JPS637756B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel thermophilic fumarase, a method for producing the same, a microorganism capable of producing the enzyme, and a method for producing L-malic acid using the enzyme. L-malic acid is an important substance that is used as a component of infusions and is increasingly being used as an acidulant for refreshing drinking water. Currently, L-malic acid is produced chemically from maleic acid.
There are methods such as optical resolution of malic acid, methods using microorganism-derived furamase, and direct fermentation of microorganisms, but the method using microorganism-derived fumarase is the most practical. Examples of using fumarase derived from microorganisms include the method by Kitahara et al. in which the lactic acid bacterium Lactobacillus brevis acts on calcium fumarate to precipitate it as calcium L-malate (Special Publication Publication No. 37-4511), and the method using yeast to convert fumaric acid into enzymes. Enzymatic production of L-malic acid from fumaric acid using Brevibacterium ammoniagenes
Method of producing malic acid (European Journal of Applied Microbiology,
Volume 3, page 169, 1976).
The method described in Japanese Patent Publication No. 37-4511 has drawbacks such as by-production of gypsum and poor growth, while the latter two have practical difficulties because the enzymatic reaction is carried out at room temperature. Being able to perform fermentation operations, enzyme reactions, etc. under high temperature conditions is extremely advantageous in terms of preventing bacterial contamination, speeding up enzyme reactions, and saving cooling energy. The present inventors investigated the fumarase activity of various thermophilic bacteria for the purpose of obtaining thermostable fumarase, and found that highly thermophilic bacteria belonging to the genus Thermus and Bacillus have thermophilic fumarase activity. He has completed his invention. Accordingly, the present invention relates to a novel thermophilic fumarase, a method for producing the same, a microorganism capable of producing the enzyme, and a method for producing L-malic acid using the enzyme, which will be explained in detail below. The highly thermophilic fumarase according to the present invention can be produced by culturing a microorganism capable of producing the enzyme in a nutrient medium, accumulating the enzyme in the culture, and collecting the enzyme from the culture. can do. The microorganism used in the present invention may be any strain as long as it has the ability to produce highly thermophilic fumarase, but specifically, strains belonging to the genus Thermus or Bacillus, such as Thermus aquaticus. No. 104 (Feikoken Bibori No. 5150, ATCC31558), Thermus lacteus knob sp.
nov.sp.) No. 108 (Feikoken Bibori No. 5149,
ATCC31557), Thermus rubens nov.sp. No. 102 (Feikoken Bacteria No. 5148, ATCC31556), Thermus aquatakes ATCC25104, Thermus aquatakes ATCC25105, Thermus thermophilus ( Thermus thermophilus ATCC27634, Bacillus licheniformis
T-511 (Microtechnology Research Institute No. 5151, NRRL B-
12024), Bacillus pumilus
pumilus) T-530 (Feikoken Bibori No. 5152,
NRRLB-12025), Bacillus subtilis ATCC6051, Bacillus licheniformis ATCC11560, Bacillus stearothermophilus
Examples include ATCC12016 and Bacillus coagulans ATCC7050. The mycological properties of the above microorganisms are described in the following documents. Thermus Aquatakes Bergey′s
Manual of Determinative Bacte―
riology 8th edition page 285 Thermus thermophilus JP-A-51-115988
Issue Publication Bacillus licheniformis Bergey′s
Manual of Determinative Bacte―
riology 8th edition, p. 534 Bacillus pumilus ditto, p. 533-534 Bacillus subtilis dit., p. 531-533 Bacillus stearothermofulis dit.
Pages 539-540 Bacillus coagulans Same as above, pages 540-541 In addition, Thermus aquatakes No. 104, Thermus lacteus knob sp. No. 108, and Thermus rubens knob sp. No. 1 isolated by the present inventors. The mycological properties of 102 and the basis for identification are shown below.

【table】

【table】

[Table] Based on the above-mentioned mycological properties, Burgee's Manual of Determinative Bacteriology, 8th edition and Journal of Bacteriology, Vol. 98, pp. 289-297 (1969), International Journal・Of Systematics・
Bacteriology Vol. 24, pp. 102-112 (1974), 25
With reference to Vol. 357-364 (1975) and Agricultural Biological Chemistry Vol. 36, pp. 2357-2366, known bacterial strains and their differences were investigated. The three strains mentioned above are Gram-negative, nonspore-free, non-motile, aerobic rods or filamentous rods, and are obligate thermophiles with an optimum growth temperature of 60°C or higher, so they are considered to belong to the genus Thermus. It will be done. Bacterium No. 104 was identified as Thermus aquatakes because it almost completely matched the experimental results for the standard strain of Thermus aquatakes used as a control strain in the identification experiment or the description in the aforementioned paper. Strain No. 108 can be distinguished from Thermus aquatakes by (1) cream-colored colonies, (2) different acid production from galactose, lactose, and glycerol, and (3) no requirement for glutamate. It was considered a new species and was named Thermus lacteus knob sp. No.102 bacteria is
(1) The colony is reddish-orange, (2) The optimum temperature for growth is low, (3) The optimum pH is low, and (4) Thermus differs in acid production from glycerol and mannitol.
It could be distinguished from Aquatakes, and it was considered a new species and named Thermus rubens knob sp. The culture of these strains will be described. In culturing these strains, conventional culturing methods for thermophilic bacteria are generally used. Carbon sources include sucrose, mannose,
Carbohydrates and sugar alcohols such as starch, molasses, glycerin, and mannitol are used alone or in combination. Depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used. As a nitrogen source, inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium nitrate, sodium nitrate, urea, etc., or organic nitrogen sources such as polypeptone, peptone, yeast extract, corn steep liquor, amino acids, etc. can be used alone or in combination. can. Also, organic acids such as fumaric acid, Mn ⁺⁺ , Co ⁺⁺ , Mg ⁺
The growth of bacteria and the production of enzymes are promoted by the addition of various salts including ⁺ and Mo ⁺⁺ . A liquid culture method is suitable as a culture method. It is desirable to culture at a culture temperature of 50 to 80°C and a pH around neutral. After culturing, thermophilic fumarase can be collected from the culture solution using a general enzyme collection method. Since this enzyme is an intracellular enzyme, it can be isolated, for example, by the following method. First, the obtained bacterial cells are subjected to Dyno Mill (Shinmaru Enterprise) at a concentration of about 50 g/min to obtain a cell-free extract. This is salted out with 40-70% ammonium sulfate for 24 hours. The buffer used is 0.1M Tris/hydrochloric acid solution. Next, the active fraction is adsorbed onto a DEAE/Sephadex G-25 (manufactured by Seikagaku Kogyo Co., Ltd.) column and eluted with a concentration gradient of 0 to 0.6M saline to collect the active fraction. This fraction is then saturated with ammonium sulfate to 70%, and the resulting precipitate is collected and dialyzed against a phosphate buffer with a pH of about 6.5.
In addition, DEAE/cellulose (manufactured by Seikagaku Corporation),
This enzyme is obtained by adsorption and elution with DEAE/Sephadex G-50 (manufactured by Seikagaku Kogyo Co., Ltd.) and finally hydroxyapatate (manufactured by Seikagaku Kogyo Co., Ltd.). The activity of this enzyme is measured as follows. 0.1 M sodium fumarate is brought into contact with the present enzyme, bacterial cells, or treated bacterial cells (10 g/DCW equivalent), and a reaction is carried out at pH 7.0 and a temperature of 40 to 70°C for 30 minutes.
The reaction solution is diluted with L-malic acid to a concentration of about 5 to 80 gamma. Collect 1 ml of the diluted solution, add 6 ml of concentrated sulfuric acid, stir thoroughly, add 0.1 ml of 2,7-naphthalene diol (1 g/100 ml, concentrated sulfuric acid), and immerse in boiling water for 20 minutes. After cooling, the amount of L-malic acid is calculated from the absorption value at 390 nm, and the enzyme activity is calculated from this value.
Other methods that can be used include high performance liquid chromatography, Marizquez enzyme method, and fumaric acid determination method using potassium permanganate. In either case, enzyme activity is expressed in international units (unit/g·min). Next, the properties of the obtained enzyme will be described. The enzymes produced by Thermus genus and Bacillus genus have slightly different properties, so they will be described separately. Thermus spp. (Thermus Aquatakes No.
The enzyme obtained from 104 is shown as a representative) (1) Action and substrate specificity Acts only on fumaric acid and L-malic acid,
Catalyzes the reaction that produces fumaric acid and water from L-malic acid and its reverse reaction. (2) Optimum PH Activity in 0.2M phosphate buffer (PH6-8) and 0.2M Tris-HCl buffer (PH8-9) at 60℃10
The measurement was performed after a minute treatment. The results are shown in Figure 1, and the optimum pH is around PH7.2. In general, fumarases of animal origin and produced by thermophilic bacteria have an optimum pH on the alkaline side, while thermophilic fumarases generally have an optimum pH on the neutral or slightly acidic side. (3) Stable PH range 1M sodium fumarate (PH7 .2) was added, reacted at 60°C for 30 minutes, and measured the activity. The results are shown in FIG. At PH8.0, there was a difference of about 2 times in the activity due to the buffer, but as shown in the figure, the PH
Values between 5.5 and 7.5 seem to be relatively stable. (4) Suitable temperature range for action The reaction was carried out for 10 minutes at PH7.2 in the range of 50 to 85°C. The results are shown in Figure 3, and the optimum temperature is around 70°C. (5) Temperature stability This enzyme was dissolved in 0.1M phosphate buffer (PH7.2) at 50-80% temperature.
After treatment at 60°C for 30 to 120 minutes, 1M sodium fumarate was added and reacted at 60°C for 10 minutes to measure activity. The results are shown in FIG. up to 60℃
100% stable. (6) Inhibition, activation and stabilization This enzyme shows some activation by certain divalent metal ions. The order is Co ⁺⁺ > Zn ⁺⁺ > Mg ⁺⁺ , with Cu ⁺⁺ showing a slight inhibitory effect. It is inhibited by metal chelating reagents such as 0-phenanthroline and α,α'-dipyridyl. (7) Molecular weight The molecular weight was approximately 180,000 as a result of gel filtration using Cephadex G-200 using cytochrome-C, ovalpmin, and γ-globulin as internal standard proteins. (8) Amino acid composition: Lysine: 6.00 Arginine: 5.80 Histidine: 1.56 Aspartic acid: 7.80 Alanine: 12.40 Threonine: 5.06 Serine: 4.80 Glutamic acid: 12.20 Proline: 6.60 Glycine: 10.10 Valine: 6.70 Methionine: 0 Isoleucine: 3. 60 Leucine: 10.60 Tyrosine : 2.50 Phenylalanine: 4.20 Tryptophan: Cystine which cannot be measured due to trace amount: 0 (9) Protein denaturation temperature (Tm value): 90℃ Bacillus genus (Bacillus licheniformis T-
The enzyme obtained from 511 is shown as a representative) (1) Action and substrate specificity Acts only on fumaric acid and L-malic acid,
Catalyzes the reaction that produces fumaric acid and water from L-malic acid and its reverse reaction. (2) Optimal PH This enzyme was added to 0.2M acetate buffer (PH5-6.5), 0.2M
The activity in phosphate buffer (PH6.5-8.5) and 0.2M Tris/HCl buffer (PH8.5-10.0) was measured by treatment at 50°C for 30 minutes. The results are as shown in FIG. 5, and the optimum pH is around PH7.0. (3) Stable PH range 16 in the above buffer at 50℃ in the PH range 5.0 to 10.0
After time treatment, activity was measured at 60°C and pH 7.0. The results are shown in Figure 6, and the stable PH range is PH7.0 ~
It is stable at 7.8. (4) Suitable temperature range for action The reaction was carried out for 10 minutes at PH7.0 in the range of 40 to 80°C. The results are shown in Figure 7, and the optimum temperature is between 50 and 60°C. (5) Temperature stability After treating the enzyme solution at 40-80℃ with pH7.0 for 30-120 minutes, add 1M sodium fumarate and heat at 50℃.
The activity was measured by reacting for 10 minutes. As shown in Figure 8, it is 100% stable up to 60°C. (6) Inhibition, activation, and stabilization This enzyme shows some activation by divalent metal ions such as Co ⁺⁺ , Zn ⁺⁺ , Mg ⁺⁺ , Mo ⁺⁺ , etc. A stabilizing effect is recognized in the presence of the substrate fumarate. Cu ⁺⁺ and O-phenanthroline, α, α′-
Metal chelating reagents such as dipyridyl exhibit some inhibitory effects. (7) Molecular weight As a result of gel filtration using Cephadex G-200 using cytochrome C, ovalbumin, and γ-globulin as internal standard proteins, the molecular weight was approximately 180,000. By allowing the present enzyme, bacterial cells containing the present enzyme, or processed products of the bacterial cells to act on fumaric acid, L.
- Can produce malic acid. The reaction is usually
Perform at 40-80℃ and pH6.0-8.0. The appropriate fumaric acid concentration during the reaction is about 0.2 to 1.7 mol.
Further, the appropriate enzyme concentration is about 5 to 50 units/ml. During the reaction, fumaric acid is usually used as a salt. Examples of the salts used include sodium salts, potassium salts, ammonium salts, magnesium salts, and zinc salts. When using conventional fumarase, the use was limited to monovalent metal salts due to solubility, but the use of the heat-stable fumarase of the present invention makes it possible to use divalent metal salts, and L-apple The conversion rate to acid was significantly improved. During the reaction, L-malic acid can be produced more practically by using immobilized enzymes, bacterial cells, and processed bacterial cells. These immobilizations can be carried out by general methods used for immobilizing enzymes, bacterial cells, etc. For example, the enzyme solution may be brought into contact with a general anion exchange resin that can adsorb fumarase, and for the immobilization of bacterial cells, ethyl cellulose,
Microencapsulation with cellulose acetate/butyrate, etc., gel entrapment with acrylamide monomer, entrapment with polyvinyl alcohol, membrane entrapment with collagen, etc. are used. Alternatively, there are methods in which bacterial cells are simply mixed with a filling mixture and treated with a crosslinking agent, etc., such as gelatin/glutaraldehyde method, chitosan/glutaraldehyde method, carrageenan/glutaraldehyde method, etc.
Glutaraldehyde method, albumin/dialdehyde starch method, etc. can be used. L-malic acid can be collected from the reaction solution by treating the reaction solution with an ion exchange resin and activated carbon in a conventional manner, followed by concentration and crystallization. Example 1 Polypeptone 0.8g/dl, yeast extract 0.4
g/dl, with a composition of sodium chloride 0.2g/dl
In a 300ml volumetric flask containing 30ml of medium adjusted to PH7.0, add Thermus Aquatakes No. 104 bacteria, Thermus Lacteus Knob Sp. No. 108 bacteria, Thermus Rubens Knob Sp. No. 102 bacteria, Thermus Aqua. Takes ATCC25104,
ATCC25105, Thermus thermophilus
Inoculate ATCC27634 and culture with shaking at 50-80°C for 16 hours. Next, transfer the entire amount of this culture to a medium with the same composition.
Inoculate the cells into a 2-capacity flask containing 300 ml and culture with shaking at the same temperature for 26 hours. This culture solution was further inoculated into a 5-capacity jar fermentor containing medium 3 of the same composition, and the temperature was 50-80°C with an aeration rate of 0.8VV.
m., after 24 hours of culture with stirring at 300 r.pm.
The amounts of bacterial cells shown in the table were obtained. The obtained bacterial cells
The mixture was centrifuged at 10,000G, washed twice, and then freeze-dried. The dried bacterial cells were brought into contact with 1 mol of sodium fumarate at 50-80°C for 30
When the fumarase activity was measured from the L-malic acid produced by the reaction, the results were as shown in Table 2.

[Table] Example 2 Add 0.2 g/fumaric acid to the same medium as in Example 1.
dl was added and Thermus Rubens Knob Sp. No. 102 bacteria was cultured at 60°C for 24 hours. When the fumarase activity was measured, 900 units/g·cell of bacterial cells were obtained. The cells were freeze-dried, and the freeze-dried cells were used to create immobilized microorganisms. Dissolve 7.2 g of Emulgen-985 (manufactured by Kao Atlas Co., Ltd.) and 3 g of Celogen-PR (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd.) as dispersants in 600 ml of water as a dispersion medium, and heat at 5 to 10°C with strong stirring.
Leave to cool. On the other hand, 15g of freeze-dried bacterial cells is 0.9%
Uniformly suspend the cells in 50 ml of physiological saline and 15 ml of 1% chitosan water, which is a bacterial flocculant. A mixture of 15 g of cellulose acetate/butyrate 381-20 (manufactured by Eastman Chemical International Ltd.) and 3 g of Arlacel-83 (manufactured by Kao Atlas Co., Ltd.) as a dispersant was dissolved in 135 g of isobutyl acetate and stirred thoroughly to form a uniform mixture. Make it a W/O emulsion. This W/O emulsion of the bacterial cell suspension and cellulose acetate/butyrate is added dropwise to the previously cooled and stirred dispersion medium using a funnel, and after the dropwise addition, 1200 ml of n-hexane is gradually added using a metering pump. As a result, solid and uniformly sized immobilized microorganisms are precipitated into the liquid, with bacterial bodies encapsulated in cellulose acetate/butyrate. Take this with a cloth, wash it thoroughly, and use it for the reaction. Using the obtained immobilized microorganisms, 1M sodium fumarate was converted to L-malic acid by continuous column operation at 60°C and a flow rate of SV = 0.5, and the conversion rate was 75% or more. Production of malic acid was possible for more than one month. Furthermore, magnesium fumarate, which is difficult to use at room temperature, is available at a concentration of 1 molar.
It is soluble at 60°C, and when used, stable magnesium L-malate was obtained with a conversion rate of 85% or more. By using a magnesium salt that is difficult to use at room temperature, the reaction rate increased by more than 10%, the process for recovering unreacted fumaric acid was reduced, and the purification process was simplified. Example 3 Thermus Aquatakes No. 104 bacteria Example 1
When cultured at 70℃ for 36 hours in a medium supplemented with 0.2g/dl of ammonium fumarate.
3 g of bacterial cells having fumarase activity of 200 units/g·cell were obtained. The cells were thoroughly washed and freeze-dried, and the freeze-dried cells were sonicated at a concentration of 50 g/ml to prepare a crude enzyme solution. This crude enzyme solution was brought into contact with an adsorption carrier, which is an anion exchange resin, to prepare an immobilized enzyme. First, Duolite-A7 (manufactured by Diamond Shamlok Chemical Co., USA) is thoroughly washed with resin water and then buffered with 0.1M phosphate buffer (PH6.0). The crude enzyme solution prepared above is loaded at an activity level of 100 to 200 units/ml of the resin at 65 to 75°C, and the mixture is adsorbed and immobilized for 16 hours. Protein adsorption amount is 90
% or more. Later as an enzyme stabilizer
0.1% sodium fumarate, 0.2% as a drafting agent
Glutaraldehyde dissolved in 0.1% phosphate buffer was reacted for 30 minutes, and thoroughly washed to obtain an immobilized enzyme. When 30 ml of this immobilized enzyme was packed into a 50 ml cone-type fluidized bed and 1 M sodium fumarate was converted to L-malic acid by continuous column operation, the SV = 0.4 at 70°C.
Continuous production of L-malic acid was possible with a conversion rate of 80% for 20 days. The reaction solution is completely clear,
There was no enzyme leakage, and because of the high temperature operation, very efficient production of L-malic acid was possible without bacterial contamination. Example 4 Thermus Lacteus Knob SP No.108
When the bacteria were cultured in the same medium as in Example 3 at 70°C for 24 hours, cells with fumarase activity of 300 units/g·cell were obtained. The obtained microbial cells were thoroughly mixed with a collagen fibril solution and casted on a Teflon sheet to obtain an immobilized microorganism. This immobilized microorganism was shredded into chips, packed into a column, and 1M magnesium fumarate was passed through it at SV = 0.5, resulting in 90%
Stable production of L-malic acid for more than 10 days was confirmed at the above conversion rate. High performance liquid chromatography Shodex Ionpakc
When the reaction solution was analyzed using -811 (manufactured by Showa Denko), only L-malic acid and fumaric acid were detected.
There were no other impurities. Example 5 Bacillus licheniformis T-511, Bacillus pumilus T-530, Bacillus subtilis ATCC6051 and Bacillus licheniformis activated in 1/2 concentration broth medium
ATCC11560 was inoculated into a 300 ml volumetric flask containing 30 ml of a medium with the composition of polypeptone 0.8 g/dl, yeast extract 0.4 g/dl, and salt 0.2 g/dl and adjusted to pH 7.0, and heated at the temperature shown in Table 3. Incubate for 24 hours. Next, the total amount of this culture was added to glucose 3g/dl, peptone 1g/dl, yeast extract 0.25g/dl, phosphate-potassium 0.1g/dl, dipotassium phosphate 0.1
g/dl, magnesium sulfate 0.1g/dl, iron sulfate
2 containing 300 ml of a medium adjusted to pH 7.2 with a composition of 0.002 g/dl, manganese sulfate 0.00005 g/dl, zinc sulfate 0.001 g/dl, and β-alanine 0.0005 g/dl.
The cells were transplanted into a flask with a full capacity and cultured for 24 hours at the temperatures shown in Table 3. As a result, the amount of bacterial cells shown in Table 3 was obtained. The obtained bacterial cells were centrifuged at 10,000 G, washed twice, and then freeze-dried. The dried bacterial cells were brought into contact with 1 mol of sodium fumarate at a concentration of 10 g/dl and reacted at 50 to 70°C for 30 minutes, and the fumarase activity was measured from the L-malic acid produced. The results are shown in Table 3.

[Table] Example 6 Glucose 3 of Bacillus pumilus T-530 bacteria
g/dl, sodium sulfate 0.2g/dl, potassium phosphate 0.1g/dl, dipotassium phosphate 0.3g/dl,
Ammonium chloride 0.5g/dl, magnesium sulfate
0.01g/dl, ammonium nitrate 0.1g/dl, calcium chloride 0.0001g/dl, Nitsche'strace
300 with a composition of element1ml/adjusted to PH7.2
30 ml of culture medium in a 2 volume flask
ml of preculture solution (medium was the same as above) was inoculated and cultured at 45°C for 32 hours. When fumarase activity was measured, it was 255 units/
G.cell microbial cells were obtained. The freeze-dried cells were sonicated at a concentration of 50 g/cell. The thus obtained crude enzyme solution was brought into contact with an adsorption carrier, which is an anion exchange resin, to prepare an immobilized enzyme. First, Duolite-A7 is thoroughly washed with resin water and then buffered with 0.1M phosphate buffer (PH6.0). Next, the crude enzyme prepared above is loaded with an activity value of 100 to 200 units/ml/resin at 65 to 75°C and adsorbed and immobilized for 16 hours. Protein adsorption amount is 90
% or more. Later, as an enzyme stabilizer
0.1% sodium fumarate, 0.2% as crosslinker
Glutaraldehyde dissolved in 0.1% phosphate buffer was reacted for 30 minutes, and thoroughly washed to obtain an immobilized enzyme. When this immobilized enzyme was packed into a jacketed column with a capacity of 10 ml and 1 M sodium fumarate was flowed at 60°C at a flow rate of SV = 0.1, L-malic acid could be continuously produced for 10 days with a conversion rate of about 70%. It was hot. Due to the high temperature operation, there was no bacterial contamination and L-malic acid was produced in a very stable manner. Example 7 When Bacillus licheniformis T-511 was cultured at 50° C. for 48 hours in the same synthetic medium as in Example 2, about 20 g of bacterial cells were obtained. The fumarase activity of this bacterial cell was 280 units/g·cell. Next, the obtained bacterial cells were thoroughly washed and freeze-dried, and then mixed with a collagen fibril solution and cast into a Teflon sheet to prepare collagen-encompassing bacterial cells. This collagen-encompassing microbial cell was shredded into chips and packed into a column, and 1M magnesium fumarate was passed through the column at a flow rate of SV = 0.05 and 50°C. L-malic acid was stable for 7 days with a conversion rate of about 85%. Generation confirmed. When the by-products were confirmed by high performance liquid chromatography, no organic acids other than fumaric acid and L-malic acid were detected. Example 8 Thermus Rubens Knob SP No.102
The bacteria are cultured in the same manner as in Example 1. The obtained bacterial cells (50 g/) are disrupted by ultrasonication. The lysate was passed through a column packed with DEAE/Sephadex G-25, eluted with a concentration gradient of 0 to 0.6M saline, and the fumarase activity fraction was collected.
This is brought to 70% saturation with ammonium sulfate, and the resulting precipitate is dialyzed against phosphate buffer (PH6.5). Using DEAE/cellulose, this dialysate was dialysed to pH7.5.
Then, the eluate is subjected to column chromatography using hydroxyapatite and freeze-dried to obtain fumarase powder. The specific activity per protein of the obtained fumarase was approximately 50 times that of the cell-free extract. The fumarase obtained here is approximately 5 mg/protein equivalent.
1M sodium fumarate at a concentration of 60 °C, PH
7.2 and after 20 hours of contact, L-malic acid was obtained with a conversion rate of about 80%. Example 9 Bacillus pumilus T-530 bacteria was cultured at 45°C for 24 hours in the same manner as in Example 1. The resulting bacterial cells are treated in the same manner as in Example 8 to obtain fumarase powder. The specific activity per protein of the obtained fumarase was about 60 times that of the cell-free extract. The fumarase obtained here was converted to protein equivalent to 10mg/
1M sodium fumarate at a concentration of 45 °C, PH7.5
When exposed for 20 hours, the conversion rate was approximately 78%.
Malic acid is obtained. Example 10 Thermus Aquatakes No. 104 bacteria Example 1
When cultured at 70°C for 36 hours in a medium containing 2 g of ammonium fumarate, 200 units/
A bacterial cell having the fumarase activity of gdrycell was obtained. After bacterial cell isolation, the bacterial cells were thoroughly washed and made into a freeze-dried product. This product was subjected to ultrasonication at a concentration of 50 g to prepare a crude enzyme solution. This was brought into contact with an adsorption carrier made of anion exchange resin to prepare an immobilized enzyme.
First, Duolite A-7 (manufactured by Diamond Shamlok Chemical Co., USA) is thoroughly washed with resin water and then buffered with 0.1M phosphate buffer (PH6.0). Add to this the crude enzyme solution prepared above at a fumarase activity value of 65 at a ratio of 100 to 200 units/ml resin.
Load at ~75°C and allow adsorption immobilization for 16 hours. The amount of protein adsorbed was over 90%. Thereafter, 1 g of sodium fumarate as a fumarase stabilizer and 2 g of glutaraldehyde as a crosslinking agent dissolved in a 0.1% phosphate buffer were reacted for 30 minutes and thoroughly washed to obtain a sample. This immobilized enzyme was placed in a cone-shaped fluidized bed with a volume of 50ml.
When 30 ml of the flask was filled and 1 mol magnesium fumarate was continuously passed through the flask, magnesium L-malate was stably obtained at SV=0.4 and a conversion rate of 85% or more at 70°C. The reaction solution was completely clear and there was no enzyme leakage, and since the reaction was carried out at high temperatures, it was possible to efficiently produce L-malic acid without any bacterial contamination. Example 11 Thermus obtained in Example 2. Rubens. When 1 mol ammonium fumarate was applied to the freeze-dried product of Nobu SP at 60°C for 5 hours, the freeze-dried product had almost no aspartase activity.
Ammonium L-malate was obtained with a conversion of 84%.

[Brief explanation of the drawing]

Figures 1 to 4 respectively show the optimum pH, stable pH range, optimal temperature range for action, and temperature stability of this enzyme produced by Thermus genus bacteria. Figures 5 to 8 show the optimum pH, stable pH range, optimal temperature range for action, and temperature stability of the present enzyme produced by Bacillus bacteria, respectively.

Claims (1)

[Claims] 1 Belongs to the genus Thermus and has an optimal temperature of 50 to 80℃.
A method for producing fumarase, which comprises culturing a microorganism capable of producing fumarase in a nutrient medium, producing and accumulating the fumarase in the culture, and collecting the fumarase from the culture.