JP3959439B2 - Thermostable trehalase and its production method - Google Patents

Description

【００２２】
【表２】

【００２３】
【表３】

【００２４】
1 スクリーニング
1.1 スクリーニング方法１
50種類の土壌サンプルを滅菌水に懸濁し、上清液を表１のA固体培地に塗布し、65℃で３日間培養し、生育のみられた菌株をC固体培地（表３）に移し、65℃で３日間培養した。得られた菌株をB液体培地（表２）に植菌し、60℃で３日間、振盪培養した。培養液を遠心分離（8000rpm、10分間、4℃）により培養上清液と菌体とに分離した。上清液については、そのトレハラーゼ活性をDNS法により測定した結果、２菌株が高トレハラーゼ産生能を有することが確認された。また、35菌体はリン酸バッファーに懸濁し、超音波処理（１秒間に0.2秒発振、0.8秒停止、100W、10分間）し、その液を遠心分離し、得られた上清液のトレハラーゼ活性をDNS法により測定した。その結果、１菌株にトレハラーゼ活性が確認された。
1.2 スクリーニング方法２
103種類の土壌サンプルを滅菌水に懸濁し、上清液を表１のA固体培地に塗布し、65℃で３日間培養した。各培養液をB液体培地（表２）に植菌し、60℃で３日間、振盪培養した。培養液を遠心分離（8000rpm、10分間、4℃）により培養上清液と菌体とに分離し、上清液のトレハラーゼ活性をDNS法により測定した結果、３菌株が高トレハラーゼ産生能を有することが確認された。
1.3 菌の選択
前記スクリーニング方法１、２でトレハラーゼ活性を認めた菌株を純粋分離し、得られた20菌株をA液体培地に植菌し、60℃で３日間振盪培養した。培養液を遠心分離（8000rpm、10分間、4℃）により培養上清液と菌体とに分離し、上清液のトレハラーゼ活性をDNS法により測定した結果、活性の高い５株が得られたが、菌の形態からNo. 6B株およびNo. 8株を選択した。
2 菌学的性質
2.1 生理的試験
No. 6B株およびNo. 8株を表１に示したA培地組成からなる固体培地および液体培地で60℃、２日間培養し、それぞれの第１次性状（細胞形態、グラム染色、運動性、芽胞、酸素に対する態度、カタラーゼ、オキシダーゼ、OFテスト等）を調べた。また、第２次性状として、炭素源、窒素源の利用、硫化水素の生成、代謝産物、酵素活性、培養至適温度、pH等を調べた。
【００２５】
結果は表４に示したとおりである。
【００２６】
【表４】

【００２７】
また、両菌株の培養温度と増殖との関係は図１に示したとおりであり、いずれの菌株も約55℃において最も良好に増殖した。
2.2 16S rDNAの決定
No. 6B株およびNo. 8株を表１に組成を示したA培地で60℃、４日間培養し、培養液を遠心分離して集菌した。菌体からDNAを抽出し、PCR法によってDNAを増幅し、DNAシークエンサーによってそれぞれのDNA配列を決定した。その結果、No. 6B株のゲノムDNAの一部配列は配列番号１に示したとおりであり、No. 8株の配列は配列番号２に示したとおりである。これらの配列は、Bacillus subtilisと約94％の相同性を示した。
3.3 菌種の特定
前記の生理学的試験および16S rDNA配列決定の結果から、この発明のNo. 6B株およびNo. 8株はバチルス属に属する微生物であることが確認された。なお、DNA配列の相同性は、98％以上が一致する場合に同一種とすることが一般的であるため、このNo. 6B株およびNo. 8株は、Bacillus subtilisと同一ではないものの、それに極めて近縁の種であることが確認された。
3 トレハラーゼ特性
3.1 粗トレハラーゼ
No. 6B株およびNo. 8株を表1に組成を示したA培地にて60℃で４日間、振盪培養し、培養上清液に硫酸アンモニウムを加え、最終濃度を70％にしてトレハラーゼを沈殿させた。
【００２８】
この粗トレハラーゼ溶液を用いて、トレハラーゼ活性に及ぼす温度とpHの影響、耐熱性、ミハエリス定数（Km値）について検討した。なお、トレハラーゼ活性は、酵素単位として50℃で１分間に1μmolのグルコースを生成する酵素量を1Uとして、グルコースの生成をDNS法により測定し、最大値に対する相対活性として算出した。
3.2 温度の影響
様々な温度における粗トレハラーゼ溶液の酵素活性を、100mMリン酸バッファー（pH7.0）中、50℃で30分間の酵素反応を行った後、生成したグルコース量をDNS法により測定した。結果は図２に示したとおりである。No. 6B株およびNo. 8株由来のトレハラーゼは、共に45〜55℃において80%以上の相対活性を示した。そして、いずれのトレハラーゼも至適温度は50℃であった。
【００２９】
また、粗トレハラーゼ溶液を100mMリン酸バッファー（pH7.0）中で30分間、様々な温度でインキュベーションし、その後の残存活性を50℃で測定した。結果は図３に示したとおりであり、いずれのトレハラーゼも50℃までは高い安定性を有することが確認された。
3.3 pHの影響
様々なpHにおいて50℃で30分間の酵素反応を行った後、生成したグルコース量をDNS法により測定することによって、粗トレハラーゼ溶液の酵素活性におけるpHの影響を調べた。pH値の調整は、以下の50mMバッファーを用いて行った。CH₃COOH-CH₃COONa(pH4.0-5.5)、Na₂HPO₄-KH₂PO₄(pH6.0-7.0)、Na₂CO₃-H₃BO₃-KCl(pH8.0-10.0)。結果は図４に示したとおりである。No. 6B株およびNo. 8株由来のトレハラーゼは、共にpH6.0〜8.0において80%以上の相対活性を示した。そして、いずれのトレハラーゼも至適pHは7.25であった。
3.4 Km値
粗トレハラーゼのKm値を、Lineweaver-Burkプロットにより算出した。結果は図５に示したとおりであり、No. 6B株由来のトレハラーゼのKm値は7.7、Vmax値は0.17、N0. 8株由来のトレハラーゼのKm値は6.8、Vmax値は0.27であった。
4 精製トレハラーゼ
4.1 トレハラーゼの精製
粗トレハラーゼ溶液を1.5φ×40cm DEAE-セルロースカラムにのせ、50-500mM NaCl濃度勾配によりトレハラーゼを溶出した。得られた分画のトレハラーゼ活性をDNS法で測定し、活性の高かった画分No. 14, 15, 16溶液（図６）を、2.5φ×75cmセファロース-4Bカラムにのせ、リン酸バッファーを用いて溶出した。得られた画分のトレハラーゼ活性をDNS法により測定し、活性の高かった画分N0. 28（図７）を、前記と同様のDEAE-セルロースカラムを用いたクロマトグラフィーにより精製した。
【００３０】
以上の精製工程による結果は表５に示したとおりである。粗トレハラーゼから最終公知のクロマトグラフィーによって126倍に濃縮され、114U/mgもの高い酵素活性を有するトレハラーゼが得られた。なお、この酵素活性は、50℃で１分間に1μmolのグルコースを生成する酵素量を1Uとした。
【００３１】
【表５】

【００３２】
4.2 精製トレハラーゼの分子量測定
粗トレハラーゼおよび各工程で得られた精製トレハラーゼの分子量をSDS-PAGEによって決定した。結果は図８に示したとおりであり、この発明のトレハラーゼの分子量は26kDaであることが確認された。
4.3 金属イオンの影響
精製トレハラーゼを10mM Tris-HClバッファー（pH7.0）に溶解し、表６に示した各金属イオン1mMを添加して50℃、30分間インキュベートし、その後の残存活性を、無添加（コントロール）を100％として算出した。結果は表６に示したとおりである。トレハラーゼ活性は、Mn++、Co++の添加によりそれぞれ205％、130％に酵素活性が増加し、Cu++、EDTAの添加によりそれぞれ0％、57％に酵素活性が低下した。
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【表６】

【００３４】
【発明の効果】
以上詳しく説明したとおり、この出願の発明によって、中性域で高い酵素活性を有する耐熱性のトレハラーゼが提供される。このトレハラーゼによって、トレハロースを含有する製品の研究開発や品質管理に不可欠なトレハロース含有量測定のためのバイオセンサが実用化可能となる。
【００３５】
【配列表】

【図面の簡単な説明】
【図１】この発明のNo. 6B株およびNo. 8株の、培養温度と増殖の程度の関係を示すグラフである。
【図２】この発明のNo. 6B株およびNo. 8株のそれぞれに由来する粗トレハラーゼの温度と相対活性の関係を示すグラフである。
【図３】この発明のNo. 6B株およびNo. 8株のそれぞれに由来する粗トレハラーゼの温度安定性を示すグラフである。
【図４】この発明のNo. 6B株およびNo. 8株のそれぞれに由来する粗トレハラーゼのpHと相対活性の関係を示すグラフである。
【図５】この発明のNo. 6B株およびNo. 8株のそれぞれに由来する粗トレハラーゼのKm値の測定結果を示すグラフである。
【図６】 DEAE-セファロースカラムを用いたクロマトグラフィーの結果である。
【図７】セファロース-4Bカラムを用いたゲルフィルトレーションの結果である。
【図８】各精製段階のトレハラーゼのSDS-PAGEによる泳動像である。MKはマーカー、レーン１はDEAE-セファロースカラムを用いた２回目のクロマトグラフィーによる最終精製トレハラーゼ、レーン２は粗トレハラーゼ、レーン３はDEAE-セファロースカラムを用いた１回目のクロマトグラフィーによる精製トレハラーゼ、レーン４はセファロース-4Bカラムを用いたゲルフィルトレーションによる精製トレハラーゼである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel thermostable trehalase and a method for producing the same. More specifically, the present invention relates to a new thermostable enzyme trehalase useful for measurement of trehalose content, a microorganism producing this trehalase, and a method for producing trehalase using this microorganism.
[0002]
[Prior art and its problems]
Trehalose is a non-reducing sugar in which two molecules of α-D-glucose are bound, and is widely distributed in nature such as microorganisms including yeast, molds, insects, mosses, algae, lichens, and crustaceans. It plays an important role in the cold resistance and drought resistance of the living body. This trehalose has heretofore been produced by an extraction method from yeast, but has not been widely used due to its high price. However, since it has become possible to mass-produce from starch etc. at low cost using an enzyme discovered recently, trehalose is used as a sweetener etc. for many foods and creams, lotions etc. using the moisturizing effect. It is also used in cosmetics and is expected to be used for pharmaceuticals and chemical products.
[0003]
In this way, the use of trehalose is expected to increase in the future, but measurement of trehalose content is indispensable in research and development of products containing trehalose and quality control. Conventionally, trehalose content was measured by thin layer chromatography or liquid chromatography, but there were problems in terms of operability and rapidity.
[0004]
[Problems to be solved by the invention]
As a simple and highly accurate method for measuring trehalose content, a method using “trehalase” is conceivable. Trehalase is an enzyme that decomposes trehalose into two glucose molecules. Thus, trehalose is decomposed into glucose using this trehalase, and the amount of glucose that can be measured more easily is identified, whereby trehalose in the product can be measured with high accuracy.
[0005]
Examples of trehalase include chlorella-derived trehalase (Japanese Patent Laid-Open No. 7-246094: Prior Art 1), Penicillium, Aspergillus, Mucor, Pichia, Hansenula, Candida, Rhodosporidium, Known are trehalases derived from microorganisms belonging to the genus Saccharomyces, Escherichia or Klebsiella (Japanese Patent Laid-Open No. 7-51063: Prior Art 2).
[0006]
However, these prior art trehalases are enzymes used to synthesize trehalose from glucose. Moreover, the trehalase of the prior art 1 has an optimum pH of 5-6, and the optimum pH of the trehalase of the prior art 2 is 4-5. In order to measure the trehalose content more easily and with high accuracy, the development of an apparatus system (biosensor) is indispensable. And the conditions of trehalase applicable to a biosensor are that it is a stable thermostable enzyme even at a temperature of 40 ° C. or higher, and has high activity in a neutral range. That is, when an enzyme that acts in an acidic region or an alkaline region is used, it is inevitable that device parts are deteriorated (corroded or dissolved) by substances used for pH adjustment. For this reason, prior art trehalases having enzyme activity in the acidic range are not suitable for use in biosensors.
[0007]
The invention of this application was made in view of the circumstances as described above, and a new thermostable trehalase having a high enzymatic activity in a neutral range, which can be used for measurement of trehalose content by a biosensor. The issue is to provide.
[0008]
[Means for Solving the Problems]
As an invention for solving the above-mentioned problems, this application is an enzyme produced by a microorganism belonging to the genus Thermophilic Bacillus, which has the following properties:
(1) has an enzymatic activity to decompose trehalose into two glucose molecules;
(2) exhibits a relative activity of 80% or more at pH 6-8;
(3) the optimum temperature is 50 ° C; and
(4) Molecular weight is 26 kD;
A thermostable trehalase characterized by comprising:
[0009]
The thermostable trehalase of the present invention is preferably a thermostable trehalase produced by a microorganism No. 6B strain (FERM P-18412) or No. 8 strain (FERM P-18411) belonging to the thermophilic Bacillus genus.
[0010]
The invention of this application further provides microorganisms No. 6B strain (FERM P-18412) and No. 8 strain (FERM P-18411) belonging to the genus Thermophilic Bacillus as novel strains.
The invention of this application also provides a method for producing a thermostable trehalase, comprising culturing a microorganism belonging to the genus Thermophilic Bacillus, and separating and purifying the thermostable trehalase of claim 1 from the culture. In this production method, the preferred embodiment is that the microorganism belonging to the genus Thermophilic Bacillus is the No. 6B strain (FERM P-18412) or the No. 8 strain (FERM P-18411).
[0011]
Hereinafter, embodiments of the invention of this application will be described in detail.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
The thermostable trehalase of the present invention is basically an enzyme having the following properties.
(1) It has an enzyme activity that decomposes trehalose into two glucose molecules.
(2) Shows a relative activity of 80% or more at pH 6-8.
(3) It exhibits a relative activity of 80% or more at a temperature of 45 to 55 ° C.
(4) The molecular weight is about 26 kD.
[0013]
That is, the trehalase (α, α-trehalase) of the present invention specifically hydrolyzes the glucoside bond of α, α-trehalose to produce two molecules of glucose. The relative activity of “80% or more” at pH 6 to 8 and 45 to 55 ° C. means that the amount of enzyme that produces 1 μmol of glucose per minute at 50 ° C. as an enzyme unit is 1 U. It means that the production shows an activity of 80% or more with respect to the maximum value of the enzyme activity measured by the 3,5-Dinitrosalicylic acid (DNS) method.
[0014]
Such trehalase can be obtained by culturing a microorganism belonging to the genus Bacillus, and isolating and purifying it from the culture. Examples of microorganisms belonging to the genus Bacillus include preferably No. 6B strains and No. 8 strains newly isolated and identified from soil by the inventors of this application. These No. 6B shares and No. 8 shares were deposited with the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology as of July 10, 2001 under the accession numbers FERM P-18412 and FERM P-18411, respectively. Has been. The optimum pH of trehalase produced by these No. 6B strain and No. 8 strain is 7.25, and the optimum temperature is 50 ° C.
[0015]
The medium used for culturing these Bacillus bacteria may be any medium as long as this strain grows well, and two different types are used for cell growth and trehalase production. The medium may be used. As the medium, a liquid medium or a solid medium can be used, and as a medium component, in addition to the carbon source, a nitrogen source, inorganic ions, and the like may be contained.
[0016]
The carbon source in the medium for cell growth is not particularly limited, and a known carbon source can be used. However, acetic acid, malic acid, succinic acid, propionic acid, valmitic acid and other lower fatty acids, starch, sucrose, etc. Can be preferably used. These carbon sources can be added to the medium alone or in combination. On the other hand, a medium for producing trehalase can contain trehalose as its substrate.
[0017]
As the nitrogen source, inorganic nitrogen compounds such as ammonium sulfate and ammonium nitrate, and organic nitrogen sources such as peptone and yeast extract can be used.
Furthermore, examples of inorganic ions include phosphate ions, magnesium ions, calcium ions, potassium ions, iron ions, copper ions, manganese ions, and the like, and these inorganic ions may be contained in the medium in the form of inorganic salts. .
[0018]
As a method for culturing microorganisms of the present invention, a culture method using a liquid medium such as a shaking culture method, a culture method using a solid medium such as an agar medium, and the like can be used. The culture temperature is, for example, 40 to 80 ° C., preferably 50 to 70 ° C., and the pH of the medium is around neutral. The culture time during the production of trehalase can be determined according to the amount of bacterial cells, the production of trehalase, and the like, but it is suitably 0.5 days to 10 days, more preferably about 1 day to 6 days.
[0019]
After such culture, the trehalase of the present invention can be isolated and purified from the culture supernatant by known means. For example, salting out only with ammonium sulfate, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, etc. Can be mentioned.
[0020]
Hereinafter, the screening method, mycological properties, and characteristics of trehalase isolated and purified from these microorganisms will be described in detail. In addition, in culture | cultivation of these strains, any one of AC culture medium which showed each composition in Tables 1-3 was selected and used.
[0021]
[Table 1]

[0022]
[Table 2]

[0023]
[Table 3]

[0024]
1 Screening
1.1 Screening method 1
50 types of soil samples were suspended in sterilized water, the supernatant was applied to the A solid medium in Table 1, cultured at 65 ° C for 3 days, and the grown strains were transferred to the C solid medium (Table 3). The cells were cultured at 65 ° C for 3 days. The obtained strain was inoculated in a B liquid medium (Table 2) and cultured at 60 ° C. for 3 days with shaking. The culture solution was separated into culture supernatant and cells by centrifugation (8000 rpm, 10 minutes, 4 ° C.). As for the supernatant, the trehalase activity was measured by the DNS method. As a result, it was confirmed that the two strains have high trehalase producing ability. 35 cells were suspended in a phosphate buffer, sonicated (oscillated for 0.2 seconds per second, stopped for 0.8 seconds, 100W, 10 minutes), centrifuged, and the resulting supernatant trehalase. Activity was measured by DNS method. As a result, trehalase activity was confirmed in one strain.
1.2 Screening method 2
103 kinds of soil samples were suspended in sterilized water, and the supernatant was applied to the A solid medium shown in Table 1 and cultured at 65 ° C. for 3 days. Each culture solution was inoculated into a B liquid medium (Table 2) and cultured at 60 ° C. for 3 days with shaking. The culture broth was separated into a culture supernatant and cells by centrifugation (8000 rpm, 10 minutes, 4 ° C.), and the trehalase activity of the supernatant was measured by the DNS method. As a result, three strains have high trehalase production ability. It was confirmed.
1.3 Selection of Bacteria The strains that showed trehalase activity in the screening methods 1 and 2 were isolated purely, and the obtained 20 strains were inoculated into A liquid medium and cultured with shaking at 60 ° C. for 3 days. The culture broth was separated into a culture supernatant and cells by centrifugation (8000 rpm, 10 minutes, 4 ° C.), and the trehalase activity of the supernatant was measured by the DNS method. As a result, five strains with high activity were obtained. However, No. 6B strain and No. 8 strain were selected from the form of the bacteria.
2 Mycological properties
2.1 Physiological tests
The No. 6B strain and No. 8 strain were cultured at 60 ° C. for 2 days in a solid medium and a liquid medium having the composition of A medium shown in Table 1, and each primary property (cell morphology, gram staining, motility, Spores, attitudes to oxygen, catalase, oxidase, OF test, etc.) were examined. As secondary properties, the use of carbon source and nitrogen source, production of hydrogen sulfide, metabolites, enzyme activity, optimum culture temperature, pH and the like were examined.
[0025]
The results are as shown in Table 4.
[0026]
[Table 4]

[0027]
Further, the relationship between the culture temperature and growth of both strains is as shown in FIG. 1, and both strains grew best at about 55 ° C.
2.2 Determination of 16S rDNA
The No. 6B strain and the No. 8 strain were cultured in A medium having the composition shown in Table 1 at 60 ° C. for 4 days, and the culture broth was collected by centrifugation. DNA was extracted from the cells, the DNA was amplified by PCR, and each DNA sequence was determined by a DNA sequencer. As a result, the partial sequence of the genomic DNA of the No. 6B strain is as shown in SEQ ID NO: 1, and the sequence of the No. 8 strain is as shown in SEQ ID NO: 2. These sequences showed about 94% homology with Bacillus subtilis.
3.3 Identification of bacterial species From the results of the physiological test and 16S rDNA sequencing described above, it was confirmed that the No. 6B strain and the No. 8 strain of the present invention belong to the genus Bacillus. In addition, since the homology of DNA sequences is generally the same when 98% or more matches, this No. 6B strain and No. 8 strain are not identical to Bacillus subtilis, It was confirmed to be a very closely related species.
3 Trehalase characteristics
3.1 Crude trehalase
No. 6B and No. 8 strains were shaken and cultured at 60 ° C. for 4 days in the medium A shown in Table 1. Ammonium sulfate was added to the culture supernatant to precipitate trehalase at a final concentration of 70%. I let you.
[0028]
Using this crude trehalase solution, the effects of temperature and pH on trehalase activity, heat resistance, and Michaelis constant (Km value) were examined. The trehalase activity was calculated as the relative activity with respect to the maximum value by measuring the production of glucose by the DNS method with the amount of enzyme producing 1 μmol of glucose per minute at 50 ° C. as the enzyme unit.
3.2 Effect of temperature The enzyme activity of the crude trehalase solution at various temperatures was measured by the DNS method after performing an enzyme reaction in 100 mM phosphate buffer (pH 7.0) for 30 minutes at 50 ° C. . The results are as shown in FIG. The trehalases derived from the No. 6B strain and the No. 8 strain both showed a relative activity of 80% or more at 45 to 55 ° C. The optimum temperature for all trehalases was 50 ° C.
[0029]
In addition, the crude trehalase solution was incubated in 100 mM phosphate buffer (pH 7.0) for 30 minutes at various temperatures, and then the remaining activity was measured at 50 ° C. The results are as shown in FIG. 3, and it was confirmed that all trehalases had high stability up to 50 ° C.
3.3 Effect of pH After performing an enzyme reaction at 50 ° C. for 30 minutes at various pHs, the effect of pH on the enzyme activity of the crude trehalase solution was examined by measuring the amount of glucose produced by the DNS method. The pH value was adjusted using the following 50 mM buffer. CH ₃ COOH-CH ₃ COONa (pH 4.0-5.5), Na ₂ HPO ₄ -KH ₂ PO ₄ (pH 6.0-7.0), Na ₂ CO ₃ -H ₃ BO ₃ -KCl (pH 8.0-10.0) . The results are as shown in FIG. The trehalases derived from the No. 6B strain and the No. 8 strain both showed a relative activity of 80% or more at pH 6.0 to 8.0. The optimum pH of all trehalases was 7.25.
3.4 Km value The Km value of the crude trehalase was calculated by Lineweaver-Burk plot. The results are as shown in FIG. 5. The Km value of trehalase derived from No. 6B strain was 7.7, the Vmax value was 0.17, the Km value of trehalase derived from N0.8 strain was 6.8, and the Vmax value was 0.27.
4 Purified trehalase
4.1 Purification of trehalase The crude trehalase solution was placed on a 1.5φ × 40 cm DEAE-cellulose column, and trehalase was eluted with a 50-500 mM NaCl concentration gradient. The trehalase activity of the obtained fraction was measured by the DNS method, and the fraction No. 14, 15, 16 solution (FIG. 6) with high activity was placed on a 2.5φ × 75 cm Sepharose-4B column, and phosphate buffer was added. And eluted. The trehalase activity of the obtained fraction was measured by the DNS method, and the highly active fraction N0.28 (FIG. 7) was purified by chromatography using the same DEAE-cellulose column as described above.
[0030]
The results of the above purification steps are as shown in Table 5. The crude trehalase was concentrated 126 times by the last known chromatography, and a trehalase having an enzyme activity as high as 114 U / mg was obtained. In this enzyme activity, the amount of enzyme that produces 1 μmol of glucose per minute at 50 ° C. was defined as 1 U.
[0031]
[Table 5]

[0032]
4.2 Molecular weight measurement of purified trehalase The molecular weight of the crude trehalase and the purified trehalase obtained in each step were determined by SDS-PAGE. The results are as shown in FIG. 8, and it was confirmed that the trehalase of the present invention had a molecular weight of 26 kDa.
4.3 Effect of metal ions Purified trehalase is dissolved in 10 mM Tris-HCl buffer (pH 7.0), 1 mM of each metal ion shown in Table 6 is added, and incubated at 50 ° C. for 30 minutes. The addition (control) was calculated as 100%. The results are as shown in Table 6. Trehalase activity increased to 205% and 130%, respectively, with addition of Mn ++ and Co ++, and decreased to 0% and 57%, respectively, with addition of Cu ++ and EDTA.
[0033]
[Table 6]

[0034]
【The invention's effect】
As described above in detail, the invention of this application provides a thermostable trehalase having a high enzyme activity in a neutral range. This trehalase makes it possible to put to practical use a biosensor for trehalose content measurement that is indispensable for research and development and quality control of products containing trehalose.
[0035]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a graph showing the relationship between the culture temperature and the degree of growth of No. 6B strain and No. 8 strain of the present invention.
FIG. 2 is a graph showing the relationship between the temperature and relative activity of crude trehalase derived from each of No. 6B strain and No. 8 strain of the present invention.
FIG. 3 is a graph showing the temperature stability of crude trehalase derived from each of No. 6B strain and No. 8 strain of the present invention.
FIG. 4 is a graph showing the relationship between pH and relative activity of crude trehalase derived from each of No. 6B strain and No. 8 strain of the present invention.
FIG. 5 is a graph showing measurement results of Km values of crude trehalase derived from each of No. 6B strain and No. 8 strain of the present invention.
FIG. 6 shows the results of chromatography using a DEAE-Sepharose column.
FIG. 7 shows the results of gel filtration using a Sepharose-4B column.
FIG. 8 is an electrophoresis image of trehalase at each purification stage by SDS-PAGE. MK is a marker, lane 1 is the final purified trehalase by the second chromatography using DEAE-Sepharose column, lane 2 is the crude trehalase, lane 3 is the purified trehalase by the first chromatography using the DEAE-Sepharose column, lane 4 is a purified trehalase by gel filtration using a Sepharose-4B column.

Claims (6)

An enzyme produced by a microorganism belonging to the thermophilic Bacillus genus, having the following properties:
(1) has an enzymatic activity to decompose trehalose into two glucose molecules;
(2) exhibits a relative activity of 80% or more at pH 6-8;
(3) exhibit a relative activity of 80% or more at a temperature of 45-55 ° C; and
(4) a molecular weight of about 26 kD;
A thermostable trehalase characterized by comprising: The thermostable trehalase according to claim 1, which is produced by a microorganism No. 6B strain (FERM P-18412) or No. 8 strain (FERM P-18411) belonging to the thermophilic Bacillus genus. Microorganism No. 6B strain (FERM P-18412) belonging to the thermophilic Bacillus genus. Microorganism No. 8 strain (FERM P-18411) belonging to the thermophilic Bacillus genus. A method for producing a thermostable trehalase comprising culturing a microorganism belonging to the genus Thermophilic Bacillus, and separating and purifying the thermostable trehalase of claim 1 from the culture. The method according to claim 5, wherein the microorganism belonging to the genus Thermophilic Bacillus is the microorganism according to claim 3 or 4.

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