WO2015019674A1 - Procédé de production de glucose déshydrogénase se liant au flavine-adénine-dinucléotide provenant du genre mucor - Google Patents

Procédé de production de glucose déshydrogénase se liant au flavine-adénine-dinucléotide provenant du genre mucor Download PDF

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WO2015019674A1
WO2015019674A1 PCT/JP2014/063308 JP2014063308W WO2015019674A1 WO 2015019674 A1 WO2015019674 A1 WO 2015019674A1 JP 2014063308 W JP2014063308 W JP 2014063308W WO 2015019674 A1 WO2015019674 A1 WO 2015019674A1
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fad
gdh
seq
cryptococcus
gene
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Japanese (ja)
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悠 歌島
正木　和夫
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東洋紡株式会社
独立行政法人酒類総合研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/05—Oxidoreductases acting on the CH-OH group of donors (1.1) with a quinone or similar compound as acceptor (1.1.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)

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  • the present invention relates to a method for efficiently producing a flavin adenine dinucleotide-binding glucose dehydrogenase (hereinafter also referred to as FAD-GDH) derived from Mucor genus having a uniform amount of sugar chain binding using a specific microorganism as a host.
  • FAD-GDH flavin adenine dinucleotide-binding glucose dehydrogenase
  • the present invention relates to a method for producing FAD-GDH suitable for use in a biosensor for blood glucose self-measurement.
  • a biosensor used for blood glucose self-measurement generally forms an electrode layer composed of a measurement electrode, a counter electrode, and a detection electrode on an insulator substrate, and an enzyme using glucose as a substrate on these electrode substrates.
  • a reagent layer containing an electron acceptor is formed.
  • the blood glucose level of this biosensor is measured by adding blood to the biosensor, causing an enzyme using glucose in the reagent layer as a substrate to react with glucose in the blood, generating electrons, and accepting electrons by the electrons. This is done by determining the glucose concentration (blood glucose level) in the blood based on the current value resulting from the reduction and oxidation of the blood.
  • glucose oxidase (EC 1.1.3.4).
  • Glucose oxidase has an advantage of high specificity for glucose and excellent thermal stability, and thus has been used for a long time in biosensors for blood glucose self-measurement.
  • the glucose concentration in blood is obtained by passing electrons generated in the process of oxidizing glucose and converting it to D-glucono- ⁇ -lactone to the electrode via a mediator.
  • glucose oxidase easily passes protons generated in the reaction to oxygen, there is a problem that dissolved oxygen affects the measured value.
  • NAD (P) -dependent glucose dehydrogenase EC 1.1.1.147
  • pyrroloquinoline quinone-dependent glucose dehydrogenase EC 1.1.5.2
  • glucose dehydrogenase EC 1.1.5.2
  • the latter pyrroloquinoline quinone-dependent glucose dehydrogenase is poor in substrate specificity and acts on saccharides other than glucose such as maltose and lactose, and thus has the disadvantage of impairing the accuracy of the measured value.
  • Patent Document 1 proposes the use of a flavin adenine dinucleotide-binding glucose dehydrogenase (FAD-GDH) derived from the genus Aspergillus.
  • FAD-GDH flavin adenine dinucleotide-binding glucose dehydrogenase
  • This enzyme is superior to the above-mentioned glucose dehydrogenase in that it has excellent substrate specificity and is not affected by dissolved oxygen.
  • this enzyme exhibits an activity remaining rate of about 89% after treatment at 50 ° C. for 15 minutes, and is excellent in thermal stability.
  • Patent Document 2 discloses a DNA sequence encoding FAD-GDH derived from Aspergillus oryzae
  • Patent Document 3 modifies the amino acid sequence of FAD-GDH derived from Aspergillus to improve thermal stability.
  • An improved FAD-GDH is disclosed. It has been found by the present inventors that FAD-GDH of Patent Document 3 can be further improved in thermal stability by self-cloning into the Aspergillus genus to form a sugar chain-linked type.
  • these enzymes have a relatively high effect on xylose, there is room for improvement when measuring blood glucose in those who are undergoing a xylose tolerance test.
  • FAD-GDH derived from the genus Mucor has been developed one after another with relatively low activity on xylose (see Patent Document 4, Patent Document 5, and Patent Document 6).
  • Patent Document 7 when FAD-GDH derived from Mucor Purini described in Patent Document 4 is expressed in yeast Tigosaccharomyces roxy, FAD-GDH having a wide molecular weight ranging from about 150 kDa to 250 kDa is produced. It has been shown. Since the molecular weight of the polypeptide portion of FAD-GDH derived from Mucor Purini is about 80 kDa, the proportion of the sugar chain portion in the glycoprotein is estimated to be 46% to 68%. Further, the production amount when FAD-GDH derived from Mucor purini is recombinantly expressed using Tigosaccharomyces roxy as a host is about 27.6 U per 1 ml of the culture solution.
  • Patent Document 8 reported an attempt to express FAD-GDH derived from Mucor-Plini in Escherichia coli, and removing the secretory signal sequence. Shows that the production amount is improved, but the production amount is only about 0.068 U per 1 ml of the culture solution.
  • FAD-GDH derived from the genus Mucor
  • eukaryotes such as yeast and filamentous fungi instead of Escherichia coli
  • Only FAD-GDH to which the sugar chain is attached is obtained.
  • the performance of FAD-GDH may vary from lot to lot, resulting in variations in measured values. There is a fear.
  • the present invention was devised in view of the current state of the prior art, and an object of the present invention is to efficiently produce FAD-GDH that is suitable for a biosensor for blood glucose self-measurement and has a uniform sugar chain binding amount. Is to provide.
  • the present inventors have used, as a host, a microorganism classified into the genus Cryptococcus which is a kind of basidiomycetous yeast, and this microorganism is described in Patent Document 4.
  • a microorganism classified into the genus Cryptococcus which is a kind of basidiomycetous yeast
  • this microorganism is described in Patent Document 4.
  • the FAD-GDH gene derived from Mucor Prini or the FAD-GDH gene derived from Mucor Himaris described in Patent Document 6 is introduced and expressed in this microorganism, the active sugar chain content Found that high-quality FAD-GDH was produced.
  • the present inventors can optimize the codon usage of the gene according to this microorganism without forming inclusion bodies. It was found that FAD-GDH while maintaining the enzyme activity is necessary for secretory production in culture medium at a high level. Further, it has been found that productivity is further improved by optimizing the secretory signal peptide sequence added to the gene sequence body.
  • a microorganism belonging to the genus Cryptococcus is a Cryptococcus sp.
  • the method according to (1) which is S-2 (Cryptococcus sp. S-2) (Accession No. FERM BP-10961) or a variant thereof.
  • the flavin adenine dinucleotide-binding glucose dehydrogenase gene is selected from the group consisting of the following (C) to (G) on the 5 ′ side of the base sequence of (A) or (B) described in (1)
  • a flavin adenine dinucleotide-binding glucose dehydrogenase enzyme protein produced by the method according to any one of (1) to (3), having a molecular weight of 95 to 100 kDa in a state including a sugar chain.
  • a protein characterized by (5) A biosensor for self-measurement of blood glucose, wherein the flavin adenine dinucleotide-binding glucose dehydrogenase enzyme protein according to (4) is used as an enzyme having glucose as a substrate.
  • the method of the present invention expresses a codon usage of a sugar chain-binding FAD-GDH gene derived from a Mucor genus microorganism in a state optimized for expression in a specific host microorganism completely different from the microorganism. Therefore, FAD-GDH having a uniform amount of sugar chain binding can be efficiently secreted and produced outside the host cell.
  • FAD-GDH is suitable for use in a biosensor for blood glucose self-measurement.
  • FIG. 1 shows a cryptococcus sp.
  • FIG. 3 is a schematic diagram of an expression vector pCsUX2 for S-2.
  • FIG. 2 shows Cryptococcus sp. Into which each FAD-GDH gene expression contract derived from Mucor Himaris was introduced. The FAD-GDH productivity of the S-2 transformant is shown.
  • FIG. 3 shows Cryptococcus sp. Into which the FAD-GDH gene derived from Mucor Himaris has been introduced. The results of SDS-PAGE of the S-2 transformant culture and calculation of the molecular weight are shown.
  • FIG. 4 shows Cryptococcus sp. Into which each FAD-GDH gene expression contract derived from Mucor Purini has been introduced.
  • FIG. 5 shows that Cryptococcus sp. To which FAD-GDH derived from Mucor Purini was introduced. The FAD-GDH productivity when fed S-2 transformants is shown.
  • FIG. 6 shows that Cryptococcus sp. To which mucor purini-derived FAD-GDH has been introduced. The results of SDS-PAGE of the culture solution when the S-2 transformant was fed-batch cultured are shown.
  • the present invention optimizes the codon usage of the sugar chain-binding FAD-GDH gene derived from a microorganism belonging to the genus Mucor, and allows it to be expressed in the original host microorganism by expressing it in a specific microorganism that is completely different from this microorganism.
  • FAD-GDH having a uniform amount of sugar chain binding compared to FAD-GDH can be obtained efficiently.
  • a microorganism of the genus Cryptococcus which is a kind of basidiomycetous yeast is used as a host microorganism for gene expression.
  • Cryptococcus sp a specific strain called strain S-2 is used.
  • This strain is a yeast isolated by the Liquor Research Institute, an independent administrative corporation, and has been confirmed to produce a large amount of enzymes such as ⁇ -amylase, acid xylanase, and cutinase.
  • this strain is located in 1-1, Higashi 1-chome, Tsukuba City, Ibaraki, Japan, Chuo No.
  • the U-5 strain which is a uracil-requiring mutant of S-2, was obtained by UV mutation, and further, the UA1 strain, which is an adenine-requiring mutant, was obtained from the U-5 strain by gene mutation introduction, and further cells from the UA1 strain.
  • the D11 strain which is a UV mutant with reduced exopolysaccharide productivity, has been obtained.
  • D11 shares are located in 1st Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, Japan, 6th Central (Postal Code 305-8586) (currently Room 2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan, Postal Code 292-0818) ), The National Institute of Advanced Industrial Science and Technology (currently known as the Product Evaluation Technology Foundation), and the International Depositary on March 23, 2012 under the deposit number FERM BP-11482.
  • FAD-GDH having a uniform amount of sugar chain binding can be obtained by using a Cryptococcus microorganism as a host microorganism for gene expression is not yet clear, but it is unique to the Cryptococcus microorganism. This is thought to be due to the existence of a sugar chain synthesis and binding system.
  • the sugar chain-binding FAD-GDH gene to be expressed in the host microorganism is (A) represented by the nucleotide sequence of SEQ ID NO: 1 or 7.
  • These base sequences are suitable for expression in the microorganism of the genus Cryptococcus of the host, not the base sequence itself of the FAD-GDH gene derived from Mucor Himaris described in Patent Document 6 and Mucor Purini described in Patent Document 4. So that codon usage is optimized. Even if the nucleotide sequence of the wild-type gene is directly introduced into a microorganism of the genus Cryptococcus and expressed, the expression product forms an inclusion body, and only inactive FAD-GDH can be obtained. According to the method of the present invention, FAD-GDH is secreted and produced while maintaining the enzyme activity without forming inclusion bodies by optimizing the codon usage of the gene to be expressed according to the codon usage of the host microorganism. Can be made.
  • the codon usage of SEQ ID NO: 1 or 7 is Cryptococcus sp. It is optimized for the expression of S-2, and the ratio of the base of the third letter of the codon of the base sequence to G or C is 88.2%.
  • cryptococcus sp The ratios in which the third character base of the codons of the base sequences of ⁇ -amylase, cutinase, and xylanase whose expression is confirmed in S-2 are G or C are 81.5%, 81.3%, 80.%, respectively.
  • the ratio of 0% and the third character base of the codon is G or C tends to be very high. From these facts, SEQ ID NO: 1 or 7 is Cryptococcus sp.
  • Cryptococcus sp not only suitable for expression in S-2, but also Cryptococcus sp. It is also expected to be suitable for expression in other cryptococcal microorganisms (Cryptococcus lifaciens, Cryptococcus flavus, Cryptococcus flavus, Cryptococcus curvatus, etc.) having a codon usage similar to S-2.
  • the codon usage of the host microorganism can be predicted by using information provided from Codon Usage Database (http://www.kazusa.or.jp/codon/).
  • This base sequence (B) is a base sequence in an equivalent range of the base sequence (A). This is a functionally equivalent enzyme protein even if a part of the base sequence of the gene encoding the enzyme protein is mutated and as a result part of the amino acid sequence of the enzyme protein is mutated. This is because there are many cases.
  • the base sequence of (B) is, for example, Transformer Mutagenesis Kit; manufactured by Clonetech, EXOIII / Mung Bean Deletion Kit; manufactured by Stratagene, QuickChange SiteDirectedMitnessisKit; It can be obtained by modifying the base sequence described in SEQ ID NO: 1 using the method. The activity of the protein encoded by the obtained gene can be confirmed by the method described in Examples below.
  • the homology between the base sequence shown in SEQ ID NO: 7 and the base sequence shown in SEQ ID NO: 1 is 83%, and as shown in the examples described later, both base sequences are Cryptococcus sp. It can be expressed efficiently with S-2.
  • the homology between the polypeptide sequence encoded by SEQ ID NO: 1 and the polypeptide sequence encoded by SEQ ID NO: 8 is 78%. From these facts, it is appropriate that the equivalent range of SEQ ID NO: 1 or 7 has a homology of 80% or more, preferably 85% or more, more preferably 90% or more, particularly preferably 95% or more. It is appropriate to have sex.
  • the FAD-GDH gene represented by the base sequence (A) or (B) may be expressed alone in the host microorganism, but in order to increase the protein expression efficiency and the expression success rate.
  • a secretory signal peptide is present at the N-terminus of the secreted protein.
  • secretory signal peptides examples include amino acid sequences (C) to (G) represented by any of SEQ ID NOs: 2 to 6.
  • amino acid sequence (C) shown in SEQ ID NO: 2 is a secretory signal peptide sequence derived from FAD-GDH produced by Mucor Himaris.
  • All of the amino acid sequences (D) to (E) shown in SEQ ID NOs: 3 to 4 are Cryptococcus sp.
  • a secretory signal peptide sequence derived from an acid xylanase produced by S-2 wherein the amino acid sequence (D) shown in SEQ ID NO: 3 is a sequence having the secretory signal peptide sequence from the start codon to the 23rd amino acid (Xs2)
  • the amino acid sequence (E) represented by SEQ ID NO: 4 is a sequence (Xs3) in which the secretory signal peptide sequence is from the start codon to 17 amino acids.
  • the amino acid sequence of (F) shown in SEQ ID NO: 5 is Cryptococcus sp. It is a secretory signal peptide sequence (As) derived from ⁇ -amylase produced by S-2.
  • the amino acid sequence of (G) shown in SEQ ID NO: 6 is Cryptococcus sp. It is a secretory signal peptide sequence (Cs) derived from cutinase produced by S-2.
  • these secretory signal peptide sequences are extremely high in efficiency, and by using them, the production efficiency of FAD-GDH outside the cells of the genus Cryptococcus is dramatically increased. Can be increased.
  • amino acid sequences of these secretory signal peptides ((C A fusion DNA obtained by binding a base sequence encoding any one of (A) to (G)) may be expressed in a host microorganism.
  • Cryptococcus sp. which is a microorganism that can be used as a host in the method of the present invention.
  • S-2 has been confirmed to secrete and produce various types of hardly degradable enzymes, and secretes and produces enzymes such as raw starch degradable ⁇ -amylase, acid xylanase, and cutinase that degrades biodegradable plastics.
  • secretory signal peptides possessed by these secreted proteins have heretofore been found in secreted proteins, and it has been unclear whether they are the most efficient secretory signal peptides.
  • computer programs for predicting the sequence of the secretory signal peptide are provided.
  • the method of the present invention comprises the step of expressing a FAD-GDH gene represented by the base sequence (A) or (B) in a microorganism of the genus Cryptococcus. Specifically, in this step, a FAD-GDH gene is inserted into an appropriate expression vector according to a conventional method to prepare a recombinant vector, and then this recombinant expression vector is introduced into a cryptococcus microorganism and transformed. A body is prepared, and this transformant can be cultured under appropriate conditions.
  • the expression vector used in the method of the present invention is not particularly limited, and examples thereof include conventionally known vectors such as E. coli vectors.
  • E. coli vectors include pBR322, pUC19, pGEM-T, pCR-Blunt, pTA2, and pET.
  • the vector DNA comprises an auxotrophic marker, a drug resistance marker, an expression promoter DNA sequence, an expression terminator DNA sequence, and more preferably a cryptococcus sp.
  • S-2-derived orotate phosphoribosyl transfer gene, xylanase promoter, and xylanase terminator are included.
  • the combination of the expression vector and the host microorganism is not particularly limited.
  • an auxotrophic marker gene or drug resistance marker gene derived from the host into which the gene is incorporated an expression promoter DNA sequence from the host into which the gene is incorporated, and the gene
  • a combination of an expression vector containing a terminator DNA sequence derived from a host into which the vector is incorporated and an auxotrophic mutant host or a drug-sensitive host Most preferably, Cryptococcus sp.
  • the method for introducing the recombinant expression vector into the cells of the host microorganism is not particularly limited, and examples thereof include electroporation.
  • the culture form of the transformant may be appropriately selected in consideration of the nutritional physiological properties of the host. Usually, liquid culture is used in many cases, but industrially, aeration and agitation culture is advantageous. It is advantageous to select a high FAD-GDH producing cell line in advance prior to culturing.
  • the nitrogen source used for the culture may be any nitrogen compound that can be used by the host microorganism except for a special N source such as a deletion of a specific amino acid component.
  • a special N source such as a deletion of a specific amino acid component.
  • These are mainly organic nitrogen sources, and for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline decomposition product and the like are used.
  • yeast extract and soybean protein are preferable, but the present invention is not limited to this, and the transformant can also be cultured by using casein polypeptone, fermented koji extract, malt extract, or the like.
  • nutrient sources commonly used for culturing microorganisms are widely used.
  • the carbon source any carbon compound that can be assimilated may be used.
  • glucose, sucrose, lactose, maltose, xylose, molasses, pyruvic acid and the like are used.
  • phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.
  • the culture temperature can be appropriately changed within the range where the fungus grows and produces FAD-GDH enzyme protein, but cryptococcus sp. In the case of S-2, it is usually about 20 to 25 ° C.
  • the culture time varies somewhat depending on conditions, the culture may be terminated at an appropriate time in consideration of the time when the FAD-GDH enzyme protein reaches the maximum yield, and is usually about 60 to 120 hours.
  • the pH of the medium can be appropriately changed within the range in which the bacteria grow and produce FAD-GDH enzyme protein, but is usually about pH 3.0 to 9.0.
  • the FAD-GDH enzyme protein of the present invention can be used by directly collecting and using a culture solution containing bacterial cells obtained by culturing the above transformant. Generally, however, filtration and centrifugation are carried out in advance according to a conventional method. It can also be used after separating the FAD-GDH enzyme protein-containing solution and the cells by, for example.
  • the FAD-GDH enzyme protein may be purified from the FAD-GDH enzyme protein-containing solution thus obtained and used.
  • Purification methods include, for example, vacuum concentration, membrane concentration, salting out treatment such as ammonium sulfate and sodium sulfate, fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, acetone, etc., heating treatment or isoelectric point treatment, adsorbent
  • treatments such as gel filtration with a gel filtration agent, adsorption chromatography, ion exchange chromatography, hydrophobic interaction chromatography and the like.
  • the FAD-GDH enzyme protein obtained by the method of the present invention has the following properties (i) to (ii).
  • the molecular weight of FAD-GDH expressed in the conventional microorganism of the genus Mucor-Himaris of Patent Document 6 is about 85 to 107 kDa in a state including a sugar chain
  • the FAD-GDH enzyme obtained by the method of the present invention Proteins have an extremely uniform amount of sugar chain bonds compared to conventional proteins. Therefore, the FAD-GDH enzyme protein obtained by the method of the present invention can be suitably used for a biosensor for blood glucose self-measurement taking advantage of such enzymatic chemical characteristics.
  • the various enzyme chemical properties described above can be examined by using known methods for specifying various enzyme properties, for example, the methods described in the following examples.
  • Various properties of the enzyme can be examined to some extent in the culture medium of the transformant producing the FAD-GDH of the present invention and in the middle of the purification process, and more specifically, using the purified enzyme. .
  • the purified enzyme refers to an enzyme that has been separated to a state that does not substantially contain components other than the enzyme, particularly proteins other than the enzyme (contaminating protein). Specifically, for example, it refers to an enzyme having a content of contaminating protein of less than about 20%, preferably less than about 10%, more preferably less than about 5%, and even more preferably less than about 1% based on weight. .
  • FAD-GDH activity is measured under the following conditions.
  • reaction reagent The following PIPES buffer 15.6 ml, DCPIP solution 0.2 ml, and D-glucose solution 4 ml are mixed to obtain a reaction reagent. 50 mM PIPES buffer pH 6.5 (including 0.1% Triton X-100) 6.8 mM 2,6-dichlorophenolindophenol (DCPIP) solution 1 M D-glucose solution
  • ⁇ Measurement conditions Pre-warm 3 ml of reaction reagent at 37 ° C. for 5 minutes. Add 0.1 ml of FAD-GDH solution, mix gently, record the change in absorbance at 600 nm for 5 minutes with a spectrophotometer controlled at 37 ° C. with water as a control, and absorb the absorbance per minute from the straight line. The change ( ⁇ OD TEST) is measured. In the blind test, a solvent dissolving FAD-GDH is added to the reaction reagent instead of the FAD-GDH solution, and the change in absorbance per minute ( ⁇ OD BLANK) is measured in the same manner. From these values, GDH activity is determined according to the following formula (I).
  • 1 unit (U) in GDH activity is defined as the amount of enzyme that reduces 1 micromole of DCPIP per minute in the presence of 200 mM D-glucose.
  • FAD-GDH activity (U / ml) ⁇ ( ⁇ OD TEST ⁇ OD BLANK) ⁇ 3.1 ⁇ dilution factor ⁇ / ⁇ 16.3 ⁇ 0.1 ⁇ 1.0 ⁇ (I)
  • 3.1 is the amount of reaction reagent + enzyme solution (ml)
  • 16.3 is the molar molecular extinction coefficient (cm 2 / micromol) under the conditions for this activity measurement
  • 0.1 is the enzyme
  • the liquid volume (ml) of the solution, 1.0 indicates the optical path length (cm) of the cell.
  • Example 1 1. Preparation of an expression cassette for the FAD-GDH gene derived from Mucor Himalis The optimization of the codon usage of the FAD-GDH gene derived from Mucor Himalis described in Patent Document 6 is performed by cryptococcus sp-S-2. The codon usage of ⁇ -amylase gene, cutinase gene, and xylanase gene, which are known genes, was referred to. The optimized gene sequence is shown in SEQ ID NO: 1.
  • the first to 60th base sequence shown in SEQ ID NO: 1 is predicted to be a base sequence encoding the secretory signal sequence of the FAD-GDH gene derived from Mucor Himalis. Cryptococcus sp-S-2.
  • the FAD-GDH protein derived from Mucor himaris recombinantly produced in (2) is predicted to have a mature sequence from which this secretory signal sequence has been removed.
  • SEQ ID NO: 8 shows the base sequence encoding the FAD-GDH protein derived from Mucor himaris secreted as a mature form.
  • the pCsUX plasmid (5.9 kbp) containing the Xyl promoter, Xyl terminator, and URA5 gene was treated with MluI and cleaved at one position immediately downstream of the Xyl promoter, followed by dephosphorylation.
  • the FAD-GDH gene fragment of MhFADGLD (opt) was ligated to the treated plasmid to construct a recombinant plasmid (pCsUXMhFADGLD (opt)).
  • FIG. 1 A schematic diagram of the pCsUX plasmid is shown in FIG.
  • This pCsUX plasmid is Cryptococcus sp.
  • a recombinant DNA is prepared by ligating the acidic xylanase promoter (Xyl-p) derived from S-2, the acidic xylanase terminator (Xyl-t), and the orthophosphate phosphoryltransferase gene (Ura5) to a commercially available pUC19 vector by a conventional method.
  • Xyl-p acidic xylanase terminator
  • Ura5 orthophosphate phosphoryltransferase gene
  • E. coli using this recombinant DNA. It can be obtained by transforming E. coli DH5 ⁇ according to a conventional method. Transformants can be selected by ampicillin resistance.
  • inverse PCR was performed using KOD-plus Mutagenesis kit (manufactured by Toyobo Co., Ltd.), and the secretory signal peptide sequence was replaced with a sequence other than SEQ ID NO: 2.
  • the secretory signal peptide sequence was sequenced using MhFADGLD (opt-sp) F of SEQ ID NO: 11 and Xylanase (sp2) -Xyl-pro-comp of SEQ ID NO: 12 as primers. Substituted with “Xylanase secretion signal peptide sequence 2” shown in No.
  • the recombinant host introduced into S-2 is Cryptococcus sp. S-2.
  • DA25 strain was used. This strain is Cryptococcus sp., Deposited internationally as FERM BP-11482.
  • a mutant of the S-2 strain was designated as Cryptococcus sp.
  • a strain into which the ade1 gene derived from the S-2 strain has been introduced see Japanese Patent Application No. 2012-113449).
  • the present bacterium is uracil-requiring for selection of transformants. By transforming the pCsUX2 plasmid, it is possible to select transformants according to uracil-requiring requirements.
  • Electroporation buffer (270 mM sucrose, 1 mM magnesium chloride, 10 mM Tris-HCl, pH 7.6) was added to the solution after energization and spread on the selection plate.
  • the selection plate used was YNB-ura agar medium (0.67% Yeast Nitrogen Base W / O amino acid, 0.078% -ura DO supplement, 2% glucose, 1% agar powder).
  • the inoculated plate was statically cultured at 25 ° C. for 1 week, and growing colonies were selected.
  • FIG. 2 shows the measured values of FAD-GDH activity of the strain having the highest productivity among the several transformants obtained.
  • a strain (UXMhGDH (FIG. 2 UXMhGDH (SEQ ID NO: 2)) was introduced by introducing a gene optimized for codon usage, and using the secretory signal peptide sequence (SEQ ID NO: 2) of Mucor-Himaris FAD-GDH. opt)), about 12 U / mL of FAD-GDH activity was detected. From this result, the secretory signal peptide sequence of Mucor-Himaris FAD-GDH was Cryptococcus sp. It was confirmed that it functions effectively also in S-2. In addition, as a control, no FAD-GDH activity was detected in the strain (UXMhGDH (ntv) in FIG. 2) into which the wild type gene obtained from the Mucor himaris wild strain was introduced.
  • the secretory signal peptide sequence is designated as Cryptococcus sp.
  • S-2 Xylanase secretion signal peptide sequence 2 (SEQ ID NO: 3) (UXXs2MhGDH (opt) in FIG. 2), a maximum of about 63 U / mL of FAD-GDH activity was detected, and the secretory signal peptide sequence was converted to cryptococcus. sp.
  • SEQ ID NO: 4 S-2 Xylanase secretion signal peptide sequence 3 (SEQ ID NO: 4) (UXXs3MhGDH (opt) in FIG.
  • the electrophoresis sample is as follows. Lane 1: Molecular weight marker (manufactured by Invitrogen, Novex (registered trademark) Sharp Unstained Protein Standard) Lane 2: FAD-GDH culture supernatant derived from recombinant mucor himaris expressed in cryptococcus
  • the molecular weight of FAD-GDH derived from recombinant mucor Himaris expressed in Cryptococcus is 95 kDa to 100 kDa.
  • Patent Document 6 describes that FAD-GDH derived from a wild strain has a molecular weight of 85 kDa to 107 kDa. From this, it can be said that the molecular weight of FAD-GDH derived from recombinant mucor Himaris expressed in Cryptococcus is remarkably uniform as compared with FAD-GDH derived from wild type.
  • Example 2 1. Preparation of expression cassette of FAD-GDH gene derived from mucor purini
  • the optimization of the codon usage of the FAD-GDH gene derived from mucor purini described in Patent Document 4 is performed by cryptococcus sp-S-2.
  • the codon usage of ⁇ -amylase gene, cutinase gene, and xylanase gene, which are known genes, was referred to.
  • the optimized gene sequence is shown in SEQ ID NO: 7.
  • the first to 60th base sequences shown in SEQ ID NO: 7 encode the amino acid sequence shown in SEQ ID NO: 16, and are the base sequences encoding the secretory signal sequence of the FAD-GDH gene derived from Mucor Purini. It is predicted. Cryptococcus sp-S-2.
  • the FAD-GDH protein derived from Mucor Purini recombinantly produced in (2) is predicted to have a mature sequence from which this secretory signal sequence has been removed.
  • the base sequence encoding the FAD-GDH protein derived from Mucor Purini secreted as a mature form is shown in SEQ ID NO: 17.
  • the pCsUX plasmid (5.9 kbp) containing the Xyl promoter, the Xyl terminator, and the URA5 gene was treated with SpeI and cleaved at one site immediately downstream of the Xyl promoter, followed by dephosphorylation.
  • the FAD-GDH gene fragment of MpFADGLD (opt) was ligated to the treated plasmid to construct a recombinant plasmid (pCsUXMpFADGLD (opt)).
  • Cryptococcus sp Of the FAD-GDH gene derived from Mucor Purini. Introduction to S-2 Cryptococcus sp. The transformation to S-2 was carried out as described in 2. As well.
  • FIG. 4 shows the measured values of FAD-GDH activity of the strain having the highest productivity among the several transformants obtained.
  • the secretory signal peptide sequence is designated as Cryptococcus sp.
  • S-2 Xylanase secretion signal peptide sequence 2 (SEQ ID NO: 4) (UXXs2MpGDH (opt) in FIG. 2), a maximum FAD-GDH activity of about 8.5 U / mL was detected, and the secretion signal peptide sequence Cryptococcus sp.
  • S-2 Xylanase secretion signal peptide sequence 3 SEQ ID NO: 5
  • UXXs3MpGDH (opt) in FIG. 2 a maximum FAD-GDH activity of about 3.8 U / mL was detected.
  • the transformant of the UXXs2MpGDH (opt) strain having the highest FAD-GDH activity was used in a 60 ml YM medium (yeast extract 0.3%, malt extract 0.3%, polypeptone, using a 200 ml baffled Erlenmeyer flask. 0.5%, glucose 1.0%) and cultured at 25 ° C. for 48 hours to prepare a preculture solution.
  • the preculture solution was inoculated into 6 L of a medium (5% yeast extract, 0.04% adecanol) using a 10 L fermenter, and stirring culture was started at 25 ° C.
  • the culture solution obtained at 72 hours of culture was centrifuged to collect the culture supernatant, which was used as a crude enzyme solution.
  • This crude enzyme solution was used as Nu-PAGE 4-12% Bis-Tris Gel (Invitrogen).
  • the molecular weight of recombinant FAD-GDH was determined by SDS-polyacrylamide gel electrophoresis using The result is shown in FIG.
  • the electrophoresis sample is as follows. Lane 1: culture supernatant at 24 hours of culture Lane 2: culture supernatant at 48 hours of culture Lane 3: culture supernatant at 72 hours of culture Lane 4: culture medium at 88 hours of culture Supernatant
  • the molecular weight of FAD-GDH derived from recombinant mucor purini expressed in cryptococcus is 95 kDa to 100 kDa.
  • Patent Document 4 describes that the molecular weight of FAD-GDH derived from a wild-type strain is 90 kDa to 130 kDa
  • Patent Document 7 describes the molecular weight of recombinant FAD-GDH expressed in Tigo Saccharomyces. Is 150 kDa to 250 kDa, and the molecular weight of recombinant FAD-GDH expressed in Aspergillus oryzae is 80 kDa to 100 kDa. From these facts, it can be said that the molecular weight of FAD-GDH derived from recombinant mucor purini expressed in Cryptococcus is extremely uniform as compared with the case of using other recombinant expression hosts.
  • FAD-GDH derived from Mucor Himaris and FAD-GDH derived from Mucor Purini were converted into Cryptococcus sp by using a gene with optimized codon usage and modifying the secretory signal sequence. .
  • S-2 was shown to be capable of active and efficient secretory expression.
  • FAD-GDH derived from Mucor Himaris and FAD-GDH derived from Mucor Purini expressed in Cryptococcus Thus, it was found that the amount of sugar chain binding was remarkably uniform as compared with the case where it was expressed in another expression host.
  • Example 2 Cryptococcus sp. Although the xylanase-derived one (SEQ ID NO: 3 or 4) produced by the S-2 strain is used, the secretory signal peptide is involved in the protein expression efficiency and expression success rate of FAD-GDH. Since it is not involved in sugar chain binding, even if the secretory signal peptide is replaced with a peptide other than SEQ ID NO: 3 or SEQ ID NO: 4, as long as a microorganism of the genus Cryptococcus is used as a host, the amount of sugar chain binding is the same as in Example 2. It is thought that FAD-GDH with uniform is obtained.
  • FAD-GDH having the same characteristics as FAD-GDH derived from Mucor Himaris and Mucor Purini can be efficiently recombinantly produced. . Therefore, the present invention is extremely useful for producing FAD-GDH suitable as a reagent for a blood glucose self-measurement biosensor.

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Abstract

L'invention concerne un procédé de production d'une glucose déshydrogénase se liant au flavine-adénine-dinucléotide (FAD-GDH), qui est appropriée pour un biocapteur utilisable dans l'auto-évaluation de glycémie et à laquelle une chaîne sucre est liée en une quantité uniforme, avec un rendement élevé. Un procédé de production d'une FAD-GDH à laquelle une chaîne sucre est liée en une quantité uniforme, ledit procédé étant caractérisé en ce qu'il implique une étape d'expression d'un gène de FAD-GDH représenté par la séquence de nucléotides représentée par SEQ ID No. : 1 ou 7 ou une séquence de nucléotides se situant dans une plage équivalente à la séquence de nucléotides susmentionnée dans un micro-organisme appartenant au genre Cryptococcus. Si on le souhaite, une séquence de peptides de signal de sécrétion représentée par l'une quelconque des SEQ ID No. : 2 à 6 peut être liée au côté 5'-terminal du gène de FAD-GDH. Selon le procédé de la présente invention, il devient possible de produire une FAD-GDH qui possède une masse moléculaire de 95 à 100 kDa lorsque la FAD-GDH contient une chaîne sucre.
PCT/JP2014/063308 2013-08-07 2014-05-20 Procédé de production de glucose déshydrogénase se liant au flavine-adénine-dinucléotide provenant du genre mucor WO2015019674A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2016163448A1 (ja) * 2015-04-09 2018-02-08 東洋紡株式会社 グルコース測定用酵素製剤
JP2020188798A (ja) * 2015-01-16 2020-11-26 東洋紡株式会社 Ｆａｄ依存型グルコースデヒドロゲナーゼ

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010140431A1 (fr) * 2009-06-04 2010-12-09 キッコーマン株式会社 Glucose déshydrogénase liée à une flavine
WO2011004654A1 (fr) * 2009-07-10 2011-01-13 天野エンザイム株式会社 Glucose déshydrogénase dérivée du microorganisme mucor
WO2012073986A1 (fr) * 2010-12-02 2012-06-07 キッコーマン株式会社 Glucose déshydrogénase liée à la flavine, procédé de production de glucose déshydrogénase liée à la flavine, et transformant de levure utilisé dans ce procédé
WO2012073987A1 (fr) * 2010-12-01 2012-06-07 キッコーマン株式会社 Transformant de e. coli, procédé de production de glucose déshydrogénase liée à la flavine à l'aide de celui-ci, et glucose déshydrogénase mutante liée à la flavine
JP2013046610A (ja) * 2011-07-28 2013-03-07 Toyobo Co Ltd 改善された発現特性を示す改変型ペルオキシダーゼ酵素遺伝子、及びそれを使用したペルオキシダーゼの生産方法
WO2013065623A1 (fr) * 2011-10-31 2013-05-10 東洋紡株式会社 Nouvelle glucose déshydrogénase
JP2013135663A (ja) * 2011-12-02 2013-07-11 Toyobo Co Ltd フラビンアデニンジヌクレオチド結合型グルコースデヒドロゲナーゼの生産方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010140431A1 (fr) * 2009-06-04 2010-12-09 キッコーマン株式会社 Glucose déshydrogénase liée à une flavine
WO2011004654A1 (fr) * 2009-07-10 2011-01-13 天野エンザイム株式会社 Glucose déshydrogénase dérivée du microorganisme mucor
WO2012073987A1 (fr) * 2010-12-01 2012-06-07 キッコーマン株式会社 Transformant de e. coli, procédé de production de glucose déshydrogénase liée à la flavine à l'aide de celui-ci, et glucose déshydrogénase mutante liée à la flavine
WO2012073986A1 (fr) * 2010-12-02 2012-06-07 キッコーマン株式会社 Glucose déshydrogénase liée à la flavine, procédé de production de glucose déshydrogénase liée à la flavine, et transformant de levure utilisé dans ce procédé
JP2013046610A (ja) * 2011-07-28 2013-03-07 Toyobo Co Ltd 改善された発現特性を示す改変型ペルオキシダーゼ酵素遺伝子、及びそれを使用したペルオキシダーゼの生産方法
WO2013065623A1 (fr) * 2011-10-31 2013-05-10 東洋紡株式会社 Nouvelle glucose déshydrogénase
JP2013135663A (ja) * 2011-12-02 2013-07-11 Toyobo Co Ltd フラビンアデニンジヌクレオチド結合型グルコースデヒドロゲナーゼの生産方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MASAKI K. ET AL.: "Construction of a new recombinant protein expression system in the basidiomycetous yeast Cryptococcus sp. strain S-2 and enhancement of the production of a cutinase-like enzyme", APPL. MICROBIOL. BIOTECHNOL., vol. 93, no. 4, February 2012 (2012-02-01), pages 1627 - 1636 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020188798A (ja) * 2015-01-16 2020-11-26 東洋紡株式会社 Ｆａｄ依存型グルコースデヒドロゲナーゼ
JP7188421B2 (ja) 2015-01-16 2022-12-13 東洋紡株式会社 Ｆａｄ依存型グルコースデヒドロゲナーゼ
JPWO2016163448A1 (ja) * 2015-04-09 2018-02-08 東洋紡株式会社 グルコース測定用酵素製剤

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