JP2759209B2 - Recombinant DNA containing xylanase gene - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a recombinant DNA in which a DNA carrying the genetic information of a heat-resistant and alkali-resistant xylanase derived from a thermotolerant anaerobic bacterium is incorporated into a vector, and the recombinant DNA is introduced. Escherichia coli.

Xylanase is agricultural waste (straw, corn stalk, etc.)
Is an enzyme that degrades xylan, which is the main component of hemicellulose. Xylose, a constituent unit of xylan, has not been developed as a very effective surface, but xylose isomerase can convert xylose to xylulose, and it is clear that yeast can utilize it and convert it to ethanol. Its use has been attracting attention since.

[Conventional technology]

Xylanase is an important enzyme for the above uses, but many enzymes are weak to heat and alkali at present, and it is difficult to react at high temperatures. Therefore, a heat-resistant and alkali-resistant xylanase is required. If such an enzyme is used, treatment at a high temperature becomes possible, and not only the reaction time is shortened, but also the risk of contamination by various bacteria is reduced.

At present, xylanases such as those of the genera Bacteroides, Cryptococcus and Streptomyces are known, and the gene for Bacillus has also been cloned [Molecular and General Genetics (Mol. Gen. Genet.) 192, 335
(1983)], there is no report that a thermostable and alkali-resistant xylanase gene derived from anaerobic bacteria was cloned.

[Problems to be solved by the invention]

The present inventors have searched for a xylanase-producing bacterium having heat resistance and alkali resistance, and as a result of diligent research, have found a xylanase-producing strain having excellent properties in heat resistance and alkali resistance. This strain is an anaerobic bacterium, and the produced enzyme has excellent properties.However, since the production involves various problems such as culture control, the present inventors first increase the xylanase productivity. For this purpose, a DNA fragment carrying the xylanase gene of a thermotolerant anaerobic bacterium was extracted, and then this DNA fragment was inserted into a recombinant D vector.
A new Escherichia coli transfected with NA and the recombinant DNA was created. That is, the present invention relates to a thermotolerant anaerobic bacterium, more specifically, Clostridium sp.
-1 (This strain was deposited with the National Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology, and its deposit number is No.
A DNA fragment having the genetic information of a heat- and alkali-resistant xylanase derived from X-1 strain, and characterized by a restriction enzyme cleavage map as shown in FIG. E. coli.

[Means for solving the problem]

The HX-1 strain was newly isolated from the natural world by the present inventors, and is a GS medium containing 1% xylan and 1.5% agar (monopotassium phosphate 0.15%, dipotassium phosphate 0.29%,
Sodium sulfate 0.13%, yeast extract 0.45%, L-cysteine hydrochloride 0.1%, MOPS (morpholino-propanesulfonic acid) 2.09%, magnesium chloride 1%, calcium chloride 0.
Xylanase is produced by culturing 15%, ferrous sulfate 0.00125% and resazurin sodium salt 0.0002%). The morphological properties of the HX-1 strain are as follows.

Shape: Bacillus Size: 0.5 × 4.0 μm Spores: Yes Motility: Yes Gram stain: negative From the results of our studies, the HX-1 strain is considered to belong to the genus Clostridium.

The chromosomal DNA carrying the gene information of the heat- and alkali-resistant xylanase can be separated and purified from the HX-1 strain by a phenol method or the like.

After culturing and collecting the strain, lysozyme treatment, SDS treatment, and protease treatment are performed, and the phenol treatment is repeated. Thereafter, a filamentous DNA mass is removed by an ethanol precipitation method. After further ribonuclease treatment, the chromosomal DNA is taken out again by ethanol precipitation. The chromosomal DNA is completely digested with a restriction enzyme, and then ligated to a vector plasmid DNA (eg, pUC9) digested with a restriction enzyme (eg, BamHI) by the action of T4 DNA ligase.

Hereinafter, the present invention will be described using BamH I and Sau3A I as restriction enzymes and pUC9 as a vector plasmid DNA as an example. The recombinant DNA obtained by the above method is cultured in Escherichia coli having no xylanase activity, then collected, treated with calcium chloride, and incorporated.

To select Escherichia coli carrying a plasmid DNA incorporating a DNA fragment carrying the genetic information of the heat- and alkali-resistant xylanase of the HX-1 strain, the ampicillin resistance must be maintained,
Select strains that have lost β-galactosidase activity.
That is, ampicillin and 5-bromo-4-chloro-
Culture was performed in a medium containing 1.5% agar in a medium containing 1.0% of polypeptone, 0.5% of yeast extract, and 0.5% of sodium chloride (hereinafter referred to as L medium) containing 3-indolyl-β-D-galactoside. All of the strains that form white colonies that grow are inoculated on an L medium (containing 1.5% agar) containing ampicillin and xylan, and colonies that form a transparent ring are selected. For the final confirmation, this strain is cultured in L medium to extract the enzyme, and its xylanase activity is measured.

Next, the recombinant DNA is isolated from the recombinant plasmid-holding strain obtained by the above method by the following method.

After culturing, the cells are collected, treated with lysozyme, treated with SDS-NaOH, and treated with potassium acetate, and isopropanol is added to the supernatant obtained by centrifugation to extract a plasmid. Then, ribonuclease treatment, phenol treatment, chloroform / isoamyl alcohol treatment are performed,
After removing the protein, ethanol precipitation is performed to obtain a plasmid. The recombinant DNA thus obtained can be introduced again into E. coli.

Escherichia coli carrying the recombinant plasmid obtained as described above is cultured by a conventional method, and crushed with a sonicator or the like, whereby a crude enzyme solution of a heat- and alkali-resistant xylanase can be obtained.

 Hereinafter, the present invention will be further clarified by examples.

Example (1) Extraction of Chromosomal DNA Carrying Thermostable Xylanase Gene The HX-1 strain is cultured in a GS medium containing 0.3% glucose, and collected near the end of the logarithmic growth phase. still,
The cultivation is performed using a glove box (anaerobic cultivation device EAN-140, manufactured by Tabai Espec) to mix the gas phase with a mixed gas (N ₂ : H).
₂ : CO ₂ = 90: 5: 5), and a pressure-sterilized medium was used.

50 mM of pH 8.0 Tris / HCl buffer (50 mM
M EDTA), and the mixture was suspended. A lysozyme solution was added, followed by treatment at 37 ° C. for 3 minutes. Then, 50 mM pH 7.5 Tris / HCl buffer (0.4% containing 1% sodium lauryl sulfate) was added.
M EDTA, containing 1 mg / ml proteinase K)
Treat at 0 ° C for 1 hour. Further, the phenol treatment is repeated three times, and the mixture is centrifuged at 2,000 × g for 15 minutes to obtain a supernatant. 3 M sodium acetate and ethanol are added to the obtained supernatant, and the glass rod is wound up. Further, 50 mM pH 7.5 Tris / hydrochloric acid buffer (containing 1 mM EDTA) is added, and a 200 μg / ml ribonuclease solution is allowed to act at 4 ° C. for 12 hours. After the action, the mixture is treated with chloroform and further centrifuged at 1,000 × g for 15 minutes to obtain a supernatant. To the obtained supernatant was added 3M sodium acetate and ethanol and wound up with a glass rod,
Obtain DNA. The obtained DNA was dissolved in 50 mM Tris-HCl buffer (pH 7.5, containing 1 mM EDTA) to obtain a chromosomal DNA solution. 594 μg of chromosomal DNA was obtained from 500 ml of the culture solution.

(2) Partial digestion of chromosomal DNA of HX-1 strain with restriction enzyme and treatment with ligase Plasmid pUC9 was used as a vector, 10 units of BamHI restriction enzyme was added to 50 μg of this plasmid, and 10 mM of pH7.
5 Tris-HCl buffer, 50 mM sodium chloride, 10 mM magnesium chloride and 1 mM dithiothreitol solution 6
Degraded in 0μ at 37 ° C for 12 hours. After the reaction, phenol / chloroform treatment is performed once, and the plasmid is precipitated by ethanol precipitation. 1M pH 8.3 was added to the obtained plasmid.
One unit of alkaline phosphatase was added to 15 μl of Tris / hydrochloric acid buffer, treated at 60 ° C. for 1 hour, and the reaction was stopped with a solution (pH 7.5) containing 100 mM EDTA.
Perform chloroform treatment twice and centrifuge at 12,000 xg for 5 minutes. The obtained supernatant was further treated twice with chloroform, centrifuged under the same conditions as above, and the completely degraded plasmid pUC9 end-treated by ethanol precipitation from the supernatant.
Get.

Using the chromosomal DNA obtained in (1),
g of 10 g of Sau3A I restriction enzyme, and added to a solution of 6 mM pH 7.5 Tris / HCl buffer, 50 mM sodium chloride, 5 mM magnesium chloride and 1 mM dithiothreitol in 4.5 ml of a solution.
Decomposed for 1 hour at 37 ° C. After the reaction, phenol / chloroform treatment is performed once, and DNA is precipitated by ethanol precipitation. The obtained DNA is treated with phenol / chloroform for 2 hours.
Perform centrifugation at 12,000 xg for 5 minutes. The obtained supernatant was further treated twice with chloroform, centrifuged under the same conditions as above, and HX-precipitated from the supernatant by ethanol precipitation.
One strain of Sau3A I restriction enzyme digest was recovered and dried under reduced pressure.
Dissolved in 50μ of water.

4μ end-treated, completely digested plasmid pUC9 and 10μ
Was mixed with a buffer solution (660 mM pH 7.6 Tris / HCl buffer containing 66 mM magnesium chloride, 660 μM ATP and 100 mM dithiothreitol) mixed with 5 μl of the Sau3A I restriction enzyme digest of the partially decomposed HX-1 strain. And after adding 30μ of water, 10
The mixture was reacted with 00 units of T4 DNA ligase at 16 ° C for 20 hours to obtain a recombinant plasmid in which the vector and the chromosomal DNA were ligated.

(3) Transformation of Escherichia coli using recombinant plasmid Escherichia coli JM103
0 ml SOB medium (2% bactotryptone, 0.5% yeast extract)
%, Sodium chloride 10 mM, potassium chloride 2.5 mM, magnesium chloride 10 mM and magnesium sulfate 10 mM
~ 7.0 medium), and after culturing with shaking at 37 ° C for 70 minutes, the cells are collected. Bacteria cells in 100 mM cold calcium chloride solution 200
After suspending in ice and leaving it on ice for 20 minutes, the cells are collected by centrifugation. Further
After suspending in 5 ml of a 100 mM cold calcium chloride solution and allowing to stand on ice for 24 hours, 10 μm of the recombinant plasmid is added to 0.2 ml of the suspension and suspended in ice for 60 minutes. Then heat-treat at 42 ° C. for 2 minutes and cool immediately. Thereafter, 1 ml of L medium was added and 37
Incubate with shaking at 40 ° C for 40 minutes. This was added to an L medium containing 50 μg / ml of ampicillin and 50 μg / ml of 5-bromo-4-chloro-3-indolyl-β-D-galactoside (agar 1.
5%), and all of the white colony strains (3500 strains) were treated with 50 μg / ml ampicillin and 1
L medium containing 1.5% xylan (containing 1.5% agar) and 50μg
medium containing 1.5% / ml ampicillin (containing 1.5% agar)
And incubate at 37 ° C for 12 hours. Overlay the former medium with 10ml of soft agar (containing 0.5% agar, 1.5mg / ml lysozyme and 10mM Tris / HCl buffer) and leave at 37 ° C for 12 hours Then, the cells were destroyed, followed by a xylanase reaction at 60 ° C. for 8 hours to select a strain (1 strain) which formed a transparent ring.

(4) Xylanase activity produced by Escherichia coli carrying the recombinant plasmid The transformed Escherichia coli selected in (3) was cultured in an L medium containing 70 μg / ml ampicillin (37 ° C., 17 hours), and collected. Suspend in 50 mM MOPS buffer (pH 6.5) containing 50 mM calcium chloride and centrifuge. After suspending in the above buffer, the cells were disrupted with a sonicator, and centrifuged.
A crude enzyme solution was obtained, salted out with ammonium sulfate, and dialyzed to obtain an enzyme solution.
The xylanase activity was measured as follows using xylan as a substrate. 0.1 ml in 0.2 ml of xylan solution (dissolve xylan in 0.1 M succinate buffer at pH 5.9 to 1%)
Add enzyme solution and react at 60 ° C for 10 minutes.
Measured using the DNS method. The amount of the enzyme that released 1 μmole xylose per minute was defined as one unit. The amount of enzyme produced by Escherichia coli carrying the recombinant plasmid was about 110 units per 1000 ml of culture solution.

(5) Preparation of Restriction Enzyme Map of Recombinant Plasmid The transformant obtained in (3) was cultured with shaking in 2000 ml of L medium containing 100 μg / ml ampicillin at 37 ° C. for 14 hours. After completion of the culture, the cells are collected by centrifugation (1,500 × g, 10 minutes). Further, a lysozyme solution (50 mM glucose, 10 mM E
25 mM Tris containing DTA and 5 mg / ml lysozyme
Hydrochloric acid buffer (pH 8.0)], and treated in ice for 5 minutes. An SDS-NaOH solution (0.2% sodium hydroxide solution containing 1% SDS) was added and the mixture was similarly treated in ice for 5 minutes. Process,
Further, a potassium acetate solution [a mixed solution (pH 4.8) of 60 ml of a 5M potassium acetate solution, 28.5 ml of glacial acetic acid and 11.5 ml of water (pH 4.8)] is added, frozen in a dry ice / ethanol refrigerant, and then thawed at room temperature. . Centrifuge this (12,000 xg, 15 minutes)
And collect the supernatant. Further, isopropanol is added to the collected supernatant, frozen in a refrigerant of dry ice / ethanol, and then thawed at room temperature. Further, this is centrifuged (12,0
(00 × g, 5 minutes) to extract the plasmid. This is washed and dried with 70% ethanol, and further diluted with 10 mM pH 8.0 Tris.
Hydrochloric acid buffer (containing 1 mM EDTA) (hereinafter referred to as TE buffer)
Is added and suspended, and the ribonuclease solution is allowed to act at room temperature for 15 minutes. After the action, the mixture is treated with an equal volume mixture of phenol and chloroform: isoamyl alcohol = 24: 1, and centrifuged at 12,000 × g for 5 minutes to obtain a supernatant. The obtained supernatant is further treated with a mixed solution of chloroform: isoamyl alcohol = 24: 1, and centrifuged at 12,000 × g for 5 minutes to obtain a supernatant. Then 3M sodium acetate and 95%
12,000 xg after freezing and thawing by adding ethanol
For 5 minutes to obtain a precipitate. The obtained precipitate is further washed with 70% ethanol, centrifuged, dissolved in a TE buffer, and subjected to ethidium bromide-cesium chloride equilibrium density gradient centrifugation to obtain a plasmid. The resulting plasmid
Apa I, BamH I, Bal II, EcoR I, EcoR V, Hind III, Kpn I, P
It was cut with restriction enzymes such as stI, PvuII, SalI, SacI, SwaI, XbaI, and XhoI to prepare a restriction enzyme cleavage map. The result is shown in FIG. When this plasmid was introduced into another Escherichia coli JM103 strain without the xylanase gene, all the transformants that became ampicillin-resistant produced a thermotolerant and alkali-tolerant xylanase. It was confirmed that the DNA fragment responsible for the DNA was incorporated. REFERENCE EXAMPLE 1 Enzyme chemical properties of xylanase derived from HX-1 strain and recombinant A. Optimal temperature The activity of a chylianase enzyme solution obtained from HX-1 strain and recombinant was measured at 40 ° C. to 100 ° C. When the temperatures were determined, they were all about 70 ° C. as shown in FIG.

B. Thermostable xylanase obtained from HX-1 strain and recombinant
It was left in a 0 mM pH 5.9 succinate buffer at 50 ° C. to 100 ° C. for 20 minutes to measure its stability. The result is shown in FIG.
As a result, the HX-1 strain was slightly more stable at higher temperatures.

C. Optimum pH HX was determined using a universal buffer (Brighton-Robinson buffer, pH 3.0 to pH 11.0) instead of the succinate buffer in the xylanase activity measurement method described in (1) of Example 1.
-1 Optimal pH of xylanase obtained from strains and recombinants
Were determined, and as shown in FIG.
Met.

D. Effects of various metal salts Various metal salts were added to a concentration of 1 mM, and the effects were examined. Table 1 shows the results.

As described above, this enzyme is K ⁺ , Fe ²⁺ , Mg ²⁺ , Ca ²⁺ , Fe ³⁺ , Al ³⁺
Was little affected, but was somewhat inhibited by Cu ²⁺ and inhibited by Hg ²⁺ .

Enzyme chemical properties (optimal temperature, thermal stability, optimal pH, effects of metal salts, etc.) on Clostridium sp. HX
-1 and the enzyme obtained from the recombinant showed the same properties.

Reference Example 2 pH Stability of Enzyme Obtained from Recombinant Enzyme activity when treated at 4 ° C. for 24 hours and at 60 ° C. for 3 hours was measured and is shown in FIG. The enzyme produced from the recombinant is heat-resistant and alkali-resistant.

〔The invention's effect〕

According to the present invention, xylanase of a thermotolerant anaerobic bacterium which has conventionally had complicated culture conditions can be produced using Escherichia coli which is easy to culture. Further, it has become possible to produce an enzyme excellent in heat resistance and alkali resistance.

[Brief description of the drawings]

Fig. 1 carries the genetic information of thermostable and alkali-tolerant xylanase obtained from Clostridium sp. Strain HX-1.
It is a DNA fragment and is 5.6 Kb in length. FIG. 2 is a restriction map of a recombinant Escherichia coli vector in which a DNA fragment carrying genetic information of heat- and alkali-resistant xylanase has been incorporated. FIG. 3 shows the optimum temperature of xylanase,
−- indicates the HX-1 strain, and-●-indicates the recombinant. FIG. 4 shows the thermostability of xylanase;
−- indicates the HX-1 strain and-● -recombinant. FIG. 5 shows the optimum pH of xylanase,
-Indicates the HX-1 strain, and-●-indicates the recombinant. FIG. 6 shows the thermostability of the recombinant xylanase at various pHs,-○-indicates 4 ° C. for 24 hours,
-●-indicates 60 ° C for 3 hours.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:19)

Claims (2)

(57) [Claims] 1. A heat- and alkali-resistant xylanase gene contained in a 5.6 kbp gene fragment having the following restriction map obtained by treating a chromosomal DNA derived from a heat- and alkali-resistant Clostridium bacterium with Sau3A I: Recombinant DNA that can be replicated by Escherichia coli in which E. coli is integrated into an Escherichia coli-based vector. 2. The recombinant DN according to claim 1, wherein the chromosome derived from a heat- and alkali-resistant Clostridium bacterium is a chromosome derived from Clostridium sp. HX-1.
A.