JPH0565157B2 - - Google Patents

Description

[Detailed Description of the Invention] Industrial Application Field This invention relates to pullulanases with good heat resistance (hereinafter referred to as
a method for producing thermostable pullulanase);
The present invention relates to a plasmid containing such an enzyme gene and a Bacillus subtilis containing the plasmid. Prior art and problems to be solved by the present invention Pullulanase is α-1.6 in pullulan and starch.
It is an enzyme that breaks down bonds, and is often used to determine the structure of branched oligosaccharides and polysaccharides. Furthermore, if pullulanase is used together with glucoamylase in the step of producing glucose in the process of producing isomerized sugar from starch, it is advantageous to improve the yield of glucose. Since the operating conditions are 60°C to 65°C, thermostable pullulanase is an enzyme that has the thermostability to withstand this temperature.
Previously, there were few pullulanases that showed heat resistance at this temperature, and Bacillus stearothermophilus
In the case of strain TRS128, the enzyme is stable, but the production amount is low. For this reason, if it is concentrated to make an industrial enzyme, the side reactions caused by the attached enzymes will be troublesome, making it impractical. In view of the current situation, the present invention has made it possible to easily prepare heat-stable pullulanase with high purity by incorporating the gene for heat-stable pullulanase into Bacillus subtilis, which is a normal temperature bacterium, by genetic engineering techniques. An example of cloned genes for thermostable pullulanase or related enzymes is Thermoanaerobium brockii.
debranching enzyme (debranching)
Cloning of enzyme) genes in Escherichia coli and Bacillus subtilis (Journal of Bacteriology, Vol. 169, No. 4302-)
4307, 1987) and cloning of pullulanase of Thermus sp. in Escherichia coli (FEMS Microbiolgy Letters).
Letters, Volume 49, Page 385388, 1988). On the other hand, the feature of the present invention is that Bacillus subtilis, which is considered to be highly safe, is used as a host, and B.
B. subtilis is mycologically similar.
The gene was obtained from stearothermophilus.
That is, the enzyme preparation prepared according to the present invention is safer than those hitherto known, and can be used in food processing. Means and Action for Solving the Problems Bacillus stearothermophilus TRS128, which produces the thermostable pullulanase of the present invention, is a thermophilic bacterium isolated from soil in Joto Ward, Osaka City by the present inventors. It has unique mycological properties. 1. Morphological properties (in broth agar medium) 1 Cell shape and size...(0.6-0.8) x (1.5-
3.0) μ bacilli. There is no linkage and there are few capsules. The sweetened meat juice agar medium shows the same rod morphology as the meat juice agar medium. 2 Motility... Yes. 3. Spores: (0.4-0.5) x (0.5-0.6) μ oval shape, located near the end, and has a thin membrane wall. 60℃・16
Many things are formed over time. Swelling of the sporangium is observed. 4 Gram staining...Positive. 2. Growth status 1 Meat juice agar plate culture...good growth. The surface is slightly dry and almost white. 2 Meat juice agar slant culture...The result is a spread-like pattern with no glossy surface. 3 Meat juice liquid culture...good growth. The liquid becomes cloudy. 4 Gelatin puncture culture... liquefy gelatin. 5 Culture in saline broth...No growth in saline solution concentration of 7%. 6 Glucose-asparagine agar medium...poor growth. 7 Glucose nitrate agar medium...slight growth. 8 Meat juice liquid culture...Grows at PH6.8, does not grow at PH5.7. 3. Physiological properties 1 Nitrate reduction...Positive. 2 Starch hydrolysis...Positive. 3 Fermentability of citric acid...Positive. 4 Catalase production...Positive. 5 Growth range: Grows up to 70℃ in a medium containing tryptone, yeast extract, and salt. The optimal growth temperature is
65℃. 6 Fermentation of sugars...Acid is formed from glucose, lactose, and sucrose. However, it does not generate gas. 7 Production of acetylmethylcarbinol...Positive. 8 Decomposition of gelatin...Positive. The above results were summarized in Bergey's Manual of Systematic Bacteriology, Volume 2.
Bacteriology Vol. 2 (1986)), this bacterium is Bacillus stearothermophilus.
stearothermophilus). Next, the enzyme produced by this strain will be explained in detail. 1. Action and Substrate Specificity This enzyme cleaves the α-1.6 glucoside bond of pullulan and ultimately produces maltotriose. Although no increase in iodine color reaction is observed when acting on amylopectin or glycogen, maltose production increases when used in combination with β-amylase. It has no effect on isomaltose, panose, and isomaltotriose. From these results, it appears that pullulan acts on amylopectin and glycogen, which have shortened side chains, and
It is thought to be a pullulanase that cleaves the α-1.6 glucosidic bond. (ii) Action PH PH4.5-8.0 (0.05M sodium acetate-acetate buffer (PH4-6) or disodium phosphate-phosphate 1
React at 60°C for 15 minutes in 0.5% pullulan with potassium buffer (PH6-8). The reducing sugar produced is DNS
Measured by method. 3. Optimal pH for action: Approximately PH5.5 under the conditions of 2 above. 4. Temperature of action When reacted for 15 minutes at various temperatures in 0.1M phosphate buffer (PH6.0) and 0.5% pullulan, the reaction reached 75°C. (5) Optimum operating temperature: 65°C under the conditions of (4) above. 6. Thermal stability When the residual activity was measured by heating in 0.2M acetate buffer (PH6.0) at various temperatures for 60 minutes, more than 90% of the activity remained even at 6.5°C. 7. PH stability: Stable at approximately pH 6.0 to 9.0, relatively stable on the alkaline side than on the acidic side (at 60°C and 60°C using various buffer solutions)
After standing for a minute, the pH was adjusted to 7.0 and the residual activity was measured). 8. Purification method After ammonium sulfate fractionation (35-55% saturation) of the culture supernatant, dissolve the precipitate in a small amount of water. After dialysis against pure water, an enzyme showing a single band on SDS-PAGE was obtained by DEAE-cellulose, gel filtration, and affinity chromatography. 9. Molecular weight 83000 (according to SDS-PAGE). 10. Products Maltotriose was ultimately obtained from pullulan. 11. Potency measurement method The titer was measured using the following method and conditions using pullulan as a substrate. 1% pullulan (0.2M acetate buffer PH5.6) 0.25ml
Add 0.25ml of enzyme solution to and react at 60℃ for 15 minutes.
The resulting increase in reducing power was quantified by the DNS method. The amount of enzyme that produced a reducing power equivalent to 1 μmol of maltotriose per minute in the above reaction was defined as 1 unit. Based on the above physicochemical properties, this enzyme is considered to be a novel pullulanase with previously unknown heat resistance. In the present invention, the gene for such a highly thermostable pullulanase was extracted from Bacillus stearothermophilus.
Especially Bacillus stearothermophilus TRS128
It is prepared by cleaving the chromosomal DNA of using the restriction enzyme Hind. To use Hind,
This is because a gene fragment can be obtained that effectively retains the molecular weight of the enzyme and the genetic information corresponding to the carrier protein necessary for secreting the produced protein to the outside of the cell. The method of cutting is a conventional method. The cut chromosomal DNA fragments are converted into vector plasmids such as pTB522 (Journal of General Microbiology, Vol. 131) using the shotgun method.
1757, (1985)) to create a recombinant plasmid that can maintain replication using Bacillus subtilis as a host. The Hind DNA fragment in the target plasmid, which has the ability to produce heat-stable pullulanase, has a molecular weight of approximately 2.7 MDa, and the cleavage site for various restriction enzymes and the molecular weight of the DNA fragment produced by the cleavage are (a) BamHI I (BamHI ), 2 locations with fragments of 0.40, 0.85, and 1.45 MDa, respectively (b) EcoRI, 2 locations, with fragments of 0.20, 1.10, and 1.40 MDa, respectively (c) 1 location, Pvu, with fragments of 0.90 MDa each , 1.80MDa. More specifically, the DNA fragment was integrated into the above-mentioned pTB522, and the present inventors constructed pTP1.
The recombinant plasmid named
cleavage sites for various restriction enzymes, etc.). The restriction enzyme map of this plasmid pTP1 is shown in the attached Figure 1. In the present invention, pTB522 was used as a vector, but commercially available vectors pUB110, J.
Described in Bact.1981Vol.146p1091
Described in pTB53, J.Bact.1982Vol.149p824
A plasmid named pTB90 can also be used similarly. The plasmid used in the present invention is
It is not limited to pTB522. The second invention of the present application (the item described in the claims is referred to as the second invention, and the item described therein is referred to as the third invention. The same applies hereinafter) is a novel microorganism transformed with the above-mentioned recombinant plasmid. . in this case,
As the host Bacillus subtilis, any strain of normal Bacillus subtilis that does not have pullulanase-producing ability can be used, but preferably Bacillus subtilis NA-1, which is an amylase- and neutral protease-deficient strain.
(Journal of Bacteriology No. 170
Vol., p. 1554, 1988) is preferred because it greatly facilitates the production and purification of thermostable pullulanase. B.subtilisNA-1 is a commonly available B.subtilisNA-1.
This strain originates from the I. subtilis Marburg strain and has lost its amylase and protease activities through standard mutation treatment. The deletion of amylase was done to facilitate the determination when pullulanase was introduced.
The purpose of protease is to prevent the decomposition of accumulated pullulanase. B. subtilis NA-1 strain has amylase,
B. subtilis, except that it does not exhibit protease activity.
It retains the mycological properties of the Marburg strain. The third invention relates to a method for producing heat-resistant pullulanase using the Bacillus subtilis of the second invention. That is, inoculating the host bacteria of the second invention into a nutrient medium,
Thermostable pullulanase is produced and accumulated by a conventional method such as aeration and agitation culture at 37° C. for 24 to 72 hours at pH 6.5 to 7.3. Furthermore, thermostable pullulanase is recovered and purified from this culture using conventional methods such as ammonium sulfate fractionation, precipitation, alcohol precipitation, and membrane concentration. Examples Example 1 (Creation of recombinant plasmid, transformation of Bacillus subtilis, and selection of clone strains) First, Bacillus stearothermophilus
Chromosomal DNA of TRS128 is prepared as follows. Bacillus stearothermophilus TRS128
overnight in a 500 ml flask containing 100 ml L medium.
Solvent was carried out at °C. The bacteria were collected by centrifugation, washed with 20ml of TE buffer (10mM Tris, 1mM EDTA, PH8.5), and 3ml of 15% sucrose-50mM Tris (PH8.5).
8.5) It was suspended in -50M EDTA-1 mg/ml lysozyme and left standing on ice for 30 minutes. Add to this 3 ml of 1% Sarkosyl-50mM Tris (PH8.5)-50mM.
Added EDTA and mixed. Next, add 5.4g CsCl and add 0.3ml ethidium bromide solution (10mg/ml).
was added and separated in an ultracentrifugal field using a RP65T rotor (manufactured by Hitachi, Ltd.). After the ultracentrifugation, the DNA section was extracted with a syringe needle and extracted three times with n-butanol to remove ethidium bromide. This DNA sample was mixed with 10mM Tris (PH7.5) - 0.1mM
Dialyzed in EDTA and stored at 4°C. Then, a recombinant plasmid is prepared by treating with restriction enzymes and ligase as follows. That is, on the one hand, a vector plasmid pTB522 (tetracycline resistant) that can be replicated and maintained using Bacillus subtilis as a host was prepared by a conventional method, and then the chromosomal DNA of Bacillus stearothermophilus TRS128 and pTB522 were prepared by a conventional method.
is cut with the restriction enzyme Hind. The reaction conditions are as follows. (A) TRS128DNA 30 μl 10× Hind buffer 10 μl Bovine serum albumin (5 mg/ml) 2 μl Water 56 μl Hind solution 2 μl (B) pTB522 10 μl 10× Hind buffer 10 μl Bovine serum albumin (5 mg/ml) 2 μl Water 76 μl Hind solution 2 μl In the table, 10× Hind buffer means 100mM Tris (PH7.4), 100mMgCl ₂ , 500mMNaCl, 10mM
Contains dithiothreitol. React with the above solution at 37°C for 1.5 to 2 hours. Mix the two types of DNA obtained above, A and B, and use 3M
Add 20 μl of potassium acetate solution, then ethanol
0.45 ml was added, centrifuged, and the supernatant was removed. The precipitate was washed with ethanol, dehydrated and desalted, and dried in a desiccator. Add 2x ligase buffer (132mM Tris (PH7.6) - 13.2mM MgCl ₂ ) to this dried product.
100μl, 100mM dithiostreitol 20μl,
After adding and mixing 20μl of 5mM ATP and 60μl of water,
Add 1μl of DNA ligase and keep at 8℃ for 16 hours.
It was made into a DNA solution. The third step is the step of introducing the above-mentioned recombinant plasmid into Bacillus subtilis and cloning it. Bacillus subtilis NA-1 is one of the amylase/neutral protease-deficient mutant strains obtained by NTG mutation treatment of ordinary pullulanase-nonproducing Bacillus subtilis. Inoculate 1 ml into 20 ml of TFI medium in a 100 ml flask. This was cultured at 37°C, and one hour after the logarithmic growth phase had passed, 4 ml was inoculated into 36 ml of TF medium in a 500 ml flask, and cultured for 1.5 hours until it became competent.
Get a cell. The TFI and TF media are as follows.

【table】

【table】

[Table] Mix the DNA solution obtained above with 1 ml of these competent cells, culture with vigorous shaking at 37°C for 30 minutes, collect the cells by centrifugation, add 1 ml of L medium, and add 1 ml of L medium.
Incubate with shaking at ℃ for 1.5 hours. 100 μl of the culture solution was spread on agar plates containing 25 μl/ml of tetracycline and cultured overnight at 37° C. to obtain transformants. This transformant was plated on a PLL agar plate (1% pullulan, 0.2% tryptone, 0.2% yeast extract, 0.2%
% NaCl, 1.5% agar (PH7.3)), 37
After culturing overnight at °C, ethanol was poured onto the plate and left for several hours to obtain colonies forming a halo. The recombinant plasmid carried by this thermostable pullulanase clone was named pTP1. Example 2 (Production of thermostable pullulanase) Bacillus subtilis NA-1 retaining pTP1 obtained in Example 1 was placed in L medium containing 3% soluble starch (containing 25 μg/ml tetracycline)
The cells were cultured at 37°C for 24 hours. The culture solution was centrifuged, the supernatant was collected, and the supernatant was heated at 60°C for 15 minutes. At this stage, most of the proteins other than the heat-stable pullulanase were denatured and precipitated, and extremely pure heat-stable pullulanase was easily obtained. Effects of the Present Invention In the present invention, Bacillus subtilis, which is highly safe and has a system for secreting enzymes outside the bacterial body, is used as a host, so it has high application value from an industrial standpoint. Generally, bacterial strains screened from nature are
Compared to amylase and other carbohydrate-related enzymes, pullulanase is produced in lower amounts and is difficult to purify. Also,
Among pullulanases, those with high heat resistance exhibit this tendency even more strongly. In the present invention, Bacillus subtilis, which is a mesophilic bacterium and does not produce amylase, is cloned by incorporating a heat-resistant pullulanase gene, thereby making it easily heat-resistant by simply heating the culture supernatant at 60°C for 15 minutes. This method made it possible to purify commercial pullulanase. Incidentally, the cloning of the thermostable pullulanase gene has made it possible to mass-produce this enzyme by utilizing gene amplification effects, enhancement of promoter activity, etc.

[Brief explanation of the drawing]

Figure 1: Restriction enzyme map of recombinant plasmid tTP1 prepared using vector plasmid pTB522. The portion corresponding to the 2.7 MDa Hind fragment indicated by the thick line in the figure is a thermostable enzyme derived from the Bacillus stearothermophilus TRS128 chromosome. This is the part that contains the pullulanase gene. However, the numbers inside the circles represent the molecular size in MDa. E...EcoRI, Pv...Pva, H...Hind,
B...BamI, Ps...PstI.

Claims (1)

[Claims] 1. Chromosomal DNA of Bacillus stearothermophilus
heat-stable pullulanase-producing ability obtained by treating with the restriction enzyme Hind.
A recombinant plasmid characterized by ligating a DNA fragment and a vector fragment obtained by treatment with Hind. 2. The recombinant plasmid according to claim 1, wherein the Bacillus stearothermophilus is Bacillus stearothermophilus TRS128 (Feikoken Deposit No. 9610). 3 Hind Bacillus stearothermophilus or Bacillus stearothermophilus TRS128
The DNA fragment obtained by processing has a molecular weight of approx.
1. The recombinant plasmid according to claim 1, which has a molecular weight of 2.7 MDa and a cleavage map with various restriction enzymes as shown in FIG. 4 Chromosome of Bacillus stearothermophilus
1. A transformed Bacillus subtilis, which is a host replicating a plasmid containing a DNA fragment capable of producing heat-stable pullulanase obtained by treating DNA with the restriction enzyme Hind. 5. The transformed Bacillus subtilis according to claim 4, wherein the Bacillus stearothermophilus is Bacillus stearothermophilus TRS128. 6 Chromosome of Bacillus stearothermophilus
The method is characterized in that Bacillus subtilis carrying a replica of a plasmid containing a DNA fragment capable of producing heat-stable pullulanase obtained by treating DNA with the restriction enzyme Hind is cultured in a nutrient medium, and heat-stable pullulanase is recovered from the culture. A method for producing thermostable pullulanase using transformed Bacillus subtilis. 7. The method for producing heat-stable pullulanase using transformed Bacillus subtilis according to claim 6, wherein the Bacillus stearothermophilus is Bacillus stearothermophilus TRS128.