JPH0560917B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel plasmid DNA, a microorganism carrying the plasmid DNA, a method for producing a protein using the microorganism, and a novel protein obtained thereby.

More specifically, the present invention provides DNA carrying maltopentaose-generating enzyme gene information, a new plasmid incorporating the DNA, a novel microorganism incorporating the plasmid, and production of the maltopentaose-generating enzyme using the microorganism. The present invention relates to a method and a novel maltopentaose-producing enzyme obtained thereby.

(Prior technology) Maltopentaose-generating enzyme was developed in 1986.
Pseudomonas by Okamoto et al. It was first discovered in the culture solution of Pseudomonas sp., and it is an enzyme that efficiently produces maltopentaose from starches (Special Publication No. 61-
49955).

Maltopentaose obtained by the action of this enzyme on starch can be used as a reagent for measuring amylase activity.
It is not only used heavily in the fields of clinical trials, bioassay reagents, and analytical reagents, but is also extremely important as a functional food material and is also heavily used in the food field.

Therefore, the current state of the industry is that the demand for maltopentaose is increasing and mass production is strongly desired.

In order to produce maltopentaose in large quantities to meet the above-mentioned needs in the industry, a large amount of enzymes that produce maltopentaose, that is, maltopentaose-producing enzymes, are required.

(Problems to be solved by the invention) However, in the conventional method using microorganisms,
Although various microorganisms, including Pseudomonas bacteria, have been screened, the production of maltopentaose-producing enzyme is low in all of them, and it is impossible to meet the needs of the industry.

(Means for solving the problem) Therefore, in order to develop an advantageous production method for maltopentaose-generating enzyme, we conducted studies from various angles.
We found that genetic engineering is the most appropriate method.

The present invention was achieved through further research, including DNA containing a gene encoding a maltopentaose-generating enzyme, and the maltopentaose-generating enzyme gene being incorporated into various vectors and introduced into host microorganisms. This was done for the purpose of developing a new recombinant microorganism and a method for producing maltopentaose-producing enzyme by culturing a newly created new recombinant microorganism.

In order to achieve the above object, the present inventors cloned a maltopentaose-producing enzyme gene from a maltopentaose-producing enzyme producing bacterium. Furthermore, by incorporating the gene into various vectors and introducing them into host microorganisms, and culturing the resulting recombinant microorganisms, they succeeded in producing maltopentaose-producing enzymes. The present invention was completed by confirming that the enzyme is a novel enzyme, unlike the more well-known enzymes.

The present invention will be specifically explained below.

(1) Cloning of the maltopentaose-producing enzyme gene After separating and purifying the DNA of a donor microorganism that has the ability to produce maltopentaose-producing enzyme, the DNA of the microorganism is cut using ultrasound, restriction enzymes, etc., and the resulting DNA is isolated and purified. A DNA fragment and a vector fragment obtained by cutting the vector in the same manner are ligated using, for example, DNA ligase, and a recombinant gene containing the maltopentaose-generating enzyme gene is produced.
Form DNA.

Furthermore, a transformed microorganism into which the maltopentaose-producing enzyme producing ability has been introduced by genetic recombination can also be used as a donor microorganism.

As the donor microorganism, a wide variety of microorganisms having the ability to produce a maltopentaose-producing enzyme can be used. As such microorganisms, for example, bacteria of the genus Pseudomonas are used, and specifically, Pseudomonas sp. KO8940 strain (FERM P-7456)
Examples include, but are not limited to.

The DNA derived from the donor microorganism can be obtained by, for example, culturing the donor microorganism in a liquid medium with aeration for about 1 to 3 days, collecting the resulting culture by centrifugation, and then lysing it. It can be prepared. As the bacteriolytic method, for example, treatment with a cell wall lytic enzyme such as lysozyme or β-glucanase, ultrasonic treatment, etc. are used.
Further, if necessary, other enzyme agents such as protease and ribonuclease, and surfactants such as sodium lauryl sulfate are used in combination. Freeze-thawing treatment may also be applied. To isolate and purify DNA from the lysate obtained in this way,
This can be carried out by appropriately combining methods such as phenol extraction, protein removal treatment, protease treatment, ribonuclease treatment, alcohol precipitation, and centrifugation according to conventional methods.

DNA can be cut by, for example, ultrasonication, restriction enzyme treatment, or the like. After cleavage, use phosphatase or
Modifying enzymes such as DNA polymerases are used.
Furthermore, the base sequence at the end of the DNA fragment can be changed by using various linkers and adapters.

From the cut DNA fragments, only fragments of optimal length can be obtained by sucrose density gradient centrifugation, extraction from electrophoresed gels, etc.

As a vector, a phage or a plasmid that can autonomously propagate in a host microorganism is suitable.

Known phage can be used as appropriate; for example, when Escherichia coli is used as the host microorganism, λ phage, M13 phage, etc. can be used.

In addition, as a plasmid, for example, when using Escherichia coli as a host microorganism,
pBR322, PUC18 and their derivatives, e.g.
pBR-AN3 etc. can be used. Furthermore, for example,
In addition to vectors such as pHY300PLK, YIp5, and YEp13, which are capable of autonomous replication in two or more host microorganisms such as Escherichia coli and Bacillus subtilis, various known shuttle vectors can also be used. Such a vector is cleaved with a restriction enzyme or the like in the same manner as the DNA described above to obtain a vector fragment.

The method for joining the DNA fragment and the vector fragment may be any method using a known DNA ligase. For example, after annealing the DNA fragment and the vector fragment, an appropriate DNA ligase may be used in vitro.
Recombinant DNA is created by the action of ligase.
If necessary, after annealing, the DNA can be introduced into a host microorganism and converted into recombinant DNA using in-vivo DNA ligase.

Any host microorganism may be used as long as the recombinant DNA is stable, capable of autonomous growth, and expression of the recombinant DNA.

The method of introducing recombinant DNA into a host microorganism is
Known methods such as the calcium method (Lederberg,
EMand Cohen, SN, J. Bacteriol., 119 ,
1072, (1974)) etc. can be adopted.

With λ phage DNA, in vitro
Packaging method (Horn, B., Methods in
Enzymology, 68 , 299 (1979)) to form λ phage particles, and add these λ phage particles to a suspension of cultured bacteria of Escherichia coli to obtain a special transduction phage possessing the ability to produce maltopentaose-producing enzymes. be able to.

The transformed microorganism into which the recombinant DNA has been introduced is selected by a conventional method, for example, by culturing it in a liquid selection medium and measuring the maltopentaose-producing enzyme activity in the culture solution. Depending on the marker on the vector, the liquid selection medium may be minimal medium or
Antibiotic-supplemented media are used as appropriate.

To measure enzyme activity, a starch solution is added to the culture solution, kept at 45°C, and then thin-layer chromatography or high-performance liquid chromatography is used to identify and quantify the maltopentaose produced.

The resulting maltopentaose-producing enzyme-producing bacteria were cultured at 37°C in a liquid selective medium and subjected to known methods such as alkaline extraction (Birnboim, HC).
and Doly, J., Nucleic Acids Res., 7 .
1513 (1979)).

The recombinant DNA containing the maltopentaose-generating enzyme gene can be cleaved with appropriate restriction enzymes by the method described above, and can be incorporated into other vectors or introduced into other hosts.

(2) Preparation of maltopentaose-generating enzyme Maltopentaose-generating enzyme can be prepared as follows. A maltopentaose-producing enzyme-producing bacterium or a transformed microorganism that has acquired the ability to produce a maltopentaose-producing enzyme by the method described above is cultured in liquid. The medium may be any medium that is used for the normal cultivation of the microorganisms; for example, carbon sources include starch, liquefied starch, glucose, glycerin, molasses, blackstrap molasses, etc., and nitrogen sources include various types. These include protein digests, soybean flour, meat extract, peptone, urea, nitrates, ammonium salts, yeast extract, and cornstarch liquor. Other nutrients such as biotin and trace metals are used as appropriate.

After culturing, if the enzyme is present in the cells, the culture solution is centrifuged to obtain the cells, treated with ultrasound or cell wall lytic enzymes, etc., the crushed cells are removed by centrifugation, and the crude enzyme solution is obtained. do. Furthermore, if the enzyme is present in the medium, the culture solution is centrifuged to remove the bacterial cells and subsequent purification is performed.

The maltopentaose-producing enzyme can be isolated from the obtained crude enzyme solution by a general enzyme purification method such as salting out, dialysis, ion exchange resin, or affinity chromatography.

Using the purified enzyme obtained in this way, we investigated the properties of this enzyme, and found that it had a molecular weight of 65,000.
(SDS-polyacrylamide slab gel electrophoresis) is characterized by an optimum pH of 6.0 and an optimum temperature of 37-45°C.

No known maltopentaose-producing enzyme has been found to have such properties, and therefore, the present enzyme was recognized as a novel, previously unknown enzyme.

Next, the present invention will be explained in detail with reference to examples.

Example 1 Cloning of maltopentaose-generating enzyme gene Pseudomonas sp.
sp) KO-8940 (FERM P-7456) in a liquid medium containing soluble starch (1 g of soluble starch, 1 g of polypeptone, 0.3 g of meat extract, 0.2 g of yeast extract, 0.3 g of sodium chloride, 100 ml of water, PH7.5) for 40 min. The bacteria were cultured at ℃ for 6 hours, collected by centrifugation, washed, and the method of Saito and Miura (Saito, H. and
Miura, K., Biochim.Biophys.Acta, 70 , 619
(1963)), dissolve the chromosomal DNA in Tris-HCl/EDTA buffer, and use restriction enzymes.
After adding Sau3AI (manufactured by Takara Shuzo Co., Ltd.) and partially decomposing it at 37°C, the decomposed product was subjected to sucrose density gradient ultracentrifugation.
Chromosomal DNA fragments of 2Kb or more were isolated and obtained.

Phage λL47 (manufactured by Amersyam) was used as a vector, and λL47 DNA cut with Bam HI (manufactured by Takara Shuzo) and the above-mentioned Pseudomonas. SP
Chromosome of approximately 2Kb or more obtained from the KO-8940 strain
The DNA fragments were mixed and ligated by adding T4 DNA ligase (Takara Shuzo Co., Ltd.) (Weiss, B.,
Sablon, AJ, Live, TR, Fareed, GCand
Richardson, CCJBiol.Chem., 243 , 4543
(1968)). The treatment solution was added to an in vitro packaging kit (manufactured by Takara Shuzo Co., Ltd.) to perform the in vitro packaging method (Horn, B., Methods).
in Enzymology, 68 , 299, (1979)).
DNA was introduced into phage particles. The phage particles were added to a bacterial cell suspension of Escherichia coli WL95, and λ containing 1% soluble starch and 1.2% agar.
Medium (Bactotryptone 10g, sodium chloride
2.5 g of the product was added to water 1) and cultured at 37°C, and the plaques that appeared and the phages that did not undergo the iodine reaction could be isolated as maltopentaose-producing enzyme-producing phages.

The resulting maltopentaose-producing enzyme-producing phages were isolated and used in Escherichia coli WL66.
The cells were cultured together with the strain in lambda medium at 37°C. The culture solution was centrifuged to remove host cells, polyethylene glycol was added to aggregate the phage particles, and the phage particles were collected by centrifugation to obtain new phages. This phage was named λOS501.
The phage particles were dialyzed against a phage suspension buffer containing 50% formamide to obtain λOS501 phage DNA. Phage λOS501DNA
It has a size of 46.0 Kb, and its physical map (restriction enzyme cleavage map) is shown in Figure 1. Here, the white part is derived from vector λL47, and the shaded part is a chromosomal DNA fragment. All abbreviations are restriction enzymes.

The obtained λOS501 phage DNA was dissolved in Tris-HCl/EDTA buffer, and the restriction enzyme Nru I
was added and completely decomposed at 37°C. decomposed products
After agarose gel electrophoresis, a 3.5 Kb chromosomal DNA fragment was isolated and obtained, and recloned using plasmid pUC18. Plasmid pUC18 (manufactured by Takara Shuzo Co., Ltd.) is 2.7 Kb, has ampicillin resistance ( ^Apr ), and can be cut at one site with the restriction enzyme Sma I. pUC18 cut at one site with Sma I
obtained from the above λOS501 phage after treatment with alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.)
3.5 Kb chromosomal DNA fragments were mixed and ligated by adding T4 DNA ligase. Using this treatment solution and using Escherichia coli JM109 strain as a host,
Lederberg, Cohen method (Lederberg, EMand
Cohen, SN, J. Bacteriol., 119 , 1072,
(1974)), the plasmid was introduced.

3.5Kb chromosomal DNA fragment obtained as above,
In other words, the physical map of the 3.5 Kb fragment of Nru I-Nru I is as shown in FIG.

The transformed bacterial cells obtained by introducing the plasmid were transferred to L medium (10 g of bactotryptone, 5 g of yeast extract, 5 g of sodium chloride, Cultured at 37℃ in water 1, PH7.0),
An ampicillin-resistant strain was obtained. This ampicillin-resistant strain was cultured in L medium containing 50 μg/ml of ampicillin and 1% soluble starch at 37°C for 24 hours, and the presence or absence of maltopentaose-generating enzyme activity in the culture solution was examined by thin layer chromatography. Ta.

For thin film chromatography, the culture solution is incubated twice at 60°C in a solvent system of n-butanol/n-propanol/water (3/5/4), and then maltopentalysis is performed by spraying 20% methanol sulfate. By searching for the presence or absence of ose, we were able to isolate a maltopentaose-producing enzyme-producing strain.

The resulting maltopentaose-producing enzyme-producing strain was isolated and cultured at 37°C in L medium containing 50 μg/ml of ampicillin. The culture solution was centrifuged to collect the bacteria. After washing, the isolated bacterial cells were Alkaline extraction method (Bironboim, HCand Doly, J., Nucleic
Acids Res., 7 , 1513, (1979)), the plasmid was isolated and a new plasmid was obtained. This plasmid was named plasmid pOS201.

Plasmid pOS201 has a size of 6.2 Kb, and its physical map is shown in Figure 2. Here, the white part is derived from vector pUC18, the diagonal line part is the chromosomal DNA fragment part, and each abbreviation is a restriction enzyme.

Escherichia coli JM109−pOS201 transformed with plasmid pOS201
JM109-pOS201) has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (FERM P-10973).

Then, the base sequence of the NruI fragment containing the maltopentaose-generating enzyme gene was determined, and the results shown in FIG. 4 were obtained. The nucleotide sequence from that position (and the amino acid sequence predicted from the nucleotide sequence from the signal start point) is determined by the fifth
Shown in the figure.

FIG. 6 illustrates the DNA sequence and amino acid sequence surrounding the maltopentaose-generating enzyme gene. In the figure, the signals are ATG (-123 position) opposite to methionine (-41 position) and/or ATG (-69 position) corresponding to methionine (-23 position).
The structural gene for the maltopentaose-generating enzyme starts from GTG (position 1), which corresponds to valine (position 1), and starts from lysine (position 591), which corresponds to valine (position 1).
end at the AAG (position 1771) corresponding to

As can be seen from Figure 6, the maltopentaose-generating enzyme gene uses ATG as the start codon.
It uses TGA as a stop codon.

FIG. 7 and FIG. 8 illustrate the DNA sequence and amino acid sequence of the maltopentaose-generating enzyme gene, respectively.

FIG. 9 illustrates the amino acid sequence of the maltopentaose-generating enzyme gene containing a signal, and the signal cleavage point is A at position 41.
(alanine) and V (valine) at position 42.

New plasmid prepared as above
Cut pOS201DNA with Xho I (manufactured by Takara Shuzo Co., Ltd.),
You will get a 0.75Kb fragment. This fragment was labeled with ^32P , and Pseudomonas. S.P. KO-8940 strain chromosomal DNA, Escherichia coli JM109 strain chromosome
Southern hybridization (Sauthern,
EM, J.Mol.Biol., 98, 503, (1975)). E. coli did not hybridize at all with chromosomal DNA, but Pseudomonas. SP KO-8940 strain chromosomal DNA, λOS501 DNA
A 0.75 Kb band was found that specifically hybridized with the . From this, I cloned
DNA is Pseudomonas. It was confirmed that it was derived from the SP KO-8940 strain.

Example Construction of pOS321-CE (1) Construction of pOS320 Contains maltopentaose-generating enzyme gene
Plasmid pOS201 (Example 1, Figure 2) in which the SphI-KpuI 2.1Kb DNA fragment was ligated to pUC18 was
Mung Bean with the overhanging end cut with SphI
After blunting with nuclease and repairing with Klenow fragment, it was cut with KpnI to obtain a 2.1 Kb DNA fragment containing the maltopentaose-generating enzyme gene.

On the other hand, pBR-AN3 (a plasmid vector with a multi-cloning site introduced into pBR322)
was cut with BamHI, the overhanging end was blunted with Klenow fragment, cut with KpnI, and Bacterial
Alkaline Phosphatase (BAP) treated and containing the above-mentioned maltopentaose-generating enzyme gene.
After cutting the 2.1 Kb DNA and ligating with T4 ligase, E. coli JM109 was transformed.

As shown in Figure 12, the resulting plasmid pOS320 has a regenerated BamHI site and contains the maltopentaose-generating enzyme gene.
The DNA fragment can be recovered as BamHI-KpnI DNA fragment.

(2) Construction of pOS321−CE Cut pOS320 with BamHI and remove the overhanging end.
Smooth with Mung Bean Nuclease and Klenow
After repair with fragment, cut with Hind and contains maltopentaose generating enzyme gene
A 2.1Kb DNA fragment was obtained.

In addition, pKK223-3 (manufactured by Pharmacia)
After cutting with EcoR, the protruding ends were blunted with Klenow fragment, cut with Hind, treated with BAP, ligated with the above DNA fragment using T4 ligase, and E. coli JM109 was transformed. As shown in FIG. 13, the EcoR site at the ligation site of the obtained plasmid pOS321-CE is regenerated, resulting in an expression plasmid in which a 2.1 Kb DNA fragment containing the maltopentaose-generating enzyme gene is ligated downstream of the tac promoter. JM109 as a transformed strain
-pOS321-CE (FERM P-11672) was obtained.

Example 3 Linking the maltopentaose-generating enzyme gene to the tac promoter using synthetic DNA (1) Construction of pOS3410 A synthetic DNA as shown in FIG. 15A was designed. This is 19 minutes from the start codon ATG of the G5-generating amylase gene (maltopentaose-generating enzyme gene, sometimes abbreviated as G5 amy).
It consists of the base sequence up to the Hae site and an EcoR site added to the 5th side. Further, rOS320 was digested with Hae and Bcl.
A 250bp DNA fragment (B) and a 1.7Kb DNA fragment (C) obtained by digestion with Bcl and Hind were prepared. The vector used was pKK223-3.
After cleavage with EcoR and Hind, BAP treatment was performed (Figures 14 and 15).

As shown in Figure 14, after ligating the above four types of DNA fragments (A to D), E. coli
JM109 was transformed and transformants having G5-generating amylase activity were selected. The sequence of the plasmid junction portion held by this recombinant was confirmed and named pOS3410.

(2) Construction of pOS3414 After cleaving pOS3410 with EcoR, Klenow
Blunt the protruding ends with fragment and use Self
ligation. E. coli JM109 was transformed with this DNA. In the obtained plasmid,
The EcoR site disappeared and a new Vsp site was generated (Figure 14). The plasmid thus obtained was named pOS3414.

(3) Construction of pOS3406 and pOS3412 After cutting pOS3410 with EcoR, Mung Bean
The protruding ends were blunted with nuclease. this
Self ligation was performed and E. coli JM109 was transformed. The resulting plasmid was used as pOS3406.
It was named. On the other hand, an Hpa linker was introduced into the DNA with the EcoR site blunted, and E.coli
The plasmid obtained by transforming JM109
It was named pOS3412 (Figure 14).

Example 4 Construction of pOS3408 As shown in Figure 16, pOS3410 was constructed using EcoR
After cutting with Hind, smoothing the protruding part with Mung Bean Nuclease and repairing with Klenow fragment.
2.0Kb containing the G5-generating amylase gene
DNA fragments were obtained. pKK233−2 (tac
Promoter, ori: pBR322) was cleaved with Nco,
After blunting the protruding ends with Mung Bean Nuclease,
It was cut with Hind and treated with BAP. Both DNA
ligation and transform E. coli JM109 with pKK233
- Obtained G5. Cut pKK233−G5 with Vsp,
A 3.2 kb DNA fragment containing the -10 region of the tac promoter and the G5-generating amylase gene was obtained.

On the other hand, vector pKK233-3 was cut with Vsp,
3.4kb containing -35 region of ori and tac promoters
DNA fragments were obtained. ligate both DNA fragments,
pOS3408 was obtained by transforming E.coli JM109 (Figure 17) (the sequence of the junction part of pOS3408 is
pKK233−G5).

The transformant obtained in this way is
Named Escherichia coli JM109−pOS3408,
This was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (FERM P-11673).

Example 5 Production of maltopentaose-generating enzyme (1) Escherichia coli JM109-pOS201
(FERM P-10973) in L liquid medium containing Ampicirin (1 g of tryptone, 0.5 g of yeast extract,
NaCl 0.5g, Ampicirin 50μg/ml, water 100ml, PH
7.0) Cultured in 3 ml at 37°C for 12 hours, and this culture solution was used as a seed culture. Add 1 ml of seed culture to ampicillin,
L liquid medium containing IPTG (isopropyl-β-D-thiogalactopyranoside) (tryptone 1
g, yeast extract 0.5g, NaCl 0.5g, ampicillin
50μg/ml, TPTG 100mmole, water 100ml, PH
7.0) and cultured at 37°C for 24 hours, a culture supernatant fraction and a bacterial cell fraction were obtained by centrifugation. After suspending the bacterial cell fraction in 100 ml of Tris-HCl/calcium acetate buffer, the bacterial cells were disrupted by ultrasound and the supernatant fraction obtained by centrifugation was mixed with the culture supernatant fraction.
ml of crude enzyme solution. Add ammonium sulfate to the crude fermentation solution obtained at a low temperature of 4°C, collect the fraction that precipitates at 0.2 to 0.5 saturation, and add it to 10mM phosphate buffer (PH
7.5). This enzyme solution was dialyzed overnight in the same buffer. Next, it was purified by ion exchange chromatography using DEAE-Toyopearl 650M and gel filtration chromatography using Toyopearl HW55S, and it was possible to obtain a sample that showed a single band electrophoretically. In addition, Asahipak E.S.
Purification was performed by high performance liquid chromatography using -520N (manufactured by Asahi Chemical Industry Co., Ltd.) to obtain a purified enzyme.

The properties of this enzyme were investigated using the purified enzyme thus obtained, and the following results were obtained.

(1) Action The mode of action of this enzyme is as follows when oligosaccharides below maltooctaose are used as substrates.

G ₈ →G ₅ +G ₃ G ₇ →G ₅ +G ₂ G ₆ →G ₅ +G ₁ G ₅ →G ₃ +G ₂ G ₄ →G ₂ +G ₂ G ₃ , G ₂ : No effect (However, G ₁ : Glucose , _G2 : maltose,
G ₃ : maltotriose, G ₄ : maltotetraose, G ₅ : maltopentaose, G ₆ : maltohexaose, G ₇ : maltoheptaose, G ₈ : maltooctaose, respectively. ) (2) Substrate This enzyme is used in amylose, soluble starch, potato, sweet potato, corn, waxy corn,
It acts on various starches such as barley, wheat, rice, tapioca, and sago to produce maltopentaose.

(3) Optimal pH for action The reaction solution composition was as follows: Substrate (2% reduced soluble starch solution) 0.5ml Various buffers (0.1M) (acetate buffer, phosphate buffer, sodium carbonate buffer) Solution) 0.4 ml Enzyme solution (8 IU/ml) 0.1 ml The above composition solution was reacted at 45° C. for 15 minutes and the reducing power was measured. The results are shown in FIG. 10, with the highest value being expressed as 100.

As is clear from the figure, the optimal pH of this enzyme is 6.0.
It is.

(4) Optimum temperature for action The reaction solution composition was as follows: Substrate (2% reduced soluble starch solution) 0.5ml 0.1M phosphate buffer (PH6.5) 0.4ml Enzyme solution (8IU/ml) The reducing power was measured by reacting 0.1 ml of the above composition solution at various temperatures from 20 to 70 DEG C. for 15 minutes, and the results are shown in FIG. 11, with the highest value being expressed as 100.

As is clear from the figure, the optimal temperature for this enzyme is 37
~45℃.

(5) Molecular weight The molecular weight of this enzyme obtained by SDS-polyacrylamide slab gel electrophoresis is 65,000.

The present enzyme having the properties shown above is completely different from conventionally known enzymes, and is a novel enzyme that produces maltopentaose in large amounts.

Example 6 Production of maltopentaose-generating enzyme (2) Escherichia coli JM109-pOS321-CE
(FERM P-11672) was cultured in 3 ml of L medium containing ampicirin at 37°C for 12 hours, and this culture solution was used as a seed culture. Transfer 1 ml of seed culture to L medium containing ampicillin and IPTG (1 g of tryptone, 0.5 g of yeast extract,
NaCl 0.5g, ampicillin 50μ,
Inoculate IPTG100mmole, water 100ml, PH7.0),
After culturing at 37°C for 24 hours, a culture supernatant fraction and a bacterial cell fraction were obtained by centrifugation. After suspending the bacterial cell fraction in 100 ml of Tris-HCl/acetate buffer, the bacterial cells were disrupted by ultrasound, and the supernatant fraction obtained by centrifugation was mixed with the culture supernatant fraction to make 200 ml of crude enzyme. It was made into a liquid.

This crude enzyme solution 0.1μ, 2% soluble starch (Merck
) 0.5μ, 0.1M phosphate buffer (PH6.5) 0.4μ
Mix and react at 45℃ for 10 minutes.
Reducing power was measured using the Nelson method. the result,
It showed an activity of 20IU/ml (1IU is 1 minute).
This is the enzyme activity that cleaves 1 μmole of α-1,4 glucoside bond).

Example 7 Production of maltopentaose-generating enzyme (3) Escherichia coli JM109-pOS3408
(FERM P-11673) in L medium containing ampicirin.
The cells were cultured in 3 ml at 37°C for 12 hours, and this culture solution was used as a seed culture. Transfer 1 ml of seed culture to L medium containing ampicillin and IPTG (1 g of tryptone, 0.5 g of yeast extract,
NaCl 0.5g, ampicillin 50μg/ml,
IPTG 0.1mM, water 100ml, PH7.0) was inoculated at 37°C.
After culturing for 24 hours, a culture supernatant fraction and a bacterial cell fraction were obtained by centrifugation. The bacterial cell fraction was added to Tris-HCl/acetate buffer.
After suspending in 100 ml, the cells were disrupted by ultrasonication and the supernatant fraction obtained by centrifugation and the culture supernatant fraction were mixed to obtain 200 ml of crude enzyme solution.

This crude enzyme solution 0.1μ, 2% soluble starch (Merck
) 0.5μ, 0.1M phosphate buffer (PH6.5) 0.4μ
were mixed and reacted at 45℃ for 10 minutes (Somogyii-
Reducing power was measured using the Nelson method. the result,
It showed an activity of 50IU/ml (1IU is 1 minute).
This is the enzyme activity that cleaves 1 μmole of α-1,4 glucoside bond).

(Effects of the Invention) Through the present invention, the structure of the maltopentaose-generating enzyme gene has been elucidated for the first time, and it has been successfully expressed in microorganisms.

As a result, maltopentaose-generating enzyme was produced in large quantities. This enzyme according to the present invention is characterized not only by its high activity but also by being a previously unknown novel enzyme. Therefore, according to the present invention, maltopentaose can be produced in large quantities by using the present enzyme.

Maltopentaose is currently used as a substrate for measuring α-amylase activity in diagnostic agents and reagents, and by producing this enzyme at low cost according to the present invention, it can be widely used in various applications including foods. be able to. Maltopentaose has excellent solubility, lacks sweetness, and has a body texture, making it useful as an ingredient for confectionery.Since it is also easily digested and absorbed, it can be used as a nutritional food for infants, the elderly, and patients. can.

[Brief explanation of the drawing]

Figure 1 is the physical map (restriction enzyme cleavage map) of phage λOS501, Figure 2 is the plasmid
Physical map of pOS201, Figure 3 shows Nru I-Nru I inserted into plasmid pOS201.
The physical maps of the 3.5 Kb fragments are shown, and the arrows in FIGS. 2 and 3 indicate the coding region of maltopentaose-producing amylase. 1 to 3 in Figure 4 are Nru I-Nru I
The DNA sequences of the fragments are shown, and 1 to 5 in FIG. 5 illustrate the entire DNA sequence and part of the amino acid sequence (signal and enzyme genes) in FIG. 4. 1 to 3 in FIG. 6 illustrate the DNA sequence and amino acid sequence surrounding the maltopentaose-generating enzyme gene. Figure 7 1
Figures 2 to 2 and 8 illustrate the DNA sequence and amino acid sequence of the structural gene of the maltopentaose-generating enzyme, respectively. FIG. 9 illustrates the amino acid sequence of the maltopentaose-generating enzyme gene containing a signal, and the arrow in the figure indicates the expected signal cleavage point. Figures 10 and 11 are graphs showing the optimal pH and temperature of this enzyme, and in Figure 10, ○-○ are
0.1M acetic acid buffer, △-△ represent 0.1M phosphate buffer, and □-□ represent 0.1M sodium carbonate buffer, respectively. Figure 12 shows the plasmid
The construction diagram of pOS321-CE, FIG. 13, shows the physical map of the plasmid obtained thereby.
Figure 14 shows plasmids pOS3410, pOS3414, and pOS3414.
3406 and 3412, and Figure 15 shows the four species A to D used for constructing plasmid pOS3410.
Represents a DNA fragment. B... Synthetic DNA fragment, B, C...
250 bp and 1.7 Kb fragments originating from pOS320,... Plasmid pKK223-3, Figure 16 is a construction diagram of plasmid pOS3408, Figure 1
Figure 7 shows the physical map of the plasmid thus obtained.

Claims (1)

[Scope of Claims] 1. DNA containing the maltopentaose-generating enzyme gene shown in FIG. 2. DNA containing a maltopentaose-generating enzyme gene, which includes the base sequence shown in FIG. 5 and a base sequence encoding an amino acid sequence. 3. DNA containing a maltopentaose-generating enzyme gene, which contains the base sequence shown in FIG. 6 or the base sequence encoding the amino acid sequence. 4. A maltopentaose-generating enzyme gene encoding the amino acid sequence shown in FIG. 5 Maltopentaose-generating enzyme gene represented by the base sequence shown in FIG. 6. DNA containing a maltopentaose-generating enzyme gene, which contains a base sequence encoding the amino acid sequence shown in FIG. 7 The DNA containing the maltopentaose-generating enzyme gene according to any one of claims 1 to 6 is ligated with a vector fragment as shown in the restriction enzyme cleavage map of FIG. 1, FIG. 2, or FIG. A phage and/or plasmid characterized in that it is a phage and/or a plasmid. 8 pOS321-CE or pOS3408 whose plasmid is indicated by the restriction enzyme cleavage map in Figure 13 or Figure 17
The plasmid according to claim 7, characterized in that it is. 9. The donor microorganism according to any one of claims 1 to 8, wherein the donor microorganism is a microorganism belonging to the genus Pseudomonas.
DNA or genes. 10. The DNA or gene according to claim 9, wherein the donor microorganism is Pseudomonas sp. KO-8940. 11. A transformed microorganism obtained by introducing the phage or plasmid according to any one of claims 7 to 8 into a host microorganism. 12. The transformed microorganism according to claim 11, wherein the transformed microorganism is Escherichia coli. 13 The transformed microorganism is Escherichia coli JM109-pOS321-CE
(FERMP-11672) or JM109-pOS3408
(FERM P-11673), The transformed microorganism according to claim 12. 14. A method for producing a maltopentaose-producing enzyme, which comprises culturing the transformed microorganism according to any one of claims 11 to 13 and collecting the maltopentaose-producing enzyme from the culture. 15 A novel maltopentaose-generating enzyme characterized by having the following properties: (A) The molecular weight of this enzyme is 65,000 (as determined by SDS-polyacrylamide slab gel electrophoresis). (B) The optimal pH of this enzyme is 6.0. (C) The optimal temperature for this enzyme is 37-45°C.