JPH07322880A - Enzyme-encoding DNA, recombinant DNA containing the same, and transformant - Google Patents

Abstract

PURPOSE:To obtain the subject DNA, useful for sweetening a food and drink without anxiety about coloring and deteriorating and capable of coding an enzyme producing a glucide having a trehalose structure from a starch saccharide. CONSTITUTION:This DNA is capable of coding an enzyme producing a nonreducing glucide having a trehalose structure at the terminal from a reducing starch saccharide having >=3 polymerization degree, e.g. a DNA fragment of 2316 base pairs having a base sequence expressed by the formula. The exemplified DNA is obtained by searching for a chromosome of Rhizobium sp. M-11 (FERM BP-4130) using an oligonucleotide chemically synthesized based on a partial amino acid sequence of the highly purified enzyme M-11 as a probe.

Description

Detailed Description of the Invention

[0001]

This invention relates to a glucose polymerization degree of 3
A novel DN encoding an enzyme that produces a non-reducing sugar having a trehalose structure at the end from the reducing starch sugar
A, a recombinant DNA containing the DNA, and a transformant.

[0002]

BACKGROUND OF THE INVENTION Trehalose is a disaccharide in which two glucose molecules are linked to each other by reducing groups, and is naturally present in trace amounts in bacteria, fungi, algae, insects and the like. Since trehalose does not have a reducing group in the molecule, it does not cause a browning reaction even when heated in the presence of amino acids, and has an advantage that foods and drinks can be sweetened without fear of coloring or deterioration. However, it is difficult to obtain a desired amount by the conventional production method, and it has hardly been actually used for sweetening foods and drinks.

The conventional production methods are roughly classified into a method of utilizing microbial cells and a method of allowing a complex enzyme system to act on sugars. The former method is disclosed in JP-A-50-154485.
As can be seen in the publications, etc., microorganisms such as bacteria and yeast are grown in a nutrient medium, and trehalose is collected from the bacterial cells in the culture. On the other hand, the latter method, as seen in JP-A-58-216695,
Maltose is used as a substrate, and a complex enzyme system composed of maltose phosphorylase and trehalose phosphorylase is allowed to act on this, and the produced trehalose is taken out of the system. Although the former method is relatively easy for the microorganisms to grow, it requires a series of complicated steps to collect trehalose from the cells, and the trehalose contained in the cells is 15% (w / w). There was a problem that it was scarce. In the latter method, although trehalose itself is relatively easy to separate, the reaction itself is an equilibrium reaction by two kinds of enzymes, and the equilibrium is always inclined to the glucose phosphate side. In principle, it was difficult to increase the yield of trehalose by reacting with the above reaction.

In view of such a situation, the present inventor conducted an intensive search for an enzyme that produces a sugar having a trehalose structure from starch sugar, and found that Rhizobium species M-1
Microorganisms such as No. 1 and Arthrobacter species Q36 produce a completely unknown enzyme which is unknown in the past, which produces a non-reducing sugar having a trehalose structure at the end from a reducing starch sugar having a glucose polymerization degree of 3 or more. It has been found. Around this finding, this non-reducing sugar is
Similarly, it was revealed that another enzyme produced by Rhizobium species M-11 and Arthrobacter species Q36 hydrolyzes trehalose and glucose and / or maltooligosaccharides almost quantitatively. By using these enzymes in combination, a desired amount of trehalose can be obtained relatively easily from starch as a raw material, and it is expected that the above-mentioned problems relating to trehalose will be solved completely. However, Rhizobium species M
Neither -11 nor Arthrobacter sp. Q36 has a sufficient ability to produce the enzyme, and if a large-scale production of trehalose or a non-reducing sugar having a trehalose structure at the end is attempted, a large amount of microorganism must be cultured. There is a problem of not becoming.

On the other hand, recent advances in recombinant DNA technology are remarkable. Nowadays, even for an enzyme whose entire amino acid sequence has not been elucidated, the gene encoding it can be isolated, and if its nucleotide sequence can be elucidated, recombinant DNA containing the DNA encoding that enzyme can be prepared and By culturing a transformant obtained by introducing it into cells of microorganisms and animals and plants, it has become possible to obtain a desired amount of enzyme relatively easily. In view of such a situation, it is urgent to find out the genes encoding both enzymes and elucidate their nucleotide sequences.

[0006]

An object of the present invention is to provide a DNA encoding an enzyme that produces a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more. is there.

Another object of the present invention is to provide a recombinant DNA containing the DNA and a vector capable of autonomous replication.

Still another object of the present invention is to provide a transformant obtained by appropriately introducing the recombinant DNA into a host.

[0009]

Means for Solving the Problems The present invention is directed to the above-mentioned first object, which is a DNA encoding an enzyme for producing a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more. Is solved by.

The present invention is directed to the second object, which is a vector capable of autonomously replicating with a DNA encoding an enzyme that produces a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more. A reproducible recombinant DNA comprising

The present invention is directed to the third object, which is a vector capable of autonomously replicating with a DNA encoding an enzyme which produces a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more. This is solved by a transformant obtained by appropriately introducing a replicable recombinant DNA containing

[0012]

The DNA of the present invention is made into a replicable recombinant DNA by inserting it into an appropriate vector capable of autonomous replication, and this recombinant DNA does not originally produce the enzyme but can be relatively easily propagated. When it is introduced into a host capable of producing a transformant, the production of the encoded enzyme is expressed.

The recombinant DNA of the present invention, which originally does not produce the enzyme, is transformed into a transformant by introducing it into a host that can be relatively easily grown, and the transformant is cultured to obtain a code. Express the production of said enzyme.

The transformant of the present invention produces the enzyme when cultured.

The present invention will be described below with reference to experimental examples, examples, etc. The present invention produces a non-reducing sugar having a trehalose structure at the end from a reducing starch sugar having a glucose polymerization degree of 3 or more. It relates to a DNA encoding an unknown novel enzyme. Such an enzyme can be obtained from a culture of Rhizobium species M-11 or Arthrobacter species Q36 (hereinafter referred to as "enzyme M-11" or "enzyme Q36"), and the present inventor uses it as a column. When this enzyme was isolated by combining various purification methods centered on chromatography and its properties and properties were investigated, it was found that its essence is a polypeptide and has the following physicochemical properties. (1) Action A non-reducing sugar having a trehalose structure at its end is produced from a reducing starch sugar having a glucose polymerization degree of 3 or more. (2) Molecular weight about 76,000 to 87,000 daltons (SDS-polyacrylamide gel electrophoresis) (3) Isoelectric point about 3.6 to 4.6 (isoelectric focusing) (4) Optimum temperature pH7 When it is incubated at 0.0 for 60 minutes, it shows an optimum temperature around 35 to 40 ° C. (5) Optimum pH When incubated at 40 ° C. for 60 minutes, the optimum pH is shown in the vicinity of pH 6.4 to 7.2. (6) Thermostability When incubated at pH 7.0 for 60 minutes, it is stable up to around 35 to 40 ° C. (7) pH stability When incubated at 25 ° C for 16 hours, it is stable up to about pH 5.5 to 11.0.

An enzyme having such physicochemical properties has not been known yet, and it is considered to be a novel substance. In addition, Rhizobium species M-11 was separated from the soil of Okayama City, Okayama Prefecture, and since December 24, 1992, the Ministry of International Trade and Industry, the Ministry of Industry and Industry, located at 1-3-1 Higashi Tsukuba City, Ibaraki Prefecture.
It has been deposited under the deposit number "FERM BP-4130" at the Patent Microorganism Depositary Center of the Institute of Biotechnology, Institute of Biotechnology. On the other hand, Arthrobacter species Q36 was separated from the soil of Soja City, Okayama Prefecture, and after June 3, 1993, the deposit number "FERM BP
-4316 ”. Japanese Patent Application No. 5-349216 by the same applicant discloses in detail the properties and properties of the enzyme as well as the mycological properties of both microorganisms.

The present inventor has highly purified the enzyme M-11.
Of the Rhozobium species M-11 using the oligonucleotide chemically synthesized based on the partial amino acid sequence as a probe, and has the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing. , 316 base pairs were obtained. Then, when the nucleotide sequence was decoded, the enzyme M-11 was identified as shown in SEQ ID NO: 1 in the sequence listing.
It was found to be composed of 772 amino acids.

On the other hand, an oligonucleotide chemically synthesized based on the partial amino acid sequence of the enzyme Q36 is used as a probe,
Chromosomal DNA of Arthrobacter species Q36
Was similarly searched, DNA consisting of 2,325 base pairs having the base sequence shown in SEQ ID NO: 4 in the sequence listing.
Fragments were obtained. When the nucleotide sequence was decoded, it was revealed that the enzyme Q36 was composed of 775 amino acids and had the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

A series of steps leading to the elucidation of the nucleotide sequences and amino acid sequences shown in SEQ ID NOS: 1 to 4 in the sequence listing is summarized as follows. (1) separating the enzyme from the culture of the donor microorganism,
Highly purified. After partially hydrolyzing the purified enzyme with a protease, two types of peptide fragments were isolated from the hydrolyzate, and the amino acid sequences thereof were determined. (2) Separately, chromosomal DNA was isolated from the microorganism of the donor microorganism, purified, and partially digested with a restriction enzyme to collect a DNA fragment of about 3,000 to 7,000 base pairs. This DNA fragment was ligated with a plasmid vector that had been cleaved in advance with a restriction enzyme with a DNA ligase to prepare a recombinant DNA. (3) A transformant is prepared by introducing recombinant DNA into Escherichia coli and performing colony hybridization using an oligonucleotide chemically synthesized based on the partial amino acid sequence as a probe to obtain a transformant containing the DNA encoding the enzyme. Selected. (4) Recombinant DNA was collected from the transformant, annealed with the primer, extended with the action of DNA polymerase, and the resulting complementary strand DNA was analyzed by the dideoxy chain terminator method to determine the nucleotide sequence. Were determined. Then, the amino acid sequence deduced from the base sequence and the partial amino acid sequence are compared,
It was confirmed that the base sequence encoded the enzyme.

The amino acid sequence encoded by the gene of the donor microorganism is as shown in SEQ ID NO: 1 or 2 in the sequence listing. The DNA of the present invention has the amino acid sequence as shown in SEQ ID NO: 1 or 2 in the sequence listing. Of course, what is encoded includes those having an amino acid sequence homologous thereto. That is, recombinant DNA
Advances in technology have made it possible in the art to substitute one or more of its constituent amino acids with other amino acids relatively easily without substantially altering the action of the enzyme. Further, even if the same DNA, depending on the host into which it is introduced, the components / composition of the nutrient medium used for culturing the transformant containing the DNA, the culture temperature / pH, etc.,
One or more amino acids near the N-terminal in the amino acid sequence of SEQ ID NO: 1 or 2 in the sequence listing are retained, although the desired enzymatic action is retained due to modification after DNA expression by an enzyme in the host. Deleted or 1 at the N-terminus
One or two or more amino acids may be newly added to produce a mutant. In view of such state of the art, the enzyme referred to in the present invention is not limited to one having the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing as it is,
Even if one or more of the amino acids in the amino acid sequence is replaced with another amino acid, or is a mutant in which the amino acid sequence is deleted or added, it has a trehalose structure at the end from a reducing starch sugar having a glucose polymerization degree of 3 or more. As long as it produces a non-reducing sugar, it is included.

Furthermore, in the art, due to the degeneracy of the genetic code, one or two or more bases in DNA can be replaced with another base without changing the encoded amino acid sequence. Thereby, the DN of the present invention
A not only has the base sequence shown in SEQ ID NO: 3 or 4 as it is, but also encodes an enzyme having the amino acid sequence shown in SEQ ID NO: 1 or 2 or a homologous variant thereof. As long as there is,
Based on the degeneracy of the genetic code, those in which one or more bases are replaced with other bases are also included.

Further, when the present recombinant DNA technology is used, generally, if the base sequence from the 5'end is determined, the complementary base sequence is uniquely determined. Therefore,
The DNA of the present invention also includes those having a base sequence complementary to any of the above base sequences. In addition,
In order for the DNA of this invention to actually express the production of the enzyme in a host, one or two or more of the bases in the base sequence encoding the enzyme or a homologous variant thereof may be appropriately replaced with another base. Needless to say.

The DNA of the present invention is as described above. Whether the DNA of the present invention is naturally derived or artificially synthesized, as long as it has the sequence as described above. It doesn't matter. As a natural source, for example, Rhizobium species M-11 (FE
RM BP-4130), Arthrobacter species Q36 (FERM BP-4316), Brevibacterium heroborum (ATCC11822), Flavobacterium aquatile (IFO3772), Micrococcus luteus (IFO3064), Micrococcus roseus (FERO). ATCC186), Kurtobacter citreum (IFO15231), Mycobacterium smegmatis (ATCC19420), and Rhizobium, Arthrobacter, Brevibacterium, including Terrabacter tumescens (IFO12960),
Examples include microorganisms of the genus Flavobacterium, genus Micrococcus, genus Curtobacterium, genus Mycobacterium, and genus Terrabaca, and the gene containing the DNA of the present invention can be obtained from the cells of these microorganisms. That is, such a microorganism is inoculated into a nutrient medium and cultured under aerobic conditions for about 1 to 3 days, and then cells are collected from the culture to obtain lysozyme or β-.
By treating with a cell wall lysing enzyme such as glucanase or ultrasonic waves, the gene containing the DNA is eluted outside the cells. At this time, a protein hydrolase such as a protease may be used in combination with the cell wall lysing enzyme, or a surfactant such as SDS may coexist or freeze-thaw when the bacterial cells are subjected to ultrasonic treatment. The target DNA can be obtained by applying to the treated product thus obtained, for example, phenol extraction, alcohol precipitation, centrifugation, protease treatment, ribonuclease treatment and the like generally used in the art.

On the other hand, in order to artificially synthesize the DNA of the present invention, for example, it is chemically synthesized based on the nucleotide sequence shown in SEQ ID NO: 3 or 4 in the sequence listing, or is shown in SEQ ID NO: 1 or 2 in the sequence listing. DN encoding an amino acid sequence
Recombinant DN by inserting A into an appropriate vector capable of autonomous replication
A may be appropriately introduced into a host, the transformant obtained may be cultured, cells may be separated from the culture, and a plasmid containing the DNA may be collected from the cells.

By the way, the present invention relates to a replicable recombinant DNA which, when originally introduced into a cell of a microorganism or animal or plant, which does not originally produce the enzyme but can be relatively easily propagated, expresses the production of the enzyme. It is also a thing.
Such recombinant DNA usually comprises a vector capable of autonomous replication with the above-mentioned DNA, and if DNA is available, it can be generally prepared relatively easily by a general recombinant DNA technique. Examples of such vectors include:
pBR322, pUC18, Bluescript I
I SK (+), pUB110, pTZ4, pC19
4, plasmid vectors such as pHV14, TRp7, YEp7, pBS7, λgt · λC, λgt · λB, ρ
Phage vectors such as 11, φ1 and φ105 are listed. Among them, for expressing the DNA of the present invention in E. coli, pBR322, pUC18, Bluescript
II SK (+), λgt · λC and λgt · λB are preferred, while pUB11 for expression in B. subtilis
0, pTZ4, pC194, ρ11, φ1 and φ105
Is preferred. pHV14, TRp7, YEp7 and p
BS7 is useful for growing recombinant DNA in more than one host.

For inserting the DNA of the present invention into such a vector, a method generally used in the art is generally used.
Specifically, first, the gene containing the DNA of the present invention and the vector capable of autonomous replication are cleaved with a restriction enzyme and / or ultrasonic waves, and then the generated DNA fragment and the vector fragment are ligated. Restriction enzymes that act specifically on nucleotides to cleave genes and vectors, especially type II restriction enzymes, specifically Sau 3AI, Eco RI, H
ind III, Bam HI, Sal I, Xba
I, Sac I, Pst I, etc.
It becomes easy to ligate the fragment and the vector fragment. DNA
To ligate the fragment and the vector fragment, if necessary, both may be annealed, and then the DNA ligase may be allowed to act in vivo or in vitro. Recombinant D thus obtained
NA can be indefinitely replicated by appropriately transforming it into a host to prepare a transformant and culturing the transformant.

The recombinant DNA according to the present invention is E. coli,
It can be introduced into an appropriate host microorganism such as Bacillus subtilis, actinomycete and yeast. When the host is E. coli, the host may be cultured in the presence of recombinant DNA and calcium ions, while when the host is Bacillus subtilis, the competent cell method or the protoplast method may be applied. In order to clone the transformant, a colony hybridization method is applied, or the transformant is cultured in a nutrient medium containing a reducing starch sugar having a degree of glucose polymerization of 3 or more, and a non-reducing sugar having a trehalose structure at the end of the starch sugar. You can select the one that produces quality.

When the transformant thus obtained is cultured in a nutrient medium, it produces the enzyme inside and outside the cells. The nutrient medium usually contains carbon sources, nitrogen sources, minerals,
If necessary, a general liquid medium supplemented with micronutrients such as amino acids and vitamins is usually used, and examples of individual carbon sources include starch, starch hydrolysates, glucose, fructose, and sugars such as sucrose. Also, as a nitrogen source,
Examples thereof include nitrogen-containing inorganic or organic substances such as ammonia or ammonium salt, urea, nitrate, peptone, yeast extract, defatted soybean, corn steep liquor and meat extract. The transformant was inoculated into such a nutrient medium, and while maintaining the nutrient medium at a temperature of 25 to 65 ° C. and a pH of 2 to 8,
Culturing under aerobic conditions such as aeration and stirring for about 1 to 6 days gives a culture containing the enzyme. This culture can be used as an enzyme preparation as it is, but usually before use, if necessary, the cells are disrupted by ultrasonic waves or cell wall lysing enzyme, and then the enzyme is removed by filtration or centrifugation. It is separated from the disrupted cells and purified. For purification, a generally-used general method for purifying an enzyme can be adopted, and examples thereof include concentration, salting out, dialysis, fractional precipitation, gel filtration chromatography, and ion exchange chromatography in a culture in which cells or disrupted cells are removed. One or a combination of two or more types such as chromatography, hydrophobic chromatography, affinity chromatography, gel electrophoresis, isoelectric focusing may be applied.

As described above, the enzyme has a remarkable effect not found in conventional enzymes, that is, it produces a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more. . Therefore, the enzyme produced by the transformant of the present invention is maltotriose, maltotetraose, maltopentaose, maltohexaose obtained by treating starch such as starch or amylopectin, amylose with acid and / or amylase. , Α-glucosyltrehalose, α-maltosyltrehalose, α-maltotriosyltrehalose having a trehalose structure at the end by acting on a starch hydrolyzate containing a series of maltooligosaccharides having a glucose polymerization degree of 3 or more. , Α-maltotetraosyltrehalose and other corresponding non-reducing sugars are produced in good yield and efficiently. And, these non-reducing sugars are disclosed in Japanese Patent Application No. 5-34
When trehalose-releasing enzyme disclosed in No. 0343 is applied, trehalose is produced almost quantitatively.

Next, a series of experiments carried out to elucidate the physicochemical properties of an enzyme that produces a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more will be described.

[0031]

[Experimental Example 1 Preparation of purified enzyme]

[0032]

[Experimental Example 1-1 Purification of Enzyme M-11] Maltose 2.0% (w / v), peptone 0.5% (w / v), yeast extract 0.1% (w / v) were added to a 500 ml Erlenmeyer flask. ),
Liquid medium containing 0.1% (w / v) disodium hydrogen phosphate and 0.1% (w / v) potassium dihydrogen phosphate (pH)
Each 100 ml of 7.0) was sterilized by autoclaving at 120 ° C. for 20 minutes. After cooling, Rhizobium species M-11 was inoculated into the liquid medium in the Erlenmeyer flask, and seed culture was carried out at 27 ° C. for 24 hours under rotary shaking. Separately
20 liters of a liquid medium having the same composition as described above was placed in a 30-liter jar fermenter, and after sterilization, the seed culture solution obtained above was added to 1% (v
/ V) inoculate and maintain the liquid medium at pH 6 to 8
The culture was performed at 0 ° C. for 24 hours with aeration and stirring.

Next, about 18 liters of the above-obtained culture was placed in an ultra-high pressure cell crushing device, the cells were crushed, and about 16 liters of the supernatant collected by centrifugation was added with ammonium sulfate to 20% saturation, After standing at 4 ° C for 1 hour, the precipitate was removed by centrifugation. Ammonium sulfate was added to the resulting supernatant to 60% saturation, and the mixture was allowed to stand at 4 ° C. for 24 hours,
The precipitate was collected by centrifugation, dissolved in a minimum amount of 10 mM phosphate buffer (pH 7.0), dialyzed against 10 mM phosphate buffer (pH 7.0) for 24 hours, and then insoluble matter was removed by centrifugation. . The obtained supernatant was previously equilibrated with a 10 mM phosphate buffer (pH 7.0), a column for ion exchange chromatography "DEAE-" manufactured by Tosoh Corporation.
Toyopearl ”was loaded, and 10 mM phosphate buffer (pH 7.0) was passed through the column under a concentration gradient of sodium chloride increasing from 0 M to 0.5 M. Fractions containing the enzyme were collected from the eluate, dialyzed against 50 mM phosphate buffer (pH 7.0) containing 2 M ammonium sulfate for 10 hours, and then insoluble matters were removed by centrifugation. Then, the supernatant was previously added with 50 mM phosphate buffer (pH 7.
0M) and equilibrated with Tosoh's hydrophobic chromatography column "Butyl Toyopearl" and loaded with 2M
A 50 mM phosphate buffer solution (pH 7.0) was passed through the column under a concentration gradient of ammonium sulfate from 0 to 0 M.
The enzyme-containing fraction was collected from the eluate, and loaded on Toso gel filtration column chromatography column "Toyopearl HW-55", which had been equilibrated with 50 mM phosphate buffer (pH 7.0) in advance, and loaded on the column. A 50 mM phosphate buffer solution (pH 7.0) was passed through, and an enzyme-containing fraction was collected from the eluate. The specific activity of enzyme M-11 thus purified was about 195 units / mg protein and the yield was about 220 units per liter of culture.

Throughout the present invention, the activity of the enzyme is expressed as an activity value (unit) measured by the following method. That is, 4 ml of 50 mM phosphate buffer solution (pH 7.0) containing 1.25% (w / v) maltopentaose was taken, 1 ml of the enzyme solution was added thereto, and the mixture was incubated at 40 ° C. for 60 minutes to react, The reaction solution is heated at 100 ° C. for 10 minutes to stop the reaction. After diluting the reaction solution 10 times with distilled water, the reducing power is measured by the Somogyi-Nelson method. One unit of the enzyme is defined as the amount of the enzyme that reduces the reducing power corresponding to 1 μmol of maltopentaose per minute under the above conditions.

[0035]

[Experimental Example 1-2 Purification of Enzyme Q36] Arthrobacter species Q36 was cultured in the same manner as in Experimental Example 1-1,
When the culture was treated, the purified enzyme Q36 having a specific activity of about 200 units / mg protein was treated at about 295 liters of culture per liter.
Obtained in unit yield.

[0036]

Experimental Example 2 Physicochemical Properties of Enzyme In this experimental example, the purified enzyme obtained in Experimental Example 1 is used as a sample to examine the physicochemical properties of the enzyme.

[0037]

[Experimental Example 2-1] Action 50 mM phosphate buffer solution (pH: 20% (w / v) containing glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose or maltoheptaose as a substrate (pH)
To 7.0), 2 units of the purified enzyme M-11 or purified enzyme Q36 obtained in Experimental Example 1 was added per 1 g of the substrate, and the mixture was reacted at 40 ° C. for 48 hours. After desalting the reaction product by a conventional method, a column for high performance liquid chromatography “WB-T-” manufactured by Wako Pure Chemical Industries, Ltd.
330 ", and the sugar concentration of the eluate was monitored with a Tosoh differential refractometer" RI-8012 type ", and distilled water was passed through the column at a flow rate of 0.5 ml / min at room temperature. The sugar contained in the reaction product was separated. Tables 1 and 2 show the sugar composition when the enzyme M-11 and the enzyme Q36 were added, respectively. The sugars P1 to P5 in the table
Are names of sugars produced by the reaction in ascending order of glucose polymerization degree.

[0038]

[Table 1]

[0039]

[Table 2]

As is clear from the results shown in Tables 1 and 2,
Enzymes M-11 and Q36 form new sugars from reducing starch sugars having a degree of glucose polymerization of 3 or more, such as maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose. However, no new sugar is produced from glucose or maltose having a glucose polymerization degree of less than 3. The sugars produced by the reaction were only the sugars P1 to P5, respectively, and the contents of the sugars P2 to P5 were remarkably high at 85% or more per solid content.

Next, in order to separate the sugars P1 to P5, a strongly acidic cation exchange resin "XT-10 manufactured by Tokyo Organic Chemical Industry Co., Ltd."
16 (Na ⁺ type) ”was packed in three jacketed stainless steel columns having an inner diameter of 2.0 cm and a length of 1 m, and these columns were connected in series. Then, the temperature in the column is set to 55
While maintaining the temperature at 0 ° C., the reaction product containing any of the sugars P1 to P5 was separately loaded onto the column, and then distilled water at 55 ° C. was passed through the column at a flow rate of SV 0.13. Examine the sugar composition of the eluate and find out whether any of the sugars P1 to P5 has a solid content of 97.
Fractions containing more than 100% were collected and pulverized by vacuum drying.
When the reducing powers of the thus purified sugars P1 to P5 were examined by the Somogyi-Nelson method, no substantial reducing power was observed for any of the sugars.

Furthermore, in order to identify the sugars P1 to P5,
50 mg of any of these sugars was taken, dissolved in 1 ml of 50 mM acetate buffer (pH 4.5), 1 unit of glucoamylase was added, and the mixture was incubated at 40 ° C. for 6 hours. When the sugar compositions of the reaction products shown in Table 1 and Table 2 were analyzed by high performance liquid chromatography, Table 3 and Table 4 respectively.
As shown in, glucose and trehalose were detected in all the reaction products. Similarly, β for sugars P1 to P5
-When amylase was allowed to act, the sugars P1 and P2 were not affected by β-amylase, while the sugar P3 contained one molecule of maltose and sugar P1, and the sugar P4 contained 1 molecule.
Molecule maltose and saccharide P2 were given, and saccharide P5 gave two molecules of maltose and saccharide P1.

[0043]

[Table 3]

[0044]

[Table 4]

The results of Tables 3 and 4 show that the sugars P1 to P5
Strongly suggest that it is composed of 1 molecule of trehalose and 1 to 5 molecules of glucose. Further, glucoamylase specifically cleaves α-1,4 bond and α-1,6 bond in maltooligosaccharide, and β-amylase cuts α-1,4 bond in maltooligosaccharide from its end in maltose units. Since it is cleaved, carbohydrate P1
It is assumed that each of P5 to P5 has a structure in which one trehalose residue is bound to the terminal of glucose or a maltooligosaccharide having a glucose polymerization degree of 2 to 5.

Judging from the above results, the sugar P
1 to P5 were respectively identified as α-glucosyltrehalose, α-maltosyltrehalose, α-maltotriosyltrehalose, α-maltotetraosyltrehalose or α-maltopentaosyltrehalose,
This confirms that the enzyme has an action of producing a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more.

[0047]

[Experimental Example 2-2 Molecular Weight] U.K. Laemli, "Nature", Vol. 227, pp. 680-685 (1970)
SDS-polyacrylamide gel electrophoresis of the purified enzyme according to the method reported in
1. The enzyme Q36 has a molecular weight of about 76,000 to 87,
A single band was observed at a position corresponding to 000 Daltons. The molecular weight marker used at this time was myosin (20
10,000 Daltons), β-galactosidase (11
6,250 daltons), phosphorylase B (97,
400 daltons), serum albumin (66,200 daltons) and ovalbumin (45,000 daltons).

[0048]

[Experimental Example 2-3 Isoelectric point] As measured by an isoelectric focusing method, both of the enzyme M-11 and the enzyme Q36 were about 3.6.
The isoelectric points are shown in FIGS.

[0049]

[Experimental Example 2-4 Optimum temperature] A test was carried out by a conventional method under the condition of incubating in 50 mM phosphate buffer (pH 7.0) for 60 minutes, and as shown in FIG. 1 or 2, the enzyme M-11, The enzyme Q36 also showed an optimum temperature around 35 to 40 ° C.

[0050]

[Experimental Example 2-5 Optimum pH] The test was carried out by an ordinary method under the conditions of incubating at 40 ° C. for 60 minutes in 50 mM acetate buffer, phosphate buffer or sodium carbonate-sodium hydrogen carbonate buffer having different pH. As shown in FIG. 3 or FIG. 4, both the enzyme M-11 and the enzyme Q36 had a pH of 6.
The optimum pH was shown in the vicinity of 4 to 7.2.

[0051]

[Experimental Example 2-6 Thermostability] When tested by a conventional method under the condition of incubating in 50 mM phosphate buffer (pH 7.0) for 60 minutes, as shown in FIG. 5 or 6, the enzyme M-11, Both enzyme Q36 was stable up to around 35 to 40 ° C.

[0052]

[Experimental Example 2-7 pH stability] According to a conventional method, a test was carried out under the conditions of incubating at 25 ° C. for 16 hours in 50 mM acetate buffer, phosphate buffer or sodium carbonate-sodium hydrogen carbonate buffer having different pH. 7 or 8
As shown in, both the enzyme M-11 and the enzyme Q36 have pH
It was stable up to around 5.5 to 11.0.

[0053]

[Experimental example 2-8 N-terminal amino acid sequence] When analyzed using a gas phase protein sequencer "470A type" manufactured by Applied Biosystem by a conventional method, the enzyme M
-11 was found to have the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing at the N-terminus.

When analyzed in the same manner, the enzyme Q36 was
It was found to have the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing at the end.

[0055]

[Experimental Example 2-9 Partial Amino Acid Sequence] An appropriate amount of the purified enzyme M-11 obtained in Experimental Example 1-1 was taken and dialyzed against 10 mM Tris-hydrochloric acid buffer solution (pH 9.0) at 4 ° C. for 18 hours.
10 mM Tris-hydrochloric acid buffer (pH 9.0) was added to make the enzyme concentration about 1 mg / ml. Take about 1 ml of this solution, add 10 μg of lysyl endopeptidase, and add 30 μC
The enzyme was partially hydrolyzed by incubating for 22 hours. The hydrolyzate was previously equilibrated with 0.1% (v / v) trifluoroacetic acid containing 16% (v / v) aqueous acetonitrile. Shiseido reverse phase high performance liquid chromatography column "Capsule Pack C18". ],
Then, under a gradient of acetonitrile increasing from 16% (v / v) to 64% (v / v), 0.1% was applied to the column.
(V / v) trifluoroacetic acid was passed through at a flow rate of 0.9 ml / min. And, about 28 minutes after the start of liquid flow or about 40 minutes
Fractions containing the peptide fragments (hereinafter referred to as "peptide fragment A" or "peptide fragment B", respectively) that were eluted after 50 minutes were collected separately, dried under vacuum, and then containing 50% (v / v) aqueous acetonitrile. It was dissolved in 0.1% (v / v) trifluoroacetic acid. After that, the same analysis as in Experimental Example 2-8 revealed that peptide fragments A and B had the amino acid sequences shown in SEQ ID NOs: 9 and 10 in the sequence listing.

Separately, purified enzyme Q36 obtained in Experimental Example 1-2
Is partially hydrolyzed in the same manner as above, and 24% (v /
v) Japan Millipore, equilibrated with 0.1% (v / v) trifluoroacetic acid containing aqueous acetonitrile.
Loaded on a limited-phase reversed-phase high-performance liquid chromatography column "Microbonder Pack C18" at 24% (v
/ V) to 0.1% (v / v) trifluoroacetic acid at a flow rate of 0.9 ml / min under a concentration gradient of aqueous acetonitrile increasing from 44% (v / v) to 44% (v / v). Then, a fraction containing a peptide fragment (hereinafter referred to as “peptide fragment C” or “peptide fragment D”, respectively) that was eluted about 22 minutes or about 40 minutes after the start of the passage of the liquid was collected, vacuum-dried, It was dissolved in 0.1% (v / v) trifluoroacetic acid containing 50% (v / v) aqueous acetonitrile. Subsequent analysis as described above revealed that peptide fragments C and D had the amino acid sequences shown in SEQ ID NOs: 11 and 12 in the sequence listing.

The physicochemical properties of the enzyme M-11 and the enzyme Q36 are as described above. In the following examples, the replicable recombinant DNA and transformant according to the present invention were prepared based on these physicochemical properties. At the same time, the DNA encoding the enzyme contained therein is collected, and its base sequence and amino acid sequence are determined. The techniques themselves used in these examples are well known in the art, for example, J. Sambrook et al., “Molecular Cloning a Laboratory Manual”, 2nd edition, 19
In 1989, it was also described in Cold Spring Harbor Laboratory Press, etc.

[0058]

Example 1 Preparation of Recombinant DNA Containing DNA Derived from Rhizobium species M-11 and Transformant

[0059]

Example 1-1 Preparation of Chromosomal DNA Rhizobium species M-11 was inoculated into Bact Nutrient Broth medium (pH 7.0), and cultivated with shaking at 27 ° C. for 24 hours. The cells were separated from the culture by centrifugation, suspended in TES buffer (pH 8.0), 0.05% (w / v) of lysozyme was added, and the mixture was incubated at 37 ° C for 30 minutes. After freezing the treated product for 1 hour at -80 ° C, add TSS buffer (pH 9.0) and heat to 60 ° C, add TES buffer / phenol mixture, and after ice cooling, collect the supernatant by centrifugation. did. To this supernatant was added 2 volumes of cold ethanol, and the precipitated crude chromosomal DNA was collected.
After dissolution in SC buffer (pH 7.1), ribonuclease and protease were added at 7.5 μg or 125 μg, respectively, and incubated at 37 ° C. for 1 hour to react. Then, a chloroform / isoamyl alcohol mixed solution was added to the reaction product to extract chromosomal DNA, cold ethanol was added, and a precipitate containing the generated chromosomal DNA was collected. The purified chromosomal DNA thus obtained had a concentration of about 1 mg / ml.
Was dissolved in SSC buffer (pH 7.1) so that the resulting solution was frozen at -80 ° C.

[0060]

Example 1-2 Preparation of recombinant DNA pBMT7 and transformant BMT7 Purified chromosome DN obtained in Example 1-1
Approximately 1 ml of solution A is taken and the restriction enzyme Sau 3AI
Approximately 35 units were added and reacted at 37 ° C. for about 20 minutes to partially cut the chromosomal DNA, and then a DNA fragment consisting of about 3,000 to 7,000 base pairs was collected by the sucrose density gradient ultracentrifugation method. Separately, plasmid vector Bluesc
After taking 1 μg of ript II SK (+) and completely digesting it with a restriction enzyme Bam HI by a conventional method,
The DNA fragment was ligated to the vector fragment by adding 10 μg of the DNA fragment obtained above and 2 units of T4 DNA ligase and allowing to stand at 4 ° C. overnight. Then, the obtained recombinant DNA is added to Toyobo's competent cell "Epicuri
30 μl of “An coli XLI-Blue”,
After standing for 30 minutes under ice-cooling, the mixture was heated to 42 ° C, SOC broth was added, and the mixture was incubated at 37 ° C for 1 hour to introduce the recombinant DNA into Escherichia coli.

Next, the transformant obtained above was treated with 5-bromo-4-chloro-3-indolyl-β-galactoside 50.
After inoculating to an agar plate medium (pH 7.0) containing μg / ml and culturing at 37 ° C. for 18 hours, a nylon membrane was placed on the medium, and about 4,400 colonies formed on the medium were placed. It was fixed on a nylon membrane. Separately, based on the sequence represented by the 17th to 21st Pro-Glu-Trp-Glu-Lys in the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing, 5'-CCNGARTGGGAR was separately prepared.
A probe 1 having a base sequence represented by AA-3 ′ was chemically synthesized, labeled with the isotope ³² P, and then hybridized to the transformant colonies immobilized on the nylon membrane, and a remarkable association was observed. Nine types of transformants were selected.

Recombinant DNA was collected from these 9 kinds of transformants by a conventional method, and the SEQ ID NO: 10 in the sequence listing was collected.
16th to 20th Th in the amino acid sequence shown in
5'-ACNGARTTYTGGG chemically synthesized based on the sequence represented by r-Glu-Phe-Trp-Asp
The probe 2 having the nucleotide sequence represented by A-3 'was prepared by EM Southern, "Journal of Molecular Biology", Vol. 98, pp. 503-517 (1975).
Y.) and hybridized according to the method described in (1), and a recombinant DNA showing a remarkable association with probe 2 was selected. Recombinant DNA and transformants selected as described above were respectively designated as "pBMT7" or "BMT7".
I named it.

The transformant BMT7 obtained above was transformed into L-broth medium containing 100 μg / ml of ampicillin (pH 7.
0) and cultured at 37 ° C. for 24 hours under rotary shaking. After completion of the culture, cells were collected from the culture by centrifugation and the recombinant DNA was eluted out of the cells by a usual alkaline method. The processed product was purified by a conventional method and analyzed,
Recombinant DNA pBMT7 consisted of about 9,300 base pairs and had the structure represented by the restriction enzyme map shown in FIG. As shown in FIG. 9, 2, which encodes the enzyme M-11
It was revealed that the DNA consisting of 316 base pairs is located downstream of the cleavage site by the restriction enzyme PstI.

[0064]

Example 1-3 Production of enzyme by transformant BMT7: Maltose 2.0% (w / v), peptone 0.5%
(W / v), yeast extract 0.1% (w / v), disodium hydrogen phosphate 0.1% (w / v), potassium dihydrogen phosphate 0.1% (w / v). Adjust the pH to 7.0, add 50 μg / ml of ampicillin, and add 2 at 120 ° C.
After heat sterilization for 0 minutes and cooling, the transformant BMT7 obtained in Example 1-2 was inoculated and cultivated with rotation shaking at 37 ° C. for 24 hours. The culture was sonicated to disrupt the cells, the insoluble matter was removed by centrifugation, and the enzyme activity in the supernatant was measured. As a result, about 3,000 units of enzyme were calculated per 1 liter of culture. Was produced.

Separately, as a control, Escherichia coli XLI-Blu was used.
The e strain and Rhizobium species M-11 were inoculated into the same liquid medium containing no ampicillin, and in the case of Rhizobium species M-11, the culture and treatment were performed in the same manner as above except that the culture temperature was set to 30 ° C. When the activity of the treated product was measured, the production of the enzyme by Rhizobium species M-11 was about 1,500 units per liter of the culture,
It was significantly lower than that of the transformant BMT7. The Escherichia coli XLI-Blue strain used as a host did not produce the enzyme at all.

Then, the enzyme produced by the transformant BMT7 was purified in the same manner as in Experimental Example 1-1, and its properties and properties were examined. As a result, the molecular weight was about 76,000 to 87 by SDS-polyacrylamide gel electrophoresis. 2,000 Dalton, and has the same physicochemical properties as those of the enzyme M-11 obtained in Experimental Example 2, such as having an isoelectric point of about 3.6 to 4.6 by isoelectric focusing. It has been found. This suggests that the enzyme can be produced by the recombinant DNA technique, and the productivity of the enzyme is significantly improved.

[0067]

Example 2 Rhizobium species M
Preparation of complementary strand DNA derived from -11 and its nucleotide sequence,
Determination of amino acid sequence] Recombinant DNA obtained in Example 1-2
pBMT7 was digested with various restriction enzymes and subcloned into Bluescript II SK (+) according to a conventional method to obtain a DNA for nucleotide sequencing. After taking 2 μg of these DNAs for determining a base sequence and denaturing them by adding 2M aqueous sodium hydroxide solution, an appropriate amount of cold ethanol was added, and the resulting precipitate containing the template DNA was collected and vacuum dried. 5'-GTAAAACGACGGCCAGT chemically synthesized on this template DNA
-50 'for primer 1 having the base sequence represented by -3'
1 / ml, 40 mM Tris-hydrochloric acid buffer solution (pH containing 20 mM magnesium chloride and 50 mM sodium chloride)
7.5) was added and incubated at 65 ° C. for 2 minutes to anneal, followed by dATP, dGTP and d.
2 μl of an aqueous solution containing 7.5 μM each of TTP,
0.5 μl of [α- ³² P] dCTP (2 mCi / ml)
And 1 μl of 0.1 M dithiothreitol and 2 μl of 1.5 units / ml of T7 DNA polymerase were added, and 2
Primer 1 was extended from the 5'end to the 3'end by incubating at 5 ° C for 5 minutes to generate complementary strand DNA.

Next, the reaction product containing the complementary strand DNA obtained above was divided into four equal parts, and ddATP, ddCTP, d
Either 8 μM or 80 μM of dGTP or ddTTP
A 50 mM sodium chloride aqueous solution containing dNTP was added to 2.
After adding 5 μl and incubating at 37 ° C. for 5 minutes to react, 95% (v containing 20 mM EDTA, 0.05% (w / v) bromphenol blue and 0.05% (w / v) xylene cyanol / V) The reaction was stopped by adding 4 μl of an aqueous formamide solution. After heating the reaction product in a boiling water bath for 3 minutes, the reaction product was loaded on a 6% (w / v) polyacrylamide gel and electrophoresed while applying a constant voltage of about 2,000 V to separate the DNA fragments. The gel was fixed by, dried and autoradiographed.

As a result of analyzing the separated DNA fragments on the radiogram, the complementary strand DNA was found to be SEQ ID NO: 5 in the sequence listing.
It was found to contain the base sequence consisting of 2,936 base pairs shown in. The amino acid sequence deduced from this base sequence is as described in SEQ ID NO: 5 in the sequence listing, and this amino acid sequence and SEQ ID NOs: 7 and 9 in the sequence listing are shown.
Or the N-terminal amino acid sequence of the enzyme M-11 shown in 10 or 10 was compared, and the N-terminal amino acid sequence of SEQ ID NO: 7 was the first in SEQ ID NO: 5 in the sequence listing
To the 20th sequence, and the partial amino acid sequence of SEQ ID NO: 9 or 10 matched with the 486th to 506th sequences or the 606th to 626th sequences of SEQ ID NO: 5 in the sequence listing. This is because the enzyme M-11 has the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and in Rhizobium species M-11, the enzyme M-
11 is DN of the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing
It is shown to be coded by A.

[0070]

Example 3 Preparation of Recombinant DNA Containing DNA Derived from Arthrobacter species Q36 and Transformant

[0071]

Example 3-1 Preparation of chromosomal DNA Chromosomal DNA was isolated and purified from Arthrobacter species Q36 in the same manner as in Example 1-1, and SSC buffer (pH 7. Dissolved in 1), -80
Frozen at ℃.

[0072]

Example 3-2 Preparation of recombinant DNA pBQT13 and transformant BQT13 The purified chromosomal DNA solution obtained in Example 3-1 was partially cleaved in the same manner as in Example 1-2, and then sucrose density gradient ultracentrifugation method. About 3,000 to 6,000
A DNA fragment consisting of base pairs was collected. Then T4
Using DNA ligase, this DNA fragment was prepared in Example 1-
Vector Blu with restriction enzyme Bam HI as in 2
ligation to digest of escript II SK (+),
The obtained recombinant DNA was introduced into Escherichia coli XLI-Blue strain. The obtained transformant was treated in the same manner as in Example 1-2.
About 4,50 produced by culturing on an agar plate medium containing -bromo-4-chloro-3-indolyl-β-galactoside
While fixing 0 colonies on the nylon membrane, the first in the amino acid sequence shown in SEQ ID NO: 12 in the sequence listing was fixed.
1 to 16th Phe-Asp-Val-Asp-T
5'-TTYGA based on the sequence represented by rp-Asp
A probe 3 having a nucleotide sequence represented by YGTNGAYTGGGA-3 'was chemically synthesized, labeled with the isotope ³² P, and then hybridized with the colonies of the transformant immobilized on the nylon membrane, and a remarkable association was observed. Eight types of transformants were selected.

Recombinant DNAs were collected from these eight kinds of transformants in the same manner as in Example 1-2, and the 16th to 20th Thr-Glu-Phe in the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing were collected. 5'-ACNGAR chemically synthesized based on the sequence represented by -Trp-Asp
Recombinant D hybridized with probe 4 having the nucleotide sequence represented by TTYTGGGGA-3 ′ and showing a remarkable association.
NA was selected. Recombinant DN selected as described above
A and the transformant were designated as "pBQT13" or "BQT13", respectively.

Thereafter, this transformant BQT13 was cultured in L-broth medium containing ampicillin in the same manner as in Example 1-2, and recombinant DNA was eluted from the cells collected from the culture, purified and analyzed. By the way, recombinant DNA pB
QT13 consists of a pair of approximately 7,200 residues and had a structure represented by the restriction enzyme map shown in FIG. As shown in FIG. 10, it was revealed that the DNA composed of 2,325 base pairs encoding the enzyme Q36 was located downstream near the cleavage site by the restriction enzyme Xmn I.

[0075]

[Example 3-3 Production of enzyme by transformant BQT13] Maltose 2.0% (w / v), peptone 0.5%
(W / v), yeast extract 0.1% (w / v), disodium hydrogen phosphate 0.1% (w / v), potassium dihydrogen phosphate 0.1% (w / v). Adjust the pH to 7.0, add 50 μg / ml of ampicillin, and add 2 at 120 ° C.
After heat sterilization for 0 minutes and cooling, the transformant BQT13 obtained in Example 3-2 was inoculated and cultured at 37 ° C. for 24 hours with rotary shaking. The culture was sonicated to disrupt the cells, the insoluble matter was removed by centrifugation, and the enzyme activity in the supernatant was measured. As a result, about 2450 units of enzyme were calculated per 1 liter of culture. Was produced.

Separately, as a control, Escherichia coli XLI-Blu was used.
strain e and Arthrobacter species Q36 were inoculated into a liquid medium of the same composition containing no ampicillin, and in the case of Arthrobacter species Q36, the culture temperature was 3
Culture and treatment were performed in the same manner as above except that the temperature was set to 0 ° C. When the activity of the treated product was measured, the production of the enzyme by Arthrobacter species Q36 was about 1,200 units per liter of the culture, which was significantly lower than that of the transformant BQT13. E. coli X used as a host
The LI-Blue strain did not produce the enzyme.

Then, the enzyme produced by the transformant BQT13 was purified in the same manner as in Experimental Example 1-1, and its properties and properties were examined. As a result, it was confirmed by SDS-polyacrylamide gel electrophoresis that the molecular weight was about 76,000 to 87, 000 Dalton, and also shows an isoelectric point at about 3.6 to 4.6 by isoelectric focusing, and was found to have physicochemical properties similar to those of the enzyme Q36 obtained in Experimental Example 2. did. This means that the enzyme can also be produced by recombinant DNA technology,
Moreover, it is suggested that the productivity of the enzyme is significantly improved.

[0078]

Example 4 Preparation of complementary strand DNA derived from Arthrobacter species Q36 and determination of its nucleotide sequence and amino acid sequence Recombinant D obtained in Example 3-2
NA pBQT13 was treated in the same manner as in Example 2 to form a template DNA, which was annealed together with Primer 1 and then allowed to act on T7 DNA polymerase to extend Primer 1 from the 5 ′ end to the 3 ′ end. Was generated. As in Example 2, the dideoxy chain terminator method was applied to the complementary strand DNA, and the separated DNA fragments were analyzed on the radiogram. As a result, the complementary strand DNA was 3,073 shown in SEQ ID NO: 6 in the sequence listing. It was found to contain a base sequence consisting of base pairs. The amino acid sequence deduced from this base sequence is as described in SEQ ID NO: 6 in the sequence listing, and this amino acid sequence was compared with the N-terminal amino acid sequence and partial amino acid sequence shown in SEQ ID NO: 8, 11 or 12 in the sequence listing. The N-terminal amino acid sequence shown in SEQ ID NO: 8 is the 1st to 20th sequence in SEQ ID NO: 6 in the sequence listing, and the partial amino acid sequence of SEQ ID NO: 11 or 12 is the 606th to 606th sequence in SEQ ID NO: 6 in the sequence listing. 6
It matched the 25th or 110th to 129th sequences. This is because the enzyme Q36 has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and in Arthrobacter species Q36, the enzyme Q36 is encoded by the DNA of the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing. It indicates that

[0079]

INDUSTRIAL APPLICABILITY As described above, the present invention is a discovery of a novel enzyme which has not been previously known and produces a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more. It is based on. This invention paves the way for large-scale and efficient production of such enzymes by recombinant DNA technology. Moreover, the enzyme produced by the transformant according to the present invention is an enzyme whose entire amino acid sequence has been clarified, and is a non-reducing sugar having a trehalose structure or a trehalose structure at the end, which is premised on the compounding use in foods and the like. Can be used with confidence in the manufacture of.

The present invention is a significant invention that has such remarkable effects and can be said to be a great invention to contribute to the field.

[0081]

[Sequence list]

 SEQ ID NO: 1 Sequence Length: 772 Sequence Type: Amino Acid Topology: Linear Sequence Type: Polypeptide Sequence Met Arg Thr Pro Ala Ser Thr Tyr Arg Leu Gln Ile Arg Arg Gly Phe Thr 1 5 10 15 Leu Phe Asp Ala Ala Glu Thr Val Pro Tyr Leu Lys Ser Leu Gly Val Asp 20 25 30 Trp Ile Tyr Leu Ser Pro Ile Leu Lys Ala Glu Ser Gly Ser Asp His Gly 35 40 45 50 Tyr Asp Val Thr Asp Pro Ala Val Val Asp Pro Glu Arg Gly Gly Pro Glu 55 60 65 Gly Leu Ala Ala Val Ser Lys Ala Ala Arg Gly Ala Gly Met Gly Val Leu 70 75 80 85 Ile Asp Ile Val Pro Asn His Val Gly Val Ala Ser Pro Pro Gln Asn Pro 90 95 100 Trp Trp Trp Ser Leu Leu Lys Glu Gly Arg Gly Ser Pro Tyr Ala Val Ala 105 110 115 Phe Asp Val Asp Trp Asp Leu Ala Gly Gly Arg Ile Arg Ile Pro Val Leu 120 125 130 135 Gly Ser Asp Asp Asp Leu Asp Gln Leu Glu Ile Lys Asp Gly Glu Leu Arg 140 145 150 Tyr Tyr Asp His Arg Phe Pro Leu Ala Glu Gly Ser Tyr Arg Asp Gly Asp 155 160 165 170 Ser Pro Gln Asp Val His Gly Arg Gln His Tyr Glu Leu Ile Gly Trp Arg 175 180 185 Arg Ala Asp Asn Glu Leu Asn Tyr Arg Arg Phe Phe Ala Val Asn Thr Leu 190 195 200 Ala Gly Ile Arg Val Glu Val Pro Pro Val Phe Asp Glu Ala His Gln Glu 205 210 215 220 Val Val Arg Trp Phe Arg Ala Gly Leu Ala Asp Gly Leu Arg Ile Asp His 225 230 235 Pro Asp Gly Leu Ala Asp Pro Glu Gly Tyr Leu Lys Arg Leu Arg Glu Val 240 245 250 255 Thr Gly Gly Ala Tyr Leu Leu Ile Glu Lys Ile Leu Glu Pro Gly Glu Gln 260 265 270 Leu Pro Ala Ser Phe Glu Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu Ala 275 280 285 Asp Val Asp Arg Val Phe Val Asp Pro Arg Gly Gln Val Pro Leu Asp Arg 290 295 300 305 Leu Asp Ala Arg Leu Arg Gly Gly Ala Pro Ala Asp Tyr Glu Asp Met Ile 310 315 320 Arg Gly Thr Lys Arg Arg Ile Thr Asp Gly Ile Leu His Ser Glu Ile Leu 325 330 335 340 Arg Leu Ala Arg Leu Val Pro Glu Gln Thr Gly Ile Pro Gly Glu Ala Ala 345 350 355 Ala Asp Ala Ile Ala Glu Ile Ile Ala Ala Phe Pro Val Tyr Arg Ser Tyr 360 365 370 Leu Pro Glu Gly Ala Glu Ile Leu Lys Glu Ala Cys Asp Leu Ala Ala Arg 375 380 385 390 Arg Arg Pro Glu Leu Gly Gln Thr Val Gln Leu Leu Gln Pro Leu Leu Leu 395 400 405 Asp Thr Asp Leu Glu Ile Ser Arg Arg Phe Gln Gln Thr Ser Gly Met Val 410 415 420 425 Met Ala Lys Gly Val Glu Asp Thr Ala Phe Phe Arg Tyr Asn Arg Leu Gly 430 435 440 Thr Leu Thr Glu Val Gly Ala Asp Pro Thr Glu Phe Ser Leu Glu Pro Glu 445 450 455 Glu Phe His Val Arg Met Ala Arg Arg Gln Ala Glu Leu Pro Leu Ser Met 460 465 470 475 Thr Thr Leu Ser Thr His Asp Thr Lys Arg Ser Glu Asp Thr Arg Ala Arg 480 485 490 Ile Ser Val Ile Ala Glu Val Ala Pro Glu Trp Glu Lys Ala Leu Asp Arg 495 500 505 510 Leu Asn Thr Leu Ala Pro Leu Pro Asp Gly Pro Leu Ser Thr Leu Leu Trp 515 520 525 Gln Ala Ile Ala Gly Ala Trp Pro Ala Ser Arg Glu Arg Leu Gln Ser Tyr 530 535 540 Ala Leu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Ser Trp Thr Asp Pro 545 550 555 560 Asp Pro Ala Phe Glu Glu Ala Leu Ser Ala Val Val Asp Ser Ala Phe Asp 565 570 575 Asn Pro Glu Val Arg Ala Glu Leu Glu Ala Leu Val Gly Leu Leu Ala Pro 580 585 590 595 His Gly Ala Ser Asn Ser Leu Ala Ala Lys Leu Val Gln Leu Thr Met Pro 600 605 610 Gly Val Pro Asp Val Tyr Gln Gly Thr Glu Phe Trp Asp Arg Ser Leu Thr 615 620 625 Asp Pro Asp Asn Arg Arg Pro Phe Ser Phe Ala Glu Arg Ile Arg Ala Leu 630 635 640 645 Asp Gln Leu Asp Ala Gly His Arg Pro Asp Ser Phe Gln Asp Glu Ala Val 650 655 660 Lys Leu Leu Val Thr Ser Arg Ala Leu Arg Leu Arg Arg Asn Arg Pro Glu 665 670 675 680 Leu Phe Thr Gly Tyr Arg Pro Val His Ala Arg Gly Pro Ala Ala Gly His 685 690 695 Leu Val Ala Phe Asp Arg Gly Ala Gly Gly Val Leu Ala Leu Ala Thr Arg 700 705 710 Leu Pro Tyr Gly Leu Glu Gln Ser Gly Gly Trp Arg Asp Thr Ala Val Glu 715 720 725 730 Leu Glu Ala Ala Met Thr Asp Glu Leu Thr Gly Ser Thr Phe Gly Pro Gly 735 740 745 Pro Ala Ala Leu Ser Glu Val Phe Arg Ala Tyr Pro Val Ala Leu Leu Val 750 755 760 765 Pro Ala Thr Gly Gly Lys Ser 770

SEQ ID NO: 2 Sequence Length: 775 Sequence Type: Amino Acid Topology: Linear Sequence Type: Polypeptide Sequence Met Arg Thr Pro Val Ser Thr Tyr Arg Leu Gln Ile Arg Lys Gly Phe Thr 1 5 10 15 Leu Phe Asp Ala Ala Lys Thr Val Pro Tyr Leu His Ser Leu Gly Val Asp 20 25 30 Trp Val Tyr Leu Ser Pro Val Leu Thr Ala Glu Gln Gly Ser Asp His Gly 35 40 45 50 Tyr Asp Val Thr Asp Pro Ser Ala Val Asp Pro Glu Arg Gly Gly Pro Glu 55 60 65 Gly Leu Ala Ala Val Ser Lys Ala Ala Arg Ala Ala Gly Met Gly Val Leu 70 75 80 85 Ile Asp Ile Val Pro Asn His Val Gly Val Ala Thr Pro Ala Gln Asn Pro 90 95 100 Trp Trp Trp Ser Leu Leu Lys Glu Gly Arg Gln Ser Arg Tyr Ala Glu Ala 105 110 115 Phe Asp Val Asp Trp Asp Leu Ala Gly Gly Arg Ile Arg Leu Pro Val Leu 120 125 130 135 Gly Ser Asp Asp Asp Leu Asp Gln Leu Glu Ile Arg Asp Gly Glu Leu Arg 140 145 150 Tyr Tyr Asp His Arg Phe Pro Leu Ala Glu Gly Thr Tyr Ala Glu Gly Asp 155 160 165 170 Ala Pro Arg Asp Val His Ala Arg Gln His Tyr G lu Leu Ile Gly Trp Arg 175 180 185 Arg Ala Asp Asn Glu Leu Asn Tyr Arg Arg Phe Phe Ala Val Asn Thr Leu 190 195 200 Ala Gly Val Arg Val Glu Ile Pro Ala Val Phe Asp Glu Ala His Gln Glu 205 210 215 220 Val Val Arg Trp Phe Arg Glu Asp Leu Ala Asp Gly Leu Arg Ile Asp His 225 230 235 Pro Asp Gly Leu Ala Asp Pro Glu Gly Tyr Leu Lys Arg Leu Arg Glu Val 240 245 250 255 Thr Gly Gly Ala Tyr Leu Leu Ile Glu Lys Ile Leu Glu Pro Gly Glu Gln 260 265 270 Leu Pro Ala Ser Phe Glu Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu Ala 275 280 285 Asp Val Asp Arg Val Leu Val Asp Pro Arg Gly Gln Glu Pro Leu Asp Arg 290 295 300 305 Leu Asp Ala Ser Leu Arg Gly Gly Glu Pro Ala Asp Tyr Gln Asp Met Ile 310 315 320 Arg Gly Thr Lys Arg Arg Ile Thr Asp Gly Ile Leu His Ser Glu Ile Leu 325 330 335 340 Arg Leu Ala Arg Leu Val Pro Gly Asp Ala Asn Val Ser Ile Asp Ala Gly 345 350 355 Ala Asp Ala Leu Ala Glu Ile Ile Ala Ala Phe Pro Val Tyr Arg Thr Tyr 360 365 370 Leu Pro Glu Gly Ala Glu Val Leu Lys Glu Ala Cys Glu Leu Ala Ala Arg 3 75 380 385 390 Arg Arg Pro Glu Leu Asp Gln Ala Ile Gln Ala Leu Gln Pro Leu Leu Leu 395 400 405 Asp Thr Asp Leu Glu Leu Ala Arg Arg Phe Gln Gln Thr Ser Gly Met Val 410 415 420 425 Met Ala Lys Gly Val Glu Asp Thr Ala Phe Phe Arg Tyr Asn Arg Leu Gly 430 435 440 Thr Leu Thr Glu Val Gly Ala Asp Pro Thr Glu Phe Ala Val Glu Pro Asp 445 450 455 Glu Phe His Ala Arg Leu Ala Arg Arg Gln Ala Glu Leu Pro Leu Ser Met 460 465 470 475 Thr Thr Leu Ser Thr His Asp Thr Lys Arg Ser Glu Asp Thr Arg Ala Arg 480 485 490 Ile Ser Val Ile Ser Glu Val Ala Gly Asp Trp Glu Lys Ala Leu Asn Arg 495 500 505 510 Leu Arg Asp Leu Ala Pro Leu Pro Asp Gly Pro Leu Ser Ala Leu Leu Trp 515 520 525 Gln Ala Ile Ala Gly Ala Trp Pro Ala Ser Arg Glu Arg Leu Gln Tyr Tyr 530 535 540 Ala Leu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Asn Trp Thr Asp Pro 545 550 555 560 Ala Pro Ala Phe Glu Glu Lys Leu Lys Ala Ala Val Asp Ala Val Phe Asp 565 570 575 Asn Pro Ala Val Gln Ala Glu Val Glu Ala Leu Val Glu Leu Leu Glu Pro 580 585 590 595 Tyr G ly Ala Ser Asn Ser Leu Ala Ala Lys Leu Val Gln Leu Thr Met Pro 600 605 610 Gly Val Pro Asp Val Tyr Gln Gly Thr Glu Phe Trp Asp Arg Ser Leu Thr 615 620 625 Asp Pro Asp Asn Arg Arg Pro Phe Ser Phe Asp Asp Arg Arg Ala Ala Leu 630 635 640 645 Glu Gln Leu Asp Ala Gly Asp Leu Pro Ala Ser Phe Thr Asp Glu Arg Thr 650 655 660 Lys Leu Leu Val Thr Ser Arg Ala Leu Arg Leu Arg Arg Asp Arg Pro Glu 665 670 675 680 Leu Phe Thr Gly Tyr Arg Pro Val Leu Ala Ser Gly Pro Ala Ala Gly His 685 690 695 Leu Leu Ala Phe Asp Arg Gly Thr Ala Ala Ala Pro Gly Ala Leu Thr Leu 700 705 710 Ala Thr Arg Leu Pro Tyr Gly Leu Glu Gln Ser Gly Gly Trp Arg Asp Thr 715 720 725 730 Ala Val Glu Leu Asn Thr Ala Met Lys Asp Glu Leu Thr Gly Ala Gly Phe 735 740 745 Gly Pro Gly Ala Val Lys Ile Ala Asp Ile Phe Arg Ser Phe Pro Val Ala 750 755 760 765 Leu Leu Val Pro Gln Thr Gly Gly Glu Ser 770 775

[0083] SEQ ID NO: 3 sequence Length: type 2316 sequence: nucleic acid Topology: linear sequence ATGAGGACAC CCGCCTCGAC CTACCGGCTG CAGATCAGGC GGGGTTTCAC GCTGTTTGAT 60 GCCGCCGAGA CCGTGCCCTA CCTGAAGTCA CTCGGGGTGG ACTGGATCTA CCTGTCGCCC 120 ATCCTGAAGG CAGAGAGCGG CTCCGACCAC GGCTATGACG TCACCGATCC CGCCGTAGTG 180 GACCCGGAGC GCGGCGGCCC TGAAGGGCTG GCCGCGGTGT CCAAGGCGGC CCGCGGTGCC 240 GGCATGGGCG TGCTGATCGA CATCGTGCCG AACCACGTGG GCGTGGCGTC GCCGCCGCAG 300 AACCCGTGGT GGTGGTCGCT GCTCAAGGAA GGGCGCGGGT CGCCCTACGC CGTGGCGTTC 360 GACGTCGACT GGGACCTGGC GGGGGGCCGC ATCCGGATCC CCGTCCTGGG CAGCGACGAC 420 GATCTGGACC AGCTCGAAAT CAAGGACGGC GAGCTGCGGT ACTACGACCA CCGCTTCCCG 480 CTGGCCGAGG GCAGCTACCG GGACGGCGAC TCCCCGCAGG ACGTCCACGG CCGGCAGCAC 540 TACGAACTCA TCGGCTGGCG GCGCGCCGAC AATGAACTGA ACTACCGCCG GTTCTTCGCG 600 GTGAACACGC TCGCCGGCAT CCGGGTGGAG GTGCCGCCGG TCTTCGATGA AGCGCACCAG 660 GAGGTGGTGC GCTGGTTCCG TGCGGGGCTC GCCGACGGGC TGCGGATCGA CCACCCGGAC 720 GGCCTGGCCG ATCCCGAGGG GTATTTGAAG CGGCTCCGTG AG GTCACCGG GGGCGCGTAC 780 CTGCTCATCG AAAAGATCCT CGAGCCGGGC GAACAGTTGC CGGCCAGCTT CGAGTGCGAA 840 GGCACCACCG GCTACGACGC CCTCGCGGAT GTCGACAGGG TCTTCGTGGA CCCGCGGGGA 900 CAGGTGCCGC TGGACCGTCT GGACGCACGG CTGCGCGGCG GTGCGCCGGC CGACTACGAG 960 GACATGATCC GCGGGACCAA GCGCCGGATC ACCGACGGCA TCCTGCACTC CGAGATCCTG 1020 CGCCTTGCCA GGCTGGTGCC CGAGCAGACC GGAATTCCCG GGGAGGCGGC CGCGGATGCG 1080 ATCGCGGAGA TCATCGCGGC CTTCCCGGTC TACCGGTCCT ATCTTCCCGA GGGCGCGGAG 1140 ATCCTGAAGG AGGCCTGCGA CCTCGCCGCG CGGAGGCGTC CGGAACTGGG CCAGACCGTC 1200 CAGCTGCTGC AGCCGCTGCT GCTGGATACC GACCTCGAGA TTTCCCGCAG GTTCCAGCAG 1260 ACCTCGGGAA TGGTCATGGC CAAAGGCGTG GAGGACACCG CGTTCTTCCG CTACAACCGG 1320 CTGGGAACGC TCACCGAGGT GGGCGCCGAC CCCACCGAGT TCTCGCTGGA ACCGGAGGAG 1380 TTTCACGTCC GGATGGCCCG CCGGCAGGCC GAACTCCCGC TCTCCATGAC CACCCTGAGC 1440 ACGCACGACA CCAAGCGCAG CGAGGACACC CGGGCCCGGA TCTCGGTGAT CGCCGAGGTC 1500 GCGCCTGAAT GGGAAAAGGC CCTGGACAGG CTGAACACCC TCGCTCCGCT GCCGGACGGC 1560 CCGCTCTCCA CGCTGCTCTG GCAGGCGATT GCGGGGGCAT GGCCGGCCAG C CGGGAACGC 1620 CTTCAGTCCT ACGCCCTGAA AGCGGCGCGC GAAGCCGGGA ACTCGACCAG CTGGACCGAT 1680 CCGGACCCGG CATTCGAGGA GGCACTTTCC GCCGTCGTCG ACTCCGCCTT CGACAATCCG 1740 GAGGTGCGTG CGGAACTTGA GGCCCTGGTG GGCCTCCTTG CGCCGCACGG TGCGTCCAAC 1800 TCGCTCGCGG CAAAGCTTGT CCAGCTGACC ATGCCGGGCG TTCCGGACGT GTACCAGGGC 1860 ACCGAGTTCT GGGACAGGTC GCTGACCGAT CCGGACAACC GGCGCCCCTT CAGCTTCGCC 1920 GAACGGATTA GGGCCTTGGA CCAGTTGGAC GCCGGCCACC GTCCGGACTC CTTCCAGGAC 1980 GAGGCGGTCA AGCTGCTGGT CACCTCGAGG GCGCTGCGGC TGCGGCGGAA CCGGCCCGAG 2040 CTCTTCACCG GCTACCGCCC CGTGCATGCC AGGGGCCCCG CCGCCGGGCA CCTGGTGGCG 2100 TTCGACCGCG GCGCCGGGGG AGTGCTGGCG CTTGCCACCC GGCTCCCCTA CGGGCTGGAA 2160 CAGTCGGGCG GCTGGCGGGA CACCGCCGTC GAGCTTGAAG CCGCCATGAC GGACGAACTG 2220 ACCGGCTCCA CTTTCGGGCC GGGACCGGCG GCGCTGTCAG AAGTCTTCCG GGCCTACCCG 2280 GTGGCCTTGT TGGTCCCCGC GACAGGAGGC AAGTCA 2316

SEQ ID NO: 4 Sequence Length: 2325 Sequence Type: Nucleic Acid Topology: Linear Sequence ATGAGAACGC CAGTCTCCAC GTACAGGCTG CAGATCAGGA AGGGATTCAC ACTCTTCGAC 60 GCGGCCAAAA CCGTTCCGTA CCTGCCCCCGCGGCGTCGCCCT 120GTCCTGCCG CCGCGCCGCG 240 GGCATGGGCG TGCTGATCGA CATCGTGCCC AACCACGTGG GCGTCGCGAC GCCGGCGCAG 300 AACCCCTGGT GGTGGTCGCT GCTCAAGGAG GGACGCCAGT CCCGTTACGC GGAGGCGTTC 360 GACGTCGATT GGGACCTCGC CGGGGGACGC ATCCGGCTGC CGGTGCTCGG CAGCGACGAT 420 GACCTCGACC AGCTCGAAAT CAGGGACGGG GAGCTGCGGT ACTACGACCA CCGATTCCCG 480 CTCGCCGAGG GAACCTACGC CGAAGGCGAC GCCCCGCGGG ATGTCCACGC CCGGCAGCAC 540 TACGAGCTCA TCGGCTGGCG CCGCGCGGAC AACGAGCTGA ACTACCGCCG CTTTTTCGCG 600 GTGAACACGC TCGCCGGCGT CCGCGTGGAA ATCCCCGCCG TCTTCGACGA GGCACACCAG 660 GAGGTGGTGC GCTGGTTCCG CGAGGACCTT GCGGACGGCC TGCGGATCGA CCACCCGGAC 720 GGCCTCGCTG ACCCCGAGGG GTACCTGAAG CGACTCCGGG AA GTCACCGG CGGCGCTTAC 780 CTGCTGATCG AAAAGATCCT GGAGCCGGGG GAGCAGCTGC CCGCCAGCTT CGAGTGTGAA 840 GGCACCACAG GCTACGACGC CCTCGCCGAC GTCGACCGGG TTCTCGTGGA CCCGCGCGGC 900 CAGGAACCGC TGGACCGGCT TGACGCGTCC CTGCGTGGCG GCGAGCCCGC CGACTACCAG 960 GACATGATCC GCGGAACCAA GCGCCGGATC ACCGACGGTA TCCTGCACTC GGAGATCCTG 1020 CGGCTGGCCC GGCTGGTTCC GGGCGACGCC AACGTTTCAA TCGACGCCGG AGCCGACGCT 1080 CTCGCCGAAA TCATCGCCGC CTTCCCGGTC TACCGCACCT ACCTGCCGGA GGGCGCCGAG 1140 GTCCTGAAGG AGGCGTGCGA GCTTGCCGCG CGTAGGCGGC CGGAACTCGA CCAGGCCATC 1200 CAGGCTCTGC AGCCGCTGCT GCTGGACACG GACCTCGAGC TTGCCCGGCG CTTCCAGCAG 1260 ACCTCGGGCA TGGTCATGGC CAAGGGCGTG GAGGACACCG CGTTCTTCCG CTACAACCGC 1320 CTGGGCACCC TCACGGAAGT GGGCGCCGAC CCCACCGAGT TCGCCGTGGA GCCGGACGAG 1380 TTCCACGCCC GGCTGGCACG CCGGCAGGCC GAGCTTCCGC TGTCCATGAC GACGCTGAGC 1440 ACGCACGACA CCAAGCGCAG CGAGGACACC CGAGCAAGGA TTTCGGTCAT TTCCGAGGTT 1500 GCGGGTGACT GGGAAAAGGC CTTGAACCGG CTGCGCGACC TGGCCCCGCT GCCGGACGGC 1560 CCGCTGTCCG CGCTGCTCTG GCAGGCCATT GCCGGCGCCT GGCCCGCCAG C CGGGAACGC 1620 CTGCAGTACT ACGCGCTGAA GGCCGCGCGT GAAGCGGGGA ACTCGACCAA CTGGACCGAT 1680 CCGGCCCCCG CGTTCGAGGA GAAGCTGAAG GCCGCGGTCG ACGCCGTGTT CGACAATCCC 1740 GCCGTGCAGG CCGAGGTGGA AGCCCTCGTC GAGCTCCTGG AGCCGTACGG AGCTTCGAAC 1800 TCCCTCGCCG CCAAGCTCGT GCAGCTGACC ATGCCCGGCG TCCCGGACGT CTACCAGGGC 1860 ACGGAGTTCT GGGACCGGTC GCTGACGGAC CCGGACAACC GGCGGCCGTT CAGCTTCGAC 1920 GACCGCCGCG CCGCGCTGGA GCAGCTGGAT GCCGGCGACC TTCCCGCGTC ATTTACCGAT 1980 GAGCGGACGA AGCTGCTAGT GACGTCGCGC GCGCTGCGGC TGCGCCGGGA CCGTCCGGAG 2040 CTGTTCACGG GGTACCGGCC GGTCCTGGCC AGCGGGCCCG CCGCCGGGCA CCTGCTCGCG 2100 TTCGACCGCG GCACCGCGGC GGCGCCGGGT GCATTGACCC TCGCCACGCG GCTTCCCTAC 2160 GGGCTGGAAC AGTCGGGTGG ATGGCGGGAC ACCGCCGTCG AACTTAACAC CGCCATGAAA 2220 GACGAACTGA CCGGTGCCGG CTTCGGACCG GGGGCAGTGA AGATCGCCGA CATCTTCCGG 2280 TCGTTCCCCG TTGCGCTGCT GGTGCCGCAG ACAGGAGGAG AGTCA 2325

SEQ ID NO: 5 Sequence length: 2936 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Sequence features Origin Biological name: Rhizobium sp. ) Strain name: M-11 (FERM BP-4130) Sequence features Characteristic signature: 5'UTR Location: 1..564 Method of determining feature: E Characteristic signature: mat peptide Location: 565. .2880 Characteristic determination method: S Characteristic symbol: 3'UTR Location: 2881..2936 Characteristic determination method: E Sequence CGTGCTCTAC TTCAACGCGC ACGACGGCGACGCGGCTCGCAGGAGAGCGC CCCGGACCCACGCCGCACGACGGC120C GTGGTGCTCC GTGCCCACAG 180 CGCCCCGGAG GAGGAACCGG ACCACTCGGT GGCCGCCTCC CTCGCAGCGC TGACGCAGAC 240 TGCGACCGCC GAAACCGCGG CGCTCACCGC CCCCACCGTT CCGGAGCCGA GGAAGACCAA 300 GAAGGCAGGA CCGAAGCCGG AAGAGGAGGGGCGCGCCGAGA CCC GACGAGGCGG CGGCGAAGCC GGAAGAGGCT GCTTCCGACG AGGCGGCGGC 420 GAAGCCGGAA GAGAAGGCTC CCGACGAGGC GGCGGCGAAG CCGGAAGAGG CTGCTTCCGA 480 CGAGGCGGCG GCGAAGCCCG CGGGGAAGGC AGCGGCCAAA ACGGCCGGCA GGCGAGCGCC 540 AGGCAAGCAG GGCGGGACGG GCTC 564 ATG AGG ACA CCC GCC TCG ACC TAC CGG CTG CAG ATC AGG CGG GGT TTC 612 Met Arg Thr Pro Ala Ser Thr Tyr Arg Leu Gln Ile Arg Arg Gly Phe 1 5 10 15 ACG CTG TTT GAT GCC GCC GAG ACC GTG CCC TAC CTG AAG TCA CTC GGG 660 Thr Leu Phe Asp Ala Ala Glu Thr Val Pro Tyr Leu Lys Ser Leu Gly 20 25 30 GTG GAC TGG ATC TAC CTG TCG CCC ATC CTG AAG GCA GAG AGC GGC TCC 708 Val Asp Trp Ile Tyr Leu Ser Pro Ile Leu Lys Ala Glu Ser Gly Ser 35 40 45 GAC CAC GGC TAT GAC GTC ACC GAT CCC GCC GTA GTG GAC CCG GAG CGC 756 Asp His Gly Tyr Asp Val Thr Asp Pro Ala Val Val Asp Pro Glu Arg 50 55 60 GGC GGC CCT GAA GGG CTG GCC GCG GTG TCC AAG GCG GCC CGC GGT GCC 804 Gly Gly Pro Glu Gly Leu Ala Ala Val Ser Lys Ala Ala Arg Gly Ala 65 70 75 80 GGC ATG GGC GTG CTG ATC GAC ATC GTG CCG AAC CAC GTG GGC G TG GCG 852 Gly Met Gly Val Leu Ile Asp Ile Val Pro Asn His Val Gly Val Ala 85 90 95 TCG CCG CCG CAG AAC CCG TGG TGG TGG TCG CTG CTC AAG GAA GGG CGC 900 Ser Pro Pro Gln Asn Pro Trp Trp Trp Ser Leu Leu Lys Glu Gly Arg 100 105 110 GGG TCG CCC TAC GCC GTG GCG TTC GAC GTC GAC TGG GAC CTG GCG GGG 948 Gly Ser Pro Tyr Ala Val Ala Phe Asp Val Asp Trp Asp Leu Ala Gly 115 120 125 GGC CGC ATC CGG ATC CCC GTC CTG GGC AGC GAC GAC GAT CTG GAC CAG 996 Gly Arg Ile Arg Ile Pro Val Leu Gly Ser Asp Asp Asp Leu Asp Gln 130 135 140 CTC GAA ATC AAG GAC GGC GAG CTG CGG TAC TAC GAC CAC CGC TTC CCG 1044 Leu Glu Ile Lys Asp Gly Glu Leu Arg Tyr Tyr Asp His Arg Phe Pro 145 150 155 160 CTG GCC GAG GGC AGC TAC CGG GAC GGC GAC TCC CCG CAG GAC GTC CAC 1092 Leu Ala Glu Gly Ser Tyr Arg Asp Gly Asp Ser Pro Gln Asp Val His 165 170 175 GGC CGG CAG CAC TAC GAA CTC ATC GGC TGG CGG CGC GCC GAC AAT GAA 1140 Gly Arg Gln His Tyr Glu Leu Ile Gly Trp Arg Arg Ala Asp Asn Glu 180 185 190 CTG AAC TAC CGC CGG TTC TTC GCG GTG AAC ACG C TC GCC GGC ATC CGG 1188 Leu Asn Tyr Arg Arg Phe Phe Ala Val Asn Thr Leu Ala Gly Ile Arg 195 200 205 GTG GAG GTG CCG CCG GTC TTC GAT GAA GCG CAC CAG GAG GTG GTG CGC 1236 Val Glu Val Pro Pro Val Phe Asp Glu Ala His Gln Glu Val Val Arg 210 215 220 TGG TTC CGT GCG GGG CTC GCC GAC GGG CTG CGG ATC GAC CAC CCG GAC 1284 Trp Phe Arg Ala Gly Leu Ala Asp Gly Leu Arg Ile Asp His Pro Asp 225 230 235 240 GGC CTG GCC GAT CCC GAG GGG TAT TTG AAG CGG CTC CGT GAG GTC ACC 1332 Gly Leu Ala Asp Pro Glu Gly Tyr Leu Lys Arg Leu Arg Glu Val Thr 245 250 255 GGG GGC GCG TAC CTG CTC ATC GAA AAG ATC CTC GAG CCG GGC GAA CAG 1380 Gly Gly Ala Tyr Leu Leu Ile Glu Lys Ile Leu Glu Pro Gly Glu Gln 260 265 270 TTG CCG GCC AGC TTC GAG TGC GAA GGC ACC ACC GGC TAC GAC GCC CTC 1428 Leu Pro Ala Ser Phe Glu Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu 275 280 285 GCG GAT GTC GAC AGG GTC TTC GTG GAC CCG CGG GGA CAG GTG CCG CTG 1476 Ala Asp Val Asp Arg Val Phe Val Asp Pro Arg Gly Gln Val Pro Leu 290 295 300 GAC CGT CTG GAC GCA CGG CT G CGC GGC GGT GCG CCG GCC GAC TAC GAG 1524 Asp Arg Leu Asp Ala Arg Leu Arg Gly Gly Ala Pro Ala Asp Tyr Glu 305 310 315 320 GAC ATG ATC CGC GGG ACC AAG CGC CGG ATC ACC GAC GGC ATC CTG CAC 1572 Asp Met Ile Arg Gly Thr Lys Arg Arg Ile Thr Asp Gly Ile Leu His 325 330 335 TCC GAG ATC CTG CGC CTT GCC AGG CTG GTG CCC GAG CAG ACC GGA ATT 1620 Ser Glu Ile Leu Arg Leu Ala Arg Leu Val Pro Glu Gln Thr Gly Ile 340 345 350 CCC GGG GAG GCG GCC GCG GAT GCG ATC GCG GAG ATC ATC GCG GCC TTC 1668 Pro Gly Glu Ala Ala Ala Asp Ala Ile Ala Glu Ile Ile Ala Ala Phe 355 360 365 CCG GTC TAC CGG TCC TAT CTT CCC GAG GGC GCG GAG ATC CTG AAG GAG 1716 Pro Val Tyr Arg Ser Tyr Leu Pro Glu Gly Ala Glu Ile Leu Lys Glu 370 375 380 GCC TGC GAC CTC GCC GCG CGG AGG CGT CCG GAA CTG GGC CAG ACC GTC 1764 Ala Cys Asp Leu Ala Ala Arg Arg Arg Pro Glu Leu Gly Gln Thr Val 385 390 395 400 CAG CTG CTG CAG CCG CTG CTG CTG GAT ACC GAC CTC GAG ATT TCC CGC 1812 Gln Leu Leu Gln Pro Leu Leu Leu Asp Thr Asp Leu Glu Ile Ser Arg 405 410 415 AGG TTC CAG CAG ACC TCG GGA ATG GTC ATG GCC AAA GGC GTG GAG GAC 1860 Arg Phe Gln Gln Thr Ser Gly Met Val Met Ala Lys Gly Val Glu Asp 420 425 430 ACC GCG TTC TTC CGC TAC AAC CGG CTG GGA ACG CTC ACC GAG GTG GGC 1908 Thr Ala Phe Phe Arg Tyr Asn Arg Leu Gly Thr Leu Thr Glu Val Gly 435 440 445 GCC GAC CCC ACC GAG TTC TCG CTG GAA CCG GAG GAG TTT CAC GTC CGG 1956 Ala Asp Pro Thr Glu Phe Ser Leu Glu Pro Glu Glu Phe His Val Arg 450 455 460 ATG GCC CGC CGG CAG GCC GAA CTC CCG CTC TCC ATG ACC ACC CTG AGC 2004 Met Ala Arg Arg Gln Ala Glu Leu Pro Leu Ser Met Thr Thr Leu Ser 465 475 480 480 ACG CAC GAC ACC AAG CGC AGC GAG GAC ACC CGG GCC CGG ATC TCG GTG 2052 Thr His Asp Thr Lys Arg Ser Glu Asp Thr Arg Ala Arg Ile Ser Val 485 490 495 ATC GCC GAG GTC GCG CCT GAA TGG GAA AAG GCC CTG GAC AGG CTG AAC 2100 Ile Ala Glu Val Ala Pro Glu Trp Glu Lys Ala Leu Asp Arg Leu Asn 500 505 510 ACC CTC GCT CCG CTG CCG GAC GGC CCG CTC TCC ACG CTG CTC TGG CAG 2148 Thr Leu Ala Pro Leu Pro Asp Gly Pro Leu Ser Thr Leu Leu Trp Gln 515 520 525 GCG ATT GCG GGG GCA TGG CCG GCC AGC CGG GAA CGC CTT CAG TCC TAC 2196 Ala Ile Ala Gly Ala Trp Pro Ala Ser Arg Glu Arg Leu Gln Ser Tyr 530 535 540 GCC CTG AAA GCG GCG CGC GAA GCC GGG AAC TCG ACC AGC TGG ACC GAT 2244 Ala Leu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Ser Trp Thr Asp 545 550 555 560 CCG GAC CCG GCA TTC GAG GAG GCA CTT TCC GCC GTC GTC GAC TCC GCC 2292 Pro Asp Pro Ala Phe Glu Glu Ala Leu Ser Ala Val Val Asp Ser Ala 565 570 575 TTC GAC AAT CCG GAG GTG CGT GCG GAA CTT GAG GCC CTG GTG GGC CTC 2340 Phe Asp Asn Pro Glu Val Arg Ala Glu Leu Glu Ala Leu Val Gly Leu 580 585 590 CTT GCG CCG CAC GGT GCG TCC AAC TCG CTC GCG GCA AAG CTT GTC CAG 2388 Leu Ala Pro His Gly Ala Ser Asn Ser Leu Ala Ala Lys Leu Val Gln 595 600 605 CTG ACC ATG CCG GGC GTT CCG GAC GTG TAC CAG GGC ACC GAG TTC TGG 2436 Leu Thr Met Pro Gly Val Pro Asp Val Tyr Gln Gly Thr Glu Phe Trp 610 615 620 GAC AGG TCG CTG ACC GAT CCG GAC AAC CGG CGC CCC TTC AGC TTC GCC 2484 Asp Arg Ser Leu Thr Asp Pro Asp Asn Arg A rg Pro Phe Ser Phe Ala 625 630 635 640 GAA CGG ATT AGG GCC TTG GAC CAG TTG GAC GCC GGC CAC CGT CCG GAC 2532 Glu Arg Ile Arg Ala Leu Asp Gln Leu Asp Ala Gly His Arg Pro Asp 645 650 655 TCC TTC CAG GAC GAG GCG GTC AAG CTG CTG GTC ACC TCG AGG GCG CTG 2580 Ser Phe Gln Asp Glu Ala Val Lys Leu Leu Val Thr Ser Arg Ala Leu 660 665 670 CGG CTG CGG CGG AAC CGG CCC GAG CTC TTC ACC GGC TAC CGC CCC GTG 2628 Arg Leu Arg Arg Asn Arg Pro Glu Leu Phe Thr Gly Tyr Arg Pro Val 675 680 685 CAT GCC AGG GGC CCC GCC GCC GGG CAC CTG GTG GCG TTC GAC CGC GGC 2676 His Ala Arg Gly Pro Ala Ala Gly His Leu Val Ala Phe Asp Arg Gly 690 695 700 GCC GGG GGA GTG CTG GCG CTT GCC ACC CGG CTC CCC TAC GGG CTG GAA 2724 Ala Gly Gly Val Leu Ala Leu Ala Thr Arg Leu Pro Tyr Gly Leu Glu 705 710 715 720 CAG TCG GGC GGC TGG CGG GAC ACC GCC GTC GAG CTT GAA GCC GCC ATG 2772 Gln Ser Gly Gly Trp Arg Asp Thr Ala Val Glu Leu Glu Ala Ala Met 725 730 735 ACG GAC GAA CTG ACC GGC TCC ACT TTC GGG CCG GGA CCG GCG GCG CTG 2820 Thr Asp Glu Leu Th r Gly Ser Thr Phe Gly Pro Gly Pro Ala Ala Leu 740 745 750 TCA GAA GTC TTC CGG GCC TAC CCG GTG GCC TTG TTG GTC CCC GCG ACA 2868 Ser Glu Val Phe Arg Ala Tyr Pro Val Ala Leu Leu Val Pro Ala Thr 755 760 765 GGA GGC AAG TCA 2880 Gly Gly Lys Ser 770 TGACGCAGCC CAACGATGCG GCCAAGCCGG TGCAGGGAGC GGGGCGCTTC GATATC 2936

SEQ ID NO: 6 Sequence length: 3084 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: Genomic DNA Sequence features Origin Biological name: Arthrobacter species (Arthrobacte species)
r sp.) Strain name: Q36 (FERM BP-4316) Sequence features Characteristic signature: 5'UTR Location: 1..677 Method of determining feature: E Characteristic signature: mat peptide Location: 678 ..3002 Method of determining features: S Characteristic symbol: 3'UTR Location: 3003..3073 Method of determining features: E array GATCCGGACG GCAACCTCAT GTCCCCGGAG GACTGGGACA GCGGCTTCGG CCGTTCGGTG 60 GGCATGTTCCTCCAAACGGCGACGGGCATCCAGGAGGGCCGCTCATCGACCGTG CACGACGGCG ACGTCGAGTT CACGCTGCCG 180 CCGGACGAAT ACGCCCCGGC CTGGGACGTC ATCATCGACA CCGCCGGTGA AGGGGCCGAC 240 TCCAAGCCCG CGGACGCCGG AACCATCCTG TCCGTTGCGG CCAAGTCGCT GGTTGTGCTT 300 CGCGCCCACA GCGCACCGGA GGAGGAGCCT GACCATTCCG TGGCTGCTTC CCTGGCTGCA 360 CTGACGCAGA CCGCCACCGC CGAGACGGCG GCGCTCACAG CTCCTGCCGT TCCCGAGCCG 420 GCCAAGACGA AGAAGCCGGC CGCTGACCCG GTTGCTGAAC CGGCCGACCC GCCGGTTGCT 480 GACCCGGCCG ACCCGGTTGC TGACCCGGTT GCTGACCCGG CGCCGGAACC GGCTGCGGAG 540 CCTGCGAAAT CCGCAGCGGA ACCTGGTGCG GAGCC TGCGA AGGACCCGGA GGAGCAGCCG 600 GCGGAAAAGC CGGCGCGCAA GCCTGCGGCA AAGCGCGGCG GCCACCTGAG GGCGGTCAAG 660 CCCGCTGGGG AGGACGC 677 ATG AGA ACG CCA GTC TCC ACG TAC AGG CTG CAg ATG Cle Gru Tg Arle Glu TTC Arg 15 ACA CTC TTC GAC GCG GCC AAA ACC GTT CCG TAC CTG CAC TCG CTC GGC 773 Thr Leu Phe Asp Ala Ala Lys Thr Val Pro Tyr Leu His Ser Leu Gly 20 25 30 GTC GAC TGG GTC TAC CTT TCT CCG GTC CTG ACT GCC GAG CAG GGC TCC 821 Val Asp Trp Val Tyr Leu Ser Pro Val Leu Thr Ala Glu Gln Gly Ser 35 40 45 GAC CAC GGG TAC GAC GTC ACC GAT CCC TCC GCC GTC GAC CCC GAA CGC 869 Asp His Gly Tyr Asp Val Thr Asp Pro Ser Ala Val Asp Pro Glu Arg 50 55 60 GGC GGG CCG GAG GGC CTC GCG GCG GTT TCC AAG GCG GCC CGC GCC GCG 917 Gly Gly Pro Glu Gly Leu Ala Ala Val Ser Lys Ala Ala Arg Ala Ala 65 70 75 80 GGC ATG GGC GTG CTG ATC GAC ATC GTG CCC AAC CAC GTG GGC GTC GCG 965 Gly Met Gly Val Leu Ile Asp Ile Val Pro Asn His Val Gly Val Ala 85 90 95 ACG CCG GCG CAG AAC CCC T GG TGG TGG TCG CTG CTC AAG GAG GGA CGC 1013 Thr Pro Ala Gln Asn Pro Trp Trp Trp Ser Leu Leu Lys Glu Gly Arg 100 105 110 CAG TCC CGT TAC GCG GAG GCG TTC GAC GTC GAT TGG GAC CTC GCC GGG 1061 Gln Ser Arg Tyr Ala Glu Ala Phe Asp Val Asp Trp Asp Leu Ala Gly 115 120 125 GGA CGC ATC CGG CTG CCG GTG CTC GGC AGC GAC GAT GAC CTC GAC CAG 1109 Gly Arg Ile Arg Leu Pro Val Leu Gly Ser Asp Asp Asp Leu Asp Gln 130 135 140 CTC GAA ATC AGG GAC GGG GAG CTG CGG TAC TAC GAC CAC CGA TTC CCG 1157 Leu Glu Ile Arg Asp Gly Glu Leu Arg Tyr Tyr Asp His Arg Phe Pro 145 150 155 160 CTC GCC GAG GGA ACC TAC GCC GAA GGC GAC GCC CCG CGG GAT GTC CAC 1205 Leu Ala Glu Gly Thr Tyr Ala Glu Gly Asp Ala Pro Arg Asp Val His 165 170 175 GCC CGG CAG CAC TAC GAG CTC ATC GGC TGG CGC CGC GCG GAC AAC GAG 1253 Ala Arg Gln His Tyr Glu Leu Ile Gly Trp Arg Arg Ala Asp Asn Glu 180 185 190 CTG AAC TAC CGC CGC TTT TTC GCG GTG AAC ACG CTC GCC GGC GTC CGC 1301 Leu Asn Tyr Arg Arg Phe Phe Ala Val Asn Thr Leu Ala Gly Val Arg 195 200 205 GTG GA A ATC CCC GCC GTC TTC GAC GAG GCA CAC CAG GAG GTG GTG CGC 1349 Val Glu Ile Pro Ala Val Phe Asp Glu Ala His Gln Glu Val Val Arg 210 215 220 TGG TTC CGC GAG GAC CTT GCG GAC GGC CTG CGG ATC GAC CAC CCG GAC 1397 Trp Phe Arg Glu Asp Leu Ala Asp Gly Leu Arg Ile Asp His Pro Asp 225 230 235 240 GGC CTC GCT GAC CCC GAG GGG TAC CTG AAG CGA CTC CGG GAA GTC ACC 1445 Gly Leu Ala Asp Pro Glu Gly Tyr Leu Lys Arg Leu Arg Glu Val Thr 245 250 255 GGC GGC GCT TAC CTG CTG ATC GAA AAG ATC CTG GAG CCG GGG GAG CAG 1493 Gly Gly Ala Tyr Leu Leu Ile Glu Lys Ile Leu Glu Pro Gly Glu Gln 260 265 270 CTG CCC GCC AGC TTC GAG TGT GAA GGC ACC ACA GGC TAC GAC GCC CTC 1541 Leu Pro Ala Ser Phe Glu Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu 275 280 285 GCC GAC GTC GAC CGG GTT CTC GTG GAC CCG CGC GGC CAG GAA CCG CTG 1589 Ala Asp Val Asp Arg Val Leu Val Asp Pro Arg Gly Gln Glu Pro Leu 290 295 300 GAC CGG CTT GAC GCG TCC CTG CGT GGC GGC GAG CCC GCC GAC TAC CAG 1637 Asp Arg Leu Asp Ala Ser Leu Arg Gly Gly Glu Pro Ala Asp Tyr Gln 305 310 315 320 GAC ATG ATC CGC GGA ACC AAG CGC CGG ATC ACC GAC GGT ATC CTG CAC 1685 Asp Met Ile Arg Gly Thr Lys Arg Arg Ile Thr Asp Gly Ile Leu His 325 330 335 TCG GAG ATC CTG CGG CTG GCC CGG CTG GTT CCG GGC GAC GCC AAC GTT 1733 Ser Glu Ile Leu Arg Leu Ala Arg Leu Val Pro Gly Asp Ala Asn Val 340 345 350 TCA ATC GAC GCC GGA GCC GAC GCT CTC GCC GAA ATC ATC GCC GCC TTC 1781 Ser Ile Asp Ala Gly Ala Asp Ala Leu Ala Glu Ile Ile Ala Ala Phe 355 360 365 CCG GTC TAC CGC ACC TAC CTG CCG GAG GGC GCC GAG GTC CTG AAG GAG 1829 Pro Val Tyr Arg Thr Tyr Leu Pro Glu Gly Ala Glu Val Leu Lys Glu 370 375 380 GCG TGC GAG CTT GCC GCG CGT AGG CGG CCG GAA CTC GAC CAG GCC ATC 1877 Ala Cys Glu Leu Ala Ala Arg Arg Arg Pro Glu Leu Asp Gln Ala Ile 385 390 395 400 CAG GCT CTG CAG CCG CTG CTG CTG GAC ACG GAC CTC GAG CTT GCC CGG 1925 Gln Ala Leu Gln Pro Leu Leu Leu Asp Thr Asp Leu Glu Leu Ala Arg 405 410 415 CGC TTC CAG CAG ACC TCG GGC ATG GTC ATG GCC AAG GGC GTG GAG GAC 1973 Arg Phe Gln Gln Thr Ser Gly Met Val Met Ala Lys Gly Val Glu Asp 420 425 430 ACC GCG TTC TTC CGC TAC AAC CGC CTG GGC ACC CTC ACG GAA GTG GGC 2021 Thr Ala Phe Phe Arg Tyr Asn Arg Leu Gly Thr Leu Thr Glu Val Gly 435 440 445 GCC GAC CCC ACC GAG TTC GCC GTG GAG CCG GAC GAG TTC CAC GCC CGG 2069 Ala Asp Pro Thr Glu Phe Ala Val Glu Pro Asp Glu Phe His Ala Arg 450 455 460 CTG GCA CGC CGG CAG GCC GAG CTT CCG CTG TCC ATG ACG ACG CTG AGC 2117 Leu Ala Arg Arg Gln Ala Glu Leu Pro Leu Ser Met Thr Thr Leu Ser 465 470 475 480 ACG CAC GAC ACC AAG CGC AGC GAG GAC ACC CGA GCA AGG ATT TCG GTC 2165 Thr His Asp Thr Lys Arg Ser Glu Asp Thr Arg Ala Arg Ile Ser Val 485 490 495 ATT TCC GAG GTT GCG GGT GAC TGG GAA AAG GCC TTG AAC CGG CTG CGC 2213 Ile Ser Glu Val Ala Gly Asp Trp Glu Lys Ala Leu Asn Arg Leu Arg 500 505 510 GAC CTG GCC CCG CTG CCG GAC GGC CCG CTG TCC GCG CTG CTC TGG CAG 2261 Asp Leu Ala Pro Leu Pro Asp Gly Pro Leu Ser Ala Leu Leu Trp Gln 515 520 525 GCC ATT GCC GGC GCC TGG CCC GCC AGC CGG GAA CGC CTG CAG TAC TAC 2309 Ala Ile Ala Gly Ala T rp Pro Ala Ser Arg Glu Arg Leu Gln Tyr Tyr 530 535 540 GCG CTG AAG GCC GCG CGT GAA GCG GGG AAC TCG ACC AAC TGG ACC GAT 2357 Ala Leu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Asn Trp Thr Asp 545 550 555 560 CCG GCC CCC GCG TTC GAG GAG AAG CTG AAG GCC GCG GTC GAC GCC GTG 2405 Pro Ala Pro Ala Phe Glu Glu Lys Leu Lys Ala Ala Val Asp Ala Val 565 570 575 TTC GAC AAT CCC GCC GTG CAG GCC GAG GTG GAA GCC CTC GTC GAG CTC 2453 Phe Asp Asn Pro Ala Val Gln Ala Glu Val Glu Ala Leu Val Glu Leu 580 585 590 CTG GAG CCG TAC GGA GCT TCG AAC TCC CTC GCC GCC AAG CTC GTG CAG 2501 Leu Glu Pro Tyr Gly Ala Ser Asn Ser Leu Ala Ala Lys Leu Val Gln 595 600 605 CTG ACC ATG CCC GGC GTC CCG GAC GTC TAC CAG GGC ACG GAG TTC TGG 2549 Leu Thr Met Pro Gly Val Pro Asp Val Tyr Gln Gly Thr Glu Phe Trp 610 615 620 GAC CGG TCG CTG ACG GAC CCG GAC AAC CGG CGG CCG TTC AGC TTC GAC 2597 Asp Arg Ser Leu Thr Asp Pro Asp Asn Arg Arg Pro Phe Ser Phe Asp 625 630 635 640 GAC CGC CGC GCC GCG CTG GAG CAG CTG GAT GCC GGC GAC CTT CCC GCG 264 5 Asp Arg Arg Ala Ala Leu Glu Gln Leu Asp Ala Gly Asp Leu Pro Ala 645 650 655 TCA TTT ACC GAT GAG CGG ACG AAG CTG CTA GTG ACG TCG CGC GCG CTG 2693 Ser Phe Thr Asp Glu Arg Thr Lys Leu Leu Val Thr Ser Arg Ala Leu 660 665 670 CGG CTG CGC CGG GAC CGT CCG GAG CTG TTC ACG GGG TAC CGG CCG GTC 2741 Arg Leu Arg Arg Asp Arg Pro Glu Leu Phe Thr Gly Tyr Arg Pro Val 675 680 685 CTG GCC AGC GGG CCC GCC GCC GGG CAC CTG CTC GCG TTC GAC CGC GGC 2789 Leu Ala Ser Gly Pro Ala Ala Gly His Leu Leu Ala Phe Asp Arg Gly 690 695 700 ACC GCG GCG GCG CCG GGT GCA TTG ACC CTC GCC ACG CGG CTT CCC TAC 2837 Thr Ala Ala Ala Ala Pro Gly Ala Leu Thr Leu Ala Thr Arg Leu Pro Tyr 705 710 715 720 GGG CTG GAA CAG TCG GGT GGA TGG CGG GAC ACC GCC GTC GAA CTT AAC 2885 Gly Leu Glu Gln Ser Gly Gly Trp Arg Asp Thr Ala Val Glu Leu Asn 725 730 735 ACC GCC ATG AAA GAC GAA CTG ACC GGT GCC GGC TTC GGA CCG GGG GCA 2933 Thr Ala Met Lys Asp Glu Leu Thr Gly Ala Gly Phe Gly Pro Gly Ala 740 745 750 GTG AAG ATC GCC GAC ATC TTC CGG TCG TTC CCC GTT GCG CTG CTG GTG 2981 Val Lys Ile Ala Asp Ile Phe Arg Ser Phe Pro Val Ala Leu Leu Val 755 760 765 CCG CAG ACA GGA GGA GAG TCA 3002 Pro Gln Thr Gly Gly Glu Ser 770 775 TGACGCACCC CTAC CCGCGG GAAGCCGCGA AACCCGTCCT GG 3073

SEQ ID NO: 7 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Sequence Met Arg Thr Pro Ala Ser Thr Tyr Arg Leu Gln Ile Arg Arg Gly Phe Thr 1 5 10 15 Leu Phe Asp 20

SEQ ID NO: 8 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Sequence Met Arg Thr Pro Val Ser Thr Tyr Arg Leu Gln Ile Arg Lys Gly Phe Thr 1 5 10 15 Leu Phe Asp 20

SEQ ID NO: 9 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence Arg Ser Glu Asp Thr Arg Ala Arg Ile Ser Val Ile Ala Glu Val Ala Pro 1 5 10 15 Glu Trp Glu Lys 20

SEQ ID NO: 10 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence Leu Val Gln Leu Thr Met Pro Gly Val Pro Asp Val Tyr Gln Gly Thr Glu 1 5 10 15 Phe Trp Asp Arg 20

SEQ ID NO: 11 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence Leu Val Gln Leu Thr Met Pro Gly Val Pro Asp Val Tyr Gln Gly Thr Glu 1 5 10 15 Phe Trp Asp 20

SEQ ID NO: 12 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment sequence Glu Gly Arg Gln Ser Arg Tyr Ala Glu Ala Phe Asp Val Asp Trp Asp Leu 1 5 10 15 Ala Gly Gly 20

[Brief description of drawings]

FIG. 1 is a diagram showing the optimum temperature of enzyme M-11.

FIG. 2 is a diagram showing the optimum temperature of enzyme Q36.

FIG. 3 is a graph showing the optimum pH of enzyme M-11.

FIG. 4 is a diagram showing the optimum pH of enzyme Q36.

FIG. 5 is a diagram showing thermostability of enzyme M-11.

FIG. 6 shows the thermostability of enzyme Q36.

FIG. 7 is a graph showing pH stability of enzyme M-11.

FIG. 8 is a graph showing pH stability of enzyme Q36.

FIG. 9: Recombinant DNA pBMT7 according to the present invention
Is a restriction enzyme map of. It should be noted that in the figure, the bold line display portion indicates the DNA encoding the enzyme M-11.

FIG. 10: Recombinant DNA pBQT according to the present invention
13 is a restriction enzyme map of 13. It should be noted that in the figure, the bold line display portion indicates the DNA encoding the enzyme Q36.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1:41) (C12N 15/09 ZNA C12R 1:06) (C12N 15/09 ZNA C12R 1: 13) (C12N 15/09 ZNA C12R 1:20) (C12N 15/09 ZNA C12R 1: 265) (C12N 15/09 ZNA C12R 1:32) (C12N 15/09 ZNA C12R 1:01) C12R 1:41 ) (C12N 15/00 ZNA A C12R 1:06) (C12N 15/00 ZNA A C12R 1:13) (C12N 15/00 ZNA A C12R 1:20) (C12N 15/00 ZNA A C12R 1: 265) ( C12N 15/00 ZNA A C12R 1:32) (C12N 15/00 ZNA A C12R 1:01)

Claims (24)

[Claims] 1. A DNA encoding an enzyme that produces a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more. 2. The DNA according to claim 1, wherein the enzyme has the following physicochemical properties. (1) Molecular weight of about 76,000 to 87,000 daltons (SDS-polyacrylamide gel electrophoresis) (2) Isoelectric point of about 3.6 to 4.6 (isoelectric focusing) 3. The enzyme is SEQ ID NO: 1 or 2 in the sequence listing.
The DNA according to claim 1 or 2, which has the amino acid sequence shown in or an amino acid sequence homologous thereto. 4. The DNA according to claim 1, 2 or 3, wherein the DNA has a base sequence shown in SEQ ID NO: 3 or 4 in the sequence listing, a base sequence homologous thereto or a base sequence complementary thereto. 5. One or two bases in the base sequence shown in SEQ ID NO: 3 or 4 in the sequence listing without changing the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing based on the degeneracy of the genetic code. The DNA according to claim 1, 2, 3 or 4, wherein the above is substituted with another base. 6. The DNA according to claim 1, which has the base sequence shown in SEQ ID NO: 5 or 6 in the sequence listing. 7. A microorganism derived from Rhizobium, Arthrobacter, Brevibacterium, Flavobacterium, Micrococcus, Curtobacterium, Mycobacterium or Terrabactor.
The DNA according to 2, 3, 4, 5 or 6. 8. A replicable recombinant DNA comprising the DNA according to claim 1 and a vector capable of autonomous replication. 9. The replicable recombinant DN according to claim 8, wherein the enzyme encoded by the DNA has the following physicochemical properties.
A. (1) Molecular weight of about 76,000 to 87,000 daltons (SDS-polyacrylamide gel electrophoresis) (2) Isoelectric point of about 3.6 to 4.6 (isoelectric focusing) 10. The replicable recombinant DNA according to claim 8 or 9, wherein the DNA encodes an enzyme having the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing or an amino acid sequence homologous thereto. 11. The replicable set according to claim 8, 9 or 10 wherein the DNA has a base sequence shown in SEQ ID NO: 3 or 4 in the sequence listing, a base sequence homologous thereto or a base sequence complementary thereto. Replacement DNA. 12. The DNA is SEQ ID NO: 3 or 4 in the Sequence Listing without changing the amino acid sequence shown in the SEQ ID NO: 1 or 2 in the Sequence Listing based on the degeneracy of the genetic code.
The replicable recombinant DNA according to claim 8, 9, 10 or 11, wherein one or more of the bases in the base sequence shown in (1) is replaced with another base. 13. The DNA having the nucleotide sequence shown in SEQ ID NO: 5 or 6 in the sequence listing.
Or a replicable recombinant DNA according to item 12. 14. The method according to claim 8, wherein the DNA is derived from a microorganism of the genus Rhizobium, Arthrobacter, Brevibacterium, Flavobacterium, Micrococcus, Curtobacterium, Mycobacterium or Terrabactor. The replicable recombinant DNA according to 9, 10, 11, 12 or 13. 15. The replicable recombinant DNA according to claim 8, 9, 10, 11, 12, 13 or 14, wherein the autonomously replicable vector is a plasmid vector Bluescript II SK (+). 16. A transformant obtained by appropriately introducing into a host recombinant DNA comprising the DNA according to claim 1 and a vector capable of autonomous replication. 17. The transformant according to claim 16, wherein the enzyme encoded by DNA has the following physicochemical properties. (1) Molecular weight of about 76,000 to 87,000 daltons (SDS-polyacrylamide gel electrophoresis) (2) Isoelectric point of about 3.6 to 4.6 (isoelectric focusing) 18. The transformant according to claim 16 or 17, wherein the DNA encodes an enzyme having the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing or an amino acid sequence homologous thereto. 19. The DNA having a base sequence shown in SEQ ID NO: 3 or 4 in the sequence listing, a base sequence homologous thereto, or a base sequence complementary thereto.
The transformant according to 8. 20. The DNA is SEQ ID NO: 3 or 4 in the sequence listing without changing the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing based on the degeneracy of the genetic code.
The transformant according to claim 16, 17, 18 or 19, wherein one or two or more of the bases in the base sequence shown in (1) is replaced with another base. 21. The DNA sequence having the nucleotide sequence shown in SEQ ID NO: 5 or 6 in the sequence listing.
The transformant according to 19 or 20. 22. The method according to claim 16, wherein the DNA is derived from a microorganism of the genus Rhizobium, Arthrobacter, Brevibacterium, Flavobacterium, Micrococcus, Curtobacterium, Mycobacterium or Terrabactor. The transformant according to 17, 18, 19, 20, or 21. 23. The autonomously replicable vector is the plasmid vector Bluescript II SK (+), 16, 17, 18, 19, 20, 21 or 22.
The transformant according to. 24. The method according to claim 16, wherein the host is Escherichia coli.
The transformant according to 7, 18, 19, 20, 21, 22 or 23.