WO2013080881A1 - Method for producing flavin-adenine dinucleotide dependent glucose dehydrogenase - Google Patents

Abstract

Provided is a method for producing an FAD-GDH in which the amount of glycosylation is low and even. A method for producing an FAD-GDH in which the amount of glycosylation is low and even, the method being characterized by involving a step for expressing, within a microorganism belonging to the genus Cryptococcus, a fused DNA in which one of the secretion signal peptide sequences from among SEQ ID NO: 9 to 14 is bound to the 5' side of an FAD-GDH gene represented by a base sequence represented by SEQ ID NO: 8 or a base sequence having a similar range. With this method, it is possible to obtain an FAD-GDH having a molecular mass between 75 and 90kDa while containing a sugar chain.

Description

Method for producing flavin adenine dinucleotide-linked glucose dehydrogenase

The present invention relates to a method for producing a flavin adenine dinucleotide-binding glucose dehydrogenase (hereinafter also referred to as FAD-GDH) that uses a specific microorganism and has a small amount of sugar chain binding and is uniform. The present invention relates to a method for producing FAD-GDH suitable for use in various biosensors.

血糖 Blood glucose self-measurement is important for diabetics to grasp their normal blood glucose level and use it for treatment. 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.

An example of an enzyme using glucose as a substrate used in this biosensor is 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. In a biosensor for blood glucose self-measurement using glucose oxidase, the glucose concentration in blood is obtained by passing electrons generated in the process of oxidizing glucose and converting it to D-glucono-δ-lactone to the electrode via a mediator. However, since glucose oxidase easily passes protons generated in the reaction to oxygen, there is a problem that dissolved oxygen affects the measured value.

In order to avoid such a problem, for example, NAD (P) -dependent glucose dehydrogenase (EC 1.1.1.147) or pyrroloquinoline quinone-dependent glucose dehydrogenase (EC 1.1.5.2 (formerly EC 1.1. The use of glucose dehydrogenase such as 99.17) has been proposed. These enzymes are superior in that they are not affected by dissolved oxygen, but the former NAD (P) -dependent glucose dehydrogenase has the disadvantage of poor stability and requires the addition of a coenzyme, making the measurement complicated. There are drawbacks. On the other hand, 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.

As a glucose dehydrogenase that does not have these disadvantages, Patent Document 1 proposes the use of a flavin adenine dinucleotide-binding glucose dehydrogenase (FAD-GDH) derived from the genus Aspergillus. This enzyme is superior to the above-mentioned glucose dehydrogenase in that it has excellent substrate specificity and is not affected by dissolved oxygen. In addition, 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, and 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.

However, when this sugar chain-binding FAD-GDH is used in a biosensor for blood glucose self-measurement, it may not operate normally. Therefore, the present inventors sought the cause, and this FAD-GDH has a large number of sugar chains bound thereto. This sugar chain significantly increases the viscosity of the reagent solution and prevents dissolution. It turns out that it does not work properly. In fact, according to the investigation by the present inventors, when FAD-GDH of Patent Document 3 is self-cloned into Aspergillus to obtain a sugar chain-binding type, the amount of sugar chain binding per enzyme powder is about 40%. The molecular weight by SDS-PAGE was about 80 kDa to 120 kDa, and the amount of sugar chain binding was large and the fluctuation was large.

Therefore, a sugar chain-binding FAD-GDH used for a biosensor for blood glucose self-measurement has been desired to have a small amount of sugar chains and a uniform amount.

International Publication No. 2004/058958 JP 2008-237210 A JP 2008-178380 A

The present invention was devised in view of the current state of the prior art, and an object of the present invention is to provide a method for producing FAD-GDH that is suitable for a biosensor for blood glucose self-measurement and has a small amount of sugar chain binding and is uniform. It is to provide.

As a result of intensive studies to achieve the above object, 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 3. It was found that when a modified Aspergillus oryzae-derived FAD-GDH gene was introduced and expressed in this microorganism, FAD-GDH with a small amount of sugar chain binding and uniform was produced.

In addition, when expressing the FAD-GDH gene derived from Aspergillus oryzae using this microorganism as a host, the present inventors do not form inclusion bodies by optimizing the codon usage of the gene according to this microorganism. In addition, it was found that FAD-GDH having its enzyme activity maintained is necessary for secretory production in the culture medium at a high level. Further, it has been found that by optimizing the secretory signal peptide sequence added to the gene sequence body, the secretory productivity of FAD-GDH outside the host cell is further improved.

The present invention has been completed based on these findings and comprises the following (1) to (4).
(1) A method for producing a flavin adenine dinucleotide-binding glucose dehydrogenase having a small amount of sugar chain binding and uniform, and comprising the following base sequence (A) or (B) in a microorganism belonging to the genus Cryptococcus: A fusion DNA in which a base sequence encoding any amino acid sequence selected from the group consisting of the following (C) to (H) is bound to the 5 ′ side of the flavin adenine dinucleotide-binding glucose dehydrogenase gene represented by A method comprising the step of expressing:
(A) the base sequence represented by SEQ ID NO: 8;
(B) a base sequence that is 90% or more homologous to the base sequence represented by SEQ ID NO: 8 and encodes a protein having flavin adenine dinucleotide-binding glucose dehydrogenase enzyme activity;
(C) the amino acid sequence represented by SEQ ID NO: 9;
(D) the amino acid sequence represented by SEQ ID NO: 10;
(E) the amino acid sequence represented by SEQ ID NO: 11;
(F) the amino acid sequence represented by SEQ ID NO: 12;
(G) the amino acid sequence represented by SEQ ID NO: 13;
(H) The amino acid sequence represented by SEQ ID NO: 14.
(2) The microorganism is Cryptococcus sp. The method according to (1), which is S-2 (Cryptococcus sp. S-2) (Accession number FERM BP-10961).
(3) A flavin adenine dinucleotide-linked glucose dehydrogenase enzyme protein produced by the method according to (1) or (2), characterized by having a molecular weight of 75 to 90 kDa in a state including a sugar chain. Protein.
(4) A biosensor for self-monitoring of blood glucose, wherein the flavin adenine dinucleotide-binding glucose dehydrogenase enzyme protein according to (3) is used as an enzyme having glucose as a substrate.

The method of the present invention optimizes the codon usage of a sugar chain-binding FAD-GDH gene derived from a microorganism of the genus Aspergillus, and is bound to a specific secretion signal peptide in a specific microorganism that is completely different from the microorganism. Therefore, FAD-GDH having a small amount of sugar chain binding and uniform can be efficiently secreted and produced outside the host cell. Such FAD-GDH is excellent in thermal stability and easily dissolved in a reagent solution, and thus is suitable for use in a biosensor for blood glucose self-measurement.

FIG. 1 shows a cryptococcus sp. FIG. 4 is a schematic diagram of an expression vector pCsUX for S-2. FIG. 2 shows the FAD-GDH productivity of each FAD-GDH gene expression contract-introduced transformant. FIG. 3 shows the results of a heat resistance test of recombinant FAD-GDH. FIG. 4 shows the results of a pH stability test of recombinant FAD-GDH. FIG. 5 shows the results of a molecular weight test of recombinant FAD-GDH. FIG. 6 shows the results of a glycolysis test of recombinant FAD-GDH.

The present invention optimizes the codon usage of a sugar chain-binding FAD-GDH gene derived from a microorganism of the genus Aspergillus and expresses it in a specific microorganism completely different from this microorganism in a state of being bound to a specific secretion signal peptide. Thus, it is possible to efficiently obtain FAD-GDH having a small amount of sugar chain binding and uniform compared with FAD-GDH expressed in the original host microorganism.

Specifically, in the method of the present invention, a microorganism of the genus Cryptococcus which is a kind of basidiomycetous yeast is used as a host microorganism for gene expression. Preferably, among the microorganisms of the genus Cryptococcus, 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 α-amylase, acid xylanase, and cutinase. In addition, this strain is located in 1-1-1 Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, Japan, National Institute of Advanced Industrial Science and Technology (current name is National Institute of Technology and Evaluation) September 1995 Deposited domestically as FERM P-15155 on the 5th, and transferred to an international deposit as FERM BP-10961 on 25th April 2008.

In the method of the present invention, the reason why FAD-GDH having a small amount of sugar chain binding and uniform can be obtained by using a microorganism of the genus Cryptococcus as a host microorganism for gene expression is not yet clear. This is thought to be due to the existence of a unique sugar chain synthesis and binding system.

In the method of the present invention, the sugar chain-bound FAD-GDH gene to be expressed in the host microorganism is (A) represented by the nucleotide sequence of SEQ ID NO: 8. This base sequence is not the base sequence itself of the modified gene derived from the genus Aspergillus described in Patent Document 3, but its codon usage is optimized so that it is suitable for expression in the host cryptococcus microorganism. Even if the base sequence of the modified gene is introduced as it is into a microorganism of the genus Cryptococcus and expressed as it is, 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: 8 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 92.6%. On the other hand, cryptococcus sp. The ratios in which the third character base of the codons of the base sequences of α-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: 8 is 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 cryptococcus microorganisms (Cryptococcus lifaciens, 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/).

Further, in the method of the present invention, (A) instead of the base sequence represented by SEQ ID NO: 8, (B) a base sequence that is 90% or more homologous to the base sequence represented by SEQ ID NO: 8, A base sequence encoding a protein having GDH enzyme activity can also be used. 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 SiteDirected Mitsnesit Kits; It can be obtained by modifying the base sequence described in SEQ ID NO: 8 using the method. The activity of the protein encoded by the obtained gene can be confirmed by the method described in Examples below.

In the method of the present invention, the FAD-GDH gene represented by the base sequence (A) or (B) binds a secretory signal peptide to the N-terminal of the protein in order to increase the protein expression efficiency and expression success rate. To express in the state. In general, a secretory signal peptide is present at the N-terminus of the secreted protein. By replacing this existing secretory signal peptide with a highly efficient secretory signal peptide, the expression efficiency and the success rate of expression of these secreted proteins are expressed. This is because it has been reported that it can be increased.

Examples of secretory signal peptides that can be used in the method of the present invention include amino acid sequences (C) to (H) represented by any of SEQ ID NOs: 9 to 14. Among these amino acid sequences, the amino acid sequence (C) represented by SEQ ID NO: 9 is a secretory signal peptide sequence derived from FAD-GDH produced by Aspergillus oryzae. All of the amino acid sequences (D) to (F) shown in SEQ ID NOs: 10 to 12 are Cryptococcus sp. S-2 is a secretory signal peptide sequence derived from an acid xylanase produced by S-2. SEQ ID NO: 10 is a sequence (Xs1) having a secretory signal peptide sequence from the start codon to the 37th amino acid, and SEQ ID NO: 11 is a secretory signal peptide. The peptide sequence is a sequence (Xs2) from the start codon to the 23rd amino acid, and SEQ ID NO: 12 is a sequence (Xs3) from the secretory signal peptide sequence to the 17th amino acid from the start codon. The amino acid sequence of (G) shown in SEQ ID NO: 13 is Cryptococcus sp. It is a secretory signal peptide sequence (As) derived from α-amylase produced by S-2. The amino acid sequence of (H) shown in SEQ ID NO: 14 is Cryptococcus sp. It is a secretory signal peptide sequence (Cs) derived from cutinase produced by S-2.

As shown in the following examples, all of these secretory signal peptide sequences are extremely efficient, and by using them, the production efficiency of secreted FAD-GDH to the outside of the microorganism of the genus Cryptococcus is drastically improved. Can be increased.

In order to express these secretory signal peptides in a state where they are bound to the N-terminal of the protein, the amino acid sequences of these secretory signal peptides ((C The fusion DNA to which the base sequence encoding any one of (A) to (H) is linked may be expressed in the 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 α-amylase, acid xylanase, and cutinase that degrades biodegradable plastics. However, 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. In addition, computer programs for predicting the sequence of the secretory signal peptide are provided. By using these computer programs, it is possible to predict the presence of the secretory signal peptide sequence contained in the secreted protein from the amino acid sequence information. However, it is currently difficult to infer the ability of each secretory signal peptide by these computer programs. That is, whether the predicted secretory signal peptide is actually available for the expression of proteins such as secreted proteins, and whether it is more efficient that can be used for mass production of these proteins. It cannot be predicted. Furthermore, depending on the type of the secretory signal peptide to be predicted, there may be several cleavage sites, and it is difficult to predict the optimal length of the secretory signal sequence. The above-mentioned secretory signal peptide used in the method of the present invention is not simply predicted by a computer program, but its effectiveness has been confirmed by repeated experiments by the present inventors.

Next, specific steps of the method of the present invention will be described. The method of the present invention comprises the step of expressing the above-mentioned fusion DNA (FAD-GDH gene + secretory signal peptide sequence) in a microorganism of the genus Cryptococcus. Specifically, in this step, a recombinant vector is prepared by inserting the fusion DNA into an appropriate expression vector according to a conventional method, and then this recombinant expression vector is introduced into a microorganism of the genus Cryptococcus to transform the transformant. And then culturing the transformant 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. Specific examples of E. coli vectors include pBR322, pUC19, pGEM-T, pCR-Blunt, pTA2, and pET. Preferably, 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. For example, 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 And 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. An expression vector containing S-2-derived orotephosphophosphoyltransferase gene, a xylanase promoter DNA sequence, and a xylanase terminator DNA sequence, and cryptococcus sp. A combination with S-2 is mentioned.

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. 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. In particular, 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.

Other nutrient sources commonly used for culturing microorganisms are widely used. As the carbon source, any carbon compound that can be assimilated may be used. For example, glucose, sucrose, lactose, maltose, xylose, molasses, pyruvic acid and the like are used. In addition, 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. Although 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 changed as appropriate as long as 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.

Alternatively, 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 Alternatively, there may be mentioned 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 (iv).
(I) Action: Shows glucose dehydrogenase enzyme activity in the presence of an electron acceptor.
(Ii) Molecular weight: 75 to 90 kDa with sugar chain included.
(Iii) Stable pH range: pH 3.5 to 7.5.
(Iv) Thermal stability: It has a residual activity of 90% or more after heat treatment at 50 ° C. for 15 minutes.

Of these, the molecular weight is particularly noteworthy. Since the molecular weight of FAD-GDH expressed in a conventional microorganism of the genus Aspergillus in Patent Document 3 is about 80 to 120 kDa in a state including a sugar chain, the FAD-GDH enzyme protein obtained by the method of the present invention is Compared with the conventional one, the amount of sugar chain binding is remarkably small and uniform. 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. .

In the present invention, FAD-GDH activity is measured under the following conditions.

FAD-GDH activity measurement method <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 (ΔOD 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 (ΔOD BLANK) is measured in the same manner. From these values, GDH activity is determined according to the following formula (I). Here, 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) = {− (ΔOD TEST−ΔOD BLANK) × 3.1 × dilution factor} / {16.3 × 0.1 × 1.0} (I)

In Formula (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, and 0.1 is the enzyme The liquid volume (ml) of the solution, 1.0 indicates the optical path length (cm) of the cell.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

1. Expression of FAD-GDH gene The optimization of the codon usage of the modified gene derived from the genus Aspergillus described in Patent Document 3 is Cryptococcus sp-S-2. The codon usage of α-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 7th to 72nd nucleotide sequences shown in SEQ ID NO: 1 are predicted to be a nucleotide sequence encoding the secretory signal sequence of the modified FAD-GDH gene derived from the genus Aspergillus. Cryptococcus sp-S-2. The modified FAD-GDH protein derived from the genus Aspergillus that is recombinantly produced in (2) is predicted to be a mature sequence from which this secretory signal sequence has been removed. The base sequence encoding the modified FAD-GDH protein derived from the genus Aspergillus that is secreted as a mature form is shown in SEQ ID NO: 8.

Next, using the rcAOFADGDH (opt) MulI_F of SEQ ID NO: 2 and the rcAOFADGDH (opt) MulI_R of SEQ ID NO: 3 as primers, the full-length sequence of the FAD-GDH gene of SEQ ID NO: 1 was amplified by PCR, and the amplified sequence (AOFADGDH (Opt)) was treated with MluI.

On the other hand, 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 AOFADGLD (opt) was ligated to the treated plasmid to construct a recombinant plasmid (pCsUXAOFADGLD (opt)).

Next, inverse PCR was performed using a KOD-plus Mutagenesis kit (manufactured by Toyobo Co., Ltd.), and mutations were added to the gene sequence. Specifically, in the recombinant plasmid pCsUXAOFADGLD (opt), the amino acid at position 163 is substituted from G to R using rcAOFADGDDH_G163R_F of SEQ ID NO: 4 and rcAOFADGDDH_G163R_R of SEQ ID NO: 5 as primers, and then SEQ ID NO: 6. A recombinant plasmid (pCsUXAOmFADGLD (opt)) was constructed by substituting V at position 551 with C using rcAOFADDGDH_V551C_F and rcAOFADDGDH_V551C_R of SEQ ID NO: 7 as primers. The sequence amplified here corresponds to a sequence obtained by adding the secretory signal peptide sequence shown in SEQ ID NO: 9 to the 5 ′ side of the sequence of SEQ ID NO: 8.

Note that the pCsUX plasmid shown in FIG. Recombinant DNA was prepared by ligating the acidic xylanase promoter (Xyl-pro) derived from S-2, the acidic xylanase terminator (Xyl-ter), and the orthophosphate phosphoryltransferase gene (URA5) to a commercially available pUC19 vector by a conventional method. And E. coli using this recombinant DNA. It can be obtained by transforming E. coli DH5α according to a conventional method. Transformants can be selected by ampicillin resistance.

Next, 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: 9. Specifically, in the recombinant plasmid pCsUXAOmFADGLD (opt), a secretory signal peptide sequence using AOFADGLD (opt-sp) F of SEQ ID NO: 15 and Xylanase (ss) -Xyl-pro-comp of SEQ ID NO: 16 as primers. Is replaced with “Xylanase secretion signal peptide sequence 1” shown in SEQ ID NO: 10 (pCsUXXsAOmFADGLD (opt)); in the recombinant plasmid pCsUXAOmFADGLD (opt), AOFADGLD (opt-sp) F of SEQ ID NO: 15 and SEQ ID NO: 17 Xylanase2 (ss) -Xyl2-pro-comp was used as a primer, and the secretion signal peptide sequence was shown as “Xylanase secretion signal peptide shown in SEQ ID NO: 11. Column 2 ”(pCsUX2AOmFADGLD (opt)); in the recombinant plasmid pCsUXAOmFADGLD (opt), AOFADGLD (opt-sp) F of SEQ ID NO: 15 and Xylanase2 (ss) -Xyl3-pro-comp of SEQ ID NO: 18 Used as a primer, the secretory signal peptide sequence was replaced with “Xylanase secretory signal peptide sequence 3” shown in SEQ ID NO: 12 (pCsUX2Xs3AOmFADGLD (opt); in the recombinant plasmid pCsUXAOmFADGLD (opt), AOFADGLD (SEQ ID NO: 15) opt-sp) F and the secretory signal peptide sequence using Amylase (ss) -Xyl2-pro-comp of SEQ ID NO: 19 as primers In the recombinant plasmid pCsUXAOmFADGLD (opt); and in the recombinant plasmid pCsUXAOmFADGLD (opt); and the “Amylase secretion signal peptide sequence” shown in SEQ ID NO: 13; Cutinase (ss) -Xyl2-pro-comp was used as a primer, and the secretory signal peptide sequence was substituted with the “Cutinase secretory signal peptide sequence” shown in SEQ ID NO: 14 (pCsUX2CsAOmFADGLD (opt)).

2. Cryptococcus sp. Transformation into S-2 The recombinant host is Cryptococcus sp. S-2. U-5 strain was used. This strain is used to select Cryptococcus sp. S-2 was made uracil auxotrophic by natural mutation, and was obtained at the National Institute of Liquor Research with the pCsUX2 plasmid. Cryptococcus sp. Acquisition of a uracil-requiring strain from S-2 was obtained from Cryptococcus sp. This can be easily performed by irradiating S-2 with UV, culturing in a medium containing 5-fluoroorotic acid, and selecting the surviving strain.

Transformation was performed using Infect Immun. 1992 Mar; 60 (3): 1101-8. (Varma et al.). Cryptococcus sp. S-2. The U-5 strain was cultured in a 20 ml YM medium (yeast extract 0.3%, malt extract 0.3%, polypeptone 0.5%, glucose 1.0%) at 25 ° C. for 48 hours. The absorbance (OD 660 nm) of the obtained culture broth was measured and found to be 2.96 Abs. Next, 6.75 ml of this culture solution was inoculated into a 200 ml liquid medium (yeast extract 0.3%, malt extract 0.3%, polypeptone 0.5%, glucose 1.0%), and 25 ° C. for 18 hours. Cultured. The absorbance (OD 660 nm) of the obtained culture broth was measured and found to be 0.94 Abs. Next, this culture solution is centrifuged to collect the cells, and the cells are washed twice with Wash buffer (270 mM sucrose, 1 mM magnesium chloride, 4 mM DTT, 10 mM Tris-HCl pH 7.6), and Electroporation buffer is used. It was suspended in (270 mM sucrose, 1 mM magnesium chloride, 10 mM Tris-HCl pH 7.6) so that the absorbance was OD660 = 50 Abs. To 100 μl of the resulting suspension, 10 μg (˜5 μl) of a linearized plasmid previously treated with SbfI was added, transferred to a cuvette for electroporation, and then Gene Pulser Xcell (BIO-RAD). ). The energization conditions were C = 25 μF; V = 0.47 kV. 600 μl 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.

The transformant obtained by transformation was subjected to colony PCR using KOD-Fx (manufactured by Toyobo Co., Ltd.) using AOFADGLD (opt-sp) F of SEQ ID NO: 15 and rcAOFADGDDH (opt) MulI_R of SEQ ID NO: 3 as primers. The introduction of the gene is confirmed by performing the following steps, and the following 3. Was cultured.

3. 1. Confirmation of FAD-GDH activity The transformant obtained in (1) was cultured in a 3 ml liquid medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1.0% glucose) at 25 ° C., 230 rpm for 48 hours. A culture solution was obtained. 0.03 ml of the preculture was transferred to a 3 ml liquid medium (yeast extract 2%, xylose 5%) and cultured at 25 ° C., 230 rpm for 72 hours to confirm FAD-GDH activity.

FIG. 2 shows the results of measuring the FAD-GDH activity in the culture supernatant for several strains. FIG. 2 shows the measured values of FAD-GDH activity of the strain having the highest productivity among the several transformants obtained.

As shown in FIG. 2, a strain (UXAOm in FIG. 2) in which a gene with optimized codon usage was introduced and the secretory signal peptide sequence (SEQ ID NO: 9) of Aspergillus oryzae FAD-GDH was used as the secretory signal peptide sequence , FAD-GDH activity of about 34.6 U / mL was detected. From this result, the secretory signal peptide sequence of Aspergillus oryzae FAD-GDH was Cryptococcus sp. It was confirmed that it functions effectively also in S-2.

Furthermore, the secretory signal peptide sequence is designated as Cryptococcus sp. In the S-2 Xylanase secretion signal peptide sequence 1 (SEQ ID NO: 10) (UXXs1AOm in FIG. 2), a maximum of about 42.0 U / mL of FAD-GDH activity was detected, and the secretory signal peptide sequence was converted to cryptococcus sp. . In the S-2 Xylanase secretion signal peptide sequence 2 (SEQ ID NO: 11) (UXXs2AOm in FIG. 2), up to about 85.7 U / mL of FAD-GDH activity was detected, and the secretory signal peptide sequence was converted to cryptococcus sp. . In the S-2 Xylanase secretory signal peptide sequence 3 (SEQ ID NO: 12) (UXXs3AOm in FIG. 2), a maximum of about 44.5 U / mL of FAD-GDH activity was detected, and the secretory signal peptide sequence was converted to cryptococcus sp. . In the S-2 Amylase secretory signal peptide sequence (SEQ ID NO: 13) (UXAsAOm in FIG. 2), a maximum of about 21.2 U / mL of FAD-GDH activity was detected, and the secretory signal peptide sequence was converted to Cryptococcus sp. In the S-2 cutinase secretion signal peptide sequence (SEQ ID NO: 14) (UXCsAOm in FIG. 2), a maximum FAD-GDH activity of about 36.0 U / mL was detected.
From the above results, it is possible to efficiently secrete and produce FAD-GDH outside the cells of the genus Cryptococcus by using the six secretory signal peptide sequences described above.

4). 2. Obtain FAD-GDH enzyme protein. The UXXs2AOm strain transformant having the highest FAD-GDH activity was cultured in a medium (5% yeast extract, 5% xylose) at 25 ° C. for 68 hours using a 10 L jar fermenter. After filtering the cultured cells, the culture supernatant was collected and used as a crude enzyme solution in the following experiments.

5. Purification of enzyme FAD-GDH enzyme protein was isolated and purified from the above crude enzyme solution by the following steps (1) to (3).

(1) Concentration / desalting The crude enzyme solution was concentrated with a ultrafiltration membrane having a fractional molecular weight of 30000 and replaced with 20 mM potassium phosphate buffer (pH 6.0) to obtain a crude enzyme concentrate.

(2) Purification by Phenyl-Sepharose FastFlow (GE Healthcare) The active fraction was adjusted to 60% ammonium sulfate saturation (pH 6.0) and then centrifuged to obtain a supernatant. The supernatant was passed through a Phenyl-Sepharose FastFlow column pre-equilibrated with 50 mM potassium phosphate buffer (pH 6.0) 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 buffer to 50 mM potassium phosphate buffer (pH 6.0), and the active fraction was collected. The active fraction was concentrated with an ultrafiltration membrane having a molecular weight cut-off of 10,000 and replaced with 50 mM potassium phosphate buffer.
(3) Purification by DEAE Sepharose (manufactured by GE Healthcare) The crude enzyme concentrate was passed through a DEAE-sepharose FastFlow column pre-equilibrated with 50 mM potassium phosphate buffer (pH 6.0), and the permeation activity image Minutes were collected. The specific activity of the purified enzyme was about 400 U / mg, and the purification rate of the enzyme was about 20 times that of the crude purified enzyme.

6). Characterization of FAD-GDH enzyme protein 5. The characteristics of the purified FAD-GDH derived from Aspergillus oryzae recombinantly produced using Cryptococcus as a host (hereinafter referred to as Cryptococcus recombination modified FAD-GDH) were evaluated. Also, as a control, purification of Aspergillus oryzae-derived modified FAD-GDH (hereinafter referred to as Aspergillus oryzae recombinant modified FAD-GDH) described in Patent Document 3, which was recombinantly produced using Aspergillus oryzae as a host. The characteristics of the enzyme were similarly evaluated.

(1) Thermal stability Each purified enzyme was adjusted to a concentration of 100 U / ml with 50 mM phosphate buffer (pH 6.0), treated at a temperature of 20 ° C. to 65 ° C. for 15 minutes, and the residual activity was calculated. The result is shown in FIG. As is clear from FIG. 3, both Aspergillus oryzae recombinant modified FAD-GDH and Cryptococcus recombinant modified FAD-GDH maintained 90% or more activity up to 50 ° C.

(2) pH stability Each purified enzyme was mixed with 100 mM acetate buffer (pH 3.5 to 5.5), 100 mM potassium phosphate buffer (pH 6.0 to 8.0), 100 mM MPIPES-NaOH buffer (pH 6.5). -7.5), 100 mM Tris-HCl buffer (pH 7.5-9.0), adjusted to a concentration of 20 U / ml, treated at 25 ° C. for 16 hours, and the residual activity was calculated. The result is shown in FIG. As is clear from FIG. 4, both Aspergillus oryzae recombinant modified FAD-GDH and Cryptococcus recombinant modified FAD-GDH maintained an activity of 90% or more in the pH range of 3.5 to 7.5. .

(3) Molecular Weight The molecular weight of each purified enzyme was determined by SDS-polyacrylamide gel electrophoresis using Nu-PAGE 4-12% Bis-Tris Gel (manufactured by Invitrogen). The results are shown 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: molecular weight marker (Invitrogen, Benchmark Protein ladder)
Lane 3: Cryptococcus recombination modified FAD-GDH purified enzyme.
Lane 4: Aspergillus oryzae recombinant modified FAD-GDH purified enzyme.
Lane 5: molecular weight marker (Invitrogen, Benchmark Protein ladder)
Lane 6: Molecular weight marker (Invitrogen, Novex (registered trademark) Sharp Unstained Protein Standard)

Further, each purified enzyme was deglycosylated using a deglycosylase (Roche, endoglycosidase H), and the molecular weight after removal of the sugar chain was determined by electrophoresis in the same manner. The results are shown in FIG. The electrophoresis sample is as follows.
Lane 1: molecular weight marker (Invitrogen, Mark 12)
Lane 2: Cryptococcus recombination modified FAD-GDH purified enzyme.
Lane 3: Cryptococcal recombination modified FAD-GDH purified enzyme after deglycosylation treatment.
Lane 4: Aspergillus oryzae recombinant modified FAD-GDH purified enzyme.
Lane 5: Aspergillus oryzae recombinant modified FAD-GDH purified enzyme after deglycosylation treatment.

As can be seen from FIG. 5, the molecular weight of Cryptococcus recombination modified FAD-GDH is 75 kDa to 90 kDa, which is uniform compared to the molecular weight of Aspergillus oryzae recombination modified FAD-GDH (80 kDa to 120 kDa).

Further, as can be seen from the comparison between lanes 2 and 3 in FIG. 6, the molecular weight of Cryptococcus recombinant modified FAD-GDH was reduced to 60 kDa by deglycosylation treatment. The sugar chain is predicted to be uniform at 15 kDa to 30 kDa, and the amount of sugar chain binding per protein is estimated to be about 20% on average. In addition, as can be seen from the comparison between lanes 4 and 5 in FIG. 6, the molecular weight of Aspergillus oryzae recombinant modified FAD-GDH was reduced to 60 kDa by deglycosylation treatment. The sugar chain of the modified FAD-GDH is predicted to be heterogeneous with about 20 kDa to 60 kDa, and the amount of sugar chain binding per protein is estimated to be about 40% on average.

(4) Amount of sugar chain bound The amount of sugar chain bound per enzyme protein was measured by the phenol-sulfuric acid method using freeze-dried Aspergillus oryzae recombinant modified FAD-GDH and cryptococcal recombinant modified FAD-GDH. did. The lyophilized FAD-GDH powder was dissolved in ion exchange water at 0.2 g / L to prepare a FAD-GDH solution. Next, 0.5 ml of this FAD-GDH solution, 0.5 ml of 5% (w / v) phenol solution, and 2.5 ml of 97% sulfuric acid were mixed and cooled to room temperature, and then the absorbance at 490 nm was measured. A calibration curve was prepared with 0-200 mg / L D-mannose standard solution, and the amount of sugar chain bound per enzyme powder weight was measured. As a result, the amount of sugar chains bound by Aspergillus oryzae recombinant modified FAD-GDH was 40%, whereas the amount of sugar chains bound by Cryptococcus recombinant modified FAD-GDH was 18%.

From the above results, the cryptococcal recombinant modified FAD-GDH obtained in this example has almost the same characteristics as the Aspergillus oryzae recombinant modified FAD-GDH, and has a small amount of sugar chain binding and is uniform. It turned out to be.

In this example, the secretory signal peptide of SEQ ID NO: 11 is used. However, the secretory signal peptide is involved in the protein expression efficiency and expression success rate of FAD-GDH, and sugar chain binding to the expressed protein. Therefore, even if the secretory signal peptide is replaced with a peptide other than SEQ ID NO: 11, as long as a microorganism of the genus Cryptococcus is used as a host, the amount of sugar chain binding is small and uniform as in this example. -It is thought that GDH is obtained.

According to the present invention, FAD-GDH having characteristics equivalent to those of modified FAD-GDH derived from Aspergillus oryzae and having a small amount of sugar chain binding and being uniform 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.

Claims (4)

1. A method for producing a flavin adenine dinucleotide-linked glucose dehydrogenase having a small amount of sugar chain binding and uniform, which is represented by the following base sequence (A) or (B) in a microorganism belonging to the genus Cryptococcus: A step of expressing a fusion DNA in which a nucleotide sequence encoding any amino acid sequence selected from the group consisting of the following (C) to (H) is bound to the 5 ′ side of a flavin adenine dinucleotide-binding glucose dehydrogenase gene: A method characterized by comprising:
   (A) the base sequence represented by SEQ ID NO: 8;
   (B) a base sequence that is 90% or more homologous to the base sequence represented by SEQ ID NO: 8 and encodes a protein having flavin adenine dinucleotide-binding glucose dehydrogenase enzyme activity;
   (C) the amino acid sequence represented by SEQ ID NO: 9;
   (D) the amino acid sequence represented by SEQ ID NO: 10;
   (E) the amino acid sequence represented by SEQ ID NO: 11;
   (F) the amino acid sequence represented by SEQ ID NO: 12;
   (G) the amino acid sequence represented by SEQ ID NO: 13;
   (H) The amino acid sequence represented by SEQ ID NO: 14.
2. Microorganism is Cryptococcus sp. The method according to claim 1, wherein the method is S-2 (Cryptococcus sp. S-2) (Accession number FERM BP-10961).
3. A flavin adenine dinucleotide-binding glucose dehydrogenase enzyme protein produced by the method according to claim 1 or 2, wherein the protein has a molecular weight of 75 to 90 kDa in a state containing a sugar chain.
4. A biosensor for self-monitoring of blood glucose, wherein the flavin adenine dinucleotide-binding glucose dehydrogenase enzyme protein according to claim 3 is used as an enzyme having glucose as a substrate.

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