JP2582112B2 - Thermostable trehalase gene DNA, recombinant plasmid containing the DNA, transformant, and method for producing thermostable trehalase - Google Patents

Description

The present invention relates to a heat-resistant trehalase gene DNA, a recombinant plasmid obtained by ligating the DNA to a vector plasmid, a transformant into which the plasmid has been introduced, and a transformant It relates to a method for producing a thermostable trehalase.

[Conventional technology]

Trehalase is widely distributed in nature in higher animals, insects, microorganisms, and the like. In particular, microorganisms that produce trehalase include Aspergillus (KH)
orikoshi, J. Bacteriol, 1966, 91, 1883), genus Trichoderuma (P. Vijiyakuumar, Can. J. Microbil)
o., 1978,24,1280), Saccharomyce
s) The genus (A. Panek, J. Biol. Chem., 1964, 239, 1671) and the like are known. However, the production of these trehalases is small and unstable to heat, so purification requires a great deal of labor.

The present inventors have previously found that thermophilic microorganisms belonging to the genus Corynebacterium produce heat-stable trehalase in and out of the cells, and have filed a patent application for the thermostable trehalase (Japanese Patent Application Laid-Open No. 62-275682). The trehalase was heat-stable and produced outside the cells, which facilitated purification.

However, the thermostable trehalase is usually obtained by culturing a microorganism belonging to the genus Corynebacterium at a temperature of 50 to 60 ° C. However, it is energetically favorable to culture the microorganism at a lower temperature.

[Problems to be solved by the invention]

Therefore, an object of the present invention is to provide a method capable of obtaining the thermostable trehalase by culturing at a lower temperature.

The present inventors cultivated Escherichia coli obtained by introducing, via a vector, DNA corresponding to the gene information of a thermostable trehalase derived from a microorganism belonging to the genus Corynebacterium into Escherichia coli, which is a mesophilic bacterium. The heat-resistant trehalase was successfully obtained industrially from the obtained cells, and the present invention was completed.

[Means for solving the problem]

The present invention relates to the thermophilic bacterium Corynebacterium (Cory).
nebacterium sp.), two restriction enzyme Hind III recognition sites, one restriction enzyme EcoR V recognition site, two restriction enzyme BamHI recognition sites, one restriction enzyme Sma I recognition site, and one restriction enzyme. The present invention relates to a thermostable trehalase gene DNA having three Nru I recognition sites and one EcoR I recognition site.

Further, the present invention relates to a restriction enzyme HindIII derived from a thermophilic bacterium Corynebacterium sp.
Two recognition sites, one restriction site EcoR V recognition site,
Two restriction sites for BamHI restriction enzyme, one for SmaI restriction enzyme, three for NruI restriction enzyme and Eco
The present invention relates to a recombinant plasmid in which a thermostable trehalase gene DNA having one RI recognition site is linked to a vector plasmid.

Further, the present invention relates to a restriction enzyme HindIII derived from a thermophilic bacterium Corynebacterium sp.
Two recognition sites, one restriction site EcoR V recognition site,
Two restriction sites for BamHI restriction enzyme, one for SmaI restriction enzyme, three for NruI restriction enzyme and Eco
The present invention relates to a microorganism of the genus Escherichia transformed with a recombinant plasmid in which a heat-resistant trehalase gene DNA having one RI recognition site is linked to a vector plasmid.

 Hereinafter, the present invention will be described in detail.

DNA carrying the genetic information of thermostable trehalase (hereinafter referred to as chromosomal DNA) is isolated and purified from a microorganism belonging to the thermophilic Corynebacterium genus. Examples of microorganisms belonging to the thermophilic corynebacterium genus include K-502 (FERM).
P-8484), K-512 (FERM P-8485) and K-522 (FERM
MP-8486) can be used. Isolation and purification of a DNA carrying the genetic information of thermostable trehalase from a microorganism belonging to the genus Corynebacterium can be carried out by a conventional method, for example, Cyto. The method of Miura (Saito & Miura) (Biochim. Biophys.
Acta72.p619-629 (1963)). That is, after the cells are lysed by Lidoteam (manufactured by Seikagaku Corporation), the DNA is treated with an alkaline buffer containing SDS and phenol.
Is extracted. The RNA is further decomposed with RNase and the DNA is
Can be isolated and purified.

The integration of vector DNA into the obtained chromosomal DNA can be performed as follows. Chromosomal DNA and vector DNA
Is cut with a restriction enzyme to prepare a chromosomal DNA fragment and a vector DNA fragment. Then, the mixture of both is treated with DNA ligase (manufactured by Toyobo). Examples of the vector DNA to be used include pBR322, pUC18, pUC19, pHSG398, pHSG399 (manufactured by Takara Shuzo), pDR540, pKK233-3, and the like (manufactured by Pharmacia). In particular, pHSG399 is preferably used. In addition, Hind III, EcoR I, Pst
I and BamH I (Toyobo products). More DNA
As the ligase, DNA ligase derived from T4 phage (manufactured by Toyobo) is preferably used.

Introduction of the obtained recombinant into Escherichia coli, for example, Proc.N
Acad. Sci. USA. 69. p2110-2114 (1972). I.e. 30
The recombinant DNA was mixed with competent cells of Escherichia coli JM101 strain (manufactured by Toyobo Co., Ltd., trade name EPICURIAN COLIJM1-01) in the presence of mM CaCl _2, placed on ice for 1 hour, and then placed on ice for 4 hours.
Heat shock at 2 ° C for 60-75 seconds allows foreign DNA
Can be introduced into E. coli. Commercially available cells can also be used as the competent cells.

The method for selecting a strain having a vector DNA incorporating a DNA fragment carrying the thermostable trehalase genetic information depends on the type of restriction enzyme or vector DNA used in preparing the recombinant DNA, but, for example, as a restriction enzyme P as vector DNA using BamHI (product of Toyobo)
When HSG399 (product of Takara Shuzo Co., Ltd.) is used, it can be performed as follows. That is, Escherichia Kori K
-12 strains such as JM101 (manufactured by Toyobo, trademark EPICURIAN COLI
A transformant strain using the JM1-01) strain as a host was
The cells are cultured on a medium containing yeast extract, NaCl, chloramphenicol, and agar (hereinafter referred to as TYC agar medium), and a heat-resistant trehalase-producing strain is selected from chloramphenicol-resistant colonies appearing on the plate.

The recombinant DNA is isolated from the recombinant DNA-containing strain obtained by the above method.

The production of the thermostable trehalase according to the present invention is performed as follows.

First, the novel recombinant E. coli obtained as described above was purified using L-broth containing 50 μg / ml of chloramphenicol.
After culturing at 37 ° C. for 5 to 24 hours, the cells are collected, and the cells are lysed with lysozyme in an ultrasonic crush zone and centrifuged. Centrifuge supernatant at 60 ℃ 1
Heat treatment for an hour, and then centrifuge. Then, the trehalase fraction of the centrifuged supernatant is fractionated using a commercially available ion exchange resin, for example, DEAE-Sephadex, DEAE-Cellulose (Pharmacia P-LG Biochemicals), DEAE-Toyopearl 650M (Tosoh). . Further, column chromatography for gel filtration, for example, Sephacryl S-300 (manufactured by Pharmacia) can be used. Thus, the desired thermostable trehalase can be produced.

〔The invention's effect〕

According to the present invention, a thermostable trehalase gene DNA, the DNA
, A transformant microorganism transformed with the recombinant plasmid, and a method for industrially producing a thermostable trehalase by culturing the transformant microorganism.

 Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1 Classification of thermostable trehalase gene The chromosomal DNA of Corynebacterium sp. K-512 (FERM P-8485) was obtained by the method of Cyto and Miura [Biochim. Biophys. Acta72. P619 to 629 (1963)]. ]. 100 μg of the obtained chromosomal DNA was partially digested with a restriction enzyme Sau3A I (manufactured by Toyobo) to obtain 80 μg of a chromosomal DNA fragment.

Separately, 1 μg of vector pHSG399 (Takara Shuzo Co., Ltd .: shown in FIG. 1) was completely digested with restriction enzyme BamHI and treated with bacterial alkaline phosphatase (Takara Shuzo Co., Ltd.) to obtain 0.8 μg of vector DNA fragment. . A ligation reaction was performed between 0.1 μg of the obtained chromosomal DNA fragment and 0.28 μg of the vector DNA fragment using T4 phage-derived DNA ligase (Takara Shuzo) at 16 ° C. for 30 minutes to obtain a recombinant DNA. E. coli JMIO I competent cells
The mixture was mixed with 200 µm (manufactured by Toyobo Co., Ltd.), and left still at 0 ° C for 1 hour. The mixture was further heated at 42 ° C. for 70 seconds to perform transformation.

The resulting transformant was inoculated into 3 ml of L-broth and incubated at 37 ° C.
After culturing for 5 hours under the following conditions, the culture solution was spread on a TYC agar plate to obtain a chloramphenicol-resistant strain. This strain was cultured overnight after inoculation with L-broth containing 50 μg / ml of chloramphenicol, and the cells were collected and washed with physiological saline. After suspending in a small amount of 10 mM phosphate buffer (pH 7.0) and sonicating,
Further, the mixture was heated at 60 ° C. for 1 hour, and the centrifugal supernatant was subjected to trehalase activity measurement (55 ° C. for 10 minutes). As a result, it was confirmed that the cells were transformed, indicating 23 units / ml.

Plasmid was converted to alkaline-SDS from the obtained transformant.
This plasmid was prepared by the method described above, and this plasmid was named pKTR360 (shown in FIG. 1).

After pKTR360 was digested with Pst I and Kpn I, the fragment inserted into the vector was recovered by agarose gel electrophoresis, and this was subjected to biotin-labeled Southern hybridization by nick translation (manufactured by Bio-Rad). As a result, the DNA fragment inserted into the plasmid pKTR360 was indeed Corynebacterium sp.
It was shown to be derived from 12 chromosomal DNAs.

The purified thermostable trehalase gene is a restriction enzyme.
Two BamH I recognition sites, two restriction enzyme Hind III recognition sites, one restriction enzyme EcoR V site, one restriction enzyme Nru I
It possessed three recognition sites, one restriction enzyme Sma I recognition site and one EcoR I recognition site. (The commercially available restriction enzyme was used).

Example 2 Method for producing thermostable trehalase in Escherichia coli Plasmid pKTR360 prepared according to Example 1 was transformed into Ec
oli JM101, the transformed strain E. coli JM101 (p
(KTR360) was inoculated into 300 ml of L-broth medium containing 50 μg / ml of chloramphenicol, and then cultured overnight. L-broth medium containing 50 μg / ml chloramphenicol
After inoculating 5% to 5000 ml, the cells were cultured at 37 ° C. for 15 hours and then collected. The obtained cells are washed with physiological saline and 100 ml of 50 mM.
After suspending in a phosphate buffer (pH 7.0), the suspension was sonicated. 6 more
After heat treatment at 0 ° C. for 1 hour, the mixture was centrifuged, and the heat-resistant trehalase activity of the supernatant from which cell debris was removed was measured. As a result, the activity was 100,000 units after 15 hours of culture. Thereafter, ion exchange chromatography using DEAE-Toyopearl 650M and gel filtration using Asahi Pack GS-520P (product of Asahi Kasei Corporation) were performed, and 20,000 units of thermostable trehalase were recovered.

When the optimal action temperature and the optimal action pH of the obtained trehalase were measured, the former was 55 ° C. and the latter was pH 7.0, and these were corynebacterium bacteria K-512FERM.
It was recognized that the pH was the same as that of the enzyme derived from P-8485, and that it had the optimum pH of once.

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

That is, 1% dissolved in 0.1 M phosphate buffer (pH 7.0)
After mixing 0.1 ml of the enzyme solution with 1.0 ml of (W / V) trehalose solution and reacting at 55 ° C. for 10 minutes, the enzyme is inactivated by heating in a boiling water bath for 10 minutes. Next, D-glucose produced by the hydrolysis is quantified using a glucose measurement reagent, iatrochrome GLU-LQ (manufactured by Yatron). The unit of the enzyme activity is 1 μmol / min under the above conditions.
The amount of the enzyme required to produce the glucose is expressed as one unit.

BRIEF DESCRIPTION OF THE DRAWINGS The figure shows the plasmid pHSG399 used as a vector and the plasmid pKTR360 containing the thermostable trehalase gene incorporated in the plasmid pHSG399. The thick line in the figure is DNA derived from Corynebacterium sp.K-512FERM P-8485,
The abbreviations in the figure indicate the following restriction enzymes. HI: Hind III, SM: Sma I, SP: Sph I, EV: EcoR V PS: Pst I, NR: Nru I, SL: Sal I, SA: Sau3A I XB: Xba I, KP: Kpn I, EL: EcoR I, SC: Sac I BA: BamH I

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Claims (8)

(57) [Claims] 1. The thermophilic bacterium Corynebacterium (Cory)
nebacterium sp.), two restriction enzyme Hind III recognition sites, one restriction enzyme EcoR V recognition site, two restriction enzyme BamHI recognition sites, one restriction enzyme Sma I recognition site, and one restriction enzyme. Thermostable trehalase gene DN possessing three Nru I recognition sites and one EcoR I recognition site
A. 2. The thermophilic bacterium Corynebacterium (Cory)
nebacterium sp.), K-502 (FERM P-8484), K-51
The DNA according to claim 1, which is 2 (FERM P-8485) or K-522 (FERM P-8486). 3. The thermostable trehalase gene according to claim 1.
A recombinant plasmid in which DNA is linked to a vector plasmid. 4. The recombinant plasmid according to claim 3, wherein the vector plasmid is an Escherichia coli vector plasmid. 5. The vector plasmid is pDR540, pUC18, pUC
The recombinant plasmid according to claim 3, which is 19, pBR322, pHSG398, pHSG399, or pKK233-3. 6. A microorganism of the genus Escherichia transformed with the recombinant plasmid of claim 3, 4 or 5. 7. The microorganism according to claim 6, wherein the microorganism is Escherichia coli. 8. A method for producing a thermostable trehalase, comprising culturing the microorganism according to claim 6 in a medium, and collecting the thermostable trehalase from the culture.