JPH03219879A - Gene of maltopentaose-forming enzyme, microorganism containing the gene, production of maltopentaose-forming enzyme using the microorganism and novel enzyme produced thereby - Google Patents

Abstract

PURPOSE:To improve the productivity of maltopentaose-producing enzyme by integrating a cloned maltopentaose-producing enzyme gene into a vector, introducing the vector into a host microoganism and culturing the transformant. CONSTITUTION:DNA of a donor microorganism capable of producing maltopentaose-producing enzyme is incised with ultrasonic wave, restriction enzyme, etc., to obtain a vector fragment. Separately, another vector fragment is produced by incising a vector such as pBR322 by the similar method. Both vector fragments are linked together using a DNA ligase, etc., to obtain a recombinant DNA containing a maltopentaose-producing enzyme gene. The recombinant DNA is introduced into a host microorganism such as Escherichia coli, the obtained transformant is cultured in liquid medium and centrifuged, the separated microbial cells are disintegrated, the broken cells are removed and the remaining component is purified to obtain a maltopentaose enzyme having a molecular weight of 65,000 (SDS-polyacrylamide slab gel electrophoresis), an optimum pH of 6.0 and an optimum temperature of 37-45 deg.C.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention provides novel plasmid DNA,
A microorganism possessing the microorganism, a method for producing 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 producing a maltopentaose-generating enzyme using the microorganism. The present invention relates to a method and a novel maltopentaose-producing enzyme obtained thereby.

(Prior art) Maltopentaose-generating enzyme was developed by Okamo in 1986.
t.

It was first discovered in the culture solution of Pseudomonas sp., and is an enzyme that efficiently produces maltopentaose from starches (Special Publication No. 61-49).
955).

Maltopentaose obtained by allowing this enzyme to act on starch can be used as a reagent for measuring amylase activity.

It is not only heavily used in the fields of clinical reagents, 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 in response to 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 genus were screened, the production potential of maltopentaose-producing enzyme was low in all of them, and it did not meet the needs of the industry. This is something that cannot be answered at all.

(Means for solving the problem) Therefore, an advantageous method for producing maltopentaose-generating enzyme'
In order to develop this, we investigated various aspects and found that genetic engineering is the most appropriate method.

The present invention was achieved through further research, and included DNA containing a gene encoding a maltopentaose-generating enzyme, and the maltopentaose-generating enzyme gene 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 maltopentaose-producing enzyme gene After separating and purifying the DNA of a donor microorganism capable of producing maltopentaose-producing enzyme, for example,
A DNA fragment obtained by cutting with ultrasound, a restriction enzyme, etc. and a vector fragment obtained by cutting the vector in the same manner are ligated using, for example, DNA ligase, etc., to obtain a DNA fragment containing the maltopentaose-generating enzyme gene. Form recombinant 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. KO8
940 strain (FERM P-7456) is exemplified, but is not limited thereto.

DNA derived from the donor microorganism is prepared 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 the culture. be able to. 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 separate and purify DNA from the lysate obtained in this way,
This can be carried out according to conventional methods, such as by appropriately combining methods such as phenol extraction, protein removal treatment, protease treatment, ribonuclease treatment, alcohol precipitation, and centrifugation.

DNA can be cut by, for example, ultrasonication, restriction enzyme treatment, or the like. After the cleavage, a modifying enzyme such as phosphatase or DNA polymerase is used as necessary. 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 gel electrophoresis, or the like.

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

Known phages can be used as appropriate, such as Escherichia coli (Escherichia coli).
li) as the host microorganism, λ phage or M1
3 phages etc. can be used.

In addition, as a plasmid, for example, when Escherichia coli is used as a host microorganism, ρBR322, pUc
18 and derivatives thereof, such as pBR-AN3, can be used. Furthermore, for example, pi (Y300PLK, YIp5
In addition to vectors such as , YEp 13, 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 DNA fragments and vector fragments is as follows:
Any method using a known DNA ligase may be used. For example, after annealing a DNA fragment and a vector fragment,
Recombinant DNA is extracted in vitro by the action of an appropriate NA ligase.
Create A. If necessary, after annealing, introduce into the host microorganism.

Recombinant DNA can also be produced using in-vivo DNA ligase.

Any host microorganism may be used as long as the recombinant DNA can stably and autonomously proliferate and express its characteristics.

The recombinant DNA can be introduced into the host microorganism using known methods, for example, when the host microorganism is Escherichia coli, the calcium method (Lederberg, E, M.

and Cohen, S., N., J., Bacte.
riol, 119.1072° (1974)), etc. can be adopted.

For λ phage DNA, in vitro backsizing method (Horn, B., Methods inE
zymology, 68.299 (1979)), and the λ phage particles were added to a culture suspension of Escherichia coli to produce a special transducing phage that has the ability to produce maltopentaose-producing enzymes. Obtainable.

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.

As the liquid selection medium, a minimal medium or an antibiotic-supplemented medium is appropriately used depending on the marker on the vector.

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

The resulting maltopentaose-producing enzyme-producing bacteria were cultured at 37°C in a liquid selective medium and subjected to known methods such as the alkaline extraction method (Birnboim, H, C, and Dol).
y.

J., Nucleic Ac1ds Res.
1513. (1979)).

Recombinant DNA containing maltopentaose producing enzyme gene
can be cleaved with appropriate restriction enzymes using the method described above, and can be incorporated into other vectors or introduced into other hosts.

(2) Preparation of maltopentaose-producing enzyme Maltopentaose-producing 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 above method 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 bacterial cells, the culture solution is centrifuged to obtain the bacterial cells, which are then treated with ultrasound, cell wall lytic enzymes, etc.
The crushed bacterial cells are removed by centrifugation to obtain a crude enzyme solution. Also,
If the enzyme is present in the medium, the culture solution is centrifuged to remove bacterial cells and subsequent purification is performed.

From the obtained crude enzyme solution, salting out, dialysis, ion exchange resin,
The maltopentaose-generating enzyme can be isolated by affinity chromatography, etc.-Funatsuri enzyme purification method.

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 (SOS
-Polyacrylamide slab gel electrophoresis), optimal pH
is 6.0, and the optimum temperature is 37 to 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 producing 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 mQ of water, pH 7.5) )
Cultured at 40℃ for 6 hours, collected by centrifugation, washed,
From the obtained bacteria, 5aito, N1ura's method (Sa
ito, H, and Miura, K., Bio
chim.

Biophys, Acta, 72.619 (196
3) Separate chromosomal DNA, dissolve it in Tris-HCl/EDTA buffer, and add restriction enzyme 5au3AI (
(manufactured by Zenshuzo Co., Ltd.) and partially decomposed at 37°C, a chromosomal DNA fragment of approximately 2 Kb or more was separated and obtained from the first decomposed product by sucrose density gradient ultracentrifugation.

Phage λL47 (manufactured by Amarjam) was used as a vector, and λL47 cut with Baa HI (manufactured by Zenshuzo Co., Ltd.) was used.
DNA and the aforementioned Pseudomonas sp. KO-89
Chromosomal DNA fragments of approximately 2 Kb or more obtained from 40 strains were mixed and ligated by adding T4 DNA ligase (manufactured by Zenshuzo Co., Ltd.) (Weiss, B., 5ablon,
A. J., Live, T.

R, ,Fareed, G, C, and Richa.
rdson, C,C,J.

Biol, Chem, 243.4543 (196
8)). The treatment solution was added to an in vitro packaging kit (manufactured by Zenshuzo Co., Ltd.), and the in vitro packaging pufferfish method (t(orn, B, Methods i) was performed.
n Enzymology, 68.29L (1979
)) The 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 was added.
Medium (Bactodryptone 10g, sodium chloride 2.5
g, water 112) and cultured at 37°C. Plaques that appeared and phages that did not undergo iodine reaction could be isolated as maltopentaose-producing enzyme-producing phages.

The resulting maltopentaose-producing enzyme-producing phage was isolated and cultured together with Escherichia coli strain 66 in λ 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 a new phage. This phage was λ0550
It's named 1.

The phage particles were dialyzed against a phage suspension buffer containing 50% formamide to obtain λ08501 phage DNA. Phage λ03501 DNA is 46. O
The physical map (restriction enzyme cleavage map) of the Kb size is shown in Figure 1. Here, the white part is derived from vector λL47, and the diagonally shaded part is a chromosomal DNA fragment. All abbreviations are restriction enzymes.

The λ03501 phage DNA obtained here was dissolved in Tris-HCl/EDTA buffer, restriction enzyme Nru I was added, and it was completely digested at 37°C. Agarose the decomposed product
After gel electrophoresis, a 3.5 Kb chromosomal DNA fragment was separated and obtained, and recloned using plasmid PUC18. Plasmid pUc1g (manufactured by Zenshuzo Co., Ltd.) is 2.7 Kb, has ampicillin resistance (Apr), and is cleaved at one site by the restriction enzyme Sma I.

After treating pUclg cut at one site with 5IlaI with alkaline phosphatase (manufactured by Zenshuzo Co., Ltd.), the above λ08
3.5 Kb chromosomal DNA obtained from phage 501
Mix the pieces.

T4 DNA ligase was added for ligation. Using this treatment solution and using Escherichia coli JM109 as a host, the Lederberg and Cohen method (Lederberg, Cohen) was used.
rberg, E, M.

and Cohen, S. N., J. Bact.
The plasmid was introduced by Eriol, 119.1072° (1974)).

The physical map of the 3.5 Kb chromosomal DNA fragment obtained above, that is, the 3.5 Kb Nru I-Nru I fragment, is shown in FIG.

The transformed bacterial cells obtained by introducing the plasmid were treated with a selection medium of 1% soluble starch and 50 μg of ampicillin.
/mQ, L medium containing 1.5% agar (10 g of bactodrypton, 5 g of yeast extract, 5 g of sodium chloride, 1 Ω of water)
, pH 7.0) at 37°C to obtain an ampicillin-resistant strain. This ampicillin-resistant strain was treated with ampicillin 50
The cells were cultured for 24 hours at 37° C. in L medium containing μg/mQ and 1% soluble starch, and the presence or absence of maltopentaose-generating enzyme activity in the culture solution was examined by thin layer chromatography.

Thin film chromatography uses n-butanol/n
- 60℃5 in a solvent system of propatool/water (315/4)
After performing upward development twice, the presence or absence of maltopentaose was detected by spraying with 20% methanol sulfate, and a maltopentaose-producing enzyme-producing strain was successfully isolated.

The resulting maltopentaose-producing enzyme-producing strain was isolated, cultured at 37°C in a medium containing 50 μg/mQ of ampicillin, the culture solution was centrifuged to collect the bacteria, and after washing, the isolated bacterial cells were Alkaline extraction method (Bironboim,
H., C., and Doly, J., Nuclei.
c Ac1dsRes,, ? , 1513. (19
79)) to obtain a new plasmid. This plasmid was named plasmid pO9201.

Plasmid pOS201 has a size of 6.2 mm and its physical map is shown in FIG. Here, the white part is derived from vector pLlc18, and the diagonal line part is derived from chromosome D.
It is an NA fragment part, and each abbreviation is a restriction enzyme.

Escherichia coli JM109-pOS201 transformed with plasmid pOS201 (Escherich
ia coli 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 starting point) is shown in FIG.

Figure 6 shows the D around the maltopentaose-generating enzyme gene.
FIG. 2 is a diagram illustrating the NA sequence and amino acid sequence. In the figure, the signal is A opposite to methionine (position -41)
It starts from ATG (-69 position) corresponding to TG (-123 position) and/or methionine (-23 position II), and the structural gene of the maltopentaose-forming enzyme corresponds to valine (position 1). AAG (177) starting from GTG (l position) and corresponding to lysine (591 position)
1 position).

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

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

FIG. 9 shows the amino acid sequence of the maltopentaose-generating enzyme gene containing a signal, and the signal break points are A (alanine) at position 41 and V (valine) at position 42. expected to be between

New plasmid ρ05201DN prepared as above
Cut A with Xho B (manufactured by Takara Shuzo Co., Ltd.) to obtain fragments o and b at 75. This fragment was labeled with 32P and subjected to Southern hybridization (Southern,
E, M,, J, Mo1. Biol.

98, 503. (1975)). Although it did not hybridize with Escherichia coli chromosomal DNA at all, a b band was found at 0 and 75 that specifically hybridized with Pseudomonas sp. strain 0-8940 chromosomal DNA and λ03501 DNA. This confirmed that the cloned DNA was derived from Pseudomonas sp. strain 0-8940.

Example 2 Construction of ρ03321-CE (1) Construction of pOS320 5ph1-K containing maltopentaose-generating enzyme gene
pu I 2. IKb DNA fragment to pUc1
Plasmid pOS201 (Example 1, 2nd
) was cut with sph l and the protruding end was cut with Mung Bea.
Smoothed by n Nuclease, Klenov f
ragment and then cut with pnl to contain the maltopentaose-generating enzyme gene. 2. IKbDN
Fragment A was obtained.

On the other hand, pBR-AN3 (a plasmid vector with a multi-cloning site introduced into pBR322) was
Cut with Hl, blunt the protruding ends with Klenow fragment, cut with Kpn I, and incubate with Bacter.
ial Alkaline Phosphatase (B
2. AP) processed and containing the maltopentaose-generating enzyme gene described above. After cutting the IKb DNA and ligating with T4 ligase, E. coli JM109 was transformed.

As shown in Figure 12, the resulting plasmid pOS320 has the BamHI site regenerated at the ligation site, and the DNA fragment containing the maltopentaose-generating enzyme gene is the BamH site.
It becomes possible to collect the pn I D N A fragment in I-.

(2) Construction of ρ05321-CE Cut pOS320 with BamHI and cut the protruding end with Hun.
Smooth with gBean Nuclease and use leno
2.1 which contains the maltopentaose-generating enzyme gene after repairing with w fragment and cutting with Hind m.
) (bDNA fragment was obtained.

In addition, pKK223-3 (manufactured by Pharmacia) was
After cutting with RI, the protruding ends were blunted with Klenow frag+ent, cut with Hind m, treated with BAP, and ligated with the above DNA fragment using T4 ligase.
E. coli JM109 was transformed. As shown in Figure 13, the resulting plasmid pOS321-CE has the EcoRI site at the ligation site regenerated and contains the maltopentaose-generating enzyme gene downstream of the tac promoter.2. This becomes an expression plasmid in which the IKb DNA fragments are ligated. JM109-pOS321-G as a transformed strain
E (FERM P-11672) was obtained.

Example 3 Linking the maltopentaose-generating enzyme gene to the tac promoter using synthetic NA (1) Construction of pOs3410 A synthetic NA as shown in FIG. 15 (a) was designed.

This is Hae IIIJ, which is 19 bases from the start codon ATG of the G5-generating amylase gene (maltopentaose-generating enzyme gene, sometimes abbreviated as G5 any).
This is the base sequence up to the top and an EcoRI site added to the 5th side. Furthermore, ρ03320 is Hae II,
A 250 bp fragment (b) obtained by digestion with BclI and a 1 and 7 Kb DNA fragment (c) obtained by digestion with the same BclI and 1 (ind III) were prepared.The vector was pKK223-3 ( d) using EcoR
I,) BAP treatment was performed after cutting with lindm (
14, 15).

As shown in Figure 14, the above four types of DNA fragments (
After ligating E. coli J old 09, transformants having G5-generating amylase activity were selected. Confirm the 5 sequence of the junction part of plas++id held by this recombinant, and
It was named O53410.

(2) Construction of PO53414 After cutting PO53410 with EcoRI, Klenow
The protruding ends are blunted using fragment, and 5elf
ligation L/ta.

E. coli JM109 was transformed with this DNA. In the resulting plasmid, EcoRI 5it
e disappeared and Vsp I 5ite was newly generated (Fig. 14). The plasmid thus obtained was used as PO534.
It was named 14.

(3) Construction of PO83406 and PO33412 PO5
After cutting 3410 with EcoRI, Mung Bean
The protruding ends were blunted with Nuclease. Se this
lfligation L/, E, coli JM1
09 was transformed. The resulting plasmid was named PO53406. On the other hand, EcoRI 5ite
Hpa 1 linker was introduced into the blunted DNA, and E
The plasmid obtained by transforming E. coli JM109 was named PO53412 (Fig. 14).

Example 4 Construction of PO53408 As shown in FIG. 16, PO53410 was cut with EcoRl and the protruding part was
2. Blunted with se, repaired with Klenoiz fragment, cut with Hind m, and contains the G5-generating amylase gene. A DNA fragment of OKb was obtained. pKK23
3-2 (tac Promoter, ori: pB
R322) was cut with NcoI, and the protruding end was cut with Mung.
After smoothing with Bean Nuclease, )find m
It was cut and treated with BAP. Ligation of both DNAs
L/, E. coli JM109 was transformed and PK
K233-G5 was obtained. pKK233-G5 to Vsp
A 3.2 kb DNA fragment containing the 110 region of the tac promoter and the 65-generating amylase gene was obtained.

On the other hand, vector pKK233-3 was cut with Vsp I,
3. Contains one 35 region of the ori and tac promoters.
A 4kb DNA fragment was obtained. Ligate both DNA fragments
ion L/, E.

E. coli JM109 was transformed to obtain PO53408 (Fig. 17) (the 5 sequence of the junction part of PO33408 is the same as pKK 233-G5).

The transformants obtained in this way are Escherichi
acoli JM109-PO33408, and it was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology (FE
RM P-11673).

Example 5 Production of maltopentaose-generating enzyme (1) Escherichia coli JM 109-pOS201 (FER
N P-10973), L containing Ampicirin
Liquid medium (tryptone 1g, yeast extract 0.5g, N
aCu 0.5g, Ampicirin 50μg/m
12, 100 ml of water, pH 7.0) 3 rnQ was cultured at 37° C. for 12 hours, and this culture solution was used as a seed culture.

Seed culture 1 vaQ was transferred to L liquid medium containing ampicillin and IPTG (isopropyl-β-D-thiogalactopyranoside) (1 g of tryptone, 0.5 g of yeast extract, Na
Cf1 O, 5 g, ampicillin 50 p g/mQ,
IPTG 100+on+ole, water 100mQ, p
H7.0) and cultured at 37°C for 24 hours, the culture supernatant fraction and cell surface fraction were obtained by centrifugation. After suspending the bacterial cell fraction in 100 mQ of Tris-hydrochloric acid/calcium 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 mQ of crude. It was made into an enzyme solution. Ammonium sulfate is added to the crude fermentation solution obtained at a low temperature of 4° C., and the fractions precipitated at 0.2 to 0.5 saturation are collected and dissolved in 10 mM phosphate buffer (pH 7.5). This enzyme solution was dialyzed overnight in the same buffer. Then DEAE
- It was purified by ion exchange chromatography using Toyopearl 650M and gel filtration chromatography using Toyopearl HW55S, and a sample showing a single band electrophoretically could be obtained. Additionally, Asahipa
Purification was performed by high performance liquid chromatography using k ES-52ON (manufactured by Asahi Chemical Industries, 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 an oligosaccharide below maltooctaose is used as a substrate.

G8 → G, +G.

C7 → G, +G.

G6 → Gs 101 G, → G3 102 G, → G2 10 G2 G3, G2: Does not work (However, G4 nigercose, G2 = maltose, G,:
Maltotriose, G4: Maltotetraose, G:
Maltopentaose, G6: maltohexaose, C7
: represents maltoheptaose, Gll: represents maltooctaose, respectively. (2) Substrate This enzyme produces maltopentaose by acting on various starches such as amylose, soluble starch, potato, sweet potato, corn, waxy corn, barley, wheat, rice, tapioca, and sago.

(3) Optimum pH for action The reaction solution composition was as follows: Substrate (2% reduced soluble starch solution) 0.5++++
1 Various buffer solutions (0,1M) (acetate buffer, phosphate buffer, sodium carbonate buffer)
0.4mQ enzyme solution (8II/
mQ) 0.1 engineering Q The above composition solution was reacted at 45° C. for 15 minutes, the reducing power was measured, and the results are shown in FIG. 10, with the highest value being expressed as 100.

As is clear from the figure, the optimal P)I of this enzyme is 6.0.

(4) Optimal temperature for action The reaction solution composition was as follows: Substrate (2% reduced soluble starch solution) 0.5+nQO
0IN phosphate buffer (pH 6,5) 0.4
mff enzyme solution (8IU/mfl)
O, 1 mff The above composition solution is heated at various temperatures from 20 to 70°C.
The reducing power was measured after reacting for 5 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
It is ℃.

(5) Molecular Weight The molecular weight of this enzyme obtained by 5DS-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) Production of maltopentaose-generating enzyme Escherichia coli JM
109-PO5321, CE (FERM P-116
72) in 3 mQ of L medium containing ampicirin.
The cells were cultured at ℃ for 12 hours, and this culture solution was used as a seed culture. 1 ml of seed culture was added to a medium containing ampicillin and IPTG (
Tryptone 1g, yeast extract 0.5g, NaC10,
5g, ampicillin 50μg, IPTG 100mmo
le, water 100m12. pi17.0), 37
After culturing at °C for 24 hours, a culture supernatant fraction and a bacterial cell fraction were obtained by centrifugation. The bacterial cell fraction was added to 100ml of Tris-HCl/acetate buffer.
After suspending in 12% of the culture, the cells were disrupted by ultrasonic waves, and the supernatant obtained by centrifugation was mixed with the culture supernatant fraction.
It was made into mN crude enzyme solution.

0.1 μQ of this crude enzyme solution, 2% soluble starch (Merc
K) 0.5μu, 0.1M phosphate buffer (pH 6,
5) 0.4 uQ was mixed and reacted at 45°C for 10 minutes, and the reducing power was measured by the Somogyii-Nelson method. As a result, it showed an activity of 20IU/n+9 (
IU is the enzyme activity that cleaves 1 μmole of a-1,4 glucoside bond per minute).

Example 7 Production of maltopentaose-generating enzyme (3) Production of maltopentaose-generating enzyme Escherichia coli J
M109-pOS3408 (FERN P-11673
) 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. Seed culture 1
m12 in L medium containing ampicillin and IPTG (1 g of tryptone, 0.5 g of yeast extract, NaCQo,
5 g, ampicillin 50 μg/mQ, IPTG 0
．． 1mM, water 100mQ, pH 7,0), 37
After culturing at °C for 24 hours, a culture supernatant fraction and a bacterial cell fraction were obtained by centrifugation. The bacterial cell fraction was added to 100ml of Tris-HCl/acetate buffer.
After suspending in A, the cells were disrupted by ultrasonication, and both supernatants obtained by centrifugation were mixed with the culture supernatant fraction, and
This was used as a crude enzyme solution of Q.

0.1 μQ of this crude enzyme solution, 2% soluble starch (Merck
) 0.5 μQ, 0.1 M phosphate buffer (pH 6,5
) 0.4 μQ was mixed and reacted at 45°C for 10 minutes.
The reducing power was measured by the mogyii-Nelson method. The results showed an activity of 50 IU/m12 (I IU
is the enzyme activity that cleaves !-1.4 glucosidic bonds in 1 μmole per minute.

(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 novel enzyme previously unknown. Therefore, according to the present invention, maltopentaose can be produced in large quantities by utilizing the present enzyme.

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

[Brief explanation of drawings]

Figure 1 shows the physical map (restriction enzyme cleavage map) of phage λ03501, Figure 2 shows the physical map of plasmid pOS201, and Figure 3 shows the physical map of the Nru I-Nru I 3.5 Kb fragment inserted into plasmid pOs201. The arrows in FIGS. 2 and 3 indicate the coding region of maltopentaose-producing amylase. 1 to 3 in Fig. 4 are D of the Nru I-Nru I fragment.
5 shows the entire DNA sequence and a portion of the amino acid sequence (signal and enzyme genes) of FIG. 4. 1 to 8 in FIG. 6 illustrate the DNA and amino acid sequences surrounding the maltopentaose-generating enzyme gene. 1 to 2 of FIG. 7 and FIG. 8 illustrate the DNA sequence and amino acid sequence of the structural gene of the maltopentaose-generating enzyme, respectively. FIG. 9 diagrammatically shows the amino acid sequence of the maltopentaose-generating enzyme gene containing a signal, and the arrow in the figure indicates the predicted signal cleavage point. Figures 10 and 11 are graphs showing the optimal pH and temperature of this enzyme, and in Figure 10, 〇-〇 is 0.
．． 1M acetate buffer, Δ-Δ represents 0.1M phosphate buffer, and Rollo represents 0.1M sodium carbonate buffer, respectively. Figure 12 is a diagram of the construction of plasmid pOS321-CE;
FIG. 13 shows a physical map of the plasmid thus obtained. Figure 14 shows plasmids PO33410, PO33414,
This is a construction diagram of 3406 and 3412, and Figure 15 is
(a) to (a) used for constructing plasmid pOS3410
D) Represents four types of DNA fragments. (a) Synthetic NA fragment (b), (c) 250bp and 1 originating from pOS320
．． 7 Kb fragment (d) Plasmid pKK223-3 Figure 16 is a diagram of the construction of plasmid pOS3408.
Figure 7 shows the physical map of the plasmid thus obtained.

Claims (16)

[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 includes the base sequence shown in FIG. 6 or a base sequence encoding an 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) DN containing a maltopentaose-generating enzyme gene containing a nucleotide sequence encoding the amino acid sequence shown in FIG.
A. (7) DNA containing the maltopentaose-generating enzyme gene
and a vector fragment. (8) The phage and/or plasmid according to claim 7, which is shown in the restriction enzyme cleavage map of FIG. 1, FIG. 2, or FIG. 3. (9) pOS321-CE or pOS340 where the plasmid is shown on the restriction enzyme cleavage map in Figure 13 or Figure 17
8. The plasmid according to claim 7, wherein the plasmid is 8. (10) The donor microorganism is Pseudomonas
The DNA or gene according to any one of claims 1 to 9, which is a microorganism belonging to the genus Monas. (11) The donor microorganism is Pseudomonas sp.
seudomonas. The DNA or gene according to claim 10, which is sp) KO-8940. (12) A transformed microorganism obtained by introducing the phage or plasmid according to any one of claims 7 to 9 into a host microorganism. (13) The transformed microorganism is Escherichia coli (Esc
13. The transformed microorganism according to claim 12, which is a transformed microorganism. (14) The transformed microorganism is Escherichia coli (Esc
herichia coli) JM109-pOS321
-CE (FERMP-11672) or JH109-
The transformed microorganism according to claim 13, which is pOS3408 (FERMP-11673). (15) A method for producing a maltopentaose-producing enzyme, which comprises culturing the transformed microorganism according to any one of claims 12 to 14 and collecting the maltopentaose-producing enzyme from the culture. (16) 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 optimum pH of this enzyme is 6.0. (C) The optimum temperature of this enzyme is 37 to 45°C.