JP7495083B2 - Recombinant production of peroxidase - Google Patents

Description

Technologies for recombinant production of peroxidase are disclosed.

Peroxidase is an enzyme abundantly found in horseradish (Aromoracia rusutica) and is used in a wide range of fields, including analysis, clinical testing, immunochemistry, histochemistry, and cytochemistry. In recent years, demand for peroxidase has been expanding not only for analytical chemistry applications, but also for its usefulness in wastewater treatment and environmental purification. However, when extracting peroxidase from wasabi radish, it is not easy to obtain peroxidase of consistent quality, since the peroxidase content and isoenzyme abundance ratio vary significantly depending on various factors such as variety, cultivation conditions, and climate.

To efficiently produce peroxidase of stable quality, production of peroxidase by gene recombination technology has been proposed. For example, Patent Document 1 attempts to produce peroxidase using Saccharomyces cerevisiae or Pichia pastris as a host. In this case, the introduction of a mutation into the peroxidase gene improves productivity to a certain extent, but there is an increase in molecular weight due to excessive sugar chain binding called hypermannose, an associated decrease in specific activity, and a change in enzyme properties due to the introduction of the mutation.

Patent Document 2 describes a method for producing a heterologous protein using a Cryptococcus sp. S-2 strain as a host with reduced extracellular polysaccharide production. Patent Document 3 describes the production of peroxidase using this host expression system. However, the recombinant peroxidase obtained by large-scale culture using a fermenter still had hypermannose sugar chain binding, and had not yet been put to practical use.

JP 2003-503005 A Patent No. 5588578 Japanese Patent No. 5245060

One challenge is to produce a peroxidase through recombinant production that has a molecular weight including glycans equivalent to that of the peroxidase obtained from horseradish.

It has been found that by using a mutant strain of Cryptococcus in which a specific sugar chain synthesis enzyme of the Cryptococcus genus has been disrupted, it is possible to produce horseradish-derived peroxidase having a molecular weight (including sugar chain) equivalent to that of the peroxidase produced by horseradish. Further research and investigation have been conducted based on this finding, and the following representative means are provided.
Item 1.
A mutant Cryptococcus strain in which the ALG3, ALG11, and OCH1 genes have been disrupted.
Item 2.
Item 2. The Cryptococcus mutant according to Item 1, which is transformed with a gene encoding horseradish peroxidase.
Item 3.
Item 3. The Cryptococcus mutant according to Item 1 or 2, further comprising a disrupted URA5 gene, an ADE1 gene, and/or a disrupted KU70 gene.
Item 4.
Item 4. The Cryptococcus mutant according to any one of Items 1 to 3, which produces horseradish peroxidase having a molecular weight of about 45 kDa as measured by SDS-PAGE.
Item 5.
A method for producing a mutant strain of Cryptococcus sp., comprising disrupting the ALG3 gene, the ALG11 gene, and the OCH1 gene of Cryptococcus sp.
Item 6.
Item 6. The method according to Item 5, further comprising transforming a Cryptococcus sp. bacterium with a gene encoding horseradish peroxidase.
Item 7.
Item 6. The method according to Item 5, wherein the Cryptococcus sp. is transformed with a gene encoding horseradish peroxidase.
Item 8.
8. The method according to any one of Items 5 to 7, comprising disrupting the URA5 gene and/or the ADE1 gene before disrupting the ALG3 gene, the ALG11 gene, and the OCH1 gene.
Item 9.
Item 9. The method according to any one of Items 5 to 8, further comprising disrupting the ALG gene, the ADE1 gene, and/or the KU70 gene.
Item 10.
Item 10. The method according to any one of Items 5 to 9, wherein the mutant strain of Cryptococcus is the mutant strain of Cryptococcus according to any one of Items 1 to 4.
Item 11.
Item 5. A method for producing horseradish peroxidase, comprising culturing the mutant strain of Cryptococcus according to any one of Items 1 to 4.

In one embodiment, a Cryptococcus bacterium is provided that has a controlled ability to add glycosylation. In one embodiment, it is possible to recombinantly produce a peroxidase that contains a glycosylation chain and has a molecular weight equivalent to that of the peroxidase produced by horseradish.

The base sequence of the ALG3 gene of Cryptococcus sp. S-2 strain and the base sequences surrounding it are shown. The base sequence of the ALG11 gene of Cryptococcus sp. S-2 strain and the base sequences surrounding it are shown. The base sequence of the OCH1 gene of Cryptococcus sp. S-2 strain and the base sequences surrounding it are shown. An example of the breeding process for a glycosyltransferase gene disruptant is shown. The molecular weights of recombinantly produced peroxidases as determined by SDS-PAGE are shown. The results of measuring the optimum temperature of recombinantly produced peroxidase are shown. 1 shows the results of measuring the optimum pH of recombinantly produced peroxidase. 1 shows the results of measuring the temperature stability of recombinantly produced peroxidase. The results of measuring the optimum temperature of recombinantly produced peroxidase are shown. The results of measuring the Km value of recombinantly produced peroxidase for hydrogen peroxide are shown. The figure shows a comparison of reaction curves when measuring creatinine using hydrogen peroxide produced by recombinant peroxidase and peroxidase derived from horseradish.

1. Cryptococcus mutant strain The Cryptococcus mutant strain preferably has the ALG3 gene, the ALG11 gene, and the OCH1 gene disrupted. The ALG3 gene, the ALG11 gene, and the OCH1 gene disrupted means that these genes are not expressed. Any method for disrupting these genes may be used, and is not particularly limited. In one embodiment, gene disruption is preferably performed using homologous recombination, and can be performed, for example, by the method disclosed in JP 2015-100327 A.

The ALG3 gene encodes α1,3 mannosyltransferase involved in the synthesis of lipid-linked glycans. α1,3 mannosyltransferase uses dolichol phosphate mannose as its donor substrate. An example of the base sequence of the ALG3 gene is shown in Figure 1. The ALG11 gene encodes α1,2 mannosyltransferase involved in the synthesis of lipid-linked glycans. α1,2 mannosyltransferase uses GDP mannose as its donor substrate. An example of the base sequence of the ALG11 gene is shown in Figure 2. The OCH1 gene encodes α1,6 mannosyltransferase that works in the Golgi apparatus. An example of the base sequence of the OCH1 gene is shown in Figure 3.

By disrupting the ALG3, ALG11, and OCH1 genes of Cryptococcus, the addition of sugar chains to proteins is controlled, and when horseradish-derived peroxidase is recombinantly produced using this as a host, the molecular weight including the sugar chain is equivalent to the molecular weight including the sugar chain of the peroxidase produced in horseradish. In one embodiment, the horseradish-derived peroxidase recombinantly produced using a mutant Cryptococcus as a host preferably has a molecular weight including the sugar chain (measured by SDS-PAGE) of about 45 kDa. In addition, the various enzymatic properties of the peroxidase recombinantly produced in this manner are equivalent to those of the peroxidase produced by horseradish. Therefore, the Cryptococcus mutant is useful as a host for producing peroxidase equivalent to the peroxidase produced by horseradish.

In one embodiment, the Cryptococcus mutant is preferably transformed with a gene encoding horseradish peroxidase so that it can produce the horseradish peroxidase. The base sequence (SEQ ID NO: 10) of the gene encoding horseradish peroxidase is publicly known. In one embodiment, the base sequence of the gene encoding horseradish peroxidase is preferably optimized in terms of codon usage so as to be suitable for expression in a Cryptococcus host, as described in Patent Document 2.

In one embodiment, the base sequence of the gene encoding horseradish peroxidase preferably has an identity of 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the base sequence of SEQ ID NO: 10. Here, "identity" can be calculated using an analysis tool that is commercially available or available via a telecommunication line (Internet). For example, specifically, in Advanced BLAST 2.1, the identity value (%) of the nucleotide sequence can be calculated by searching using the program blastn with various parameters set to default values.

In one embodiment, the gene encoding the horseradish-derived peroxidase preferably has a signal sequence upstream so that the peroxidase produced by its expression is secreted outside the bacterial cell. As the signal peptide, for example, those described in Patent Document 2 or 3 can be used. In one embodiment, the signal peptide is preferably a secretory signal peptide of acid xylanase of Cryptococcus sp. S-2 strain. In addition, in one embodiment, the gene encoding the horseradish-derived peroxidase is preferably one in which the vacuolar retention signal peptide has been removed so that the amount remaining in the bacterial cell is reduced.

The method of transformation is not particularly limited, and can be performed by incorporating DNA having the base sequence into an appropriate vector and introducing it into a Cryptococcus bacterium. Transformation can also be performed using homologous recombination. In one embodiment, transformation with a gene encoding horseradish peroxidase can also be performed after disrupting the ALG3 gene, the ALG11 gene, and the OCH1 gene.

The Cryptococcus bacterium (hereinafter also referred to as "gene-undisrupted Cryptococcus bacterium") in which the ALG3 gene, ALG11 gene, and OCH1 gene are to be disrupted is arbitrary and is not particularly limited. Specific examples of gene-undisrupted Cryptococcus bacterium include Cryptococcus sp. S-2, Cryptococcus liquefaciens, Cryptococcus flavus, Cryptococcus curvatus, and the like. In one embodiment, a preferred gene-undisrupted Cryptococcus bacterium is Cryptococcus sp. S-2. Cryptococcus sp. The S-2 strain was internationally deposited at the International Patent Biological Depositary Center under the deposit number FERM BP-10961 on September 5, 1995.

In one embodiment, the gene-undisrupted Cryptococcus bacteria are preferably uracil-requiring. Gene disruption can be performed efficiently by utilizing uracil requirement. Uracil-requiring Cryptococcus bacteria can be obtained by any method. For example, uracil-requiring Cryptococcus bacteria can be obtained by irradiating Cryptococcus bacteria with ultraviolet light to cause mutations, as described in Patent Document 1 or 2. Specifically, Cryptococcus bacteria can be obtained by irradiating Cryptococcus bacteria with ultraviolet light, culturing them in a medium containing 5-fluoroorotic acid, and selecting a strain that grows. In one embodiment, a preferred uracil-requiring gene-undisrupted Cryptococcus bacteria is the Cryptococcus sp. S-2 U5 strain.

In one embodiment, the gene-undisrupted Cryptococcus bacterium preferably has a disrupted KU70 gene. By disrupting the KU70 gene, the frequency of homologous recombination can be increased, and the ALG3 gene, the ALG11 gene, and the OCH1 gene can be disrupted more efficiently by homologous recombination. Disruption of the KU70 gene can be performed, for example, by the method described in Patent Document 3.

In one embodiment, the gene-undisrupted Cryptococcus bacterium is preferably adenine-requiring. Gene disruption can be performed efficiently by utilizing adenine requirement. Adenine-requiring Cryptococcus bacterium can be obtained by any method. For example, adenine-requiring Cryptococcus bacterium can be obtained by disrupting the ADE1 gene encoding phosphoribosyl-aminoimidazole synthetase, as described in Patent Document 2 or 3. In one embodiment, a preferred adenine-requiring gene-undisrupted Cryptococcus bacterium is the Cryptococcus sp. S-2 D11 strain. The Cryptococcus sp. S-2 D11 strain has been internationally deposited at the International Patent Biological Depositary Center under the deposit number FERM BP-11482.

In one embodiment, the gene-undisrupted Cryptococcus bacterium is uracil-requiring, adenine-requiring, and preferably has a disrupted KU70 gene.

2. Method for producing mutant strain of Cryptococcus A mutant strain of Cryptococcus can be produced by disrupting the ALG3 gene, ALG11 gene, and OCH1 gene of Cryptococcus. The ALG3 gene, ALG11 gene, and OCH1 gene, Cryptococcus bacteria, and the method for disrupting the genes are as described above. The gene disruption may be performed on Cryptococcus bacteria transformed with a gene encoding horseradish-derived peroxidase, or on Cryptococcus bacteria that have not been transformed. In the latter case, the Cryptococcus bacteria can be transformed with a gene encoding horseradish-derived peroxidase during or after the gene disruption process.

In one embodiment, as described above, it is preferable that the Cryptococcus bacterium to be subjected to the gene disruption has a disruption in uracil requirement, adenine requirement, and/or the KU70 gene.

3. Method for Producing Horseradish Peroxidase Horseradish peroxidase can be produced by culturing the above-mentioned Cryptococcus mutant transformed with a gene encoding horseradish peroxidase.

Culture conditions may be appropriately selected taking into consideration the nutritional and physiological properties of the host. Liquid culture is usually used, and aeration and agitation culture is preferred. Nutrient sources that are commonly used in the culture of microorganisms can be widely used. Examples of nitrogen sources that can be used include peptone, meat extract, yeast extract, casein hydrolysate, and alkaline hydrolysate of soybean meal.

Carbon sources include, for example, glucose, sucrose, lactose, maltose, xylose, molasses, pyruvic acid, etc. Other nutrient sources include salts of phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, zinc, etc., amino acids, and vitamins.

The culture temperature can be appropriately selected within the range at which the Cryptococcus mutant strain grows and produces peroxidase, and is usually around 20 to 25°C. The culture time can be terminated at an appropriate time when the peroxidase yield reaches its maximum, and is usually around 60 to 120 hours. The pH of the medium can be appropriately selected within the range at which the Cryptococcus mutant strain grows and produces peroxidase, and is usually around pH 3.0 to 9.0.

The culture medium containing the fungal cells obtained by culturing the above-mentioned Cryptococcus mutant strain can be collected as is and used as peroxidase, but it is generally preferable to separate the peroxidase in advance by filtering or centrifuging the culture medium in accordance with conventional methods.

In one embodiment, it is preferable to purify peroxidase from a peroxidase-containing culture solution and use it. Examples of purification methods include vacuum concentration, membrane concentration, salting out with ammonium sulfate, sodium sulfate, etc., fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, acetone, etc., heating treatment, isoelectric point treatment, gel filtration with an adsorbent or gel filtration agent, adsorption chromatography, ion exchange chromatography, hydrophobic interaction chromatography, etc.

In one embodiment, the horseradish peroxidase produced using a Cryptococcus mutant as a host has properties equivalent to those of the peroxidase produced by horseradish. Here, the properties include the molecular weight including the glycan, the optimum activity temperature, the optimum activity pH, the temperature stability, the pH stability, and the Km value. For example, the horseradish peroxidase produced using a Cryptococcus mutant as a host has a molecular weight including the glycan (measured by SDS-PAGE) of about 45 kDa, an optimum activity temperature of about 45° C., an optimum activity pH of about 6.5, and is stable at temperatures below 50° C. and at a pH of 5.0 to 10.0. In one embodiment, the horseradish peroxidase preferably has the amino acid sequence of SEQ ID NO: 11. The amino acid sequence of SEQ ID NO: 11 does not include a secretory signal peptide and a vacuolar retention signal peptide.

The present invention will be described in further detail below with reference to examples, but the present invention is not limited to these.

Test 1. Acquisition of sugar chain synthesis genes (ALG3 gene, ALG11 gene, OCH1 gene) of Cryptococcus sp. S-2 strain 1-1. Preparation of Cryptococcus sp. S-2 strain genomic DNA 50 ml of YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% polypeptone, 1.0% glucose) was placed in a 500 mL Sakaguchi flask and sterilized by autoclave to prepare a medium. Cryptococcus sp. S-2 strain, which had been restored in advance with YM plate medium, was inoculated into the YM medium with a platinum loop, cultured with shaking at 25 ° C. and 180 rpm for 2 days, and filtered with Miracloth (Merck Co., Ltd.) to obtain the bacterial cells. The obtained cells were freeze-fractured in liquid nitrogen, 5 ml of a mixture of phenol:chloroform:isoamyl alcohol (25:24:1, pH 7.2) was added, thoroughly suspended, and centrifuged at 3000 rpm for 20 minutes at room temperature, and about 4 ml of the aqueous layer was used as a sample. Genomic DNA was extracted from this sample using MagExtractor-PlantGenome- (manufactured by Toyobo).

1-2. Determination of genome sequence of Cryptococcus sp. S-2 strain The genomic DNA obtained in 1-1 was fragmented using Nextera XT Sample prep kit (manufactured by Illumina), and tagmentation was performed. The tagmented fragmented genomic DNA was clustered on a flow cell using Miseq Reagent kit v2 300 cycles (Illumina), and data acquisition was performed using Miseq (Illumina). Based on the obtained FASTQ file, de novo assembly was performed according to the standard method using CLC GENOMICS Workbench Ver7.5 to obtain whole genome information of Cryptococcus sp. S-2 strain.

1-3. Acquisition of sugar chain synthesis genes (ALG3 gene, ALG11 gene, OCH1 gene) derived from Cryptococcus sp. S-2 strain Based on the whole genome information acquired in 1-2, a BLAST search was performed using existing sugar chain synthesis genes derived from Aspergillus oryzae as a query, and the base sequences of the ALG3 gene, ALG11 gene, and OCH1 gene were identified. The base sequence of the ALG3 gene (SEQ ID NO: 1) and its upstream and downstream base sequences on the genomic DNA are shown in FIG. 1. The ALG11 gene (SEQ ID NO: 2) and its upstream and downstream base sequences on the genomic DNA are shown in FIG. 2. The OCH1 gene (SEQ ID NO: 3) and its upstream and downstream base sequences on the genomic DNA are shown in FIG. 3.

Test 2. Acquisition of a glycan synthesis enzyme gene disruptant The breeding process for acquiring the recombinant peroxidase producing strain, Cryptococcus sp. OC106, is shown in FIG. 4. First, the Cryptococcus sp. S-2 strain was subjected to a mutation treatment by ultraviolet irradiation to obtain the Cryptococcus sp. U5 strain, which became uracil auxotrophic. The ADE1 gene was removed from the Cryptococcus sp. U5 strain by the Pop in-Pop out method described in JP 2015-100327 A to obtain the Cryptococcus sp. A1U5 strain. Cryptococcus sp. In order to increase the efficiency of homologous recombination, the KU70 gene was removed from the A1U5 strain by the Pop in-Pop out method using a uracil marker as a target, and the AK223 strain was obtained. The horseradish peroxidase gene was introduced into the AK223 strain by the Pop in method using an adenine marker as a target, and the ADE1-PEO strain was obtained. The ALG3 gene and the ALG11 gene were removed from the ADE1-PEO strain by the Pop in-Pop out method using a uracil marker as a target, and the AK223 strain was obtained. AK223 (Δalg3, Δalg11) strain was obtained. Cryptococcus sp. AK223 (Δalg3, Δalg11) strain was subjected to a mutation treatment by ultraviolet irradiation, and the productivity of peroxidase was used as an indicator to obtain Cryptococcus sp. 3u-4P11G strain. For Cryptococcus sp. 3u-4P11G strain, the OCH1 gene was disrupted by the Pop-in method using a uracil marker as a target, and the Cryptococcus sp. OC106 strain was obtained, using the sugar chain content imparted to peroxidase as an indicator.

Test 3. Obtaining recombinant peroxidase Cryptococcus sp. OC106 strain was cultured on SC (-ura) agar medium (1.34% Yeast Nitrogen Base w/o Amino acids, 0.154%-ura DO supplement, 4% D-Glucose, 3% agar powder) at 25 ° C. for 3 days to obtain colonies. The obtained colonies were cultured in 60 mL liquid medium (1% Glufinal, 0.3% Bacto Yeast Extract, 0.5% Bacto Peptone, 0.3% Malt Extract, 0.04% Adecanol LG-126, 0.002% Tetracycline Hydrochloride) at 25 ° C. for 48 hours to obtain a seed culture solution. The seed culture was cultured in a 10 L jar fermenter in a production medium (7% Meest P1G, 0.011% ferrous sulfate, 0.035% Hemron 2HiWS, 0.04% Adekanol LG-126, 4.0% xylose, 0.002% tetracycline hydrochloride, 50% xylose fed at 6 mL/hr) at 25° C. for 6 days. The cultured cells were filtered, and the culture supernatant was obtained as a crude enzyme solution.

The recombinant peroxidase was isolated and purified from the above crude enzyme solution by the following steps (1) to (4).
(1) Concentration and desalting The crude enzyme solution was passed through an ultrafiltration membrane with a molecular weight cutoff of 100,000, and further concentrated using an ultrafiltration membrane with a molecular weight cutoff of 30,000. The solution was then replaced with 10 mM sodium acetate buffer (pH 4.5) to obtain a crude enzyme concentrate.

(2) Purification by SP Sepharose (GE Healthcare) The crude enzyme concentrate was passed through an SP-Sepharose FastFlow column pre-equilibrated with 10 mM sodium acetate buffer (pH 4.5) to adsorb the enzyme. After washing the column with the same buffer, the enzyme was eluted by gradient elution from the same buffer to 10 mM sodium acetate buffer (pH 4.5) containing 100 mM sodium chloride, and active fractions were collected.

(3) Purification by Octyl-sepharose FastFlow (GE Healthcare) The active fraction was adjusted to 60% ammonium sulfate saturation (pH 4.5), and then centrifuged to obtain a supernatant. The supernatant was passed through an Octyl-sepharose FastFlow column that had been pre-equilibrated with 10 mM sodium acetate buffer (pH 4.5) containing 60% saturated ammonium sulfate to adsorb the enzyme. After washing the column with the same buffer, the enzyme was eluted by gradient elution from the same buffer to 10 mM sodium acetate buffer (pH 4.5), and the active fraction was collected.

(4) Concentration and desalting The active fraction was concentrated using an ultrafiltration membrane with a molecular weight cutoff of 10,000, and the concentrate was replaced with ion-exchanged water.

Test 4. Evaluation of Enzyme Characteristics The enzymatic characteristics of the recombinant peroxidase purified in Test 3 were evaluated. As a comparative control, the characteristics of wild-type peroxidase derived from horseradish (PEO-302, manufactured by Toyobo Co., Ltd.) were also evaluated in the same manner.

Method for measuring peroxidase activity: Prepare a mixed solution of 1.5 ml of 1 mM ABTS/100 mM sodium acetate buffer (pH 4.5) and 1.5 ml of 5.8 mM H ₂ O ₂ aqueous solution in a test tube, and preheat at 25°C for about 5 minutes. Add 0.1 ml of peroxidase enzyme solution appropriately diluted with (50 mM phosphate buffer (pH 6.0)/0.1% Triton-X100) to this reaction solution, gently mix, and then record the absorbance change at 405 nm for 5 minutes using a spectrophotometer controlled at 25°C for water, and measure the absorbance change per minute (ΔOD _test ) from the linear portion. For the blank test, add a solution for dissolving and diluting peroxidase to the reagent mixture instead of the POD solution, and measure the absorbance change per minute (ΔOD _blank ) in the same manner. From these values, calculate the peroxidase activity according to the following formula. Here, one unit (U) of peroxidase activity is defined as the amount of enzyme that oxidizes 1 μmole of ABTS per minute under conditions of pH 4.5 and 25° C.

U/ml=(ΔOD _test −ΔOD _blank )×dilution ratio×3.1/(34.7×0.1×1.0)

In the formula, 3.1 represents the volume (ml) of the reaction reagent + enzyme solution, 34.7 represents the millimolar absorption coefficient ( ^cm2 /micromole) under these activity measurement conditions, 0.1 represents the volume (ml) of the enzyme solution, and 1.0 represents the optical path length (cm) of the cell.

The molecular weight of each purified enzyme was determined by SDS-polyacrylamide gel electrophoresis using Nu-PAGE 4-12% Bis-Tris Gel (Invitrogen). The electrophoretic samples are as follows:
Lane 1: Molecular weight marker (Thermo Scientific, Benchmark Protein Ladder)
Lane 2: PEO-302 (Toyobo Co., Ltd., purified wild-type peroxidase enzyme derived from horseradish)
Lane 3: rPEO (recombinant peroxidase purified enzyme, derived from ADE1-PEO strain)
Lane 4: rPEO (recombinant peroxidase purified enzyme, derived from Cryptococcus sp. OC106 strain)

As shown in Figure 5, the molecular weight including the glycan of the recombinant peroxidase derived from Cryptococcus sp. OC106 strain was approximately 45 kDa, which was found to be equivalent to the molecular weight of wild-type peroxidase derived from horseradish. On the other hand, the molecular weight including the glycan of the recombinant peroxidase derived from Cryptococcus sp. ADE1-PEO strain, in which the glycan synthesis gene was not destroyed, was approximately 55 kDa, suggesting the possibility that excess glycan had been attached to increase the molecular weight.

The enzyme activity was measured at 20° C. to 70° C. As a result, as shown in FIG. 6, the optimum temperature for both the wild-type peroxidase derived from horseradish and the recombinant peroxidase derived from Cryptococcus sp. OC106 strain was 45° C.

4-3. Optimal pH
The wild-type peroxidase derived from horseradish and the recombinant peroxidase derived from Cryptococcus sp. OC106 were dissolved in 100 mM acetate buffer (pH 3.0-6.0) and 100 mM potassium phosphate buffer (pH 6.0-8.0), respectively. The relative activity was calculated after adjusting the concentration to 1 U/ml with 500 mL of water. As a result, as shown in FIG. 7, the optimal pH of the wild-type peroxidase derived from horseradish and the recombinant peroxidase derived from Cryptococcus sp. The pH was in the range of 6.5.

4-4. Thermal stability Horseradish wild-type peroxidase and Cryptococcus sp. OC106 recombinant peroxidase were each prepared in 50 mM potassium phosphate buffer (pH 6.0) at a concentration of 2 U/ml, and treated at temperatures of 37° C. to 80° C. for 10 minutes to calculate the residual activity. As a result, as shown in FIG. 8, both horseradish wild-type peroxidase and Cryptococcus sp. OC106 recombinant peroxidase maintained 90% or more of activity up to 50° C.

4-5. pH Stability The wild-type peroxidase derived from horseradish and the recombinant peroxidase derived from Cryptococcus sp. OC106 strain were prepared at a concentration of 2 U/ml in 100 mM acetate buffer (pH 3.5-6.0), 100 mM potassium phosphate buffer (pH 6.5-8.0), and 100 mM glycine-NaOH buffer (pH 9.0-12.0), respectively, and treated at 25° C. for 20 hours, and the residual activity was calculated. As a result, as shown in FIG. 9, both the wild-type peroxidase derived from horseradish and the recombinant peroxidase derived from Cryptococcus sp. OC106 strain maintained 90% or more of activity in the pH range of 5.0 to 10.0.

4-6. Km value for hydrogen peroxide The Km value of each purified enzyme was calculated by appropriately adjusting the concentration of hydrogen peroxide. As a result, as shown in Figure 10, the Km value of the wild-type peroxidase derived from horseradish was 1.5 mM, while the Km value of the recombinant peroxidase derived from Cryptococcus sp. OC106 strain was 1.6 mM, which was equivalent.

OC106-derived recombinant peroxidase and horseradish-derived wild-type peroxidase were adjusted to 10 U/mL with 25 mM PIPES-NaOH (pH 7.5) and then stored at 4° C. for 1 week and at 37° C. for 3 weeks. Then, the following reagents were prepared and 5 mg/dL creatinine was measured.

<First Reagent>
25 mM PIPES-NaOH (pH 7.5)
0.39g/dL EDTA/3Na
3.3 g/dL NaCl
1.0 g/dL Triton X-100
50 U/mL creatine amidinohydrolase (Toyobo CRH-221)
12 U/mL sarcosine oxidase (Toyobo SAO-351)

<Second Reagent>
25 mM PIPES-NaOH (pH 7.5)
0.25g/dL NaN3
0.045g/dL Potassium Ferrocyanide
1.0 g/dL Triton X-100
300 U/mL creatinine amide hydrolase (Toyobo CNH-311)
0.0-1.2 U/mL peroxidase

A Hitachi 7060-type automatic analyzer was used. 270 μL of the first reagent was added to 6 μL of sample and incubated at 37° C. for 5 minutes to prepare the first reaction. Then, 90 μL of the second reagent was added and incubated for 5 minutes to prepare the second reaction. The absorbance at 546 nm was measured using a two-endpoint method in which the absorbance of the first reaction and the second reaction were corrected for the liquid volume and the difference in absorbance was taken. As a result, the reaction curves when creatinine was measured using the creatinine reagent prepared after storing each enzyme at 4° C. for 1 week were equivalent as shown in FIG. 11A. The reaction curves were also equivalent when the creatinine reagent prepared after storing each enzyme at 37° C. for 3 weeks was used, as shown in FIG. 11B.

Based on this, we believe that the wild-type peroxidase derived from horseradish and the recombinant peroxidase derived from Cryptococcus sp. OC106 strain have equivalent performance.

Claims (4)

A mutant strain of the genus Cryptococcus in which the ALG3 gene, the ALG11 gene, and the OCH1 gene have been disrupted and which has been transformed with a gene encoding a horseradish-derived peroxidase having 90% or more identity to the base sequence of SEQ ID NO: 10. A method for producing a mutant strain of Cryptococcus, comprising disrupting the ALG3 gene, ALG11 gene, and OCH1 gene of Cryptococcus, and transforming Cryptococcus with a gene encoding a horseradish peroxidase having 90% or more identity to the base sequence of SEQ ID NO: 10. A method for producing a mutant strain of Cryptococcus, comprising disrupting the ALG3 gene, the ALG11 gene, and the OCH1 gene of Cryptococcus, wherein the Cryptococcus is transformed with a gene encoding a horseradish-derived peroxidase having 90% or more identity to the base sequence of SEQ ID NO: 10. A method for producing horseradish peroxidase, comprising culturing the mutant strain of Cryptococcus sp. according to claim 1.

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