JPH02268684A - Dna having genetic information of thermostable peroxidase and use thereof - Google Patents

Abstract

PURPOSE:To obtain a DNA useful for mass producing thermostable peroxidase in terms of genetic engineering, containing a base sequence to code thermostable peroxidase and a polypeptide having the same biological properties as those of thermostable peroxidase. CONSTITUTION:A thermostable peroxidase gene of Bacillus stearothermophilus IAM110001 is cloned and the DNA sequence of the gene is determined. A plasmid having the DNA sequence is prepared, a host cell is transformed with the plasmid and cultured to mass produce thermostable peroxidase. The thermostable peroxidase has >=95% activity even by retention at 60 deg.C for 10 minutes.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to DNA having genetic information for thermostable peroxidase and its uses, and more specifically to DNA containing I) NA and HDDNA encoding thermostable peroxidase. The present invention relates to a recombinant plasmid, a transformant transformed with the plasmid, and a method for producing thermostable peroxidase using the transformant.

(Prior art) Peroxidase has traditionally been an enzyme that oxidizes various compounds in the presence of hydrogen peroxide, and in recent years it has been used together with various oxidases for the determination of body fluid components such as glucose, cholesterol, phospholipids, and uric acid as clinical diagnostic agents. It is also used as a labeling enzyme in enzyme immunoassays.

The peroxidase mentioned here is the enzyme number EC1゜1.
1.17, strain name Donor: hydrog
en-peroxide oxldoreduetas
It is about e. Plants such as horseradish and radish are widely known as sources of peroxidase. However, since these plant-derived peroxidases contain isozymes with different properties, it is necessary to separate the isozymes in order to obtain pure enzymes for use in diagnostic reagents, which is a problem in that it is very time-consuming.

Various peroxidases of microbial origin are also known, including those of the genus Ofdfodendron (
JP-A-59-179075), Arthromyces genus (
Japanese Patent Publication No. 81-43987)
-128887).

In addition, the present inventors extensively searched for peroxidases with excellent thermostability and found Bacillus stearothermophilus (Ba.
c.lusstearothermophyl 1us)
95% even after heating for 10 minutes at 60°C
We have already discovered and proposed a heat-stable peroxidase that suppresses the residual peroxidase activity of
-207384).

(Problems to be Solved by the Invention) However, the productivity of peroxidase originating from microorganisms is still low - it has not yet been cultured. It is desired to develop a method to increase productivity.

(Means for Solving the Problems) The present inventors have made extensive studies to produce large amounts of thermostable peroxidase using recombinant DNA technology.

The present inventors cloned the thermostable peroxidase gene possessed by Bacillus stearothermophilus IAMI100I, and determined the DNA sequence of the gene:r. Next, a plasmid for the DNA G fl' is constructed, cells are transformed with the plasmid to create a transformant, and high production is achieved by applying a method for producing thermostable peroxidase using the transformant. We have perfected a manufacturing method that is unique to our products.

Zunawaji The present invention is characterized in that it is a base sequence encoding a thermostable peroxidase and a polypeptide having biologically equivalent properties. A recombinant plasmid characterized by containing a DNA encoding a polypeptide having properties, a recombinant plasmid containing a thermostable peroxidase and a polypeptide having biologically equivalent properties thereof; A transformant transformed with a plasmid and a transformant transformed with a recombinant plasmid containing a DNA encoding a thermostable peroxidase and a polypeptide encoding a biologically equivalent property thereof are cultured. This is a method for producing thermostable peroxidase, which is characterized by collecting thermostable peroxidase and a polypeptide having biologically equivalent properties thereto from a culture.

The thermostable peroxidase of the present invention is an enzyme that maintains a peroxidase activity of 95% or more even when heated at 60° C. for 10 minutes. Other physical and chemical properties of the thermostable peroxidase of the present invention are disclosed in JP-A-83-20738.
It is stated in the No.

The thermostable peroxidase of the present invention has the amino acid sequence shown in FIGS. 1a-d, which was derived from the DNA sequence obtained from Bacillus sterothermophilus IAMI100I. I) N encoding the thermostable peroxidase of the present invention
A encodes the amino acid sequence shown in Figures 1a-d. As such l) NA, for example, Figure 2 a-d
There is a base sequence shown in

As is well known, many polypeptides have multiple I)NA sequences encoding them.

The base sequence is not uniquely determined and may have many possibilities. In the case of the DNA encoding the amino acid sequence of the thermostable peroxidase of Bacillus stearothermophilus strain IAMI1001, which was revealed by the present inventors, there are many possibilities for the base sequence other than the base sequence of the natural gene. However, the DNA of the present invention is not limited to the original DNA of the bacterial strain described above, but also includes other DNA encoding the amino acid sequence of the thermostable peroxidase revealed by the present invention.

In addition, according to recombinant I) NA technology, a specific site of the basic DNA is artificially mutated in a manner that does not change the basic characteristics of what is encoded by WDNA or improves its characteristics. can be caused. Regarding DNA having a natural base sequence encoding the thermostable peroxidase provided by the present invention, or DNA encoding the same amino acid sequence but different from the natural one, artificial insertion of I) NA can be similarly performed. , deletion, and substitution, it is possible to obtain a polypeptide having biologically equivalent properties to the thermostable peroxidase of the present invention, and the present invention also includes such mutant genes. .

As a method for obtaining a recombinant plasmid carrying the DNA of the present invention, first, for example, Bacillus stearothermophilus strain IAM11001 is cultured in a nutrient medium, and then the Recombnant DNA Technique is used from the culture.
es (Rodrlnguez R,L, et at
, AddlsonllesleyPublish
Chromosome 1) NA is obtained according to the method described in NG Company, 1983). Next, this chromosomal DNA is cut with a restriction enzyme, and the vector and the cut chromosome 1) NA are linked together at the V loop or sticky end of the 1) NA with I) NA ligase, etc., and recombined. The resulting DNA vector is transformed into a replicable host cell and cultured to create a gene library. A colony containing the desired DNA is collected by a method of expressing peroxidase activity in colony-L obtained by culturing the transformant, and the recombinant of the present invention is obtained from the culture obtained by culturing the colony. Purify the plasmid by minutes.

Furthermore, expression vectors such as the expression vector pUc18 (Toyobo) carrying the lac promoter, the tac promoter which is a strong E. coli promoter, and rrnB
Expression vector pKK223-3 (Pharmacia+J:) carrying a liposomal RNA terminator, trr
) Expression vector pDR720 carrying the promoter (
) 7.11777 1), inducible expression vector pP
The above DNA is added to L-Lambda (Pharmacia) etc.
A recombinant plasmid can be constructed to produce the thermostable peroxidase of the present invention in microorganisms such as Escherichia coli. Furthermore, for example, shuttle vector pHY300PLK (Toyobo) between Bacillus bacilli and Escherichia coli, plasmid vector p, UB 110 (J,
Baeterol, -LJ YuA, 3 18-32
9.1978) etc., it is possible to construct a recombinant plasmid that produces thermostable peroxidase of Bacillus stearothermophilus IAMIIO01 in a dead microbial cell and in a culture solution. can.

By introducing a recombinant plasmid containing DNA encoding the thermostable peroxidase of the present invention into cells,
It is possible to obtain a transformant that produces thermostable peroxidase inside and outside the bacterial cell. As the cells, bacteria such as Escherichia coli and Bacillus subtilis, yeast, animal cells, etc. can be used.

By culturing the transformant thus produced under appropriate medium conditions, it is possible to produce thermostable peroxidase in large quantities. In this case, for example, the production of thermostable peroxidase can be advantageously carried out by adding a inducing agent such as isoprobyl thiogalactone at the early stage of the culture.

Isolation of thermostable peroxidase after culturing can be carried out, for example, by treating the bacterial cells with lysozyme, disrupting them using means such as ultrasound, or extracting, separating, and purifying them from the culture solution.

In addition to the host-vector system of E. coli and Bacillus subtilis,
Host-vector systems such as yeast, Pseudomonas, or Actinobacteria can be used, and thermostable peroxidase can be mass-produced by taking advantage of the characteristics of each host t-vector system.

〔Example〕

EXAMPLES Below, examples will be given to further explain the present invention in detail.

The present invention is not limited only to the following examples.

Example 1 (1) Bacillus stearothermophilus IAMt
Preparation of chromosomal DNA of ioo I strain Bacillus stearothermophilus IAMII001 strain was transformed into Lurla-B
ertanl (LB) medium (peptone 1%, yeast extract 0.5%, salt 1%, pH 7.0) was inoculated into 100 m9, cultured with shaking at 55°C for 12 hours, and the culture solution was transformed into j-
It was. This culture was centrifuged at 12,000 rpm for 10 minutes to obtain about 1 g of wet bacterial cells.
jnant DNA Techniques (Rodr
lHuez R,L, et al, Addlson4e
sleyPubltshlng Company, 19
Approximately 750 ug of chromosomal DNA was prepared by the method described in 83).

Next, 100 μg of this chromosomal DNA was digested with restriction enzyme RI (
Cutting was carried out using 50 units (manufactured by Toyobo Co., Ltd.) at 37°C for 13 hours.

After cutting, perform 5-20% sucrose density gradient centrifugation.
Approximately 10 μg of l) NA fragment of the ~10 kb fraction was prepared.

■ Preparation of recombinant plasmid containing DNA encoding thermostable peroxidase Plasmid vector pBR322 (manufactured by Toyobo) 1 u
After cutting g to f, □ RI 5-L = notebook at 37°C for 2 hours, chromosomal DNA 2 prepared in (1)
1) and reacted with T41) NA ligase (manufactured by Toyobo) unit for 12 hours at 16°C to ligate the NA. Next, using the obtained recombinant plasmid, Mo1
ecular Clonlng (Manlatls
T,et al,, ColdSpring tlar
bor, 1982), L, +9-
L''-UM228 was transformed.

Use 1) Approximately 1X10' colonies of transformants were obtained per 18 g of NA. The search for a recombinant plasmid containing f) NA encoding a thermostable peroxidase was carried out by colony assay.

That is, a reaction solution having the following composition was prepared.

4mM 4-aminoantipyrine 0.5m1i
'24.6mM 2.4-dichlorophenol 0.
5□Q8.111mM H2O21, OmQ100d
Phosphate buffer, pH 8,01,0□q then -1-
The reaction solution was filtered using filter paper (Advantec'l 131 90
, , ) and contacted with colonies of transformants grown on the LB agar culture medium, and colonies showing strong coloration were selected.

When searching from 1.500 colonies, two colonies showing strong coloration were obtained. Molecular Cloning (Manla
tlsT,et al,,Cold Spring H
When the recombinant plasmid was extracted by the method described in J. Arbor, 1982) and cut with r'' and ~RI, an approximately 3, 1 kb I)NA fragment common to the two recombinant plasmids was found. This approximately 3゜lkb fragment? )1 and cut with LLLRI again to open the ring pB R322 [
Ligate using the method described in 1i11 to create the recombinant plasmid pOD1.
I got 0. This approximately 3, lkb LLLRI fragment was characterized with various restriction enzymes (see Figure 3). pOD
lo encodes a biologically active thermostable peroxidase.

- The approximately 3.1 kb LLLRI fragment was ligated to M13rnp18 phage DNA (manufactured by Toyobo) cut with LLLR1, and then 1. Delaying was performed with LIL31 nuclease (manufactured by Toyobo), and DNA fragments of various sizes were prepared according to conventional methods.

For the t'J single strand 1-)NA, M13.
M13 Sequencin
The base sequence was determined using the nucleotide sequence g Kit (manufactured by Toyobo).

The determined nucleotide and amino acid sequences are shown in Figures 2 ad and Figures 1 ad.

(3) Code for thermostable peroxidase I) N
Preparation of transformants using recombinant plasmids containing A.

pODlo, a recombinant plasmid containing I)NA encoding the above-mentioned thermostable peroxidase, was used to transform 4E-0-transformed A-U M 228 according to the method described by Alan Clonlng, above. Lllj-0M228 (pOD IO) was obtained. It was confirmed that the transformant produced thermostable peroxidase.

Next, Table 1 shows the productivity of heat-stable peroxidase by each strain. The activity of thermostable peroxidase was measured according to the following method.

4mM 4-aminoantipyrine 0.51. l
Q24JmM 2,4-dichlorophenol 0.5m
(!8.8IIM H20□ 1
．． OJroomM phosphate buffer, pH 6,01
,0Illll! After prewarming at 50°C for about 5 minutes, 10 μQ of enzyme solution was added, and the increase in absorbance at 500 nm was measured. The molecular extinction coefficient is 1.38X10
'M-'cm-' was used.

In addition, the culture was carried out in the above-mentioned LB medium, and L and LLLL were set at 37°.
C1 Bacillus stearothermophilus IAM1100
1 was cultured with shaking at 55°C for 12 hours to determine intracellular enzyme activity.
'llll+ determined.

Table 1 ■・, From the data -L danΔL1j−UM228(p.

1,) 10) is clearly producing thermostable peroxidase.

(Φ f= LLL” M 228 (p OD 10
) production of thermostable peroxidase and purification of the enzyme.

Dispense the LB medium 6Q into a 1 (l) jar fermenter, autoclave at 121°C for 15 minutes, and then let it cool.
6 mQ of 50-g/mQ ampicillin (manufactured by Nacalai Tesque) was added aseptically. Into this medium, 1 L and LLLL cells were cultured with shaking at 37°C for 16 hours in a medium with the same composition as above.
80 mQ of a culture of UM 228 (pOD 10) was inoculated and cultured with aeration at 37°C for 16 hours. The thermostable peroxidase activity at the end of the culture was 1.5 U/mf7.

Culture solution 6Q was collected by centrifugation, and 20 m M'J
The suspension was suspended in acid buffer pH 7.0, disrupted by ultrasonication using a conventional method, and then centrifuged at 15 + OOOrpl for 20 minutes to obtain a crude enzyme solution of thermostable peroxidase. The crude enzyme solution thus obtained was subjected to ammonium sulfate fractionation (35% saturation to 55% saturation) to obtain active fraction 361Q, which was desalted by dialysis against 100 mM phosphate buffer pH 7.0.

Furthermore, it was adsorbed onto I) EAE-Sephadex A-50 (manufactured by Pharmacia) buffered with 20mM'J acid buffer pH 7.0. After washing with 20mM phosphate buffer MpH7,0, O~0.5M NaCl! Elute with 20mM'J acid buffer pH 7.0 containing 310+
m(' was obtained.i[f After desalting, DEAE-Sephadex A50 chromatography was performed, and the active fraction 200
I got raQ.

Furthermore, molecular separation was performed using Toyopearl HW55 (manufactured by Toyo Soda) buffered with 20 m M 'J acid buffer pH 7.0, and an active fraction of 60. I got Q. Then TSK-DEA
E-3SW (manufactured by Toyo Soda) was adsorbed and eluted using high performance liquid chromatography to obtain a highly pure product 128, 11Q. The obtained enzyme solution was 14.77U, /+w[! -Specific activity 9.9
It was U/-g protein and the yield was 21%.

I. The physicochemical properties of the thermostable peroxidase obtained by the purification steps described above are as follows: r) The NA donor strain Bacillus.
The physicochemical properties were exactly the same as those of the thermostable peroxidase produced by Stearothermophilus IAMI1001 strain.

(Effects of the Invention) As is clear from the above, by culturing a transformant using a recombinant plasmid carrying the DNA encoding the thermostable peroxidase of the present invention in a nutrient medium, thermostable peroxidase can be obtained. Peroxidase can be obtained efficiently (in large quantities).

[Brief explanation of drawings]

In Figure 1, a-d are Bacillus stearothermophilus I.
The amino acid sequence corresponding to the DNA sequence encoding the thermostable peroxidase of AMI100I strain is shown. Figures 2 a-d show l) NA encoding the thermostable peroxidase of Bacillus stearothermophilus strain IAMI100I. Figure 3 shows Bacillus stearothermophilus IAMI1
00I strain (approximately 3.1 kbr) NA fragment and the structure of pODlo from plasmid pBR322 are shown.

Claims (4)

[Claims] (1) DNA characterized by being a base sequence encoding a thermostable peroxidase and a polypeptide having biologically equivalent properties thereof. (2) A recombinant plasmid characterized by containing DNA encoding a thermostable peroxidase and a polypeptide having biologically equivalent properties thereof. (3) A transformant transformed with a recombinant plasmid containing DNA encoding a thermostable peroxidase and a polypeptide having biologically equivalent properties. (4) A transformant transformed with a recombinant plasmid containing a DNA encoding a thermostable peroxidase and a polypeptide having biologically equivalent properties thereof is cultured, and from the culture, a thermostable peroxidase and its A method for producing thermostable peroxidase, which comprises collecting a polypeptide having biologically equivalent properties.