tgcgttgaca gattgctccg acgttatccc ttcggccgtt 900 agtaataacg ctgctccagt cattccagga ggtttgactg tcgatgatat agaggtatct 960 tgcccgtctg aaccatttcc agaaatcgcc actgcatccg gtccattgcc atcacttgct 1020 cctgctccat aa 1032 <210> 15 <211> 1032 <212> DNA <213> Artificial Sequence <220> <223> gene sequence encoding CiP protein variant of sequence No: 6 <400> 15 caaggtcctg gtggtggtgg atctgttact tgtccaggag gtcaaagcac atcaaattct 60 cagtgctgtg tttggtttga tgtactggac gatcttcaaa ccaacttcta ccaagggtca 120 aaatgcgaat cacctgtcag aaaaatcttg cgtatcgtct tccatgatgc catcggattt 180 agtccagctt taacggctgc cggtcaattc ggtggaggtg gtgcggatgg atctattatc 240 gcccattcta acatcgaact tgcattccct gcaaacggtg gattgaccga tacaattgaa 300 gcattacgtg cagtcggtat taatcacggt gtctctttcg gtgatttaat ccagttcgct 360 accgcggttg gtatgtccaa ttgtccgggt tcacccagat tagaatttct gaccggaaga 420 agtaactcgt cccaaccatc acctccgagt ctgatcccag gtcctggtaa tacggttact 480 gccattctag acaggatggg cgatgctgga ttttctcccg atgaggtcgt cgatttgcta 540 gcagcacatt ctttagcgtc tcaagagggt ttgaacagtg ccatatttag gtccccgttg 600 gatagtaccc cccaggtatt cgacacacag ttctatatcg aaaccctgtt gaagggtact 660 actcaacccg gtccttcatt gggtgttgca gaagaattgt ctccgttccc tggagaattt 720 agaatgagat cggatgctct tttggcaaga gactccagaa ctgcgtgtag gtggcaatcc 780 atgacttcaa gtaacgaggt tatgggtcaa aggtatagag cagccatggc gaaaatgtca 840 gtgttgggtt tcgacagaaa tgcgttgaca gattgctccg acgttatccc ttcggccgtt 900 agtaataacg ctgctccagt cattccagga ggtttgactg tcgatgatat agaggtatct 960 tgcccgtctg aaccatttcc agaaatcgcc actgcatccg gtccattgcc atcacttgct 1020 cctgctccat aa 1032 <210> 16 <211> 1032 <212> DNA <213> Artificial Sequence <220> <223> gene sequence encoding CiP protein variant of sequence No: 7 <400> 16 caaggtcctg gtggtggtgg atctgttact tgtccaggag gtcaaagcac atcaaattct 60 cagtgctgtg tttggtttga tgtactggac gatcttcaaa ccaacttcta ccaagggtca 120 aaatgcgaat cacctgtcag aaaaatcttg cgtatcgtct tccatgatgc catcggattt 180 agtccagctt taacggctgc cggtcaattc ggtggaggtg gtgcggatgg atctattatc 240 gcccattcta acatcgaact tgcattccct gcaaacggtg gattgaccga tacaattgaa 300 gcattacgtg cagtcggtat taatcacggt gtctctttcg gtgatttaat ccagttcgct 360 accgcggttg gtatgtccaa ttgtccgggt tcacccagat tagaatttct gaccggaaga 420 agtaactcgt cccaaccatc acctccgagt ctgatcccag gtcctggtaa tacggttact 480 gccattctag acaggatggg cgatgctgga ttttctcccg atgaggtcgt cgatttgcta 540 gcagcacatt ctttagcgtc tcaagagggt ttgaacagtg ccatatttag gtccccgttg 600 gatagtaccc cccaggtatt cgacacacag ttctatatcg aaaccctgtt gaagggtact 660 actcaacccg gtccttcatt gggtcatgca gaagaattgt ctccgttccc tggagaattt 720 agaatgagat cggatgctct tttggcaaga gactccagaa ctgcgtgtag gtggcaatcc 780 atgacttcaa gtaacgaggt tatgggtcaa aggtatagag cagccatggc gaaaatgtca 840 gtgttgggtt tcgacagaaa tgcgttgaca gattgctccg acgttatccc ttcggccgtt 900 agtaataacg ctgctccagt cattccagga ggtttgactg tcgatgatat agaggtatct 960 tgcccgtctg aaccatttcc agaaatcgcc actgcatccg gtccattgcc atcacttgct 1020 cctgctccat aa 1032 <210> 17 <211> 1032 <212> DNA <213> Artificial Sequence <220> <223> gene sequence encoding CiP protein variant of sequence No: 8 <400> 17 caaggtcctg gtggtggtgg atctgttact tgtccaggag gtcaaagcac atcaaattct 60 cagtgctgtg tttggtttga tgtactggac gatcttcaaa ccaacttcta ccaagggtca 120 aaatgcgaat cacctgtcag aaaaatcttg cgtatcgtct tccatgatgc catcggattt 180 agtccagctt taacggctgc cggtcaattc ggtggaggtg gtgcggatgg atctattatc 240 gcccattcta acatcgaact tgcattccct gcaaacggtg gattgaccga tacaattgaa 300 gcattacgtg cagtcggtat taatcacggt gtctctttcg gtgatttaat ccagttcgct 360 accgcggttg gtatgtccaa ttgtccgggt tcacccagat tagaatttct gaccggaaga 420 agtaactcgt cccaaccatc acctccgagt ctgatcccag gtcctggtaa tacggttact 480 gccattctag acaggatggg cgatgctgga ttttctcccg atgaggtcgt cgatttgcta 540 gcagcacatt ctttagcgtc tcaagagggt ttgaacagtg ccatatttag gtccccgttg 600 gatagtaccc cccaggtatt cgacacacag ttctatatcg aaaccctgtt gaagggtact 660 actcaacccg gtccttcatt gggttacgca gaagaattgt ctccgttccc tggagaattt 720 agaatgagat cggatgctct tttggcaaga gactccagaa ctgcgtgtag gtggcaatcc 780 atgacttcaa gtaacgaggt tatgggtcaa aggtatagag cagccatggc gaaaatgtca 840 gtgttgggtt tcgacagaaa tgcgttgaca gattgctccg acgttatccc ttcggccgtt 900 agtaataacg ctgctccagt cattccagga ggtttgactg tcgatgatat agaggtatct 960 tgcccgtctg aaccatttcc agaaatcgcc actgcatccg gtccattgcc atcacttgct 1020 cctgctccat aa 1032 <210> 18 <211> 1032 <212> DNA <213> Artificial Sequence <220> <223> gene sequence encoding CiP protein variant of sequence No: 9 <400> 18 caaggtcctg gtggtggtgg atctgttact tgtccaggag gtcaaagcac atcaaattct 60 cagtgctgtg tttggtttga tgtactggac gatcttcaaa ccaacttcta ccaagggtca 120 aaatgcgaat cacctgtcag aaaaatcttg cgtatcgtct tccatgatgc catcggattt 180 agtccagctt taacggctgc cggtcaattc ggtggaggtg gtgcggatgg atctattatc 240 gcccattcta acatcgaact tgcattccct gcaaacggtg gattgaccga tacaattgaa 300 gcattacgtg cagtcggtat taatcacggt gtctctttcg gtgatttaat ccagttcgct 360 accgcggttg gtatgtccaa ttgtccgggt tcacccagat tagaatttct gaccggaaga 420 agtaactcgt cccaaccatc acctccgagt ctgatcccag gtcctggtaa tacggttact 480 gccattctag acaggatggg cgatgctgga ttttctcccg atgaggtcgt cgatttgcta 540 gcagcacatt ctttagcgtc tcaagagggt ttgaacagtg ccatatttag gtccccgttg 600 gatagtaccc cccaggtatt cgacacacag ttctatatcg aaaccctgtt gaagggtact 660 actcaacccg gtccttcatt gggttgggca gaagaattgt ctccgttccc tggagaattt 720 agaatgagat cggatgctct tttggcaaga gactccagaa ctgcgtgtag gtggcaatcc 780 atgacttcaa gtaacgaggt tatgggtcaa aggtatagag cagccatggc gaaaatgtca 840 gtgttgggtt tcgacagaaa tgcgttgaca gattgctccg acgttatccc ttcggccgtt 900 agtaataacg ctgctccagt cattccagga ggtttgactg tcgatgatat agaggtatct 960 tgcccgtctg aaccatttcc agaaatcgcc actgcatccg gtccattgcc atcacttgct 1020 cctgctccat aa 1032 <210> 19 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer for rCiP (rCiP-N-EcoRI) <400> 19 cggaattcca gggtcctgga ggaggcgggt cag 33 <210> 20 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer for rCiP (rCiP-C-NotI) <400> 20 acgcgtcgac tcaaggagca ggagcgaggg agg 33 <210> 21 <211> 35 <212> DNA <213> Artificial Sequence <220> 5 'flanking primer (EcoRI) <400> 21 gcgcgaattc caaggtcctg gtggtggtgg atctg 35 <210> 22 <211> 37 <212> DNA <213> Artificial Sequence <220> 3 'flanking primer (NotI) <400> 22 gcgcgcggcc gcttatggag caggagcaag tgatggc 37 <210> 23 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229A (M1-F) <400> 23 cccggtcctt cattgggtgc tgcagaagaa ttgtctccg 39 <210> 24 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229A (M1-R) <400> 24 cggagacaat tcttctgcag cacccaatga aggaccggg 39 <210> 25 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229I (M3-F) <400> 25 cccggtcctt cattgggtat tgcagaagaa ttgtctccg 39 <210> 26 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229I (M3-R) <400> 26 cggagacaat tcttctgcaa tacccaatga aggaccggg 39 <210> 27 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229L (M2-F) <400> 27 cccggtcctt cattgggttt ggcagaagaa ttgtctccg 39 <210> 28 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229L (M2-R) <400> 28 cggagacaat tcttctgcca aacccaatga aggaccggg 39 <210> 29 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229G (M8-F) <400> 29 cccggtcctt cattgggtgg tgcagaagaa ttgtctccg 39 <210> 30 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229G (M8-R) <400> 30 cggagacaat tcttctgcac cacccaatga aggaccggg 39 <210> 31 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229V (M4-F) <400> 31 cccggtcctt cattgggtgt tgcagaagaa ttgtctccg 39 <210> 32 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229V (M4-R) <400> 32 cggagacaat tcttctgcaa cacccaatga aggaccggg 39 <210> 33 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229H (M7-F) <400> 33 cccggtcctt cattgggtca tgcagaagaa ttgtctccg 39 <210> 34 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229H (M7-R) <400> 34 cggagacaat tcttctgcat gacccaatga aggaccggg 39 <210> 35 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229Y (M5-F) <400> 35 cccggtcctt cattgggtta cgcagaagaa ttgtctccg 39 <210> 36 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229Y (M5-R) <400> 36 cggagacaat tcttctgcgt aacccaatga aggaccggg 39 <210> 37 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229W (M6-F) <400> 37 cccggtcctt cattgggttg ggcagaagaa ttgtctccg 39 <210> 38 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> mutagenic primer for F229W (M6-R) <400> 38 cggagacaat tcttctgccc aacccaatga aggaccggg 39  

Claims (7)

Consisting of a protein represented by the amino acid sequence of SEQ ID NOs: 2 to 4 substituted with an amino acid at position 229 of a peroxidase (CiP) protein derived from Coprinus cinereus represented by the amino acid sequence of SEQ ID NO: 1 CiP protein variant, characterized in that selected from the group. Gene encoding the CiP protein variant sequence according to claim 1. The method of claim 2, Gene is characterized in that the gene is selected from the group consisting of the gene represented by the nucleotide sequence of SEQ ID NO: 11 to 13. Recombinant vector comprising the gene according to claim 2. The method of claim 4, wherein Recombinant vector, characterized in that the recombinant vector is prepared by inserting the gene into the pPICZαA vector. A host cell comprising the recombinant vector of claim 4. The method of claim 6, A host cell wherein said host cell is Pichia pastoris.

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