KR102398203B1 - Novel oxygenase from fungi Podospora anserina S mat+ and method for preparing thereof - Google Patents

Abstract

The present invention relates to a novel oxygenase derived from fungus Podospora anserine S mat+ and a method for producing the same, and more specifically, to a novel oxygenase, polynucleotide encoding the same, an expression vector, a transformant, and a method for producing an oxygenase using the same. The novel oxygenase derived from fungus Podospora anserine S mat+ does not require expensive electron transfer systems that require coenzymes or reductases, and exhibits various oxidation reactions and wide substrate specificity, and thus has the advantage of producing C-H products in an inexpensive manner by adding oxygen to C-H bonds with respect to a variety of substrates.

Description

Novel oxygenase from fungi Podospora anserina S mat+ and method for preparing thereof

The present invention relates to a novel oxygenation enzyme derived from the fungus Podospora anserina S mat+ and a production method thereof, and more particularly, to an oxygenation enzyme and a polynucleotide encoding the same, an expression vector, a transformant, and an oxygenation enzyme using the same It is about production methods.

Unspecific peroxygenase (UPO), an enzyme that exists only in fungi or mushrooms and is secreted out of cells, catalyzes a wide variety of oxidation reactions such as CH hydroxylation for a wide variety of substances in and outside the body. It exhibits the same reaction diversity as the P450 enzyme (Cytochrome P450) that was found to be The existing CH-bonded oxygen-activated P450 enzyme was limited in its development as an industrial enzyme because of its dependence on expensive coenzyme and reductase. On the other hand, UPO catalysis does not require an expensive electron transfer system (coenzyme or reductase) compared to the catalysis of the P450 enzyme and catalyzes the reaction only with inexpensive hydrogen peroxide (H ₂ O ₂ ). It is very inexpensive and has the advantage of exhibiting high activity and productivity. In addition, UPO is very important industrially because of its high applicability to various substances such as fine chemicals, biofuels, drug metabolites, and bioactive compounds. Recently, there have been several attempts to develop new oxygenating enzymes such as UPO that can be used in various industries, but industrially produced oxygenating enzymes have not yet been identified.

Korean Patent No. 10-1459817 Korean Patent No. 10-1803174

An object of the present invention is to provide a novel oxygenating enzyme derived from the fungus Podospora anserina S mat+.

Another object of the present invention is to provide a polynucleotide encoding the above oxygenation enzyme.

Another object of the present invention is to provide an expression vector comprising the polynucleotide.

Another object of the present invention is to provide a transformant into which the expression vector is introduced.

Another object of the present invention is to provide a method for producing an oxygenation enzyme using the transformant.

As a result of research to develop a new oxygenation enzyme, the present inventors discovered a novel oxygenation enzyme using a non-specific peroxygenase gene present in the mold Podospora anserina S mat+, and the oxygenation enzyme The present invention was completed by developing a transformant capable of mass-producing and confirming that the oxygenation enzyme has various oxidation reactions and broad substrate specificity.

One aspect of the present invention provides an oxygenation enzyme comprising the amino acid sequence of SEQ ID NO: 5.

In the present invention, “oxygenase” refers to an enzyme that adds molecular oxygen (O ₂ ) to a substrate among oxidoreductases that catalyze oxidation or reduction reactions of organic compounds. Aromatic peroxygenase, mushroom peroxygenase, haloperoxidase-peroxygenase, agrocybe erita peroxygenase present in mushrooms ( Agrocybe aegerita peroxidase) refers to non-specific peroxidase (UPO). Since this non-specific peroxygenase catalyzes an enzymatic reaction using hydrogen peroxide (H ₂ O ₂ ) as shown in Scheme 1 below, it is also called hydrogen peroxide oxidoreductase, and contains heme and thiolate such as the P450 enzyme. It belongs to the heme-thiolate protein.

[Scheme 1]

According to one embodiment of the present invention, the oxygenation enzyme may be peroxygenase, preferably non-specific peroxygenase.

According to one embodiment of the present invention, the oxygenation enzyme may be derived from the fungus Podospora anserina , preferably from Podospora anserina S mat+.

The Podospora anserina S mat + is a Podospora anserina belonging to the pseudohomothallic ascomyceum, and is an S strain that produces dikaryotic ascospores having mat +. and a specific peroxygenase gene. This non-specific peroxygenase gene is SEQ ID NO: 2 by applying codon optimization to increase protein synthesis in E. coli into which the non-specific peroxygenase gene is introduced for mass production of a novel oxygenation enzyme. It may be represented by the nucleotide sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 3, and by removing the signal peptide associated with the extracellular secretion of the fungus to increase the expression of the synthesized protein, the amino acid sequence of SEQ ID NO: 5 can

The oxygenation enzyme comprising the amino acid sequence of SEQ ID NO: 5 in the present invention is a variant of an amino acid having a different sequence by deletion, insertion, substitution, or a combination of amino acid residues within the range that does not affect the function of the protein. , or fragments. Amino acid exchange at the protein and peptide level that does not entirely alter the activity of the oxygenating enzyme is known in the art. In some cases, it may be transformed into phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, and the like. Accordingly, the present invention includes a protein having an amino acid sequence substantially identical to the protein comprising the amino acid sequence of SEQ ID NO: 5, and a mutant or active fragment thereof. The 'substantially identical protein' refers to, but is not limited to, those having amino acid sequence homology of at least 80%, preferably at least 90%, and most preferably at least 95%. Here, 'homology' refers to a degree of similarity in the nucleotide sequence or amino acid sequence of a gene encoding a protein, and when the homology is sufficiently high, the expression product of the gene may have the same or similar activity.

The amino acid sequence of SEQ ID NO: 5 may be encoded by a gene consisting of the nucleotide sequence of SEQ ID NO: 4, and as long as it can encode the protein of the present invention having the same amino acid sequence, substantially the same as the nucleotide sequence of SEQ ID NO: 4 It may be encoded by a gene consisting of the same different base sequence. The base sequence may be single-stranded or double-stranded, and may be a DNA molecule or an RNA molecule.

The oxygenation enzyme in the present invention catalyzes the reaction of adding oxygen using various kinds of internal and external substances as substrates, and in this case, a coenzyme or a reductase is not separately required.

According to one embodiment of the present invention, the oxygenation enzyme is ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), toluene (Toluene), atorvastatin (Atorvastatin), progesterone (Progesterone), Monacolin (Monacolin) J, phloretin (Phloretin) and naringin (Naringin) may have activity on one substrate selected from the group consisting of DC.

According to an embodiment of the present invention, the oxygenation enzyme exhibits various enzyme activities depending on the type of substrate and reaction conditions, for example, the productivity is 44.3 mM for ABTS, and it is confirmed that the k _cat is 7371 sec ^-1 did

In addition, another aspect of the present invention provides a polynucleotide encoding the oxygenation enzyme.

The polynucleotide may include a nucleotide sequence encoding an oxygenation enzyme comprising the amino acid sequence of SEQ ID NO: 5.

According to one embodiment of the present invention, the polynucleotide may include the nucleotide sequence of SEQ ID NO: 4.

The polynucleotide including the nucleotide sequence of SEQ ID NO: 4 may encode a novel oxygenation enzyme not known in the prior art.

In addition, another aspect of the present invention provides an expression vector comprising the polynucleotide.

In the present invention, the term "expression vector" refers to a gene construct including a gene insert to allow expression of a target protein in an appropriate host cell, and essential regulatory elements operably linked to express the gene insert. The expression vector includes expression control elements such as a start codon, a stop codon, a promoter, and an operator, and the start codon and stop codon are generally considered to be part of a nucleotide sequence encoding a polypeptide, and when the gene construct is inserted or administered It must exhibit action in the subject and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inducible.

Here, 'operably linked' refers to a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the coding sequence. The operative linkage with the expression vector can be prepared using a genetic recombination technique well known in the art, and site-specific DNA cleavage and ligation can use enzymes generally known in the art.

The type of the expression vector is not particularly limited as long as it functions to express a desired gene and produce a desired protein in various host cells of prokaryotic and eukaryotic cells, but it is in a natural state while retaining a promoter showing strong activity and strong expression power. A vector capable of producing a large amount of a foreign protein in a form similar to that of Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII, λgt10, λgt11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pDZ vectors, pBR systems, and pUC systems may be used as plasmid vectors. , pBluescript II-based, pGEM-based, pTZ-based, pCL-based, pET-based and the like can be used. The usable vector is not particularly limited, and a known expression vector may be used.

According to an embodiment of the present invention, the pET28a vector may be used as the expression vector.

In addition, the expression vector may include a selection marker, the selection marker is for selecting a transformant (host cell) transformed with the vector, the selection marker in the medium treated with the selection marker Since only expressing cells can survive, selection of transformed cells is possible. Representative examples of the selection marker include, but are not limited to, kanamycin, streptomycin, and chloramphenicol.

In addition, another aspect of the present invention provides a transformant introduced with the expression vector.

As used herein, “transformant” refers to a cell transfected with a vector having a gene encoding one or more target proteins introduced into a host cell to express the target protein. Vector introduction is electroporation, heat-shock, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof.

The host cells include cells transfected, transformed, or transfected with the expression vector or polynucleotide of the present invention in vitro. A host cell comprising the expression vector of the present invention is a recombinant host cell, a recombinant cell or a recombinant microorganism. In this case, the host cell may be any cell such as a eukaryotic cell, a prokaryotic cell, etc., but is not particularly limited thereto, E. coli, Streptomyces, Salmonella typhimurium Bacterial cells such as; yeast cells; fungal cells such as Pichia pastoris ; insect cells such as Drosophila and Spodoptera Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bow melanoma cells; or plant cells.

According to one embodiment of the present invention, the transformant is preferably E. coli.

In addition, another aspect of the present invention provides a method for producing an oxygenation enzyme comprising the step of culturing the transformant.

The culture may be made according to an appropriate medium and culture conditions known in the art, and those skilled in the art can easily adjust the medium and culture conditions for use. Specifically, the medium may be a liquid medium, but is not limited thereto. The culture method may include, for example, batch culture, continuous culture, fed-batch culture, or a combination culture thereof, but is not limited thereto. The medium should meet the requirements of a particular strain in an appropriate manner, and may be appropriately modified by a person skilled in the art.

According to one embodiment of the present invention, the medium may include various carbon sources, nitrogen sources and trace element components. Carbon sources that can be used include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, fatty acids such as linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture, but are not limited thereto. Nitrogen sources that can be used include peptone, yeast extract, broth, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen source may also be used individually or as a mixture, but is not limited thereto. Sources of phosphorus that may be used include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth, but is not limited thereto. In addition, essential growth substances such as amino acids and vitamins may be included. In addition, precursors suitable for the culture medium may be used. The medium or individual components may be added batchwise or continuously by an appropriate method to the culture medium during the culturing process, but is not limited thereto.

According to one embodiment of the present invention, it is possible to adjust the pH of the culture medium by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the microorganism culture medium in an appropriate manner during culture. In addition, it is possible to suppress the formation of bubbles by using an antifoaming agent such as fatty acid polyglycol ester during culture. Additionally, in order to maintain the aerobic state of the culture medium, oxygen or oxygen-containing gas (eg, air) may be injected into the culture medium. The temperature of the culture medium may be usually 20 °C to 45 °C, for example, 25 °C to 40 °C. The incubation period may be continued until a useful substance is obtained in a desired production amount, and may be, for example, 10 to 160 hours.

According to one embodiment of the present invention, after the transformant culturing step, it may further include the step of recovering the oxygenated enzyme from the cultured transformant and the medium containing the same.

In the recovery step, the oxygenated enzyme produced from the medium may be collected or recovered using a suitable method known in the art depending on the culture method. e.g. centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g. ammonium sulfate precipitation), chromatography (e.g. ion exchange, affinity, hydrophobicity and size exclusion) may be used, but the present invention is not limited thereto.

According to one embodiment of the present invention, in the step of recovering the oxygenated enzyme, the biomass is removed by centrifuging the culture medium at low speed, and the obtained supernatant may be separated through ion exchange chromatography.

According to one embodiment of the present invention, the step of recovering the oxygenation enzyme may include a step of purifying the oxygenation enzyme.

The novel oxygenating enzyme derived from the fungus Podospora anserina S mat+ according to the present invention does not require an expensive electron transport system requiring coenzyme or reductase, and exhibits various oxidation reactions and broad substrate specificity, thereby There is an advantage that the CH product can be produced inexpensively by adding oxygen.

1 shows a vector map cloned UPO gene derived from the fungus Podospora anserina S mat+ according to an embodiment of the present invention.
Figure 2 is a graph measuring the CO spectrum of the whole cell by centrifugation after culturing a transformant producing PAUPO according to an embodiment of the present invention.
Figure 3 is a graph measuring the CO spectrum with the supernatant obtained by lysis (lysis) of the transformant producing PAUPO according to an embodiment of the present invention.
Figure 4 is a graph measuring the CO spectrum after purifying the expressed PAUPO according to an embodiment of the present invention using a His-tag.
5 is a graph measuring UV spectrum after purification of expressed PAUPO according to an embodiment of the present invention using His-tag.
6 is an SDS-PAGE result confirming whether the expressed PAUPO was purified according to an embodiment of the present invention.
7 is a graph measuring PAUPO activity with respect to toluene according to an embodiment of the present invention.
8 is a graph measuring PAUPO activity for atorvastatin according to an embodiment of the present invention.
9 is a graph measuring PAUPO activity for progesterone according to an embodiment of the present invention.
10 is a graph measuring PAUPO activity for monacolin J according to an embodiment of the present invention.
11 is a graph measuring PAUPO activity for phloretin according to an embodiment of the present invention.
12 is a graph measuring PAUPO activity against naringin DC according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail. However, these descriptions are provided for illustrative purposes only to help the understanding of the present invention, and the scope of the present invention is not limited by these illustrative descriptions.

Example 1. Mold Podospora anserina Construction of transformant containing UPO gene derived from S mat+

In order to construct a transformant capable of producing a novel oxygenation enzyme, the UPO gene and E. coli C2566 derived from the fungus Podospora anserina S mat+ containing the nucleotide sequence shown in SEQ ID NO: 1 were used.

First, in order to express the Podospora anserina S mat+ UPO gene in E. coli, codon optimization was performed for E. coli (SEQ ID NO: 2). Thereafter, a polynucleotide (SEQ ID NO: 4) was prepared as an insert for insertion into E. coli by deleting a codon corresponding to a signal peptide from the codon-optimized nucleotide sequence. Gibson assembly was used to clone the insert into the pET28a vector. Four primers were prepared and used so that 17 base pairs and 17 base pairs of the polynucleotide overlap at the vector site. The primers used are shown in Table 1 below, and a vector map including the four primers is shown in FIG. 1 .

Primer name Primer sequence (5' > 3') SEQ ID NO: F3 (Forward 3) GTGCCGCGCGGCAGCCATATGGCTGAGTTGGATT SEQ ID NO: 6 R3 (Reverse 3) AATCCAACTCAGCCATATGGCTGCCGCGCGGCAC SEQ ID NO: 7 F2 (Forward 2) CATATTTTGGGCATATAACTCGAGCACCACCACCA SEQ ID NO: 8 R2 (Reverse 2) TGGTGGTGGTGCTCGAGTTATATGCCCAAATATG SEQ ID NO: 9

The vector and primers F2 and R3, the insert and primers F3 and R2 were respectively added and amplified by PCR (amplification conditions: 30 cycles; 98°C for 10 seconds, 55°C for 15 seconds, 72°C for 5 seconds). Restriction enzyme Dpn I was treated to remove the parental plasmid only when the vector was amplified among the amplified PCR products. After gel extraction to obtain a PCR product, it was cloned by Gibson PCR. After that, the Gibson PCR product was transformed into E. coli C2566 by electroporation, and 20 uL was smeared on an LK plate. Transformants were obtained by culturing them at 37°C overnight. A colony of the transformant was collected and placed in 3-5 mL of LB liquid medium (LK liquid medium) containing kanamycin and cultured at 37° C., 200 rpm for 17 hours (overnight). After DNA was extracted from the cultured colonies with mini prep, the size of the DNA was checked by electrophoresis, and the DNA of the right size was isolated.

Example 2. Mold Podospora anserina Expression and purification of S mat+-derived UPO (PAUPO)

The cell stock of the cloned transformant was placed in 10 mL of LK broth at 0.1% and incubated for about 17 hours (overnight). Then, the culture medium was inoculated (seeding) at 2% in 250 mL of LK liquid medium, and main culture was performed at 37 ° C., 200 rpm for 2 to 2 hours and 30 minutes. When the OD value of the culture medium was 0.4 ~ 0.8, 0.5 mM ALA (5-aminolevulinic acid) and 0.25 mM IPTG (isopropylthio-β-D-galactopyranoside) were added to induce expression of the desired protein (PAUPO). When it was cultured at 16° C. and 160 rpm, a CO spectrum peak was observed around 40 to 48 hours (see FIG. 2).

Cells were recovered by centrifugation of the culture medium (4999 g, 20 minutes, 4° C.). The recovered cell pellet was washed with 1XPBS (washing). In a cold room in advance, 20% EtOH filled in the column is drained, washed twice with 10 mL of sterile (autoclaved) tertiary water, and then washed once with 10 mL of Lysis buffer. was prepared. The prepared column was always kept dry. The cell pellet was resuspended in lysis buffer 5mL (containing 0.5M NaCl and 20mM Tris[pH7.5]) and the cells were lysed using a sonicator (Misonix, Inx., Farmingdale, NY). The cell lysate was centrifuged at 14,000 rpm, 20 minutes, and 4°C. After centrifugation, only the supernatant was recovered and injected into the His-tag column prepared to collect the flow-through. At this time, a little was left in a crude state. Wash buffer (containing lysis buffer, and 10 mM or 20 mM imidazole) was flowed twice. When the wash buffer flows down, 2 mL of the elution buffer is added and the flowing liquid is collected. After transferring to a centrifuge filter 10KD according to the cell size, centrifugation (3,000rpm, 20 minutes, 4℃) was performed. KPi (pH7.4), which is an enzyme activity buffer, was added, and the cell conditions were changed by repeating 3 times at 3.000rpm, 20 minutes, and 4°C. Only the liquid collected in the filter was recovered, and enzyme activity was measured using a CO spectrum (see FIG. 4 ), and a typical Soret peak of a heme protein was confirmed using a UV spectrum (see FIG. 5 ). Thereafter, it was confirmed by SDS-PAGE whether the purification was done properly (see FIG. 6 ), and finally PAUPO was obtained.

Example 3. Confirmation of PAUPO activity using various substrates

In order to confirm the enzymatic activity of PAUPO, ABTS, toluene, atorvastatin, progesterone, monacolin J, phloretin and naringin DC were used as substrates.

3-1. ABTS

In a cuvette, 0.69 mL of H ₂ O, 0.1 mL of 1M sodium acetate buffer (pH 4.0), 0.1 mL of 2 mM ABTS, and 1 mM H ₂ O ₂ were added. A cuvette was placed in a UV spectrum measuring instrument and measurement was started at 436 nm. Then, 0.1mL of PAUPO was added, mixed well, and the measurement was continued for 300 seconds to obtain the productivity and k _cat value of PAUPO.

As a result, the productivity of PAUPO was 44.3mM, and k _cat was found to be 7371 sec ^-1 .

3-2. toluene

To prepare a reaction mixture of 250uL, put 25uL of 1M sodium acetate buffer (pH 4.0), 5mmol of substrate, and 100pmol of PAUPO in a test tube, and then add H ₂ O ₂ so that the total volume becomes 250uL. H ₂ O was added. Thereafter, 2mM H ₂ O ₂ was added and reacted for 10 minutes. 0.6 mL of ethyl acetate was added and the reaction was terminated by vortexing for 30 seconds. After centrifugation at 3,000 rpm for 10 minutes, 180uL of the supernatant was placed in an HPLC tube. Samples (30 μl) were injected onto a Gemini C18 column (4.6 mm×150 mm, 5 μm, Phenomenex, Torrance, CA). DW for mobile phase A and MeOH containing 0.1% acetic acid for mobile phase B were used. The mobile phase A/B (65/35, v/v) was flowed at a rate of 1 ml·min ⁻¹ using a gradient pump (LC-20AD, Shimadzu, Kyoto, Japan). The eluate was measured with UV of 254 nm.

As a result, as shown in Table 2 and FIG. 7 below, PAUPO was found to have enzymatic activity with respect to toluene.

temperament
(substrate) reaction conditions
(Reaction condition) product
(product) k _cat
(turnover number) (min ^-1 ) toluene pH 4.0 o-Cresol 7.26

3-3. Atorvastatin, Progesterone and Monacolin J

To prepare a reaction mixture of 250uL, 25uL of 1M sodium acetate buffer (pH 4.0), 25mmol of substrate, and 25pmol of PAUPO are added to a test tube, and the total volume including H ₂ O ₂ is 200uL. H ₂ O was added. Thereafter, 2mM H ₂ O ₂ was added at 37° C. and reacted for 30 minutes. Atorvastatin CH ₂ CL ₂ 0.6mL was added, and progesterone and monacolin J were added 0.6mL ethyl acetate and mixed (vortexing) for 30 seconds to terminate the reaction. After centrifugation at 3,000 rpm for 10 minutes, 180 uL of the organic solvent layer was separated, evaporated under nitrogen gas, and analyzed by HPLC. Samples (30 μl) were injected onto a Gemini C18 column (4.6 mm×150 mm, 5 μm, Phenomenex, Torrance, CA). The mobile phase was flowed at a rate of 1 ml·min ^-1 and the eluate was measured by UV (atorvastatin: 287 nm, progesterone: 240 nm, monacolin J: 238 nm).

As a result, as shown in Table 3 and FIGS. 8 to 10, PAUPO was found to have enzymatic activity against atorvastatin, progesterone and monacolin J.

temperament
(substrate) reaction conditions
(Reaction condition) product
(product) k _cat
(turnover number) (min ^-1 ) Atorvastatin pH 4.0 4-OH atorvastatin 0.020 progesterone pH 6.0 Product1 0.10 pH 4.0 Product1 0.070 Monacolin J pH 4.0 Product1 0.13

3-4. Phloretin and Naringin DC

To prepare 250uL of reaction mixture, 25uL of 1M sodium acetate buffer (pH 4.0), substrate (0.5mM phloretin, 0.2mM naringin DC) and 16.4ul of PAUPO were put in a test tube, and then H ₂ O ₂ Including H ₂ O was added so that the total volume (volume) was 250 uL. Thereafter, 2mM H ₂ O ₂ was added at 37° C. and reacted for 30 minutes. The reaction was terminated by adding 0.6 mL of ethyl acetate and mixing (vortexing) for 30 seconds. After centrifugation at 3,000 rpm for 10 minutes, 0.35 mL of the organic solvent layer was separated, evaporated under nitrogen gas, and analyzed by HPLC. Samples (30 μl) were injected onto a Gemini C18 column (4.6 mm×150 mm, 5 μm, Phenomenex, Torrance, CA). The mobile phase was flowed at a rate of 1 ml·min ^-1 , and the eluate was measured by UV with a wavelength of 285 nm.

As a result, as shown in Table 4 and FIGS. 11 and 12 below, PAUPO was found to have enzymatic activity against phloretin and naringin DC.

temperament
(substrate) reaction conditions
(Reaction condition) product
(product) k _cat
(turnover number) (min ^-1 ) phloretin pH 6.0 3-OH phloretin 0.96 pH 4.0 (Product1) 3-OH phloretin 0.27 pH 4.0 (Product2) Product2 0.050 Naringin DC pH 6.0 Neoeriocitrin DC 2.39 pH 4.0 Neoeriocitrin DC 3.6

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Novel oxygenase from fungi Podospora anserina S mat+ and method for preparing <130> PN200362 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 807 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide sequence of UPO gene from fungi Podospora anseina S mat+ <400> 1 atgttgggca ttcgcctggt atctctgctg gccttcactg gctcagccct cgcagaactc 60 gacttctcca agtggaagac tcgccagccc ggcgagctcc gcgctccctg tccggccatg 120 aacagtcttg ccaaccacgg cttcatccag cgtgatggca agaatatcac cgtcgagggc 180 ctcacccctg tcctcaagga ggtcttccat ctcagtcacg agctcgcctt caccgtgtct 240 cagctgggcc tctttacggc gcttgaccca tccaaaggtg tctttactct tcaagacctc 300 acggatcgtc acaacgtctt tgagcacgat gcttccctgt ctcgcgaaga tgccaagttc 360 ggcggtgacc agtctgttct tcataaagga cagttccaaa agttcatgga tcacttcaag 420 ggagaaaagt acatctcctt cgaggccgct gctaaggccc gctatgccat ggtccaggac 480 tcgcgcaaga gaaaccccga cttcacctac gatgtcacgc accgcatcac cagctacggc 540 gaaaccatca agtacctccg taccattgtc gagccctcta cagggaagtg cccggttgac 600 tggatcaaga tcctttttga gcaagagcgt cttccctaca acgagggctg gaggccccct 660 accaatgagc tcagcggctt cagtctcgcg agcgaggttt tggagctggc tttgatcact 720 cctgagaaac ttcccgtcga tgagtgcttg ggcaagggca agggtaaagg aaactgcaag 780 agacggcgca gctaccttgg tatctaa 807 <210> 2 <211> 807 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide sequence of UPO gene - codon optimization <400> 2 atgcttggca ttcgtttagt tagcttgctt gctttcacgg gttccgcgtt ggctgagttg 60 gatttttcta aatggaagac tcggcagcct ggtgagctgc gtgcgccatg tcccgcgatg 120 aatagcttgg ctaaccacgg gtttatccag agagatggga agaatattac agttgaaggc 180 cttactccag tcttaaaaga ggtctttcat cttagccatg aacttgcatt tactgtgtct 240 caattaggtc tgtttactgc gttagatcca tcaaagggag tttttactct tcaggattta 300 accgatagac acaacgtatt tgagcatgac gcgtccttgt cgcgcgaaga cgccaaattc 360 gggggcgatc aatccgtctt acataaaggt caatttcaga agtttatgga tcactttaaa 420 ggagagaaat atataagttt cgaggcggcg gcaaaagcac ggtacgcgat ggttcaagac 480 agtagaaaaa gaaatccaga cttcacttac gatgtaacgc accgtataac ctcgtatggt 540 gagacgatca agtatcttag aactatcgtc gaaccttcca cagggaagtg ccctgttgat 600 tggataaaga tactttttga acaggagcgg cttccgtaca acgaagggtg gcggccccca 660 acgaatgagc tgtcaggatt tagtctggca tctgaggtct tggaattggc tttgattacc 720 cctgagaagt tgcccgtgga tgaatgtttg ggaaaaggaa aaggcaaagg caactgtaag 780 agacggcgtt catatttggg catataa 807 <210> 3 <211> 268 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of UPO gene - codon optimization <400> 3 Met Leu Gly Ile Arg Leu Val Ser Leu Leu Ala Phe Thr Gly Ser Ala 1 5 10 15 Leu Ala Glu Leu Asp Phe Ser Lys Trp Lys Thr Arg Gln Pro Gly Glu 20 25 30 Leu Arg Ala Pro Cys Pro Ala Met Asn Ser Leu Ala Asn His Gly Phe 35 40 45 Ile Gln Arg Asp Gly Lys Asn Ile Thr Val Glu Gly Leu Thr Pro Val 50 55 60 Leu Lys Glu Val Phe His Leu Ser His Glu Leu Ala Phe Thr Val Ser 65 70 75 80 Gln Leu Gly Leu Phe Thr Ala Leu Asp Pro Ser Lys Gly Val Phe Thr 85 90 95 Leu Gln Asp Leu Thr Asp Arg His Asn Val Phe Glu His Asp Ala Ser 100 105 110 Leu Ser Arg Glu Asp Ala Lys Phe Gly Gly Asp Gln Ser Val Leu His 115 120 125 Lys Gly Gln Phe Gln Lys Phe Met Asp His Phe Lys Gly Glu Lys Tyr 130 135 140 Ile Ser Phe Glu Ala Ala Ala Lys Ala Arg Tyr Ala Met Val Gln Asp 145 150 155 160 Ser Arg Lys Arg Asn Pro Asp Phe Thr Tyr Asp Val Thr His Arg Ile 165 170 175 Thr Ser Tyr Gly Glu Thr Ile Lys Tyr Leu Arg Thr Ile Val Glu Pro 180 185 190 Ser Thr Gly Lys Cys Pro Val Asp Trp Ile Lys Ile Leu Phe Glu Gln 195 200 205 Glu Arg Leu Pro Tyr Asn Glu Gly Trp Arg Pro Pro Thr Asn Glu Leu 210 215 220 Ser Gly Phe Ser Leu Ala Ser Glu Val Leu Glu Leu Ala Leu Ile Thr 225 230 235 240 Pro Glu Lys Leu Pro Val Asp Glu Cys Leu Gly Lys Gly Lys Gly Lys 245 250 255 Gly Asn Cys Lys Arg Arg Arg Ser Tyr Leu Gly Ile 260 265 <210> 4 <211> 759 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide sequence of UPO gene - no signal peptide <400> 4 atggctgagt tggatttttc taaatggaag actcggcagc ctggtgagct gcgtgcgcca 60 tgtcccgcga tgaatagctt ggctaaccac gggtttatcc agagagatgg gaagaatatt 120 acagttgaag gccttactcc agtcttaaaa gaggtctttc atcttagcca tgaacttgca 180 tttactgtgt ctcaattagg tctgtttact gcgttagatc catcaaaggg agtttttact 240 cttcaggatt taaccgatag acacaacgta tttgagcatg acgcgtcctt gtcgcgcgaa 300 gacgccaaat tcgggggcga tcaatccgtc ttacataaag gtcaatttca gaagtttatg 360 gatcacttta aaggagagaa atatataagt ttcgaggcgg cggcaaaagc acggtacgcg 420 atggttcaag acagtagaaa aagaaatcca gacttcactt acgatgtaac gcaccgtata 480 acctcgtatg gtgagacgat caagtatctt agaactatcg tcgaaccttc cacagggaag 540 tgccctgttg attggataaa gatacttttt gaacaggagc ggcttccgta caacgaaggg 600 tggcggcccc caacgaatga gctgtcagga tttagtctgg catctgaggt cttggaattg 660 gctttgatta cccctgagaa gttgcccgtg gatgaatgtt tgggaaaagg aaaaggcaaa 720 ggcaactgta agagacggcg ttcatatttg ggcatataa 759 <210> 5 <211> 252 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of UPO gene - no signal peptide <400> 5 Met Ala Glu Leu Asp Phe Ser Lys Trp Lys Thr Arg Gln Pro Gly Glu 1 5 10 15 Leu Arg Ala Pro Cys Pro Ala Met Asn Ser Leu Ala Asn His Gly Phe 20 25 30 Ile Gln Arg Asp Gly Lys Asn Ile Thr Val Glu Gly Leu Thr Pro Val 35 40 45 Leu Lys Glu Val Phe His Leu Ser His Glu Leu Ala Phe Thr Val Ser 50 55 60 Gln Leu Gly Leu Phe Thr Ala Leu Asp Pro Ser Lys Gly Val Phe Thr 65 70 75 80 Leu Gln Asp Leu Thr Asp Arg His Asn Val Phe Glu His Asp Ala Ser 85 90 95 Leu Ser Arg Glu Asp Ala Lys Phe Gly Gly Asp Gln Ser Val Leu His 100 105 110 Lys Gly Gln Phe Gln Lys Phe Met Asp His Phe Lys Gly Glu Lys Tyr 115 120 125 Ile Ser Phe Glu Ala Ala Ala Lys Ala Arg Tyr Ala Met Val Gln Asp 130 135 140 Ser Arg Lys Arg Asn Pro Asp Phe Thr Tyr Asp Val Thr His Arg Ile 145 150 155 160 Thr Ser Tyr Gly Glu Thr Ile Lys Tyr Leu Arg Thr Ile Val Glu Pro 165 170 175 Ser Thr Gly Lys Cys Pro Val Asp Trp Ile Lys Ile Leu Phe Glu Gln 180 185 190 Glu Arg Leu Pro Tyr Asn Glu Gly Trp Arg Pro Pro Thr Asn Glu Leu 195 200 205 Ser Gly Phe Ser Leu Ala Ser Glu Val Leu Glu Leu Ala Leu Ile Thr 210 215 220 Pro Glu Lys Leu Pro Val Asp Glu Cys Leu Gly Lys Gly Lys Gly Lys 225 230 235 240 Gly Asn Cys Lys Arg Arg Arg Ser Tyr Leu Gly Ile 245 250 <210> 6 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer - F3 <400> 6 gtgccgcgcg gcagccatat ggctgagttg gatt 34 <210> 7 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer - R3 <400> 7 aatccaactc agccatatgg ctgccgcgcg gcac 34 <210> 8 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer - F2 <400> 8 catatttggg catataactc gagcaccacc acca 34 <210> 9 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer - R2 <400> 9 tggtggtggt gctcgagtta tatgcccaaa tatg 34

Claims (8)

An oxygenation enzyme comprising the amino acid sequence of SEQ ID NO: 5.
The method according to claim 1,
The oxygenation enzyme is a fungus Podospora anserina ( Podospora anserina ) Oxygenation enzyme derived from S mat +.
The method according to claim 1,
The oxygenation enzyme is toluene (Toluene), atorvastatin (atorvastatin), progesterone (Progesterone), monacolin (monacolin) J, phloretin (Phloretin) and naringin (Naringin) to oxygen to one substrate selected from the group consisting of DC Oxygenation enzyme to be added.
A polynucleotide encoding the oxygenation enzyme of claim 1 .
An expression vector comprising the polynucleotide of claim 4.
A transformant into which the expression vector of claim 5 is introduced.
7. The method of claim 6,
The transformant is an E. coli transformant.
A method for producing an oxygenation enzyme comprising the step of culturing the transformant of claim 6.

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