JPH0998779A - Trehalose synthetase, its production and production of trehalose using the enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new trehalose synthetase acting on maltose to produce trehalose useful for pharmaceuticals, foods, cosmetics, etc., by culturing a microorganism belonging to the genus Pseudomonas and having trehalose synthetase producing capacity in a culture medium. SOLUTION: The objective new trehalose synthetase is produced by culturing a microorganism belonging to the genus Pseudomonas and capable of producing a trehalose synthetase [e.g. Pseudomonas sp.F-1 (FERM P-14747)] in a medium and collecting the product from the cultured material. The trehalose synthetase acts on maltose to form trehalose, acts on trehalose to form maltose, has optimum pH of 6.0-10.0 at 37 deg.C, optimum temperature of 45 deg.C at pH7.0, pH stability of 5.0-11.0 at 37 deg.C, heat stability of 40-50 deg.C at pH7.0, isoelectric point of 5.0-6.0 and molecular weight of 40,000-80,000 (SDS.PAGE), is inactivated with Co<++> , Zn<++> , Ni<++> , Hg<++> , Cu<++> and Cd<++> and has an amino-terminal amino acid sequence expressed by the formula.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel trehalose synthase having a hitherto unknown catalytic action for converting maltose into trehalose or trehalose into maltose. This novel trehalose synthase can easily produce trehalose (O-α-D-glycopyranosyl- (1 → 1) -α-D-glycopyranoside) by acting it on a maltose substrate.

[0002]

2. Description of the Related Art Trehalose has two glucose molecules α
It is a non-reducing (1,1) linked disaccharide and is widely distributed in insects, yeasts, molds, bacteria, basidiomycetes, red algae, lichens, plants and the like in the natural world. Since this trehalose has a structure similar to that of overlapping water molecules, it replaces water, which is indispensable for life in the living body, and has a biological protection function of protecting cells, proteins, nucleic acids, etc. from drying and freezing. Therefore, trehalose is a useful substance such as a drug, a diagnostic agent, a hormone, a fertilized egg, a sperm, a physiologically active substance, an enzyme, a microorganism, or a food,
Can be used in cosmetics.

Heretofore, as a method for producing trehalose, a method of extracting from a microorganism such as yeast, a method of fermentation using a microorganism, and a method of synthesizing trehalose by an enzymatic method have been known. Among them, a method for synthesizing trehalose by an enzymatic method has been attracting attention as an inexpensive mass production method. Enzymatic methods include maltose phosphorylase derived from Lactobacillus brevis and Euglena gracili.
s) -derived trehalose phosphorylase from maltose (JP-A-63-60998), α
There is known a method for producing trehalose by reacting trehalose phosphorylase with -glucose-1-phosphate and glucose (JP-A-6-189779). However, in the above-mentioned enzymatic method, a plurality of enzymes from a plurality of microorganisms are involved, the reaction is very complicated, and there is a problem in supplying a large amount of the enzyme.

[0004]

Therefore, an object of the present invention is to provide a novel trehalose synthase capable of producing trehalose using maltose as a substrate, and to produce trehalose more easily and efficiently than ever before. Is to provide a method.

[0005]

[Means for Solving the Problems] Therefore, the inventors of the present invention conducted extensive research to solve the above problems, and searched various natural strains for strains having an activity of producing trehalose using maltose as a substrate. As a result, it was found that a certain microorganism belonging to the genus Pseudomonas produces an enzyme which converts maltose into trehalose in the cells. This enzyme alone has a catalytic action for directly converting maltose into trehalose, and such an enzyme is a novel enzyme that has never been known so far. That is,
The present invention is a trehalose synthase having the following enzymatic chemical properties.

(1) Action: This enzyme acts on maltose to produce trehalose, and also acts on trehalose to produce maltose. (2) Optimum pH: 6.0 to 10.0 at 37 ° C (3) Optimum temperature: 45 ° C at pH 7.0 (4) pH stability: 5.0 to 11.0 at 37 ° C (5) Thermal stability: 40 to at pH 7.0 50 ℃ (6) Molecular weight: 40,000-80,000 (SDS ・ PAGE) (7) Isoelectric point: 5.0 to 6.0 (8) Inhibition of activity: Co ⁺⁺ , Zn ⁺⁺ , Ni ⁺⁺ , Hg ⁺⁺ , Cu ⁺⁺ , Is blocked by Cd ⁺⁺ . Not inhibited by Tris buffer. (9) Amino-terminal amino acid sequence: Thr → Gln → Pro → Asp → P
ro → Ser → Tyr → Val → Lys → Trp → Leu → Glu → Asp → Arg → A
la → Met → Leu → Lys → Ala → Ser → Gln → Ala → Arg → Ala → S
er → Leu → Tyr

Furthermore, the present invention is a method for producing trehalose synthase, which comprises culturing a microorganism belonging to the genus Pseudomonas and having the ability to produce trehalose synthase in a medium and collecting trehalose synthase from the culture. Furthermore, the present invention is a method for producing trehalose, which comprises reacting maltose with trehalose synthase to produce trehalose.

Hereinafter, the present invention will be described in detail. The microorganism used in the present invention may be any strain as long as it belongs to the genus Pseudomonas and has the ability to produce a novel trehalose synthase that converts maltose into trehalose. Such a microorganism may be a known one, or may be a fungus or the like newly isolated from soil, seawater or the like, and a mutant having improved trehalose synthesizing ability by subjecting those organisms to mutation treatment. May be Specific examples of preferable microorganisms include Psedomonas sp.F-1 newly isolated by the present inventors from soil. This strain is Pseudomonas sp. F-1 (Psedo
monas sp.F-1) was deposited at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, and the deposit number is FERM P-14747.
It is.

Any medium can be used as a medium for culturing the above-mentioned microorganisms, as long as it is a medium generally used in this field, and carbon sources include glucose, lactose, maltose, starch, crude sugar, molasses and the like. They can be used alone or as a mixture. As a nitrogen source, peptone, meat extract,
In addition to organic nitrogen sources such as yeast extract, constea liquor and urea, inorganic nitrogen sources such as ammonium salts of sulfuric acid, nitric acid and phosphoric acid can be used alone or in combination.
As inorganic salts, potassium, sodium, calcium,
Sulfates, carbonates, nitrates and phosphates of magnesium, manganese, iron and the like can be used alone or in combination.

The culture can be carried out under aeration conditions using shaking culture or a jar-famenter. The cells obtained by culturing are separated from the culture solution by filtration such as centrifugation, and the novel trehalose synthase is separated and purified from the cells by conventional means. For example, the microbial cells are disrupted by a method such as ultrasonic disruption, then the disrupted bacterial cells and the extracted enzyme solution are separated, and then a precipitate is obtained by salting out with ammonium sulfate or the like. This precipitate can be dissolved, dialyzed and purified by column chromatography such as ion exchange chromatography, hydrophobic chromatography and gel filtration chromatography. The enzymatic chemical properties of the novel trehalose synthase produced by the above procedure are as follows.

(1) Action: This enzyme acts on maltose to produce trehalose, and also acts on trehalose to produce maltose. (2) Optimum pH: 6.0 to 10.0 at 37 ° C (3) Optimum temperature: 45 ° C at pH 7.0 (4) pH stability: 5.0 to 11.0 at 37 ° C (5) Thermal stability: 40 to at pH 7.0 50 ℃ (6) Molecular weight: 40,000-80,000 (SDS ・ PAGE) (7) Isoelectric point: 5.0 to 6.0 (8) Inhibition of activity: Co ⁺⁺ , Zn ⁺⁺ , Ni ⁺⁺ , Hg ⁺⁺ , Cu ⁺⁺ , Is blocked by Cd ⁺⁺ . Not inhibited by Tris buffer. (9) Amino-terminal amino acid sequence: Thr → Gln → Pro → Asp → P
ro → Ser → Tyr → Val → Lys → Trp → Leu → Glu → Asp → Arg → A
la → Met → Leu → Lys → Ala → Ser → Gln → Ala → Arg → Ala → S
er → Leu → Tyr

According to the present invention, it is possible to provide a novel trehalose synthase which produces trehalose using maltose as a substrate. Furthermore, the novel trehalose synthase can synthesize trehalose either while it is retained inside the cells, or it is extracted from the cells or further purified to act on the substrate maltose. it can.

The feature of the present invention is that, unlike the method of extracting and purifying trehalose from the culture of the microorganism or the cells of the microorganism, trehalose can be obtained using the substrate maltose as a raw material. Most of them are sugars, and the separation and purification of trehalose from the reaction solution can be carried out very easily. Furthermore, since a single enzyme can directly convert maltose to trehalose, there is no need to carry out the reaction by taking out different enzymes from different microorganisms as in the conventional enzyme method,
Trehalose can be produced efficiently and in high yield.

The maltose concentration as a substrate in this reaction is preferably 10 mM to 2M. The reaction temperature is 30 to 6
The temperature is 0 ° C., the pH is preferably 6 to 10, and the reaction is preferably performed for 1 to 200 hours. A buffer is preferably used to keep the pH constant. When it is used as immobilized cells or immobilized enzyme, it can be carried out by a continuous method. For example, they are packed in a column and continuously passed as a bioreactor under the reaction conditions similar to those of the batch system for 1 month to 1 year. As a result, trehalose can be inexpensively and continuously mass-produced.

After completion of the reaction, the bacterial cells or the treated product of bacterial cells are removed from the reaction solution by centrifugation or filtration separation such as ultrafiltration. The obtained supernatant or filtrate may be directly concentrated to crystallize trehalose, or trehalose may be crystallized by purifying with activated carbon or an ion exchange resin and then concentrating. If necessary, recrystallization can be performed for purification.

In the present invention, by immobilizing bacterial cells or crudely purified or purified enzyme on a carrier, the immobilized bacterial cells or immobilized enzyme can be repeatedly used, and a large amount of trehalose can be continuously produced from maltose. You can

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to these Examples. Example 1 Preparation of novel trehalose synthase (preparation of bacterial cells) Pseudomonas sp. F-1 FER as inoculum
Use M P-14747. 1 mol of molasses, 500 g of skipjack extract,
Medium containing 10 g of magnesium sulfate and 10 g of potassium phosphate 1
28 in 50 liters with 100 liter jars
It was cultured at ℃ for 17 hours. After culturing, the bacterial cells were recovered by centrifugation, the bacterial cells were further suspended in water, and the operation of collecting the bacterial cells by centrifugation was repeated twice, and the bacterial cells were washed to obtain 653 g of bacterial cells.
I got

(Method for measuring enzyme activity) A final concentration of 100 mM maltose, 100 mM phosphate buffer solution (pH 7.0) in 0.02 g of bacterial cells,
Prepare the reaction mixture by mixing so that it becomes 5 mM EDTA, 37 ℃
The reaction was carried out. After completion of the reaction, a part of the reaction solution was analyzed by high performance liquid chromatography (HPLC) to quantify the produced trehalose. The HPLC analysis conditions were as follows.

Column: Liclosolve NH ₂ (manufactured by Merck) Solvent: Acetonitrile: Water: Formic acid = 80: 20: 1 Flow rate: 1 ml / min Temperature: 40 ° C. Detector: Differential refractometer Incidentally, trehalose synthase activity is 37 ° C. The enzyme titer for producing 1 μmol of trehalose per minute using maltose as a substrate at pH 7.0 was set to 1 U (unit). As a result, the trehalose synthase activity in the cells was 1.16 U / g.
Met.

(Preparation of crude enzyme) 50 g of the obtained bacterial cells was added to 100
The suspension was suspended in mM phosphate buffer to make 300 ml. This suspension was subjected to an ultrasonic disruption device to disrupt the cells. The crushed solution was centrifuged to obtain a supernatant. A protamine solution was added to the supernatant and the generated precipitate was removed by centrifugation. Ammonium sulfate (40% saturation) was added to the obtained supernatant and salted out to collect the precipitate by centrifugation. The precipitate was suspended in 20 ml of 50 mM Tris buffer and 5 mM EDTA (pH 7.4), placed in a dialysis tube, and dialyzed with 3 liters of the buffer as a dialysate to obtain 27 ml of a crude enzyme solution.

Example 2 Purification of Trehalose Synthase (Preparation of Purified Enzyme) 27 ml of the crude enzyme solution obtained in Example 1 was adsorbed on a DEAE-Separ (Pharmacia Biotech) column equilibrated with the same buffer solution. , 0 to 0.3M NaCl
The trehalose synthesis active fraction was eluted with a gradient of the same buffer solution containing. The eluted fraction (53 ml) was concentrated by ultrafiltration and dialyzed with 1 liter of the same buffer containing 0.5 M ammonium sulfate as a dialysate. The dialyzed enzyme suspension was adsorbed on a Phenyl-Sepharose (Pharmacia Biotech) column equilibrated with the same buffer containing 0.5 M ammonium sulfate, and trehalose synthesis was performed by a gradient of the same buffer containing 0.5 to 0 M ammonium sulfate. The active fraction was eluted. 24 ml of active fraction obtained
Ammonium sulfate (40% saturation) was added to and salted out, and the precipitate was collected by centrifugation. The precipitate was dissolved in 50 mM Tris buffer 5 mM EDTA (pH7.4) and equilibrated with the same Su.
Gel filtration was performed with a perdex 200 (Pharmacia Biotech) column. The obtained purified enzyme was confirmed to be a single protein by SDS polyacrylamide gel electrophoresis. The enzyme activity was 56.4 U / mg (protein). The enzymatic properties of this enzyme are as follows.

(Molecular weight) The molecular weight of the purified enzyme was measured by SDS.
-Polyacrylamide gel electrophoresis was performed for measurement. Thyroglobulin (670,000), γ-globulin (158,000), ovalbumin (44,000), myoblobin (17,000), and vitamin B ₁₂ (1,350) were used as molecular weight markers. As a result, the molecular weight was 40,000-80.000. (Isoelectric point) The isoelectric point was measured by ampholine isoelectric focusing. As a result, the isoelectric point was 5.0 to 6.0.

(Optimal pH) The optimal pH of the purified enzyme was measured.
Citrate buffer between pH 3-6, phosphate buffer between pH 6-8, Tris-maleate buffer between pH 6-7.5,
Glycine buffer was used between pH 8.5 and 10.5, and phosphate buffer was used between pH 10.0 and 12.0, and the activity was measured in each buffer to measure the activity. As a result, the optimum pH was 6.0 to 10.0 (see Fig. 1).

(Optimal temperature) The optimal reaction temperature of the purified enzyme was measured. The activity was measured at each temperature of 40 ° C to 80 ° C to measure the activity. As a result, the optimum temperature was 45 ° C (Fig. 2
reference). (PH stability) The stability of the purified enzyme at various pH was measured. Citrate buffer between pH 3-6, phosphate buffer between pH 6-8, Tris-maleate buffer between pH 6-8.5, glycine buffer between pH 8.5 and 10.5, pH 10.0 -1
A phosphate buffer was used during 2.0, and the activity of the purified enzyme was measured after leaving the purified enzyme in each buffer at 37 ° C. for 1 hour. As a result, pH stability was pH 5.0 to 11.0 (see FIG. 3).

(Thermal Stability) The stability of the purified enzyme at various temperatures was measured. After leaving the purified enzyme at each temperature of 4 ° C to 80 ° C for 30 minutes, its residual activity was measured. As a result, the thermal stability was 50 ° C. or lower (see FIG. 4). (Inhibition of activity) The inhibition of the activity of the purified enzyme by various metal ions is measured. The purified enzyme previously dialyzed against 10 mM Tris-HCl buffer was subjected to activity measurement in a reaction solution containing 20 mM Tris buffer (pH 7.1), 100 mM maltose, and 10 mM various metal ions. The results are as follows.

[0026]

(Substrate Specificity) The substrate specificity of the purified enzyme was measured. As a substrate, maltose, trehalose, isomaltose, nigerose, lactose, sucrose,
Gentiobiose and cellobiose were used, and the activity was measured under the same conditions after replacing the substrate maltose of the enzyme activity measurement method with these substrates. The results are as follows.

[0028]

From this table, it can be seen that there is substrate specificity for maltose and trehalose. (Amino-terminal amino acid sequence) The amino-terminal amino acid sequence of the purified enzyme was determined. After performing SDS-polyacrylamide gel electrophoresis of the purified enzyme according to a standard method, PVDF was used.
The membrane was transferred and stained with Coomassie blue. The membrane of the target band of 67 kDa was cut out and washed thoroughly with water. Cut film with 0.5% polyvinylpyrrolidone 1
Time processed. The membrane was thoroughly washed with water and then subjected to analysis of amino terminal amino acid sequence. The resulting array is Thr → Gln
→ Pro → Asp → Pro → Ser → Tyr → Val → Lys → Trp → Leu → Glu
→ Asp → Arg → Ala → Met → Leu → Lys → Ala → Ser → Gln → Ala
→ Arg → Ala → Ser → Leu → Tyr.

Example 3 Trehalose Synthesis Using Crude Enzyme 8 g of the ammonium sulfate-precipitated crude enzyme prepared in Example 1 was suspended in 30 ml of 50 mM phosphate buffer pH 7.0 5 mM EDTA and placed in a dialysis tube, and the same buffer solution 3.5 was added. Dialysis was performed using 1 liter of dialysate to obtain 35 ml (0.8 U / ml) of crude enzyme solution. After washing 50 ml of enzyme-immobilized carrier Chitovar BCW3503 (Fuji Boseki) with 1 liter of distilled water, 500 ml of 50 mM phosphate buffer (pH 7.0)
It was washed with 5 mM EDTA. To the immobilized carrier from which water was removed with a glass filter, 150 ml of 2.5% glutaraldehyde aqueous solution and 50 ml of 50 mM phosphate buffer (pH 7.0) 5 mM EDTA were added and gently shaken at room temperature for 2 hours. After the reaction was completed, it was washed with 500 ml of the same buffer solution to prepare an activated immobilization carrier. The crude enzyme was added to this activated immobilization carrier and 4
Immobilize enzyme (0.78U / ml) by shaking reaction overnight at ℃
Was prepared.

To 50 ml of the obtained immobilized enzyme, 120 g of maltose, 25 ml of 0.5 M phosphate buffer pH 7.050 mM EDTA and distilled water were added to make 250 ml, and trehalose synthesis was carried out at 37 ° C. for 5 days. The reaction solution was quantified by high performance liquid chromatography. As a result, 70 g of trehalose was produced in 5 days. The conversion rate was about 58% (see FIGS. 5-9).

[0032]

Industrial Applicability According to the present invention, a novel trehalose synthase can be provided.

According to the present invention, trehalose can be easily and efficiently produced, and trehalose can be mass-produced inexpensively by using a bioreactor.

[Brief description of drawings]

FIG. 1 is a graph showing the optimum pH of a novel trehalose synthase.

FIG. 2 is a graph showing the optimum temperature of a novel trehalose synthase.

FIG. 3 is a diagram showing pH stability of a novel trehalose synthase.

FIG. 4 is a diagram showing the thermal stability of a novel trehalose synthase.

FIG. 5: NM of trehalose obtained in the reaction of Example 3
The figure which shows R chart.

FIG. 6 NM of trehalose obtained in the reaction of Example 3
The figure which shows R chart.

FIG. 7 is a diagram showing an NMR chart of a trehalose reagent (manufactured by Sigma).

FIG. 8 is a diagram showing an NMR chart of a trehalose reagent (manufactured by Sigma).

FIG. 9 is a diagram showing the time course of trehalose production in the reaction of Example 3.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tadashi Wada 25-1 Seiichiki Shizuoka City, Shizuoka Prefecture Dias Kawaguchi B201 (72) Inventor Takafumi Sano 1759-4 Minamimatsuno, Fujikawa-cho, Anbara-gun, Shizuoka Prefecture Inventor Kazuo Oishi 7-1 Sanen-cho, Numazu-shi, Shizuoka 7-1 Occupation 303 (72) Masaaki Yamagishi 256-2 Ogawa, Yaizu-shi Shizuoka (72) Inventor Toshiya Ota 178-3 Kakita, Shimizu-cho, Sunto-gun Shizuoka 301

Claims (3)

[Claims] 1. A trehalose synthase having the following enzymatic chemistry. (1) Action: This enzyme acts on maltose to produce trehalose, and also acts on trehalose to produce maltose. (2) Optimum pH: 6.0 to 10.0 at 37 ° C (3) Optimum temperature: 45 ° C at pH 7.0 (4) pH stability: 5.0 to 11.0 at 37 ° C (5) Thermal stability: 40 to at pH 7.0 50 ℃ (6) Molecular weight: 40,000-80,000 (SDS ・ PAGE) (7) Isoelectric point: 5.0 to 6.0 (8) Inhibition of activity: Co ⁺⁺ , Zn ⁺⁺ , Ni ⁺⁺ , Hg ⁺⁺ , Cu ⁺⁺ , Is blocked by Cd ⁺⁺ . Not inhibited by Tris buffer. (9) Amino-terminal amino acid sequence: Thr → Gln → Pro → Asp → P
ro → Ser → Tyr → Val → Lys → Trp → Leu → Glu → Asp → Arg → A
la → Met → Leu → Lys → Ala → Ser → Gln → Ala → Arg → Ala → S
er → Leu → Tyr 2. A method for producing trehalose synthase, which comprises culturing in a medium a microorganism belonging to the genus Pseudomonas and capable of producing trehalose synthase, and collecting trehalose synthase from the culture. 3. A method for producing trehalose, which comprises reacting maltose with trehalose synthase to produce trehalose.