JPH0712309B2 - New trehalase and its manufacturing method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel trehalase and a method for producing the same. More specifically, it relates to a thermostable trehalase outside and inside the cells obtained by culturing a novel thermophilic microorganism belonging to the genus Corynebacterium, and to the production thereof.

BACKGROUND ART Generally, trehalose is composed of two molecules of D-glucose, 1,1.
-It is a kind of non-reducing dimonosaccharide in a linked form, and depending on the binding mode, α, α-, α, β- and β, β-
There are isomers of the species. In addition, only α, α-forms exist in nature, and they are widely distributed in ergot, mold, yeast, red algae, lichens and many insects. Not only does it have a function as an antifreezing agent, but it is known that insects control the concentration depending on the season and acquire cold resistance.

Trehalase is known as an enzyme that hydrolyzes such trehalose into two molecules of glucose. It is a higher animal (rat small intestine, pig small intestine, etc.), insect (silkworm, etc.),
It is widely distributed in fungi (for example, Aspergillus nigar), animals such as yeast, plants, and microorganisms. In particular, it has been suggested that this trehalase is strongly involved in the transport of the crucose in higher animals, and it is expected that this trehalase is greatly involved in the energy acquisition and transformation during flight in insects. There is. On the other hand, it is known that microorganisms are involved in regulation of energy acquisition during spore germination. As described above, trehalase has been attracting attention as an important physiologically active substance from the viewpoint of physiological action of each organism, and research has been advanced.

As a microorganism that produces this trehalase, aspergillus (Aspergillus) genus [K. K. Horikoshi, Journal of Bacteriology (J. Bacteriol.), 1966, 91 , 1883]; Neurospora (N.
eurospora) genus [eye. Etch. Lanny,
Journal of Bacteriology (J. Bacterio
l.), 1973, 115, 582]; Humicola genus [A. R. S. ARS Prasad, Biosimikae Biophysical Actor. (Biochim.Biop
hys.Acta.), 1978, 525, 162]; Phycomyces (Phycom
yces) [J. A. Van Ash (JAVanA
ssche), Bioshikae Biophysical Actor. (Biochim.Biophys.Acta.), 1975, 391, 154]; Trichoderma genus [Pe. Fijayakumar, Canadian Journal of Microbiology, Can.J.Microbiol., 1978,
24, 1280]; Pururaria (Pullularia) genus [e. Meldinger, Journal of Bacteriology, 1971, 106, 1034]; genus Chaetomium [Invention of Japanese Patent Publication No. 60-22917]; genus Cochliobolus [em. Matsubara, Chemical & Pharmaceutical Bulletin (Chem.Pharm.Bull.), 1981, 29 , 372
3]; genus Dictyosterium [K. A. KAKillick, Achievements of Biochemistry & Biophysics (Ar
ch.Biochem.Biophys.), 1983, 222, 561] and the like, in yeast, the genus Saccharomyces [A. A. Panek, Journal of Biological Chemistry (J. Biol. Chem.), 1964, 239, 1671], etc. are known and are being studied in detail. In addition, although there are few examples of bacteria and actinomycetes, mycobacteria (Mycobacter
ium) genus [Bee. W. Patterson (BWPatters
on), Method in Enzymology (Methed in
Enzymology), 1972, 28 , 996]; Streptomyces (St
reptomyces) genus [A. E. AEHey, Journal of Bacteriology (J. Bacteriol.), 196
8, 96 , 105] are known.

However, as the location of this trehalase,
The genus Trichoderma [Pe. Fijayakmar, Canadian Journal
Of Microbiology, Can.J.Microbiol., 197
8, 24 , 1280] and is produced extracellularly by
It has only been confirmed that trehalase is released from the ostium of the angiosperm in the genus omium) (Invention of Japanese Patent Publication No. 60-22917), and other microorganisms mainly produce spores and intracellular cells, and trehalase The preparation required a lot of labor.

Also, conventional trehalase is unstable to heat,
The above Humicola genus, Saccharomyces (Sa
ccharomyces) [Pea. Jay. Kelly (PJKell
y), Analytical Biochemistry (Anal.Bioc
hem.), 1976, 72 , 353], only a study on the heat stabilizing protective substance was conducted.

Problems to be Solved by the Invention The fact that a large amount of various industrially or medically useful enzymes can be obtained means that useful enzymes can be used to obtain useful substances and to study physiological actions. Is important in. As described above, in order to mass-produce a specific enzyme, it is necessary to discover and search for a microorganism that enables extracellular production.

Further, in order to effectively utilize the enzyme thus obtained in a bioreactor or the like, it is important to have high stability, particularly high stability to temperature. That is,
To enable repeated use in bioreactors, etc., it is generally necessary to immobilize the enzyme, and at that time, the enzyme may be exposed to a relatively constant temperature for immobilization. It is highly likely that it will be deactivated during operation. Moreover, the reaction rate is increased by carrying out the enzymatic reaction at a high temperature, and the target product can be mass-produced.

Further, although trehalase is widely distributed in animals, plants, and microorganisms, the content of trehalase in these is extremely low, which makes the purified trehalase enzyme extremely expensive.

Therefore, it is eagerly desired to develop a trehalase having good mass productivity and good thermostability and a method for producing the trehalase, and the object of the present invention is also in this point, and a novel thermostable trehalase and a method for producing the same are provided. To do.

Means for Solving the Problems In view of the current state of the art conventional methods for trehalase enzyme, the present inventors have found the ability to generate and accumulate trehalase stable in heat and easy to purify in an external culture solution. As a result of diligent search to obtain the microorganisms, the growth temperature is moderate to high, and Corynebacterium (corynebacteriu)
The present invention has been completed based on the finding that a certain type of thermophilic bacterium belonging to the genus m) has the above-described requirements and that it also produces and accumulates the target enzyme in the cells.

That is, the present invention firstly provides a novel thermostable trehalase enzyme, which has the following physicochemical properties.

(A) Action-specifically hydrolyzes the glycoside bond of α, α-trehalose to produce two molecules of glucose.

(B) Substrate specificity: It acts specifically on α, α-trehalose and does not act on the same disaccharides sucrose, lactose, melibiose, maltose, cellobiose and gentiose.

(C) Optimum pH and stable pH range: The optimum pH is 6.5 to 7.5, and it is stable within the pH range of 5.0 to 10.0 under heating conditions of 60 ° C for 30 minutes.

(D) Temperature stability: It is stable for 15 hours or more under heating conditions of pH 7.0 and 65 ℃.

(E) Optimum working temperature range: Optimal working temperature is around 60 ° C.

(F) Deactivation condition: pH 4.0 under treatment conditions of 60 ° C for 30 minutes
And completely deactivates at 11.0. Also, it is completely deactivated at 85 ° C under the treatment conditions of pH 7.0 and 15 minutes.

(G) Inhibition and activation: Deactivated by mercuric chloride and phloretin, and activated by heat treatment at 60 ° C for 2 hours.

(H) Molecular weight: 55,000 to 65,000. And (i) Isoelectric point ... 4.4.

The present invention also relates to a method for producing the above novel thermostable trehalase, wherein the trehalase belongs to the genus Corynebacterium, cultivates a microorganism that produces the above trehalase extracellularly and intracellularly, and in the culture solution or the intracellular body. It can be obtained by a method characterized in that the enzyme is produced / accumulated in, and then separated / purified.

The novel extracellular and intracellular thermostable trehalase-producing strains used in the method of the present invention have been newly searched and isolated from the natural world by the present inventors. These strains were designated as Bergey's Mannual of D.
Identified according to eterminative Bacteriology), 8th edition, they are all aerobic ascospores, have no motility, and are positive for Gram stain and catalase test. However, since it grows well at high temperatures around 60 ° C, it was considered to be a new strain that is taxonomically different from known Corynebacterium.

The following Table 1 shows the mycological properties of the isolated three types of extracellular and intracellular thermostable trehalase-producing bacteria.

In addition, the above-mentioned fungus was sent to the Institute of Industrial Technology
FERM P-8484 (K-502), FERM P-8485 (K-51
2) and FERM P-8486 (K-522).

The method for producing the novel extracellular and intracellular thermostable trehalase of the present invention will be described in more detail. First, the extracellular thermotolerant trehase has the above-mentioned physicochemical properties. This is inoculation of the above-mentioned extracellular and intracellular thermostable trehalase-producing bacteria into an appropriate medium, and at 55 to 65 ° C,
The culture is performed aerobically for 48 to 72 hours, and the medium contains a carbon source, a nitrogen source, and if necessary, inorganic salts and micronutrients.

First, various conventionally known materials can be used as the carbon source, and typical examples thereof include glucose, starch syrup, and soluble starch. The nitrogen source is not particularly limited, and yeast extract, peptone, meat extract, corn steep liquor, amino acid solution, organic nitrogen such as soybean meal, or ammonium sulfate, urea, ammonium nitrate, inorganic nitrogen such as ammonium chloride, etc. It can be illustrated as being cheap and easily available. Needless to say, the organic nitrogen source serves as a carbon source. Furthermore, such a carbon source,
In addition to the nitrogen source, various commonly used salts, for example, inorganic salts such as magnesium salts, potassium salts, phosphates, iron salts, and vitamins can be added.

Suitable media for use in the method of the invention include, for example, 1% soluble starch, 1% glucose, 1% polypeptone, 0.5% yeast extract, 0.1% K ₂ HPO ₄ and
It may be a liquid medium containing MgSO ₄ · 7H ₂ O 0.02%.

Moreover, since the growth pH of the microorganism used in the method of the present invention is within the range of weak acid to basicity, it is necessary to maintain the above-mentioned predetermined pH value by using an appropriate buffer solution. As a buffer solution useful for that purpose, a phosphate buffer solution can be mentioned as a typical example, but the buffer solution is not limited to this and any known buffer solution can be used.

Since all of the bacteria used in the above method of the present invention produce trehalase extracellularly, the produced trehalase is released into the culture medium and accumulated therein. Cultivation of these bacteria can be obtained by either a batch system or a continuous system, and the produced enzyme can be separated and purified, for example, as follows.

That is, it is possible to first remove the bacterial cells in the culture solution by centrifugation, filtration, etc., and then apply the resulting supernatant (crude enzyme solution) as it is to the hydrolysis reaction of trehalose, which is economical. Is advantageous to. Further, it can be further purified before use. Therefore, it can be purified by, for example, salting out with ammonium sulfate or the like, a solvent precipitation method with ethanol, acetone, isopropanol or the like, an ultrafiltration method, a gel filtration method, or a general enzyme purification method with an ion exchange resin or the like.

Hereinafter, an example of a preferable purification method of the extracellularly produced trehalase of the present invention will be described.

For example, a K-502 strain belonging to the genus of thermophilic Corynebacterium is inoculated into the above medium and aerobically cultured at 55 ° C. for 72 hours to obtain a culture solution at 10,000 rpm, 0 rpm.
The cells are removed by centrifugation at 0 ° C for 10 minutes to obtain 3 supernatants. Then, the supernatant was concentrated about 10 times using an ultrafiltration membrane having an average molecular weight cut-off of 10,000, and a 10 mM phosphate buffer solution (pH 7.
Dialyse overnight at 0). The precipitate generated after this dialysis was removed by centrifugation, and the resulting supernatant was washed with 10 mM phosphate buffer (pH 7.
0) Adsorbed on a DEAE-cellulose column equilibrated with
The enzyme is eluted by the same buffer gradient method as above containing 0-0.8 M NaCl. The eluted active fractions are collected, concentrated using an ultrafiltration membrane, and then dialyzed overnight using the same phosphate buffer as above. Then, the dialysis enzyme was similarly adsorbed on the gluconate-Sepharose 4B column equilibrated with the phosphate buffer, washed with the buffer containing 0.1 M NaCl, and then the active fraction was added using the same buffer containing 1 M NaCl. Was eluted. This active fraction is dialyzed overnight in the same buffer containing 0.1 M NaCl. Subsequently, the dialysis enzyme is adsorbed to a DEAE-Toyopearl column equilibrated with the above buffer solution containing 0.1 M NaCl, and the enzyme is eluted by the concentration gradient method of the above buffer solution containing 0.1 to 0.5 M NaCl. The eluted active fractions are collected, concentrated using an ultrafiltration membrane, and dialyzed overnight in the above buffer containing 0.1 M NaCl. Further, the thus dialyzed enzyme is concentrated using an ultrafiltration membrane. 0.1M of this concentrated enzyme
Biogel P- equilibrated with the above buffer containing NaCl
Load into a 150 gel filtration column and elute with the same buffer as above containing 0.1 M NaCl. The active fraction thus obtained is concentrated,
Polyacrylamide gel disk electrophoresis (gel concentration 7.
15 mg of a single enzyme preparation was obtained at 5%, pH 4.5).
The activity yield was 18%. Incidentally, K-512 bacteria and K-522 bacteria useful in the method of the present invention can be purified in the same manner as above except that the gel filtration resin is appropriately changed.

The thermostable trehalase-producing bacterium used in the present invention also produces and accumulates trehalase in the cells. The thermostable trehalase obtained in this case also has the same physicochemical properties as the above-mentioned extracellularly produced trehalase. This trehalase can be produced according to almost the same method as the above extracellular trehalase. That is, a thermostable trehalase-producing bacterium as described above is inoculated into an appropriate medium,
Incubate aerobically at ~ 65 ° C for 48-72 hours. The medium used in this case may be the same as that used for the production of extracellular trehalase.

Since the intracellular trehalase is produced in the bacterial cells in any case, the steps of disrupting the bacterial cells and removing the DNA are required. Examples of the method include lysozyme, French press, disruption of cells by ultrasonic treatment, and removal of DNA by treatment with polyethyleneimine or DNase. However, the method is not limited to such a method, and any other known arbitrary means can be used.

For example, first, collect the bacterial cells in the culture solution by centrifugation, filtration, etc., and then use the resulting bacterial cells with lysozyme (2 mg)
Lysis at ℃, further remove the precipitate generated by the treatment with polyethyleneimine by centrifugation or the like, the supernatant obtained is a crude enzyme solution. It is also possible to use this supernatant as it is for the hydrolysis reaction of trehalose, which is economically advantageous. Further, it can be further purified before use. Therefore, for example, it can be purified by salting out with ammonium sulfate or the like, a solvent precipitation method with ethanol, acetone, isopropanol, etc., an ultrafiltration method, a gel filtration method, or a general enzyme purification method with an ion exchange resin or the like.

Hereinafter, one example of a preferred method for purifying trehalase of the present invention will be described.

That is, for example, K-, which belongs to the genus of thermophilic Corynebacterium
For example, the culture solution obtained by inoculating the 502 strain in the above-mentioned medium and aerobically culturing at 55 ° C. for 72 hours is 10,000 rp.
Centrifuge at 0 ° C. for 10 minutes to obtain 10 g of bacterial cells. The cells obtained are thoroughly washed with 10 mM phosphate buffer containing 0.85% NaCl, lysed with lysozyme, and polyethyleneimine is further added to remove the resulting precipitate by centrifugation. Then, the supernatant was washed with an ultrafiltration membrane having an average molecular weight cut-off of 10,000 to obtain about 10
Concentrate twice and dialyze overnight against 10 mM phosphate buffer (pH 7.0). The precipitate generated after this dialysis was removed by centrifugation, and the resulting supernatant was equilibrated with 10 mM phosphate buffer (pH 7.0).
The enzyme is adsorbed on an E-cellulose column, and the enzyme is eluted by the same concentration gradient method of a buffer solution as described above, in which 0 to 0.8 M NaCl is added. The active fraction thus eluted is collected and dialyzed overnight using an ultrafiltration membrane. Then, the dialysis enzyme was similarly adsorbed on the gluconate-Sepharose 4B column equilibrated with the phosphate buffer, and after washing with the equilibration solution containing 0.1 M NaCl,
The active fraction was eluted with the same buffer containing 1M NaCl.
This active fraction is dialyzed overnight against the same buffer containing 0.1 M NaCl. Subsequently, the dialysis enzyme is adsorbed on a DEAE-Toyopearl column equilibrated with the buffer containing 0.1 M NaCl, and the enzyme is eluted by a concentration gradient method of the buffer containing 0.1 to 0.5 M NaCl. . Collect the eluted active fractions,
Concentrate using an ultrafiltration membrane and dialyze overnight against the above buffer containing 0.1 M NaCl. Further, the thus dialyzed enzyme is concentrated using an ultrafiltration membrane. The concentrated enzyme is packed in a gel filtration column of Biogel P-150 equilibrated with the above buffer solution containing 0.1 M NaCl and eluted with the same buffer solution containing 0.1 N NaCl. The active fraction thus obtained was concentrated and subjected to polyacrylamide gel disk electrophoresis (gel concentration 7.5%, pH
In 4.5) 3 mg of a single enzyme preparation was obtained. The activity yield was 18%. Incidentally, K- which is useful in the method of the present invention
512 bacteria and K-522 bacteria can be purified in the same manner as above except that the gel filtration resin is appropriately changed.

The method for measuring the activity of trehalase and the method for displaying the activity are as follows.

That is, 1% dissolved in 0.1M phosphate buffer (pH 7.0)
0.1 ml of (W / V) trehalose solution is mixed with 0.1 ml of enzyme solution, reacted at 60 ° C. for 10 minutes, and then heated in a boiling water bath for 10 minutes to inactivate the enzyme. Next, the D-glucose produced by hydrolysis was analyzed by the Somogy-Nelson method [Method in Carbohydrate Chemistry (Method i
Carbohydrate Chemistry), 1962, 1 , 386]. In addition, the unit of enzyme activity is expressed as one unit of the amount of enzyme required to produce 1 μmol glucose per minute under the above-mentioned conditions.

In addition to the physicochemical and enzymatic chemical properties of thermostable trehalase I obtained by the method of the present invention, in addition to the above, each strain K-50
2, the molecular weights of the enzymes obtained from K-512 and K-522 are 55,000 ± 2,000, 60,000 ± 2,000 and 65,000 ± 2,000, respectively.
It is 0. In addition, these molecular weights were obtained by the gel filtration method. Table 2 compares the physicochemical and enzymatic chemical properties of the trehalase produced extracellularly and intracellularly of the present invention and the trehalase derived from conventionally known microorganisms.

In order to obtain a large amount of trehalase, which is widely distributed in animals, plants, and microorganisms and plays an important role in the physiological actions of each organism by a simple method, a microorganism or microbial cell that extracellularly produces this trehalase enzyme It is important to search for microorganisms produced outside and inside the cells, and by obtaining such microorganisms, trehalase can be supplied inexpensively and stably.

From an industrial point of view, it is advantageous that the enzyme extracted from the microorganism has high heat resistance. For example, performing hydrolysis of trehalose using this at high temperature accelerates its reaction rate, It is important for mass production of degradation products, and the enzyme is immobilized for use in bioreactors, etc. It is advantageous to use.

For this purpose, there are some cases in which the relationship between the growth temperature of microorganisms and heat resistance was examined, but the desired results could not be obtained [Bio-Simicae Biophysica Acta. (Biochim.Biophys.Acta.), 1978, 525, 162]. Therefore, in order to obtain a thermostable enzyme, it is necessary to develop a new microorganism that produces it. It is also known to have excellent heat resistance [Journal of Bact (J.Bacteriol.), 1973, 115- 2,582 ], but this is a microorganism having the intracellular production of the enzyme of interest, i.e. Neurospora (Neurospora) obtained from mycelium and ascospores, and the heat resistance is maintained by increasing the enzyme concentration or adding a polyol. This is not preferable because the amount is limited and the separation and purification are complicated.

By the way, the method of the present invention, by using a certain novel bacterium belonging to the genus Corynebacterium of thermophilic and enzyme extracellular production, the problem of heat resistance and the problem of mass productivity, further, Both separation and purification problems could be solved advantageously.

Further, the bacterium belonging to the genus Corynebacterium used in the present invention not only produces trehalase extracellularly but also intracellularly as described above.

Therefore, in this case, the productivity of trehalase in a single lot is greatly improved although the separation and purification are as complicated as the conventionally known microorganisms. Moreover, since trehalase produced in the cells is also highly thermostable, it can be advantageously used in various applications requiring thermostability. The most important advantage is that useful enzymes can be recovered from both the bacterial cells and the culture broth cultured under the same conditions.

Examples Hereinafter, the novel thermostable trehalase of the present invention and the method for producing the same will be described more specifically according to Examples. However, the scope of the present invention is not limited by the following examples.

Example 1 Thermophilic bacterium Corynebacterium No.-K-502 (Coryneba
cterium sp. K-502: FERM P-8484) in a 1-volume Erlenmeyer flask with 2% soluble starch, 1% polypeptone,
Inoculate into 300 ml of medium (pH 7.2) containing 0.5% yeast extract, 0.1% K ₂ HPO ₄ and 0.02% MgSO ₄ .7H ₂ O, 72 hours at 55 ° C, 250r
Rotate with shaking at pm and centrifuge the culture to 0.28 units.
A crude enzyme solution of / ml was obtained. The enzyme activity was measured according to the method described above (the same applies hereinafter).

Example 2 Thermophilic bacterium Corynebacterium No.-K-512 (Coryneba
cterium sp. K-512: FERM P-8485) was inoculated into 300 ml of a medium containing 2% glucose instead of the soluble starch having the medium composition described in Example 1, and shaken under the same conditions as in Example 1. After culturing, 0.37 unit / ml of crude enzyme solution was obtained.

Example 3 Thermophilic bacterium Corynebacterium No.-K-522 (Coryneba
cterium sp. K-522: FERM P-8484) was inoculated into the medium having the composition described in Example 1 and cultivated at 60 ° C. for 48 hours with rotary shaking at 250 rpm, and the culture solution was centrifuged to obtain 0.33. A unit / ml crude enzyme solution was obtained.

Example 4 The thermophilic bacterium Corynebacterium No.-K-502 (Coryneba
cterium sp. K-502: FERM P-8484) in a 1-volume Erlenmeyer flask with 2% soluble starch, 1% polypeptone,
Inoculate into 300 ml of medium (pH 7.2) containing 0.5% yeast extract, 0.1% K ₂ HPO ₄ and 0.02% MgSO ₄ .7H ₂ O, 72 hours at 55 ° C, 250r
Culturing was carried out by rotary shaking at pm, and the culture solution was centrifuged to collect bacterial cells to obtain 10 ml of 0.5 unit / ml crude enzyme solution by the method described above.

Example 5 Thermophilic bacterium Corynebacterium No.-K-512 (Coryneba
cterium sp. K-512: FERM P-8485) was inoculated into 300 ml of a medium containing 2% glucose instead of the soluble starch having the medium composition described in Example 4, and shaken under the same conditions as in Example 4. After culturing, 10 ml of a crude enzyme solution containing 1.25 units / ml was obtained.

Example 6 Thermophilic bacterium Corynebacterium No.-K-522 (Coryneba
cterium sp. K-522: FERM P-8486) was inoculated into the medium having the composition described in Example 4 and cultivated at 60 ° C. for 48 hours with rotary shaking at 250 rpm, and the culture solution was centrifuged to give 0.95. 10 ml of a crude enzyme solution having a unit / ml was obtained.

Effects of the Invention As described in detail above, according to the present invention, a novel thermophilic microorganism, which is based on the use of a bacterium belonging to Corynebacterium having extracellular and intracellular productivity of trehalase, A trehalase enzyme having high thermostability can be advantageously produced with high productivity. In addition, since the use of the microorganisms that can be produced extracellularly makes the separation and purification operations extremely easy, it has become possible to inexpensively and stably supply trehalase, which was expensive in the past.

Further, the trehalase-producing bacterium used in the present invention also has intracellular productivity, and therefore, thermostable trehalase can be recovered from the bacterial cells themselves obtained when the culture solution is recovered. From this fact, the yield of trehalase is further improved.

Furthermore, since the trehalase of the present invention has a high thermostability, it is possible to carry out the hydrolysis reaction of trehalose at a high temperature, and as a result, the reaction rate can be greatly improved, so that the hydrolysis product can be mass-produced. Becomes Further, it is possible to maintain sufficient enzyme activity even when exposed to a high temperature during immobilization, and it becomes possible to obtain an excellent immobilized enzyme for a bioreactor.

Front Page Continuation (72) Inventor Hiroki Horikoshi 4-39-8 Sakuradai, Nerima-ku, Tokyo (72) Inventor Nobuyuki Nakamura 1-4-23 National City, Tokyo

Claims (7)

[Claims] 1. A novel trehalase having the following physicochemical properties. (A) Action-specifically hydrolyzes the glycoside bond of α, α-trehalose to produce two molecules of glucose. (B) Substrate specificity: It acts specifically on α, α-trehalose and does not act on the same disaccharides sucrose, lactose, melibiose, maltose, cellobiose and gentiose. (C) Optimum pH and stable pH range: The optimum pH is 6.5 to 7.5, and it is stable within the pH range of 5.0 to 10.0 under heating conditions of 60 ° C for 30 minutes. (D) Temperature stability: It is stable for 15 hours or more under heating conditions of pH 7.0 and 65 ℃. (E) Optimum working temperature range: Optimal working temperature is around 60 ° C. (F) Deactivation condition: pH 4.0 under treatment conditions of 60 ° C for 30 minutes
And completely deactivates at 11.0. Also, it is completely deactivated at 85 ° C under the treatment conditions of pH 7.0 and 15 minutes. (G) Inhibition and activation: Deactivated by mercuric chloride and phloretin, and activated by heat treatment at 60 ° C for 2 hours. (H) Molecular weight: 55,000 to 65,000. (I) Isoelectric point ... 4.4. 2. Belonging to the genus Corynebacterium, the following physicochemical properties are as follows: (a) Action: .alpha.,. Alpha.-trehalose glycoside bond is specifically hydrolyzed to produce two molecules of glucose; (B) Substrate specificity: It acts specifically on α, α-trehalose and does not act on the same disaccharides sucrose, lactose, melibiose, maltose, cellobiose and gentiose; (c) Optimum pH and stability pH range ... Optimum pH is 6.5-7.5, and it is stable within the range of pH 5.0-1.0 under heating conditions of 60 ° C. for 30 minutes; (d) Stability against temperature ... pH 7.0, It is stable for more than 15 hours under heating conditions of 65 ° C; (e) Optimum temperature range of action ... Optimum action temperature near 60 ° C; (f) Deactivation condition ... Treatment conditions of 60 ° C for 30 minutes Then pH 4.0
And 11.0, and completely inactivated at 85 ° C under the treatment conditions of pH 7.0 and 15 minutes; (g) Inhibition and activation: Inactivated by mercuric chloride and phloretin, Activated by heat treatment at 60 ° C. for 2 hours; (h) culturing a microorganism having a novel trehalase-producing ability having a molecular weight of 55,000 to 65,000; (i) isoelectric point of 4.4; and culturing the trehalase. A method for producing the above-mentioned novel extracellular trehalase, which comprises producing and accumulating in a liquid and collecting this. 3. The method for producing extracellular trehalase according to claim 2, wherein the culturing is aerobically carried out at a temperature within the range of 55 to 65 ° C. 4. The method for producing extracellular trehalase according to claim 2 or 3, characterized in that the pH of the culture solution is in the range of 5 to 9. 5. Belonging to the genus Corynebacterium, the following physicochemical properties are as follows: (a) Action: .alpha.,. Alpha.-trehalose glycoside bond is specifically hydrolyzed to produce two molecules of glucose; (B) Substrate specificity: It acts specifically on α, α-trehalose and does not act on the same disaccharides sucrose, lactose, melibiose, maltose, cellobiose and gentiose; (c) Optimum pH and stability pH range: Optimum pH is 6.5 to 7.5, stable at pH 5.0 to 10.0 at 60 ° C for 30 minutes; (d) Stability against temperature: pH 7.0, heating at 65 ° C It is stable for more than 15 hours under the conditions; (e) Optimum temperature range of action ... Optimum action temperature in the vicinity of 60 ° C; (f) Deactivation condition: pH4.0 under treatment conditions of 60 ° C for 30 minutes And completely deactivate at 11.0; (g) Inhibition and activation ... It is inactivated by dimercury and phloretin, and activated by heat treatment at 60 ° C for 2 hours. (H) Molecular weight: 55,000-65,000; (I) Isoelectric point: 4.4; A method for producing the novel intracellular trehalase, which comprises culturing a microorganism, producing and accumulating the trehalase in the intracellular body, and collecting the trehalase. 6. The method for producing intracellular trehalase according to claim 5, wherein the culture is aerobically carried out at a temperature within the range of 55 to 65 ° C. 7. The method for producing intracellular trehalase according to claim 5 or 6, wherein the culture solution has a pH within the range of 5 to 9.