JP5277482B2 - Method for producing flavin adenine dinucleotide-linked glucose dehydrogenase - Google Patents
Method for producing flavin adenine dinucleotide-linked glucose dehydrogenase Download PDFInfo
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- JP5277482B2 JP5277482B2 JP2012255852A JP2012255852A JP5277482B2 JP 5277482 B2 JP5277482 B2 JP 5277482B2 JP 2012255852 A JP2012255852 A JP 2012255852A JP 2012255852 A JP2012255852 A JP 2012255852A JP 5277482 B2 JP5277482 B2 JP 5277482B2
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- 235000019162 flavin adenine dinucleotide Nutrition 0.000 title claims description 13
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Classifications
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Abstract
Description
本発明は、特定の微生物を利用した、糖鎖結合量が少なくかつ均一である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(以下、FAD−GDHとも記載する)の生産方法に関し、特に、血糖自己測定用のバイオセンサに使用するのに好適なFAD−GDHの生産方法に関する。 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.
血糖自己測定は、糖尿病患者が通常の自分の血糖値を把握して治療に生かすために重要である。血糖自己測定に用いられるバイオセンサは、一般的に、絶縁体基板上に、測定電極、対電極及び検知電極からなる電極層を形成し、これらの電極基板上に、グルコースを基質とする酵素と電子受容体を含む試薬層を形成してなる。このバイオセンサの血糖値の測定は、バイオセンサに血液を添加して、試薬層中のグルコースを基質とする酵素を血液中のグルコースと反応させて、電子を生成させ、この電子による電子受容体の還元及び酸化から生じる電流値に基づいて血液中のグルコース濃度(血糖値)を求めることによって行われる。 Blood glucose self-measurement is important for a diabetic patient to grasp his or her 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.
このバイオセンサで使用されるグルコースを基質とする酵素の例としては、グルコースオキシダーゼ(EC 1.1.3.4)が挙げられる。グルコースオキシダーゼは、グルコースに対する特異性が高く、熱安定性に優れているという利点を有していることから、血糖自己測定用のバイオセンサにおいて古くから利用されている。グルコースオキシダーゼを利用した血糖自己測定用のバイオセンサにおいては、グルコースを酸化してD−グルコノ−δ−ラクトンに変換する過程で生じる電子がメディエーターを介して電極に渡されることで血液中のグルコース濃度の測定がなされるが、グルコースオキシダーゼは反応で生じたプロトンを酸素に渡しやすいため、溶存酸素が測定値に影響してしまうという問題があった。 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 self-measurement of blood glucose using glucose oxidase, the concentration of glucose in blood is obtained by passing electrons generated in the process of oxidizing glucose and converting it to D-glucono-δ-lactone via an 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.
このような問題を回避するために、例えばNAD(P)依存型グルコースデヒドロゲナーゼ(EC1.1.1.47)あるいはピロロキノリンキノン依存型グルコースデヒドロゲナーゼ(EC1.1.5.2(旧EC1.1.99.17))といったグルコースデヒドロゲナーゼの使用が提案されている。これらの酵素は溶存酸素の影響を受けない点で優位であるが、前者のNAD(P)依存型グルコースデヒドロゲナーゼは、安定性が乏しいという欠点や補酵素の添加が必要で測定が煩雑になるという欠点がある。一方、後者のピロロキノリンキノン依存型グルコースデヒドロゲナーゼは、基質特異性に乏しく、マルトースやラクトースといったグルコース以外の糖類にも作用するため、測定値の正確性を損ねてしまうという欠点がある。 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.
これらの欠点を有さないグルコースデヒドロゲナーゼとして、特許文献1には、アスペルギルス属由来の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(FAD−GDH)の使用が提案されている。この酵素は、基質特異性に優れかつ溶存酸素の影響を受けない点で上述のグルコースデヒドロゲナーゼより優位である。また、この酵素は、50℃、15分処理で89%程度の活性残存率を示し、熱安定性についても優れている。特許文献2には、アスペルギルス・オリゼ由来のFAD−GDHをコードするDNA配列が開示されており、特許文献3には、アスペルギルス属由来のFAD−GDHのアミノ酸配列に改変を加えて熱安定性を向上させたFAD−GDHが開示されている。この特許文献3のFAD−GDHは、アスペルギルス属にセルフクローニングして糖鎖結合型にすることによって、熱安定性をさらに高めることができることが本発明者らによって判明している。 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 improves the thermal stability by modifying the amino acid sequence of Aspergillus derived FAD-GDH. An improved FAD-GDH is disclosed. It has been found by the present inventors that the FAD-GDH of Patent Document 3 can be further improved in thermal stability by self-cloning into Aspergillus genus to form a sugar chain binding type.
しかしながら、この糖鎖結合型FAD−GDHを血糖自己測定用のバイオセンサに使用すると、正常に作動しない場合があった。そこで本発明者らがその原因を追求したところ、このFAD−GDHには多数の糖鎖が結合しており、この糖鎖が試薬溶液の粘度を著しく増加させ、溶解を妨げるため、バイオセンサが正常に作動しなくなることが判明した。実際、本発明者らの調査によれば、特許文献3のFAD−GDHをアスペルギルス属にセルフクローニングして糖鎖結合型とした場合、酵素粉末あたりの糖鎖結合量は約40%程度であり、SDS−PAGEによる分子量は約80kDaから120kDaであり、糖鎖結合量が多い上に変動が大きかった。 However, when this sugar chain-binding FAD-GDH is used for a biosensor for blood glucose self-measurement, it may not operate normally. Therefore, the present inventors pursued the cause, and this FAD-GDH has a large number of sugar chains bound thereto, and 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 the genus 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 sugar chain binding amount was large and the fluctuation was large.
従って、血糖自己測定用のバイオセンサに使用する糖鎖結合型FAD−GDHとしては、糖鎖結合量が少なくかつ均一であるものが望まれていた。 Therefore, it has been desired that the sugar chain-binding FAD-GDH used for the biosensor for self-monitoring blood sugar has a small amount of sugar chain binding and is uniform.
本発明は、かかる従来技術の現状に鑑み創案されたものであり、その目的は、血糖自己測定用のバイオセンサに好適な、糖鎖結合量が少なくかつ均一であるFAD−GDHの生産方法を提供することにある。 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.
本発明者らは、かかる目的を達成するために鋭意検討した結果、担子菌酵母の一種であるクリプトコッカス属に分類される微生物を宿主として使用し、この微生物に、特許文献3に記載されている改変型アスペルギルス・オリゼ由来FAD−GDH遺伝子を導入してこの微生物中でこの遺伝子を発現させると、糖鎖結合量が少なくかつ均一であるFAD−GDHが生産されることを見出した。 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 has been found that when a modified Aspergillus oryzae-derived FAD-GDH gene is introduced and expressed in this microorganism, FAD-GDH having a small amount of sugar chain binding and uniform is produced.
また、本発明者らは、この微生物を宿主としてアスペルギルス・オリゼ由来のFAD−GDH遺伝子を発現させる場合、遺伝子のコドンユーセージをこの微生物に合わせて最適化することが、封入体を形成せずに酵素活性を維持したままのFAD−GDHを培養液中に高レベルで分泌生産させるために必要であることを見出した。また、遺伝子配列本体に付加される分泌シグナルペプチド配列を最適化することにより、宿主細胞外へのFAD−GDHの分泌生産性がさらに飛躍的に向上されることを見出した。 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 to the outside of the host cell is further improved.
本発明は、これらの知見に基づいて完成されたものであり、以下の(1)〜(4)から構成されるものである。
(1)糖鎖結合量が少なくかつ均一である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であって、クリプトコッカス(Cryptococcus)属の微生物中で、以下の(A)または(B)の塩基配列で表わされる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遺伝子の5′側に以下の(C)〜(H)からなる群より選択されるいずれかのアミノ酸配列をコードする塩基配列が結合された融合DNAを発現させる工程を含むことを特徴とする方法:
(A)配列番号8で示される塩基配列;
(B)配列番号8で示される塩基配列に対して90%以上相同な塩基配列であって、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酵素活性を持つタンパク質をコードする塩基配列;
(C)配列番号9で示されるアミノ酸配列;
(D)配列番号10で示されるアミノ酸配列;
(E)配列番号11で示されるアミノ酸配列;
(F)配列番号12で示されるアミノ酸配列;
(G)配列番号13で示されるアミノ酸配列;
(H)配列番号14で示されるアミノ酸配列。
(2)微生物が、クリプトコッカスsp.S−2(Cryptococcus sp.S−2)(受託番号FERM BP−10961)であることを特徴とする(1)に記載の方法。
(3)(1)または(2)に記載の方法によって生産される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酵素タンパク質であって、糖鎖を含んだ状態で75〜90kDaの分子量を有することを特徴とするタンパク質。
(4)グルコースを基質とする酵素として、(3)に記載の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酵素タンパク質を使用することを特徴とする血糖自己測定用バイオセンサ。
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-linked glucose dehydrogenase having a small amount of sugar chain binding and uniform, wherein the following base sequence (A) or (B) is used in a microorganism belonging to the genus Cryptococcus: 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 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 No. FERM BP-10961).
(3) A flavin adenine dinucleotide-binding 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.
本発明の方法は、アスペルギルス属の微生物由来の糖鎖結合型FAD−GDH遺伝子のコドンユーセージを最適化し、この微生物とは全く異なる特定の微生物中で、特定の分泌シグナルペプチドと結合された状態で発現させるので、糖鎖結合量が少なくかつ均一であるFAD−GDHを宿主細胞外へ効率的に分泌生産することができる。かかるFAD−GDHは、熱安定性に優れかつ試薬溶液に容易に溶解するため、血糖自己測定用のバイオセンサに使用するのに好適である。 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, it is possible to efficiently secrete and produce FAD-GDH having a small amount of sugar chain binding and uniform to the outside of 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.
本発明は、アスペルギルス属の微生物由来の糖鎖結合型FAD−GDH遺伝子のコドンユーセージを最適化し、この微生物とは全く異なる特定の微生物中で、特定の分泌シグナルペプチドと結合された状態で発現させることにより、本来の宿主微生物中で発現させたFAD−GDHと比べて糖鎖結合量が少なくかつ均一であるFAD−GDHを効率的に得ることを特徴とするものである。 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 bound to a specific secretion signal peptide. Thus, it is characterized in that FAD-GDH having a small amount of sugar chain binding and uniform is efficiently obtained as compared with FAD-GDH expressed in the original host microorganism.
具体的には、本発明の方法では、遺伝子発現の宿主微生物として、担子菌酵母の一種であるクリプトコッカス属の微生物を使用する。好ましくは、クリプトコッカス属の微生物の中でも、クリプトコッカスsp.S−2株という特定の菌株を使用する。この菌株は、独立行政法人酒類総合研究所において単離された酵母であり、αアミラーゼ、酸性キシラナーゼ、クチナーゼなどの酵素を大量に生産することが確認されている。なお、この菌株は、日本国茨城県つくば市東1丁目1番地1 中央第6の独立行政法人産業技術総合研究所(現在の名称は、製品評価技術基盤機構) 特許生物寄託センターに1995年9月5日にFERM P−15155として国内寄託し、2008年4月25日にFERM BP−10961として国際寄託に移管済である。 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 S-2 strain 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 6th 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 April 25, 2008.
本発明の方法において、クリプトコッカス属の微生物を遺伝子発現の宿主微生物として使用することにより糖鎖結合量が少なくかつ均一であるFAD−GDHが得られる理由は未だ明確ではないが、クリプトコッカス属の微生物に特有の糖鎖合成及び結合システムが存在するためであると考えられる。 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 Cryptococcus microorganism 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.
本発明の方法では、宿主微生物中で発現させる糖鎖結合型FAD−GDH遺伝子は、(A)配列番号8の塩基配列で表わされるものである。この塩基配列は、特許文献3に記載のアスペルギルス属由来の改変型遺伝子の塩基配列そのものではなく、宿主のクリプトコッカス属の微生物中での発現に適するようにそのコドンユーセージが最適化されている。改変型遺伝子の塩基配列をクリプトコッカス属の微生物中にそのまま導入して発現させても、発現産物は封入体を形成してしまい、不活性なFAD−GDHしか得られない。本発明の方法によれば、発現させる遺伝子のコドンユーセージを宿主微生物のコドンユーセージに合わせて最適化することにより、封入体を形成させずに酵素活性を維持したままFAD−GDHを分泌生産させることができる。 In the method of the present invention, the sugar chain-binding FAD-GDH gene expressed in the host microorganism is represented by (A) 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 directly 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 in accordance with the codon usage of the host microorganism. Can be made.
配列番号8のコドンユーセージは、クリプトコッカスsp.S−2の発現に最適化されたものであり、その塩基配列のコドンの3文字目の塩基がGもしくはCである比率は92.6%である。一方、クリプトコッカスsp.S−2で発現が確認されている、αアミラーゼ、クチナーゼ、キシラナーゼの塩基配列のコドンの3文字目の塩基がGもしくはCである比率はそれぞれ、81.5%、81.3%、80.0%であり、コドンの3文字目の塩基がGもしくはCである比率は非常に高い傾向がある。これらのことから、配列番号8はクリプトコッカスsp.S−2中での発現に適するだけでなく、クリプトコッカスsp.S−2と類似のコドンユーセージを有する他のクリプトコッカス属の微生物(Cryptococcus liquefaciens, Cryptococcus flavus, Cryptococcus curvatusなど)中での発現にも適すると予測される。なお、宿主微生物のコドンユーセージは、Codon Usage Database(http://www.kazusa.or.jp/codon/)から提供される情報を用いることで予測することが可能である。 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 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/).
また、本発明の方法では、(A)配列番号8で示される塩基配列の代わりに、(B)配列番号8で示される塩基配列に対して90%以上相同な塩基配列であって、FAD−GDH酵素活性を持つタンパク質をコードする塩基配列も使用できる。この(B)の塩基配列は、(A)の塩基配列の均等の範囲の塩基配列である。これは、酵素タンパク質をコードする遺伝子の塩基配列の一部に変異が生じたり、またその結果として酵素タンパク質のアミノ酸配列の一部に変異が生じても、機能的には同等の酵素タンパク質であることが多いからである。(B)の塩基配列は、例えばTransformerMutagenesis Kit;Clonetech製、EXOIII/Mung Bean Deletion Kit;Stratagene製、QuickChange Site Directed Mutagenesis Kit;Stratagene製、KOD−Plus−Mutagenesis Kit;東洋紡製などの市販のキットやPCR法を利用して配列番号8に記載の塩基配列を改変することによって得ることができる。得られた遺伝子によってコードされるタンパク質の活性は、後述の実施例に記載の方法によって確認することができる。 In addition, 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 Selection Kit; manufactured by Stratagene; 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.
本発明の方法では、(A)または(B)の塩基配列で表されるFAD−GDH遺伝子は、タンパク質の発現効率及び発現成功率を高めるため、タンパク質のN末満に分泌シグナルペプチドを結合させた状態で発現させる。一般的に、分泌タンパク質のN末端には、分泌シグナルペプチドが存在しており、この既存の分泌シグナルペプチドを高効率な分泌シグナルペプチドと置換することで、これら分泌タンパク質の発現効率及び発現成功率を上げることができることが報告されているからである。 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 expression efficiency and success rate of the protein. To be expressed in a wet 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.
本発明の方法で使用できる分泌シグナルペプチドとして、配列番号9〜14のいずれかで示されるアミノ酸配列(C)〜(H)を挙げることができる。これらのアミノ酸配列のうち、配列番号9で示される(C)のアミノ酸配列は、アスペルギルス・オリゼが生産するFAD−GDHに由来する分泌シグナルペプチド配列である。配列番号10〜12で示される(D)〜(F)のアミノ酸配列は、いずれもクリプトコッカスsp.S−2が生産する酸性キシラナーゼに由来する分泌シグナルペプチド配列であり、配列番号10は分泌シグナルペプチド配列を開始コドンから37番目のアミノ酸までとした配列(Xs1)であり、配列番号11は分泌シグナルペプチド配列を開始コドンから23番目のアミノ酸までとした配列(Xs2)であり、配列番号12は分泌シグナルペプチド配列を開始コドンから17アミノ酸までとした配列(Xs3)である。配列番号13で示される(G)のアミノ酸配列は、クリプトコッカスsp.S−2が生産するα―アミラーゼに由来する分泌シグナルペプチド配列(As)である。配列番号14で示される(H)のアミノ酸配列は、クリプトコッカスsp.S−2が生産するクチナーゼに由来する分泌シグナルペプチド配列(Cs)である。 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) represented by 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 the secretory signal peptide sequence from the start codon to the 37th amino acid, and SEQ ID NO: 11 is a secretion signal 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.
以下の実施例で示すように、これらの分泌シグナルペプチド配列はいずれも、極めて高効率であり、これらを用いることにより、クリプトコッカス属の微生物の細胞外へのFAD−GDHの分泌生産効率を飛躍的に増大させることができる。 As shown in the following examples, all of these secretory signal peptide sequences are extremely efficient, and by using them, the efficiency of secretory production of FAD-GDH from the outside of the microorganism of the genus Cryptococcus is drastically improved. Can be increased.
これらの分泌シグナルペプチドをタンパク質のN末端に結合された状態で発現させるためには、上述の(A)または(B)の塩基配列の5′側にこれらの分泌シグナルペプチドのアミノ酸配列((C)〜(H)のアミノ酸配列)のいずれかをコードする塩基配列が結合された融合DNAを宿主微生物中で発現させればよい。 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 ) To (H) amino acid sequence) may be expressed in the host microorganism.
本発明の方法において宿主として使用することができる微生物であるクリプトコッカスsp.S−2は、各種難分解性酵素を分泌生産することが確認されており、生でんぷん分解性のα―アミラーゼや酸性キシラナーゼ、生分解性プラスチックを分解するクチナーゼなどの酵素を分泌生産する。しかしながら、これらの分泌タンパク質の有する分泌シグナルペプチドは、従来から分泌タンパク質に見出されており、最も高効率な分泌シグナルペプチドかどうかは不明であった。また、分泌シグナルペプチドの配列を予測するコンピュータプログラムが提供されており、これらのコンピュータプログラムを用いることで、アミノ酸配列情報から分泌タンパク質に含まれる分泌シグナルペプチドの配列の存在を予測することが可能であるが、これらのコンピュータプログラムにより各分泌シグナルペプチドの能力を推測することは現在のところ困難である。すなわち、予測された分泌シグナルペプチドが、実際に、分泌タンパク質等のタンパク質の発現に利用可能であるか否か、さらにこれらタンパク質の大量生産に利用できる、より高効率なものであるか否かは予測することができない。さらに、予測される分泌シグナルペプチドの種類によっては、切断部位が数箇所に及ぶ場合もあり、分泌シグナル配列の最適な長さに関しても、予測することは困難である。本発明の方法で使用する上述の分泌シグナルペプチドは、単にコンピュータプログラムによって予測されたものではなく、本発明者らの繰り返しの実験によってその有効性を認められたものである。 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 persistent enzymes, and secretes and produces enzymes such as raw starch-degrading 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.
次に、本発明の方法の具体的な工程について説明する。本発明の方法は、クリプトコッカス属の微生物中で、上記融合DNA(FAD−GDH遺伝子+分泌シグナルペプチド配列)を発現させる工程を含む。この工程は、具体的には、常法に従って融合DNAを適当な発現ベクターに挿入して組換えベクターを作成し、次にこの組換え発現ベクターをクリプトコッカス属の微生物に導入して形質転換体を作成し、この形質転換体を適当な条件下で培養することによって行うことができる。 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.
本発明の方法で使用する発現ベクターとしては、特に限定するものではないが、従来公知のベクター、例えば大腸菌のベクターが挙げられる。大腸菌のベクターとしては、具体的にはpBR322、pUC19、pGEM−T、pCR−Blunt、pTA2、pETなどが挙げられる。好ましくは、ベクターDNAは、栄養要求性マーカー、薬剤耐性マーカー、発現プロモーターDNA配列、発現ターミネーターDNA配列を含み、より好ましくは、クリプトコッカスsp.S−2由来のorotate phosphoribosyl transferase遺伝子、キシラナーゼプロモーター、キシラナーゼターミネーターを含む。 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. The S-2-derived orophosphate phosphoyltransferase gene, xylanase promoter, and xylanase terminator are included.
発現ベクターと宿主微生物の組み合わせとしては、特に限定するものではないが、例えば遺伝子を組み込む宿主由来の栄養要求性マーカー遺伝子または薬剤耐性マーカー遺伝子と、遺伝子を組み込む宿主由来の発現プロモーターDNA配列と、遺伝子を組み込む宿主由来のターミネーターDNA配列とを含む発現ベクターと、栄養要求性変異宿主または薬剤感受性宿主との組み合わせが挙げられる。最も好ましくは、クリプトコッカスsp.S−2由来のorotate phosphoribosyl transferase遺伝子と、キシラナーゼプロモーターDNA配列と、キシラナーゼターミネーターDNA配列とを含む発現ベクターと、クリプトコッカスsp.S−2との組み合わせが挙げられる。 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 comprising an S-2-derived orophosphate phosphoyltransferase gene, a xylanase promoter DNA sequence, and a xylanase terminator DNA sequence, and a cryptococcus sp. A combination with S-2 is mentioned.
宿主微生物の細胞に組換え発現ベクターを導入する方法としては、特に限定するものではないが、エレクトロポレーションなどの方法が挙げられる。 A method for introducing the recombinant expression vector into the cells of the host microorganism is not particularly limited, and examples thereof include electroporation.
形質転換体の培養形態は、宿主の栄養生理的性質を考慮して適宜選択すればよく、通常多くの場合は液体培養で行うが、工業的には通気攪拌培養を行うのが有利である。なお、培養に先立って、高FAD−GDH生産細胞株を予め選抜しておくことが有利である。 The culture form of the transformant may be appropriately selected in consideration of the nutritional physiological properties of the host. Usually, the culture is performed in liquid culture, 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.
培養に用いる窒素源は、特定のアミノ酸成分に欠失があるなど特殊なN源を除いて、宿主微生物が利用可能な窒素化合物であればどんなものでも良い。これらは主として有機窒素源であり、例えばペプトン、肉エキス、酵母エキス、カゼイン加水分解物、大豆粕アルカリ分解物などが使用される。特に、酵母エキスや大豆蛋白質が好ましいが、これに限定されるものではなく、カゼインポリペプトン、発酵麹エキス、麦芽抽出物などを用いることによっても、形質転換体を培養することができる。 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.
その他の栄養源としては、微生物の培養に通常用いられるものが広く使用される。炭素源としては、資化可能な炭素化合物であればよく、例えば、グルコース、シュークロース、ラクトース、マルトース、キシロース、糖蜜、ピルビン酸などが使用される。その他、リン酸塩、炭酸塩、硫酸塩、マグネシウム、カルシウム、カリウム、鉄、マンガン、亜鉛などの塩類、特定のアミノ酸、特定のビタミンなどが必要に応じて使用される。 As other nutrient sources, those 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.
培養温度は、菌が発育してFAD−GDH酵素タンパク質を生産する範囲で適宜変更しうるが、クリプトコッカスsp.S−2の場合、通常は20〜25℃程度である。培養時間は、条件によって多少異なるが、FAD−GDH酵素タンパク質が最高収量に達する時期を見計らって適当な時期に培養を終了すればよく、通常は60〜120時間程度である。培地pHは、菌が発育してFAD−GDH酵素タンパク質を生産する範囲で適宜変更しうるが、通常はpH3.0〜9.0程度である。 The culture temperature can be appropriately changed within the range in which the fungus grows and produces FAD-GDH enzyme protein. In the case of S-2, it is usually about 20-25 ° 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 medium pH 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.
本発明のFAD−GDH酵素タンパク質は、上記形質転換体を培養して得られる菌体を含む培養液をそのまま採取し利用することもできるが、一般には、常法に従って、予め、濾過、遠心分離などにより、FAD−GDH酵素タンパク質含有溶液と菌体とを分離した後に利用することもできる。 The FAD-GDH enzyme protein of the present invention can be used by directly collecting and using a culture solution containing cells obtained by culturing the above transformant, but generally, filtration and centrifugation in advance according to a conventional method. It can also be used after separating the FAD-GDH enzyme protein-containing solution and the microbial cells.
あるいは、このようにして得られたFAD−GDH酵素タンパク質含有溶液からFAD−GDH酵素タンパク質を精製して利用してもよい。精製方法としては、例えば、減圧濃縮、膜濃縮、硫酸アンモニウム、硫酸ナトリウムなどの塩析処理、親水性有機溶媒、例えばメタノール、エタノール、アセトンなどによる分別沈殿、加温処理や等電点処理、吸着剤或いはゲル濾過剤などによるゲル濾過、吸着クロマトグラフィー、イオン交換クロマトグラフィー、疎水性相互作用クロマトグラフィー等の処理を挙げることができる。 Or you may refine | purify and utilize FAD-GDH enzyme protein from the FAD-GDH enzyme protein containing solution obtained in this way. 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.
本発明の方法により得られるFAD−GDH酵素タンパク質は、以下の(i)〜(iv)の性質を有する。
(i)作用:電子受容体存在下でグルコースデヒドロゲナーゼ酵素活性を示す。
(ii)分子量:糖鎖を含んだ状態で75〜90kDaである。
(iii)安定pH範囲:pH3.5〜7.5である。
(iv)熱安定性:50℃15分間の熱処理後に90%以上の残存活性を有する。
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 in a state including a sugar chain.
(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.
このうち、特に注目すべきは、その分子量である。従来の特許文献3のアスペルギルス属の微生物中で発現されたFAD−GDHの分子量は、糖鎖を含んだ状態で約80〜120kDaであるので、本発明の方法により得られるFAD−GDH酵素タンパク質は、従来のものと比べて糖鎖結合量が著しく少なくかつ均一である。従って、本発明の方法により得られるFAD−GDH酵素タンパク質は、このような酵素化学的特徴を活かして、血糖自己測定用バイオセンサに好適に使用することができる。 Of these, the molecular weight is particularly noteworthy. Since the molecular weight of FAD-GDH expressed in a conventional microorganism of the genus Aspergillus of 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.
上記の各種の酵素化学的性質は、酵素の諸性質を特定するための公知の手法、例えば、以下の実施例に記載の方法を用いて調べることができる。酵素の諸性質は、本発明のFAD−GDHを生産する形質転換体の培養液や、精製工程の途中段階において、ある程度調べることもでき、より詳細には、精製酵素を用いて調べることができる。 The various enzyme chemical properties described above can be examined using known techniques 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, can be examined using the purified enzyme. .
精製酵素とは、当該酵素以外の成分、特に当該酵素以外のタンパク質(夾雑タンパク質)を実質的に含まない状態に分離された酵素を指す。具体的には、例えば、夾雑タンパク質の含有量が重量換算で全体の約20%未満、好ましくは約10%未満、更に好ましくは約5%未満、より一層好ましくは約1%未満の酵素を指す。 A purified enzyme refers to an enzyme that has been separated so as to be substantially free of 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. .
本発明において、FAD−GDHの活性測定は以下の条件で行う。 In the present invention, FAD-GDH activity is measured under the following conditions.
FAD−GDHの活性測定法
<反応試薬>
下記のPIPES緩衝液15.6ml、DCPIP溶液0.2ml、D―グルコース溶液4mlを混合して反応試薬とする。
・ 50mM PIPES緩衝液pH6.5(0.1%TritonX−100を含む)
・ 6.8mM 2,6−ジクロロフェノールインドフェノール(DCPIP)溶液
・ 1M D−グルコース溶液
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
<測定条件>
反応試薬3mlを37℃で5分間予備加温する。FAD−GDH溶液0.1mlを添加し、ゆるやかに混和後、水を対照にして37℃に制御された分光光度計で、600nmの吸光度変化を5分間記録し、直線部分から1分間あたりの吸光度変化(ΔOD TEST)を測定する。盲検はFAD−GDH溶液の代わりにFAD−GDHを溶解する溶媒を反応試薬に加えて同様に1分間あたりの吸光度変化(ΔOD BLANK)を測定する。これらの値から以下の式(I)に従ってGDH活性を求める。ここでGDH活性における1単位(U)とは、濃度200mMのD−グルコース存在下で1分間に1マイクロモルのDCPIPを還元する酵素量として定義される。
FAD−GDHの活性(U/ml)={−(ΔOD TEST−ΔOD BLANK)×3.1×希釈倍率}/{16.3×0.1×1.0}・・・(I)
<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, and record the change in absorbance at 600 nm for 5 minutes using a spectrophotometer controlled at 37 ° C. with water as a control. Absorbance per minute from the straight line The change (ΔOD TEST) is measured. In the blind test, a solvent that dissolves 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)
なお、式(I)中の3.1は反応試薬+酵素溶液の液量(ml)、16.3は本活性測定条件におけるミリモル分子吸光係数(cm2/マイクロモル)、0.1は酵素溶液の液量(ml)、1.0はセルの光路長(cm)を示す。 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.
以下、本発明を実施例によりさらに具体的に説明するが、本発明は、これらの実施例に限定されるものではない。 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
1.FAD−GDH遺伝子の発現
特許文献3に記載のアスペルギルス属由来の改変型遺伝子のコドンユーセージの最適化は、クリプトコッカスsp−S−2.の既知の遺伝子であるαアミラーゼ遺伝子、クチナーゼ遺伝子、キシラナーゼ遺伝子のコドンユーセージを参考に行った。最適化された遺伝子配列を配列番号1に示す。
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.
配列番号1に示される7番目から72番目の塩基配列は、アスペルギルス属由来の改変型FAD−GDH遺伝子の分泌シグナル配列をコードする塩基配列であると予測される。クリプトコッカスsp−S−2.において組換え生産されたアスペルギルス属由来の改変型FAD−GDHタンパク質は、本分泌シグナル配列が除去された成熟型の配列と予測される。成熟型として分泌されるアスペルギルス属由来の改変型FAD−GDHタンパク質をコードする塩基配列を配列番号8に示す。 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.
次に、配列番号2のrcAOFADGDH(opt)MulI_F及び配列番号3のrcAOFADGDH(opt)MulI_Rをプライマーとして用いて、配列番号1のFAD−GDH遺伝子全長配列をPCRにより増幅し、増幅された配列(AOFADGDH(opt))をMluI処理した。 Next, using the rcAOFADGDDH (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.
一方、Xylプロモーター、Xylターミネーター、及びURA5遺伝子を含むpCsUXプラスミド(5.9kbp)をMluI処理し、Xylプロモーターの直ぐ下流を一箇所切断したのち、脱リン酸化処理した。処理後のプラスミドにAOFADGLD(opt)のFAD−GDH遺伝子断片を連結し、組換えプラスミド(pCsUXAOFADGLD(opt))を構築した。 On the other hand, the pCsUX plasmid (5.9 kbp) containing the Xyl promoter, Xyl terminator, and URA5 gene was treated with MluI, cleaved at one site immediately downstream of the Xyl promoter, and then dephosphorylated. The FAD-GDH gene fragment of AOFADGLD (opt) was ligated to the treated plasmid to construct a recombinant plasmid (pCsUXAOFADGLD (opt)).
次に、KOD−plus Mutagenesis kit(東洋紡績社製)を用いて、InversePCRを行い、遺伝子配列に変異を加えた。具体的には、組換えプラスミドpCsUXAOFADGLD(opt)において、配列番号4のrcAOFADGDH_G163R_Fと配列番号5のrcAOFADGDH_G163R_Rをプライマーとして用いて、163位のアミノ酸をGからRに置換し、次に、配列番号6のrcAOFADGDH_V551C_Fと配列番号7のrcAOFADGDH_V551C_Rをプライマーとして用いて、551位のVをCに置換することにより、組換えプラスミド(pCsUXAOmFADGLD(opt))を構築した。なお、ここで増幅された配列は、配列番号8の配列の5´側に、配列番号9に示す分泌シグナルペプチド配列が付与されたものに相当する。 Next, inverse PCR was performed using 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.
なお、図1に示すpCsUXプラスミドはクリプトコッカスsp.S−2に由来する酸性キシラナーゼプロモーター(Xyl−pro)、酸性キシラナーゼターミネーター(Xyl−ter)、及びorotate phosphoribosyl transferase遺伝子(URA5)を市販のpUC19ベクターに常法により連結して組換え体DNAを作製し、この組換え体DNAを用いてE.coli DH5αを常法に従い形質転換することで取得することができる。形質転換体はアンピシリン耐性により選択できる。 Note that the pCsUX plasmid shown in FIG. Recombinant DNA was prepared by ligating the acidic xylanase promoter derived from S-2 (Xyl-pro), 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.
次に、KOD−plus Mutagenesis kit(東洋紡績社製)を用いて、InversePCRを行い、分泌シグナルペプチド配列を配列番号9以外のものに置換した。具体的には、組換えプラスミドpCsUXAOmFADGLD(opt)において、配列番号15のAOFADGLD(opt―sp)F及び配列番号16のXylanase(ss)−Xyl−pro−compをプライマーとして用いて、分泌シグナルペプチド配列を、配列番号10に示す「Xylanase分泌シグナルペプチド配列1」に置換したもの(pCsUXXsAOmFADGLD(opt));組換えプラスミドpCsUXAOmFADGLD(opt)において、配列番号15のAOFADGLD(opt―sp)F及び配列番号17のXylanase2(ss)−Xyl2−pro−compをプライマーとして用いて、分泌シグナルペプチド配列を、配列番号11に示す「Xylanase分泌シグナルペプチド配列2」に置換したもの(pCsUX2AOmFADGLD(opt));組換えプラスミドpCsUXAOmFADGLD(opt)において、配列番号15のAOFADGLD(opt―sp)F及び配列番号18のXylanase2(ss)−Xyl3−pro−compをプライマーとして用いて、分泌シグナルペプチド配列を、配列番号12に示す「Xylanase分泌シグナルペプチド配列3」に置換したもの(pCsUX2Xs3AOmFADGLD(opt);組換えプラスミドpCsUXAOmFADGLD(opt)において、配配列番号15のAOFADGLD(opt―sp)F及び配列番号19のAmylase(ss)−Xyl2−pro−compをプライマーとして用いて、分泌シグナルペプチド配列を、配列番号13に示す「Amylase分泌シグナルペプチド配列」に置換したもの(pCsUX2AsAOmFADGLD(opt));及び組換えプラスミドpCsUXAOmFADGLD(opt)において、配列番号15のAOFADGLD(opt―sp)F及び配列番号20のCutinase(ss)−Xyl2−pro−compをプライマーとして用いて、分泌シグナルペプチド配列を、配列番号14に示す「Cutinase分泌シグナルペプチド配列」に置換したもの(pCsUX2CsAOmFADGLD(opt))をそれぞれ作製した。 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 secretory signal peptide sequence was expressed as “Xylanase secretory 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; Using Cutinase (ss) -Xyl2-pro-comp as a primer, the secretory signal peptide sequence was substituted with the “Cutinase secretory signal peptide sequence” shown in SEQ ID NO: 14 (pCsUX2CsAOmFADGLD (opt)).
2.クリプトコッカスsp.S−2への形質転換
組換え宿主にはクリプトコッカスsp.S−2. U−5株を使用した。本菌株は、形質転換体の選択のために、クリプトコッカスsp.S−2を自然変異によりウラシル要求性にしたものであり、pCsUX2プラスミドと共に独立行政法人酒類総合研究所において取得されたものである。なお、クリプトコッカスsp.S−2からのウラシル要求性株の取得は、クリプトコッカスsp.S−2にUVを照射し、5−フルオロオロト酸を含む培地で培養し、生き残った株を選抜することにより容易に行うことができる。
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-requiring by natural mutation, and was obtained at the National Institute of Liquor Research with the pCsUX2 plasmid. Cryptococcus sp. Acquisition of a uracil auxotrophic strain from S-2 was obtained from Cryptococcus sp. It can be easily carried out by irradiating S-2 with UV, culturing in a medium containing 5-fluoroorotic acid, and selecting surviving strains.
形質転換は、Infect Immun. 1992 Mar;60(3):1101−8.(Varmaら)に記載の方法で実施した。クリプトコッカスsp.S−2. U−5株を20mlYM培地(酵母エキス0.3%、麦芽エキス0.3%、ポリペプトン0.5%、グルコース1.0%)で25℃、48時間培養した。得られた培養液の吸光度(OD660nm)を測定したところ、2.96Absであった。次に、この培養液6.75mlを200ml液体培地(酵母エキス0.3%、麦芽エキス0.3%、ポリペプトン0.5%、グルコース1.0%)に植菌し、25℃、18時間培養した。得られた培養液の吸光度(OD660nm)を測定したところ、0.94Absであった。次に、この培養液を遠心分離して菌体を回収し、Wash buffuer(270mMシュークロース、1mM塩化マグネシウム、4mM DTT、10mM Tris−HCl pH7.6)で菌体を2回洗浄し、Electroporation buffer(270mMシュークロース、1mM塩化マグネシウム、10mM Tris−HCl pH7.6)に吸光度OD660=50Absとなるように懸濁した。得られた懸濁液100μlに、予めSbfIによって制限酵素処理し、直鎖化したプラスミド10μg(〜5μl)を添加し、エレクトロポレーション用のキュベットに移した後、Gene Pulser Xcell (BIO−RAD社)を用いて通電した。通電条件は、C=25μF; V=0.47kVで行った。通電後の液中に600μl Electroporation buffer(270mMシュークロース、1mM塩化マグネシウム、10mM Tris−HCl pH7.6)を加え、選択プレート上に塗り広げた。選択プレートはYNB−ura寒天培地(0.67%Yeast Nitrogen Base W/O amino acid、0.078% −ura DO supplement、2%グルコース、1%寒天粉末)を用いた。植菌したプレートを25℃で1週間静置培養し、生育コロニーを選抜した。 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. The suspension 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 plasmid previously treated with restriction enzyme with SbfI and linearized 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.
形質転換で得られた形質転換体については、配列番号15のAOFADGLD(opt―sp)F及び配列番号3のrcAOFADGDH(opt)MulI_Rをプライマーとして用いてKOD−Fx(東洋紡績社製)によるコロニーPCRを行うことにより遺伝子の導入を確認し、遺伝子が導入された菌株に関して、次の3.で培養を行った。 For the transformant obtained by transformation, colony PCR by 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.FAD−GDH活性の確認
2.で取得した形質転換体を、3ml液体培地(0.3%酵母エキス、0.3%麦芽エキス、0.5%ペプトン、1.0%グルコース)で25℃、230rpm、48時間培養し、前培養液とした。前培養液0.03mlを3ml液体培地(酵母エキス2%、キシロース5%)に植え継ぎ、25℃、230rpm、72時間培養し、FAD−GDH活性を確認した。
3. 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.
数個の菌株に関して培養液上清中のFAD−GDH活性を測定した結果を、図2に示す。図2には、得られた数個の形質転換体のうち、最も生産性の高かった菌株のFAD−GDH活性測定値を示している。 The results of measuring the FAD-GDH activity in the culture supernatant for several strains are shown in FIG. FIG. 2 shows the measured FAD-GDH activity of the strain having the highest productivity among the several transformants obtained.
図2に示すとおり、コドンユーセージを最適化した遺伝子を導入し、分泌シグナルペプチド配列としてアスペルギルス・オリゼのFAD−GDHの分泌シグナルペプチド配列(配列番号9)を使用した菌株(図2のUXAOm)では、約34.6U/mLのFAD−GDH活性が検出された。本結果から、アスペルギルス・オリゼのFAD−GDHの分泌シグナルペプチド配列は、クリプトコッカスsp.S−2においても有効に機能することが確認された。 As shown in FIG. 2, a strain (UXAOm in FIG. 2) in which a gene optimized for 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. , About 34.6 U / mL of FAD-GDH activity 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.
さらに、分泌シグナルペプチド配列をクリプトコッカスsp.S−2のXylanase分泌シグナルペプチド配列1(配列番号10)に変更したもの(図2のUXXs1AOm)では、最大約42.0U/mLのFAD−GDH活性が検出され、分泌シグナルペプチド配列をクリプトコッカスsp.S−2のXylanase分泌シグナルペプチド配列2(配列番号11)に変更したもの(図2のUXXs2AOm)では、最大約85.7U/mLのFAD−GDH活性が検出され、分泌シグナルペプチド配列をクリプトコッカスsp.S−2のXylanase分泌シグナルペプチド配列3(配列番号12)に変更したもの(図2のUXXs3AOm)では、最大約44.5U/mLのFAD−GDH活性が検出され、分泌シグナルペプチド配列をクリプトコッカスsp.S−2のAmylase分泌シグナルペプチド配列(配列番号13)に変更したもの(図2のUXAsAOm)では、最大約21.2U/mLのFAD−GDH活性が検出され、分泌シグナルペプチド配列をクリプトコッカスsp.S−2のCutinase分泌シグナルペプチド配列(配列番号14)に変更したもの(図2のUXCsAOm)では、最大約36.0U/mLのFAD−GDH活性が検出された。
以上の結果から、上述する6つの分泌シグナルペプチド配列を用いることにより、FAD−GDHをクリプトコッカス属の微生物の細胞外に効率的に分泌生産することが可能である。
In addition, the secretory signal peptide sequence is designated as cryptococcus sp. In the S-2 Xylanase secretory 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), a maximum of 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 of about 36.0 U / mL of FAD-GDH activity was detected.
From the above results, FAD-GDH can be efficiently secreted and produced outside the cells of the genus Cryptococcus by using the six secretory signal peptide sequences described above.
4.FAD−GDH酵素タンパク質の取得
3.でFAD−GDH活性が最も高かったUXXs2AOm株形質転換体を、10L容ジャーファーメンターを用いて、培地(5%酵母エキス、5%キシロース、)で25℃、68時間培養した。培養菌体をろ過した後、培養上清を採取し、以下の実験で粗酵素溶液として用いた。
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.酵素の精製
上記の粗酵素溶液から、以下のステップ(1)〜(3)により、FAD−GDH酵素タンパク質を単離精製した。
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)濃縮・脱塩
粗酵素溶液を分画分子量30000の限界濾過膜で濃縮し、20mMリン酸カリウム緩衝液(pH6.0)に置換して、粗酵素濃縮液を得た。
(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)Phenyl−sepharose FastFlow(GEヘルスケア社製)による精製
活性画分を、60%硫酸アンモニウム飽和(pH6.0)になるように調整後、遠心分離し、上清を得た。60%飽和硫酸アンモニウムを含む50mMリン酸カリウム緩衝液(pH6.0)で予め平衡化させたPhenyl−sepharose FastFlowカラムにこの上清を通液して、酵素を吸着させた。カラムを同緩衝液で洗浄したのち、同緩衝液から50mMリン酸カリウム緩衝液(pH6.0)へのグラジエント溶出法で酵素を溶出させて、活性画分を回収した。活性画分を分画分子量10000の限界濾過膜で濃縮し、50mMリン酸カリウム緩衝液に置換した。
(3)DEAEセファロース(GEヘルスケア社製)による精製
50mMリン酸カリウム緩衝液(pH6.0)で予め平衡化させたDEAE−sepharose FastFlowカラムに粗酵素濃縮液を通液して、透過活性画分を回収した。精製酵素の比活性は、約400U/mgであり、酵素の精製倍率は、粗精製酵素の約20倍であった。
(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.FAD−GDH酵素タンパク質の特性評価
上記5.で単離した、クリプトコッカスを宿主として組換え生産したアスペルギルス・オリゼ由来の改変型FAD−GDH(以下、クリプトコッカス組換え改変型FAD−GDHと称する)の精製酵素の特性を評価した。また、対照として、アスペルギルス・オリゼを宿主として組換え生産した、特許文献3に記載のアスペルギルス・オリゼ由来の改変型FAD−GDH(以下、アスペルギルス・オリゼ組換え改変型FAD−GDHと称する)の精製酵素についても同様に特性を評価した。
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. Further, 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)熱安定性
各精製酵素を、50mMリン酸緩衝液(pH6.0)で100U/ml濃度に調製し、20℃〜65℃の温度で15分間処理し、残存活性を算出した。その結果を図3に示す。図3から明らかなように、アスペルギルス・オリゼ組換え改変型FAD−GDH及びクリプトコッカス組換え改変型FAD−GDHはいずれも50℃まで90%以上の活性を維持していた。
(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 apparent 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安定性
各精製酵素を、100mM酢酸緩衝液(pH3.5〜5.5)、100mMリン酸カリウム緩衝液(pH6.0〜8.0)、100mMPIPES―NaOH緩衝液(pH6.5−7.5)、100mMTris−HCl緩衝液(pH7.5〜9.0)、で20U/ml濃度に調製し、25℃で16時間処理し、残存活性を算出した。その結果を図4に示す。図4から明らかなように、アスペルギルス・オリゼ組換え改変型FAD−GDH及びクリプトコッカス組換え改変型FAD−GDHはいずれもpH3.5〜7.5の範囲で90%以上の活性を維持していた。
(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 calculated the residual activity. 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)分子量
Nu−PAGE 4−12% Bis−Tris Gel(Invitrogen社製)を用いたSDS−ポリアクリルアミドゲル電気泳動により、各精製酵素の分子量を求めた。結果を図5に示す。泳動サンプルは以下の通りである。
レーン1:分子量マーカー(Invitrogen社製、Novex(登録商標) Sharp Unstained Protein Standard)
レーン2:分子量マーカー(Invitrogen社製、Benchmark Protein ladder)
レーン3:クリプトコッカス組換え改変型FAD−GDH精製酵素。
レーン4:アスペルギルス・オリゼ組換え改変型FAD−GDH精製酵素。
レーン5:分子量マーカー(Invitrogen社製、Benchmark Protein ladder)
レーン6:分子量マーカー(Invitrogen社製、Novex(登録商標) Sharp Unstained Protein Standard)
(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)
また、脱糖鎖酵素(Roche社製、endoglycosidase H)を用いて各精製酵素を脱糖鎖処理し、同様に電気泳動により、糖鎖除去後の分子量を求めた。結果を図6に示す。泳動サンプルは以下の通りである。
レーン1:分子量マーカー(Invitrogen社製、Mark12)
レーン2:クリプトコッカス組換え改変型FAD−GDH精製酵素。
レーン3:脱糖鎖処理後のクリプトコッカス組換え改変型FAD−GDH精製酵素。
レーン4:アスペルギルス・オリゼ組換え改変型FAD−GDH精製酵素。
レーン5:脱糖鎖処理後のアスペルギルス・オリゼ組換え改変型FAD−GDH精製酵素。
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 recombinant modified FAD-GDH purified enzyme.
Lane 3: Cryptococcus 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.
図5からわかるように、クリプトコッカス組換え改変型FAD−GDHの分子量は、75kDa〜90kDaであり、アスペルギルス・オリゼ組換え改変型FAD−GDHの分子量(80kDa〜120kDa)と比較して均一である。 As can be seen from FIG. 5, the molecular weight of Cryptococcus recombinant modified FAD-GDH is 75 kDa to 90 kDa, which is uniform compared to the molecular weight of Aspergillus oryzae recombinant modified FAD-GDH (80 kDa to 120 kDa).
さらに図6のレーン2と3の対比からわかるように、クリプトコッカス組換え改変型FAD−GDHを脱糖鎖処理することによりその分子量は60kDaになったことから、クリプトコッカス組換え改変型FAD−GDHの糖鎖は15kDa〜30kDaと均一であることが予測され、タンパク質あたりの糖鎖結合量は平均して20%程度であると推定される。また、図6のレーン4と5の対比からわかるように、アスペルギルス・オリゼ組換え改変型FAD−GDHを脱糖鎖処理することによりその分子量は同じく60kDaになったことから、アスペルギルス・オリゼ組換え改変型FAD−GDHの糖鎖は約20kDa〜60kDaと不均一であることが予測され、タンパク質あたりの糖鎖結合量は平均して40%程度であると推定される。 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 the Aspergillus oryzae recombinant modified FAD-GDH was also 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)糖鎖結合量
凍結乾燥されたアスペルギルス・オリゼ組換え改変型FAD−GDH及びクリプトコッカス組換え改変型FAD−GDHを使用して、フェノール硫酸法により、酵素タンパク質あたりの糖鎖結合量を測定した。凍結乾燥したFAD―GDH粉末をイオン交換水で0.2g/Lに溶解し、FAD−GDH溶液を調製した。次に、このFAD−GDH溶液0.5mlと、5%(w/v)フェノール溶液0.5mlと、97%硫酸2.5mlを混合し、室温に冷却した後、490nmの吸光度を測定した。0−200mg/LのD−マンノース標準液で検量線を作成し、酵素粉末重量あたりの糖鎖結合量を測定した。その結果、アスペルギルス・オリゼ組換え改変型FAD−GDHの糖鎖結合量は40%であるのに対し、クリプトコッカス組換え改変型FAD−GDHの糖鎖結合量は18%であった。
(4) Amount of glycan bound The amount of glycan bound per enzyme protein is 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 a 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%.
以上の結果から、本実施例により得られたクリプトコッカス組換え改変型FAD−GDHは、アスペルギルス・オリゼ組換え改変型FAD−GDHとほぼ同等の特性を有し、しかも糖鎖結合量が少なくかつ均一であることが判明した。 From the above results, the Cryptococcus recombination modified FAD-GDH obtained in this example has almost the same characteristics as the Aspergillus oryzae recombination modified FAD-GDH, and the amount of sugar chain binding is small and uniform. It turned out to be.
なお、本実施例では分泌シグナルペプチドとして配列番号11のものを使用しているが、分泌シグナルペプチドはFAD−GDHのタンパク質発現効率及び発現成功率に関与し、発現されたタンパク質への糖鎖結合には関与しないので、分泌シグナルペプチドを配列番号11以外のものに置換しても、クリプトコッカス属の微生物を宿主として使用する限り、本実施例と同様に糖鎖結合量が少なくかつ均一であるFAD−GDHが得られると考えられる。 In addition, although the thing of sequence number 11 is used as a secretion signal peptide in a present Example, a secretion signal peptide is concerned with 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.
本発明によれば、アスペルギルス・オリゼ由来の改変型FAD−GDHと同等の特性を有し、しかも糖鎖結合量が少なくかつ均一であるFAD−GDHを効率的に組換え生産することができる。従って、本発明は、血糖自己測定バイオセンサ用の試薬として好適なFAD−GDHを生産するために極めて有用である。 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 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 (3)
(A)配列番号8で示される塩基配列;
(B)配列番号8で示される塩基配列に対して90%以上相同な塩基配列であって、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酵素活性を持つタンパク質をコードする塩基配列;
(C)配列番号9で示されるアミノ酸配列;
(D)配列番号10で示されるアミノ酸配列;
(E)配列番号11で示されるアミノ酸配列;
(F)配列番号12で示されるアミノ酸配列;
(G)配列番号13で示されるアミノ酸配列;
(H)配列番号14で示されるアミノ酸配列。 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 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 a flavin adenine dinucleotide-binding glucose dehydrogenase gene: And the microorganism is Cryptococcus sp. S-2 (Cryptococcus sp. S-2) (Accession number FERM BP-10961) :
(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.
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