JP2002017391A - Method for mass production of difructose dianhydride iv - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing difuructose dianhydride IV (DFA IV) by applying an LFTase gene which is a new gene prepared based on a new design and is mass producable the DFA IV that is a functional oligo saccharide, efficiently, since it highly expresses a highly active enzyme, to levan which can be efficiently produced in a large amount by culturing bacteria belonging to the genus Serratia (Serratia levanicum) on a sucrose-containing medium, and then possible to produce the DFA IV through a route of sucrose to levan and to DFA IV in a continuous process. SOLUTION: This method for producing the DFA IV is to apply the new LFTase to the level for producing the DFA IV efficiently in the large amount.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to difructose
The present invention relates to a method for producing dianhydride IV (hereinafter, also referred to as DFA IV).
The present invention relates to a novel and useful method for efficiently mass-producing DFA IV from levan by using a novel levanfructotransferase.

[0002]

2. Description of the Related Art DFA IV is an indigestible disaccharide having a 6,2 ': 2,6'-type fructoside bond, and has a low calorie and fructose-based cool sweetness. It also has a calcium absorption promoting effect, and has been confirmed to be useful as a functional food in addition to sweeteners.
Its mass production is desired. And DFA IV is
Levan has a levan fructotransferase (hereinafter referred to as L
(Also referred to as FFTase), but for both Levan and LFTase,
At present, the production volume is insufficient, and a satisfactory and efficient manufacturing method has not yet been developed.
High-purity levan and high-purity LFT for mass production of IV
There is a strong demand for the probability of a method of industrially producing ase in large quantities. There is also a need for the development of an efficient DFA IV recovery, separation and purification method.

[0003] For the production of DFA IV by reacting with levan, LFTase is prepared by using Arthrobacter nicotinovorans GS-9.
(Hereinafter, also referred to as GS-9 bacteria) is an inducing enzyme that is induced and expressed by levan in the medium during culturing. However, the expression level of this enzyme is very small, and it is difficult to apply it industrially. Further, the production of an LFTase enzyme solution requires levan, which complicates the process and is a major obstacle to industrial production. In addition, at this time, industrial mass production of levan itself has not been established, and the decrease in LFTase productivity by this method cannot be denied.

[0004] In addition, as described above, natural LFTase
In addition to the production of LFTase, the production of recombinant LFTase was also studied. That is, LFT derived from GS-9
The ase gene is described in Saito et al. (Saito et al .; Biosci.
Biotech. Biochem., 1997, 61 (12), 2076-2079), the primary sequence of which has been elucidated, and an expression system using E. coli as a host has been constructed using the pUC plasmid vector system. Have been. However,
The expression level is not sufficiently satisfactory, and there is still room for improvement.

[0005]

SUMMARY OF THE INVENTION In view of the state of the art described above, the present invention solves the above-mentioned disadvantages and provides a high-purity DF.
It was made for the purpose of newly developing a method for efficiently producing AIV, and a new LFTase was developed.
The purpose of the present invention is to increase the efficiency of the FA IV production method itself and to develop an efficient purification process. Also, DFA
IV can be produced by treating levan with LFTase. However, since an efficient mass production method of levan itself, which is a raw material or a substrate, has not been established, an efficient mass production method of DFA IV has been established. To establish it, mass production of levan is necessary, and in the sugar industry where the development of new uses for sucrose is expected, it would be even more convenient if a mass production method of levan using sucrose was developed. is there.

[0006]

DISCLOSURE OF THE INVENTION The present invention has been made to achieve the above object, and has been studied from all aspects such as microbial engineering, genetic engineering, and chromatography theory. A new method for the efficient production and purification of DFT IV using the modified LFTase and DFA IV has been newly developed, and finally the present invention has been completed.

[0007] In addition, for levan used as a raw material, not only succeeded in developing a new efficient mass production method, but also succeeded in developing a new method for producing levan using sucrose. Moreover, quite unexpectedly, after the production of levan, it is possible to proceed to the next LFTase treatment without isolating levan from the culture broth, followed by purification, isolation and crystallization of the crude DFA IV A very useful finding is that sucrose can be used as a raw material, and high-purity DFA can be continuously obtained without interruption.
For the first time, mass production of IV was also successful, and the present invention was completed.

As described above, the reaction proceeds smoothly without causing mutual interference in a series of processes, and even though a plurality of microorganisms and enzymes are used and various complicated treatments are performed, The fact that high-purity DFA IV can be mass-produced very efficiently is just astonishing.

Hereinafter, the present invention will be described with reference to an example of a method of producing DFA IV by a series of operations using sucrose as a starting material (FIG. 8). It is also possible to perform an intermittent operation to perform the next reaction using, for example, using commercially available levan,
It is of course possible to produce DFA IV by acting on LFTase. Hereinafter, the present invention will be described according to this flow, taking continuous processing as an example.

Levan is a β-2,6-linked polyfructan (a polysaccharide composed of a chain of β-2,6 bonds of fructofuranose), and is used for pharmaceutical purposes such as addition to reagents or blood substitutes. In addition, since it is a polysaccharide composed of a fructose chain, it is expected to be effectively used in the field of food and drink. And Leban, Bacillussu
btilis. Bacillus megatherium. Streptococcus saliva
Although it is produced by culturing rius or the like, the present invention has developed a more efficient production method.

That is, the present invention is to produce levan efficiently by culturing levanogenic bacteria belonging to the genus Serratia in a sugar-containing solution to produce levan.

In the present invention, any microorganism can be used as long as it is a levan-forming bacterium belonging to the genus Serratia.
Serratia levanicum as an example
(Hereinafter, sometimes referred to as Serratia).
Further, a successful strain was successfully screened from among Serratia strains, and one of the strains was identified as Serratiale.
vanicum NN strain (hereinafter referred to as Serratia NN strain).
(Also referred to as a bacterium) and deposited with the National Institute of Advanced Industrial Science and Technology as a FERM P-17895.

In order to carry out the present invention, it is necessary to contact sucrose with Serratia bacteria. For example, Serratia bacteria may be cultured in a liquid containing 1 to 35% sugar. The cultivation of Serratia bacterium in the present invention can be performed according to a conventional method, except that sugar is contained in the medium, and sucrose is added to commonly used carbon sources, nitrogen sources, minerals, etc. to culture Serratia bacterium. Suitable pH (6-7.5), temperature (25-35 ° C)
The culture may be adjusted to the above. The sucrose content is 1 L of medium.
Per, 10 to 400 g, preferably 100 to 300 g,
More preferably, the amount is 150 to 250 g, but if it is less than the above range, the culturing time may be extended, and it is not limited to only the above range.

As described above, the present inventors produce Levan by culturing Serratia in a sucrose-containing culture medium. Therefore, the present inventors have developed a method for producing Levan by separating and collecting Levan. It was successful for the first time. And more characteristically, the levan produced by the bacteria belonging to the genus Serratia developed by the present inventors is a linear type having fewer β-2, 1 bonds compared to levan produced by other microorganisms. It is Leban. Therefore, levan produced by a bacterium belonging to the genus Serratia is suitable as a raw material for producing oligosaccharides, and is also excellent in that oligosaccharides can be obtained in high yield. Therefore, when levan obtained by this method is used, the present invention for producing DFA IV which is a kind of oligosaccharide has a remarkable effect that the production efficiency can be further improved. The present inventors were not satisfied with this, and as a result of conducting research to further increase the production efficiency of levan, newly developed a preculture method and a cooling ripening method, and at least one of these methods was used. When combined with the method using a sucrose-containing medium as described above, the production efficiency of levan was also significantly improved. The present invention also includes these.

The pre-culture method involves culturing Serratia bacteria in a small amount of medium prior to a culture method (sometimes called a main culture method) performed in a sucrose-containing medium. Usually, the medium is cultured in a medium to which sucrose is not added (the medium may have the same composition as the medium used in the main culture method or may be different). After culturing for a certain period (about 10 to 30 hours), the whole culture (cells + medium) is inoculated directly into the main culture solution without separating or separating the cells. When precultured with a sucrose-containing solution, levan may already be produced in the culture.

The cooling ripening method is a method of raising the lowered pH and maintaining the temperature at a low temperature after completion of the main culture or after a certain period of time, and is a method created by the present inventors,
This is a new name for the cooling aging method. The end of the main culture is when the amount of levan production reaches a peak, and it is natural that the amount of levan production can be obtained by actual measurement. It may be good and / or when the pH is measured and the pH drops to around 3.5 to 5.3. In addition, since the main culture usually ends in many cases after 5 to 20 hours, the end of the main culture may be determined by the culture time, or the amount of levan production, viscosity, and pH may be determined based on the culture time. May be measured to determine the end of the culture.

After the culture is stopped or terminated in this way, the lowered pH is raised (pH 4.5-7.0), and the temperature is lowered (20 ° C. or lower, preferably 15 ° C. or lower, more preferably 1-10 ° C.). To maintain. The period may be set to the time when the production amount of levan is measured and reaches a peak in the same manner as described above, and is usually 3 days to 10 days or more, but 1 week to
About two weeks is a good guide.

After cooling and ripening in this manner, levan may be recovered according to a conventional method. For example, a solid-liquid separation of a culture is performed (by a conventional method such as filtration, centrifugation, and decantation), and the obtained product is obtained. The levan is precipitated as a white precipitate by adding 40-100%, preferably 50-70% alcohol to the resulting culture. If necessary, it is dried to obtain levan in the form of a white amorphous powder.
As the alcohol, one or more kinds of aliphatic saturated or unsaturated alcohols such as methanol, propanol, isopropanol, ethanol, butanol, sec (or tert) butanol, amyl alcohol, isoamyl alcohol, and allyl alcohol, and other various alcohols are used in combination. it can. The precipitated levan is separated and dried by ventilation drying, drying under reduced pressure, desiccator drying or other methods.

Using the purified levan once isolated in this manner, this was redissolved in a buffer, and the resulting solution was added to LFT.
ase (or an enzyme solution),
Although AIV can be synthesized, in the present invention,
Without isolating levan, for example, after cooling and aging, the temperature of the solution is adjusted to 30 to 40 ° C., the pH is adjusted to 5 to 7 while stirring at 50 to 200 rpm, and LFTase (or its enzyme solution) is dissolved. After the addition, a synthesis reaction may be performed, for example, for 12 to 36 hours to produce DFA IV from levan.

Next, L used in the DFA IV synthesis reaction
FTase will be described. In the present invention, an excellent active LFTa which does not have the above-mentioned drawbacks is used.
Since it is necessary to use se, as a result of studies from various fields, we focused on genetic engineering techniques and decided to design a system to further enhance expression efficiency.

That is, the inventors of the present invention aimed to greatly improve the expression system of the LFTase gene which Saito et al. Succeeded in constructing and to increase the expression efficiency. In order to develop a new one, an attempt was made to newly create only an open reading frame (hereinafter sometimes referred to as an ORF) of the LFTase gene, and as a result of various designs, the ORF base sequence was finally determined. Then, the ORF is to be created by the PCR method.
Determination of the base sequences of sense primers and antisense primers and their artificial synthesis were attempted and succeeded. PCR using GS-9 chromosomal DNA as a template
By carrying out, ORF only was successfully prepared.

This is ligated to a multi-cloning site of a pET-type plasmid vector, for example, a pET-3a plasmid vector, and a new expression plasmid pET /
LFTsa was constructed. ORF cutout is LFTa
The restriction enzyme NdeI and BamHI sites were created at the start and stop codons of the se gene by site-specific conversion, and the ORF was framed particularly. This plasmid vectors efficiently transfer the gene linked as a foreign gene in E. coli, the translation may, T7 La
c promoter has been introduced and Isopropyl
-Β-D-thiogalactopyranosid
e (sometimes referred to as IPTG) enables high expression and is devised so that partial purification can be performed by a simple operation.

This plasmid vector is used for E. coli BL21.
The transformant transformed into the (DE3) strain was Escher
ichia coli BL21 (DE3) -pET /
It was named LFTsa and deposited with the National Institute of Advanced Industrial Science and Technology as a FERM P-17896. The transformant thus created has a very high LFTas
e activity, and according to the present invention, the original strain (GS-
E. coli JM10 transfected with DNA molecule pBB-1, which was successfully constructed by Saito et al.
9 strains (Escherichia coli JM109 / pLFT-BBI: JP-A-1)
1-69978, FERM P-16316) 2-2
It is possible to obtain an activity as high as 0-fold, and it is possible to obtain an activity several tens of times higher than these by further screening. In addition, it was also confirmed that according to the present invention, a remarkable effect of eliminating the need for levan, which was conventionally required for enzyme production, was obtained.

By culturing the transformant thus created, LFTase can be expressed in large amounts. Since the transformant produced LFTase in the cells, after the culture was completed, the cells were disrupted, and the supernatant was used as a cell-free extract. From this cell-free extract, dialysis was performed according to a conventional method for enzyme purification. , Chromatographic treatment, concentration, ultrafiltration, etc., as appropriate, to obtain a purified enzyme.

The LFTase of the present invention is a novel enzyme having the following physicochemical properties, which has been artificially created as a result of extremely careful calculation by gene design, and has an extremely high activity. It has excellent effects such as not being an inducible enzyme that requires the presence of levan for its production. LFT thus prepared
Ase, by making this act on Levan, DF
AIV can be manufactured efficiently. At that time LF
Tase is not only isolated and purified, but also partially purified, or the above-described cell-free extract (or its concentrated solution).
Can also be used.

(1) Action The present enzyme has an action of decomposing levane, a polyfractane having a β-2,6 fructoside bond, to synthesize DFA IV.

(2) Method for measuring titer The enzyme activity was determined by the method of Saito et al. (Saito et al .; Biosci.
Biotech. Biochem., 1997, 61 (12), 2076-2079). 0.1 M sodium phosphate buffer (p
H6.0), the enzyme solution was mixed with a substrate solution in which levan was dissolved so that the final concentration became 10 g / L, and the mixture was heated at 37 ° C.
For 10 minutes. Then, the reaction solution is placed in boiling water for 5 hours.
The reaction was stopped by holding for about 1 minute. Generated DFA
IV was quantified by HPLC, and the activity value was calculated. Here, one unit of the enzyme was defined as the amount of the enzyme that produced 1 μmol of a reaction product per minute under the present enzyme reaction conditions.

(3) Substrate Specificity Carbohydrate-related substances that can be catalyzed by the present enzyme were investigated. As a result, the enzyme was found to act on levan and levan oligosaccharides having a chain length of 3 to 7. It did not act on various other carbohydrates.

(4) Optimum pH and stable pH range The effect of the present enzyme on pH was investigated. For the study of stability, the enzyme solution was maintained at 4 ° C. for 24 hours in a buffer at each pH, and then the solution was replaced with a buffer at pH 6.0, and the enzyme activity was measured according to a standard method. As a result, the optimal pH of the enzyme
Was 6.0 and stable over a wide range of pH from 4.0 to 12.0.

(5) Optimum temperature and stable temperature range The effect of the present enzyme on the temperature was investigated. Stability was examined by keeping the enzyme solution at each temperature for 20 minutes and then quickly
C., and the enzyme activity was measured according to a standard method. As a result, the optimum temperature was 50 ° C., and the temperature was stable up to 40 ° C.

(6) Deactivation Conditions Due to pH, Temperature, etc. In the pH and temperature conditions described in (4) and (5), especially in stability, deactivation conditions are in a range where the relative activity is reduced or more. Is considered. Further, disappearance of the enzyme activity after keeping the enzyme solution in boiling water for 5 minutes has been confirmed.

(7) Inhibition, activation and stabilization In the presence of Hg ²⁺ ions and Ag ²⁺ ions, the activity was significantly inhibited.

(8) Purification Method After preparing a cell-free extract of transformed E. coli,
-Toyopearl 650M (manufactured by Toso Corporation) anion exchange resin or the like can be partially purified efficiently.

(9) Method of Measuring Molecular Weight and Molecular Weight The molecular weight of the LFTase protein was measured simultaneously with Laemmli (Laemmli: Na
ture (London), 1970, 227, 680-685)
Performed by S-PAGE. The gel concentration was 7.5%. Protein staining by Coomassie Brill
lient Blue was used. As a result, the molecular weight is
It was measured at approximately 50,000 Da. This value is similar to the molecular weight of 53,153 Da calculated from the amino acid sequence deduced from the DNA base sequence of the gene, and is reliable.

(10) Identification of Reaction Product After purification of the oligosaccharide produced by the present enzyme, C ¹³ -NM
The spectrum was analyzed by R. As a result, the reaction product was identified as DFA IV.

(11) Inducibility of the enzyme Levan is not required for the induced expression of the enzyme.

The novel enzyme according to the present invention includes all enzymes having the above-mentioned physicochemical properties.
For example, the protein represented by the amino acid sequence of SEQ ID NO: 1 (FIG. 1) is also exemplified as an example. Even in the production method, the gene (the base sequence is shown in SEQ ID NO: 2, FIG.
No. 1) (FERM P-1)
7896), the novel enzyme according to the present invention can be obtained. Since one example of the novel amino acid sequence has been clarified (SEQ ID NO: 1), the enzyme can also be obtained by synthesis.

The novel LFTase thus prepared
Is a solution of the enzyme itself (for example, the above-described cell-free extract) or a levan solution (pH 5-
Adjusted to around 6.5) to decompose Levan and
AIV is synthesized.

In order to crystallize DFA IV, it is necessary to increase its content. First, the saccharide contained in the DFA IV-containing liquid is assimilated or / and removed, and is reduced as much as possible to increase the DFA IV content. Next, the DF
In order to desalt the AIV-containing solution, the solution is passed through a packed column using a strongly basic ion exchange resin and a strongly acidic ion exchange resin or a strongly basic ion exchange resin. A liquid obtained by collecting and concentrating the effluent is passed through a chromatographic column made of a salt-type cation exchange resin, and the effluent DFAIV fraction is separated and collected by chromatography to obtain high-purity DF.
A liquid IV is obtained. Then, if the DFA IV fraction is concentrated and then decocted or cooled, DFA IV crystals can be precipitated, and DFA IV crystals can be produced. In order to crystallize DFA IV efficiently, its purity (DFA IV / solid content W / W%) should be 60
% Or more.

The yeast used for assimilating the sugar in the DFA IV-containing liquid may be any yeast belonging to Saccharomyces cerevisiae, and raw yeast and dried yeast are used. The higher the concentration of the DFA IV-containing liquid to be subjected to the yeast aeration culture treatment, the higher the concentration of the stock solution to be subjected to the chromatographic separation in the subsequent step, which is preferable since the concentration cost is reduced. When producing yeast,
Normally, the concentration in the culture tank is maintained at a solid content of 0.01 W / W%, but in the case of the present invention, the concentration of the DFA IV-containing solution in the culture tank is in the range of 10 to 60 W / W% of the solid content in the yeast treatment. And preferably 30 to 50 W / W%, so that the concentration of the difference in the yeast treatment can be increased.

The temperature of the yeast treatment of the DFA IV-containing liquid is 30 to 37 ° C., and the amount of yeast added to the DFA IV-containing liquid and the treatment time are in a substantially proportional relationship. Is appropriately selected. In addition, Na-, K-, and Ca-type resins are used as the salt-type cation exchange resin used in the chromatography. As the chromatographic separation method, there are a monobet method, a pseudo-movement method, an intermediate method described above, and the like, and any method can be used.

As described above, by making full use of these technologies, the production of DFA IV from sugar has been made efficient and continuous. High purity (about 9 to 150 g or more) from 1000 g of sugar (9
(0% or more) DFA IV single fraction.

After the yeast treatment, if desired, a flocculant (for example, PAC manufactured by Mitsubishi Chemical Corporation) is added, the cells are recovered by centrifugation or the like, and then heat-treated (80 to 100 ° C.). ), May be concentrated and then proceed to desalination treatment.

Hereinafter, embodiments of the present invention will be described.

[0045]

Example 1 Serratia NN (FERM P-1789)
Levan was manufactured using 5). The culture of Serratia is described in Yokota et al. (Yokota et al .: Biosci. Biotec
h. Biochem., 1993, 57, 745-749), and Kojim
(Kojima et al .: J. Ferment. Bioeng., 1993, 7
This was improved with reference to the method of 5, 9-12). The details are described below.

Preculture method 5.0 g / L yeast extract, 1
In a 3 L Erlenmeyer flask, 1 L of a medium having a composition of 0.0 g / L polypeptone and 10.0 g / L sodium chloride (pH 7.0) (sometimes referred to as Medium 1) is prepared.
One loopful of Serratia was inoculated and rotary-cultured with shaking at 30 ° C. for 24 hours.

Main culture method 200 g / L sucrose, g / L
Yeast extract, 8.5 g / L polypeptone, 0.3 g /
A medium (sometimes referred to as medium 2) having a composition of L glucose (pH 7.0) was prepared in a 200-L stainless steel jar fermenter (Model F-02, manufactured by Hitachi, Ltd.), and cultured for pre-cultured Serratia bacteria The whole liquid was inoculated.
There is no problem even if the amounts of the yeast extract and polypeptone in the medium 2 are half. Then, at a temperature of 30 ° C. and stirring of 80 rpm
m and aeration at 1 vvm. Temperature, pH and viscosity were measured over time.

Cultivation is terminated when the culture solution viscosity has increased the most and the pH has dropped to around 4.0. The culture is stopped and the pH is adjusted to 5.5. The temperature was cooled to 4 ° C. This condition was controlled so as to be maintained for one week or more.

As described above, using Medium 1 and Medium 2,
Serratia was cultured in a 200 L fermenter to synthesize levan. The progress at that time is shown in FIG. 10
When the pH was adjusted over time and cooled and aged, levan synthesis increased without decreasing. There is a high correlation between levan synthesis and the viscosity in the medium, and the degree of levan synthesis is monitored by viscosity. The highest viscosity in the flask test is 30
Therefore, in the present example, the value was 40, and the efficiency was sufficiently improved.

The levan was precipitated with 65% ethanol. This was centrifuged to separate and collect levan.
Next, this was dried in an electric dryer at 50 ° C. for 1 hour to obtain 60 g of levan as a white powder.

[0051]

Embodiment 2 (1) Arthrobacter nic
Preparation of only the open reading frame (ORF) of the levanfructotransferase (LFTase) gene of otinovorans GS-9 strain The basic techniques for gene recombination manipulation experiments frequently used in the present specification are known in the art, and are not specifically described. The method of Sambrook et al. (Sambrook et al .:
Molecular Cloning A Laboratory Manual, 2nd Edition, 1989).

Genetic recombination was performed for the purpose of preparing only the parts necessary for the expression and activity of the LFTase gene.
Here, “only necessary parts” means “mature LFTas
e only the gene corresponding to e). Sait
o et al. (Saito et al .: Biosci. Biotech. Biochem., 19
97, 61, 2076-2079). The nucleotide sequence of the gene DNA containing the LFTase gene is shown in SEQ ID NO: 4 (FIG. 4) (where the nucleotide sequence from the 91st nucleotide to the 1644th nucleotide is the OR of the LFTase gene.
F) and the amino acid sequence of LFTase deduced therefrom is shown in SEQ ID NO: 3 (FIG. 3).

First, in the design, SEQ ID No. 4 in the sequence listing was set to include the first to 90th nucleotide sequences and the LFTase
Base sequence 91 to 189, which is a part of the structural gene of
Remove up to th. The 91st to 189th nucleotide sequences correspond to the first signal peptide sequence of LFTase (the 1st to 33rd amino acid sequences),
Even if removed, the mature part functions and there is no problem with the enzyme activity. Further, a new start codon is added. Immediately before the start codon and immediately after the stop codon, an appropriate restriction enzyme site is set by forcibly replacing the nucleotide sequence so that the gene can be arbitrarily cut.

In practice, the nucleotide sequence from the 187th to 189th nucleotides
First, a start codon sequence (atg) is added, and a restriction enzyme NdeI (catatg) site is added so that the immediately preceding codon sequence can be cleaved, and BamHI (ggatcc) is added at the stop codon.
The site was designed. Primers of about 20 bases containing the base sequence to be replaced were prepared, and a PCR reaction was performed using chromosomal DNA of GS-9 bacteria as a template. Taq polymerase was manufactured by Takara Shuzo. After the PCR reaction, the product was recovered and purified, treated with the restriction enzymes NdeI and BamHI, ligated to an appropriate plasmid vector, and the nucleotide sequence substitution was confirmed by DNA sequencing. In addition, since there are no restriction enzymes NdeI and BamHI sites in the structural gene of LFTase, it is not cleaved midway.

In summary, the substituted bases are as follows: SEQ ID NO: 186 from c to T, SEQ ID NO: 1
No. 87 from g to A, SEQ ID NO: 188 from c to T,
SEQ ID NO: 189 from c to G,

SEQ ID NO: 1646 was converted from c to G, SEQ ID NO: 1647 from c to A, and SEQ ID NO: 1649 from g to C. As shown in SEQ ID NO: 2 (FIG. 2),
Important portions used in the present invention are cut out, and include restriction enzymes NdeI (catatg) and BamHI.
A (ggatcc) site was forcibly created. The remaining array (hyphen) is not used.

The above-described design was implemented by the PCR method. To prepare only the LFTase ORF, Sa
Ito et al. (Saito et al .: Biosci. Biotech. Bioche
m., 1997, 61 (12), 2076-2079) with reference to the analyzed DNA base sequence.
Two types of synthetic oligonucleotide primers of 7 bases were prepared. These primers were artificially synthesized using a DNA synthesizer (manufactured by Applied Biosystems).

The amplified PCR product (OFT of LFTase)
RF only) can be cleaved by restriction enzyme treatment,
To enable ligation to a plasmid vector, the nucleotide sequence was forcibly replaced to create a restriction enzyme site. Restriction enzyme sites can also be distinguished between the start codon side and the stop codon side.
Two types of NdeI and BamHI were set. The sense primer and the antisense primer are shown in SEQ ID NOS: 5 and 6 (FIGS. 5 and 6), and the substituted bases are underlined in the figures.

Separately, the GS-9 chromosomal DNA prepared by the method of Saito et al. Was mixed with the above two kinds of primers and a reagent for PCR reaction, and a PCR reaction was carried out. Taq DNA polymerase used a standard product from Takara Shuzo. The PCR reaction conditions were as follows: the total solution volume was 100 microliters, the number of cycles was 35, and one cycle was 98.
1 minute at 68 ° C and 3 minutes at 68 ° C. Amplified LFT
Confirmation of the ORF fragment of ase, the DNA base sequence of about 1,500 base pairs, particularly the DNA base sequence of the substituted site,
Performed at the sequence level. The obtained DNA base sequence is shown in SEQ ID NO: 2 (FIG. 2).

(2) Ligation of ORF of LFTase and Plasmid Vector The PCR reaction solution (containing ORF fragment of LFTase) of (1) was recovered and purified by ethanol precipitation, and then the restriction enzyme Nd
Double digestion was performed with eI and BamHI. This was further recovered and purified by the ethanol precipitation method. Apart from this, pET-3a
A plasmid vector (manufactured by Takara Shuzo) was double-digested with restriction enzymes NdeI and BamHI, recovered and purified by an ethanol precipitation method, and ligated to the previously purified PCR product by a ligation reaction. The plasmid vector prepared here was named pET / LFTsa (FIG. 7). The construction of the conventional pLFT-BB1 is also shown.

(3) Plasmid vector pET / LFT
Transformation of sa Plasmid vector pET / LFT prepared in (2)
sa is Hanahan (D. Hanahan: Techniques for
transformation of E. coli, in DNA Cloning: A Pract
ical Approach, Vol.1, ed.by DM Glover, IRL Pre
ss, Oxford, 1985, 109-135), and transformed into competent cells of E. coli BL21 (DE3) strain (Takara Shuzo) by the heat shock method. After culturing in a selective medium, the unified transgenic E. coli is
li BL21 (DE3) -pET / LFTsa, which was deposited as FERM P-17896 with the National Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology.

(4) Escherichia coli BL21 (DE3) -pET / L
Production of LFTase by FTsa Escherichia coli BL21 (DE3) -pET / LFTsa (FERM P-1789
6) was cultured in 5 ml of LB medium containing 100 microgram / ml ampicillin until mid-log phase growth (37 ° C.). 0.2 ml of this bacterial solution was treated with Isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM.
Was inoculated into 100 ml of the same medium containing the mixture, and cultured with shaking at 37 ° C. for 24 hours. After the culture, the E. coli cells were centrifuged and separated into culture supernatants. The cells were washed twice with 10 mM sodium phosphate buffer (pH 6.0) and then resuspended in the same buffer.
Crushed with an ultrasonic crusher. The crushed liquid was centrifuged, and the supernatant was used as a cell-free extract. This solution is the LFTase solution.

The thus prepared E. coli BL21
(DE3) -LF of cell-free extract of PET / LFTsa
Tase activity was measured according to a standard method.
It was 5.1 units / culture solution (ml). According to the present invention, very high productivity was obtained, about 30 times that of the original strain (GS-9 bacteria) and about 5 times that of the system of Saito et al. In addition, since the recombinants produced the enzyme in the cells (GS-9 was secreted and expressed outside the cells), the enzyme units were converted into numerical values per 1 ml of the culture solution and compared (Table 1).

(Table 1) Table 1: Comparison of levanfructotransferase activities Location LFTase activity (U / ml culture) ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Original strain A. nicolinovorans GS-9 Extracellular 3.3 3.3 pLTF-BB1 / Escherichia coli JM109 17.6 pET / LFTsa / Escherichia coli BL-21 (DE3) 105.1 ━━━━━━━━━━━━━━

The expressed LFTase was partially purified from this cell-free extract. The cell-free extract was dialyzed overnight against a 10 mM sodium phosphate buffer (pH 6.0).
And DEAE Bio equilibrated with buffer as carrier
-It was subjected to anion exchange column chromatography using Gel A (manufactured by Bio-Rad). Elution of the adsorbed protein was performed by linearly changing the concentration of sodium chloride contained in the buffer solution from 0 to 0.4 M. According to a standard method, the LFTase active fraction was measured, and the active fraction was concentrated with a centrifugal ultrafiltration membrane. This crude enzyme solution was dialyzed overnight against a 10 mM sodium phosphate buffer (pH 6.0). This solution was used as a partially purified solution of the recombinant enzyme.

[0066]

Embodiment 3 DFA from Leban using LFTase
IV was produced.

(1) The solution obtained by cooling and aging the Serratia bacterium culture solution prepared above was adjusted to 37 ° C. without subjecting Levan precipitation with alcohol to pH around 6.0 while stirring at 100 rpm. Adjusted. The thus prepared LFTase was added to 200 L of the levan-containing liquid thus obtained.
Add 10 ml of solution (100 U / ml), incubate for 24 hours at 40 ° C.,
An IV synthesis reaction was performed.

Separating and recovering levan from the culture of Serratia bacteria means separating levan from surplus sugars, impurities and cells in the medium, and DFA in the next reaction.
It has been considered important in the sense that levan can be dissolved in an optimal buffer during the IV synthesis reaction. However, it was revealed that only the operation of adjusting the pH to the optimum pH at the time of the enzyme reaction at the end of cooling ripening did not cause any problem in the subsequent efficiency. As a result, the recovery of levan by ethanol precipitation becomes unnecessary, and the materials and time used for ethanol precipitation can be greatly reduced.

Further, there is a step of removing the excess sugar by adding yeast in a subsequent operation (this operation is sometimes referred to as yeast treatment), but this step can also be performed in the same tank. Met. The concentration of the enzyme reaction solution subjected to aeration culture treatment with yeast was 1 to 40% by weight, preferably 5 to 30% by weight.

(2) The enzyme reaction solution (D
To 10 L of FA IV (containing 320 g), 300 g of baker's yeast (Nitten Yeast, trade name) (water content: 66%) was added, followed by aeration culture at 30 ° C. for 24 hours. The culture was filtered and then concentrated to a Bx50. A resin column having an inner diameter of 12 cm and a height of 79 cm is filled with 7.6 L of a strong acidic cation exchange resin Amberlite CR-1310 (trade name) in the form of Na, and the concentrated solution is charged with 0.02 to 0.10 L / L. -R / 1 cycle, space velocity 0.13
The solution was passed under the conditions of ０．５0.53 and a temperature of 80 ° C., and was extruded with warm water. DFA IV The fraction with high purity is collected and DFA
The fraction was designated as IV fraction. D obtained by performing this operation 10 times
The FA IV fractions were mixed and activated carbon was added to 0.6 kg of the mixture to decolorize it. Activated carbon is removed by diatomaceous earth filtration,
The filtrate was concentrated under reduced pressure at 70 ° C. Then, cooling crystallization (7
(0 → 25 ° C.), and the crystallized product was separated by a centrifugal separator to obtain 120 g of DFA IV crystals having a purity of 99.9%.

(3) The crude DFA IV solution after the yeast treatment is passed through a cation exchange resin and then an anion exchange resin under the conditions shown in FIG. 10 to produce the effects of desalting and chromatography. Was performed. FIG. 10 shows the results of HPLC analysis of the purified DFA IV solution. As a control, the result of HPLC analysis of the crude DFA IV solution before the purification treatment is also shown in FIG.
It was shown to. As is clear from the results, it is possible to adsorb and remove impurities by the desalting effect and to separate DFA IV and impurities (mainly, the first and second peaks from the right of the dotted line in the figure) by the chromatographic effect. Understand.

The purification mechanism is as follows at present. As shown in FIG. 11, a column filled with a cation exchange resin and a column filled with an anion exchange resin were directly connected, and a crude DFA IV solution was passed through the column to desalinate. It is natural that the cations and anions in the crude DFA IV solution are removed by the present method, but the chromatographic effect by the anion exchange resin is also large, and a high purity DFA IV solution has been successfully obtained. (FIGS. 11 and 12)

By adjusting (delaying) the timing of collecting the liquid discharged from the anion exchange resin, not only A in the figure but also B (glycerol generated during the yeast treatment) can be removed. By using this method, it is possible to obtain a DFA IV solution having a purity of 90% or more with a recovery rate of 70 to 80%.

[0074]

The present invention makes it possible to produce DFA IV efficiently by applying the novel LFTase according to the present invention to levan. In addition, a purification method has been developed, and high-purity DFA IV can be produced.

The LFTase according to the present invention (as well as the enzyme gene) is a novel substance which is hitherto unknown. The enzyme is β
It has the effect of cleaving -2,6-polyfructan from the reducing end in disaccharide units. This enzyme has a very high activity, and by allowing it to act on levan, DFA IV having various functions can be efficiently produced. In addition, the enzyme is of a genetically modified type, has not only high activity, but also has the feature that it can be industrially mass-produced.

Further, according to the present invention, a novel method for producing a conventionally unknown levan using Serratia sp. Has been developed,
Leban can be produced efficiently. Furthermore, according to the present invention, it is possible for the first time to further increase the production efficiency of levan by newly using a preculture method and / or a cooling ripening method, and to succeed in the industrial production of levan for the first time. It was done.

As described above, the present invention makes it possible to mass-produce raw materials such as levan and LFTase, which makes it possible for the first time to mass-produce DFA IV, which has never been possible before. Since it was developed, it has become possible for the first time to produce high-purity DFA IV in large quantities. Therefore, further industrial application of DFA IV to functional foods, pharmaceuticals, and the like has become possible.

In addition, the present invention relates to a method for preparing DFA I from sucrose.
It has an epoch-making feature that a series of steps up to the crystallization of V can be carried out continuously without interruption, the isolation operation, the transfer to another tank and other operations are omitted, and the DFA is reduced in cost. In addition to being able to produce IV, it also opens up new ways to make effective use of sucrose, making a significant contribution to the sugar industry.

[0079]

[Sequence List] SEQUENCE LISTING <110> Nippon Tensaiseito Kabushiki Kaisha <120> Production Method of DFA IV in Large Amount <130> 6337 <141> 2000-7-6 <160> 6 <210> 1 <211> 485 <212 > PRT <213> Arthrobacter nicotinovorans <400> 1 MET Gln Ala Ser Leu Arg Ala Ile Tyr His MET Thr Pro Pro Ser 5 10 15 Gly Trp Leu Cys Asp Pro Gln Arg Pro Val His Thr Asn Gly Ala 20 25 30 Tyr Gln Leu Tyr Tyr Leu His Ser Gly Gln Asn Asn Gly Pro Gly 35 40 45 Gly Trp Asp His Ala Thr Thr Gly Asp Gly Val Ser Tyr Thr His 50 55 60 His Gly Val Val MET Pro MET Gln Pro Asp Phe Pro Val Trp Ser 65 70 75 Gly Ser Ala Val Val Asp Thr Ala Asn Thr Ala Gly Phe Gly Ala 80 85 90 Gly Ala Val Ile Ala Leu Ala Thr Gln Pro Thr Asp Gly Lys Phe 95 100 105 Gln Glu Glu Gln Tyr Leu Tyr Trp Ser Thr Asp Gly Gly Tyr Ser Phe 110 115 120 Thr Ala Leu Pro Asp Pro Val Ile Val Asn Thr Asp Gly Arg Thr 125 130 135 Ala Thr Thr Pro Ala Glu Val Glu Asn Ala Glu Trp Phe Arg Asp 140 145 150 Pro Lys Ile His Trp Asp Ala Thr Arg Asn Glu Trp Val Cys Val 155 160 165 Ile Gly Arg Ala Arg Tyr Ala Ala Phe Tyr Thr Ser Pro Asn Leu 170 175 180 Arg Asp Trp Gln Trp Lys Ser Asn Phe Asp Tyr Pro Asn His Ala 185 190 195 Leu Gly Gly Ile Glu Cys Pro Asp Leu Phe Glu MET Thr Ala Gly 200 205 210 Asp Gly Thr Arg His Trp Val Phe Gly Ala Ser MET Asp Ala Tyr 215 220 225 Ser Ile Gly Leu Pro MET Thr Phe Ala Tyr Trp Thr Gly Ser Trp 230 235 240 Asn Gly Thr Ala Phe Ile Ala Asp Asn Leu Thr Pro Gln Trp Leu 245 250 255 Asp Trp Gly Trp Asp Trp Tyr Ala Ala Val Thr Trp Pro Ala Val 260 265 270 Glu Ala Pro Glu Thr Lys Arg Leu Ala Thr Ala Trp MET Asn Asn 275 280 285 285 Trp Lys Tyr Ala Ala Arg Asn Val Pro Thr Asp Ala Ser Asp Gly 290 295 300 Tyr Asn Gly Gln Asn Ser Ile Thr Arg Glu Leu Arg Leu Glu Arg 305 310 315 Gln Ser Gly Gly Trp Tyr Thr Leu Leu Ser Thr Pro Val Pro Ala 320 325 330 Leu Ser Asn Tyr Ala Thr Ser Ser Thr Thr Leu Pro Asp Arg Thr 335 340 345 345 Val Asn Gly Ser Phe Val Leu Pro Trp Ser Gly Arg Ala Tyr Glu 350 355 360 Leu Glu Leu Asp Ile Ser Trp Asp Thr Ala Ala Asn Val Gly Val 365 370 375 Ser Val Gly Arg Ser Ser Asp Gly Ser Arg His Thr Asn Ile Gly 380 385 390 Lys Tyr Gly Asp Glu Leu Tyr Val Asp Arg Ala Ser Ser Glu Gln 395 400 405 Ser Gly Tyr Ala Leu Ala Pro Tyr Thr Arg Ala Ala Ala Pro Ile 410 415 420 Asp Ala Asn Ala Arg Ser Val His Leu Arg Ile Phe Val Asp Thr 425 430 435 Gln Ser Val Glu Val Phe Val Asn Ser Gly His Thr Val Val Ser 440 445 450 Gln Gln Val His Phe Ala Ala Gly Asp Thr Gly Ile Ser Leu Tyr 455 460 465 Ala Asp Gly Gly Pro Ala Asn Phe Thr Gly Ile Thr Ile Arg Glu 470 475 480 Phe Gly Asn Pro Ile 485 <210> 2 <211> 1467 <212> DNA <213> Arthrobacter nicotinovorans <400> 2 caTATGcagg catccctccg ggcgatctac cacatgaccc cgccctcggg ctggctatgt 60 gatccgcagc gacccgtaca tacaaacggc gcctaccagc tctactacct ccactccggc 120 cagaacaacg gaccgggcgg atgggaccac gcgaccaccg gcgacggagt gtcttacacc 180 caccatggag tggtgatgcc aatgcaaccg gacttccccg tgtggtcggg atcggcagta 240 gtggacaccg ccaacaccgc cggcttcggc gccggcgcag tcatcgcgct cgccacccaa 300 cccaccgacg gaaaattcca ggaacagtac ctttactggt ccacggatgg cggctactcc 360 ttcaccgcat tgcctgaccc ggtcattgtg aacactgatg gacggacggc caccaccccc 420 gccgaggtgg agaacgcaga atggttccgc gacccgaaaa ttcactggga cgcgacgcgc 480 aacgagtggg tctgtgtcat cggcagggcc cgctacgctg ccttctacac ctctcccaac 540 ctgcgggatt ggcaatggaa gtccaacttc gactacccca accacgccct cggcggtatc 600 gaatgcccgg atctgttcga aatgaccgca ggagacggaa cccggcactg ggtgttcggg 660 gcgagcatgg acgcctacag catcggcttg cccatgacct ttgcctactg gacaggttca 720 tggaacggca cagcattcat cgccgacaac ctcacaccac agtggcttga ctggggatgg 780 gactggtacg ccgccgtgac ctggccggcc gtggaagcac ctgagaccaa gcggcttgcc 840 acagcgtgga tgaacaactg gaaatatgcc gcccgcaacg tgcccacgga cgcgtccgat 900 ggctataacg ggcaaaattc catcacgcgc gagctcaggc tcgagcgcca atcgggcggc 960 tggtacacct tgctcagcac gcccgttccg gcgctttcga actatgccac ctccagcacc 1020 acccttccgg accgcacagt caacggcagt ttcgtacttc cgtggagcgg ccgggcgtat 1080 gaactggaac tcgatatttc atgggacacg gcagcgaacg tgggagtctc ggtgggccgc 1140 tcgtccgatg gcagccgcca tacgaacatc ggcaaatacg gtgacgagtt gtacg tcgat 1200 cgcgcatcct cggagcaaag cggttatgcg ctggcaccct acacccgcgc cgccgcgccc 1260 atcgatgcga acgccagatc cgtccacctg cgcatctttg tagacaccca aagtgttgag 1320 gtgttcgtaa attccgggca cacggtggtt tcgcagcagg tgcacttcgc ggccggggac 1380 acggggatct ccctctatgc ggacggcggt ccggccaact tcaccggcat caccatccgc 1440 gagttcggga accccatcta agGAtCc <210> 3 <211> 517 <212> PRT <213> Arthrobacter nicotinovorans <400> 3 MET Thr Tyr Asp Ile Ser Arg Arg Thr Ala Leu Gln Gly Ala Gly 5 10 15 Val Gly Ala Leu Ala Leu Phe MET Ser Asn Ala Ile Pro Val Ala 20 25 30 Ala His Ala Gln Ala Ser Leu Arg Ala Ile Tyr His MET Thr Pro 35 40 45 Pro Ser Gly Trp Leu Cys Asp Pro Gln Arg Pro Val His Thr Asn 50 55 60 Gly Ala Tyr Gln Leu Tyr Tyr Leu His Ser Gly Gln Asn Asn Gly 65 70 75 Pro Gly Gly Trp Asp His Ala Thr Thr Gly Asp Gly Val Ser Tyr 80 85 90 Thr His His Gly Val Val MET Pro MET Gln Pro Asp Phe Pro Val 95 100 105 Trp Ser Gly Ser Ala Val Val Asp Thr Ala Asn Thr Ala Gly Phe 110 115 120 Gly Ala Gly Ala Val Ile Ala Leu Ala Thr Gln Pro Thr Asp Gly 125 130 135 Lys Phe Gln Glu Gln Tyr Leu Tyr Trp Ser Thr Asp Gly Gly Tyr 140 145 150 Ser Phe Thr Ala Leu Pro Asp Pro Val Ile Val Asn Thr Asp Gly 155 160 165 Arg Thr Ala Thr Thr Pro Ala Glu Val Glu Asn Ala Glu Trp Phe 170 175 180 180 Arg Asp Pro Lys Ile His Trp Asp Ala Thr Arg Asn Glu Trp Val 185 190 195 Cys Val Ile Gly Arg Ala Arg Tyr Ala Ala Phe Tyr Thr Ser Pro 200 205 210 Asn Leu Arg Asp Trp Gln Trp Lys Ser Asn Phe Asp Tyr Pro Asn 215 220 225 His Ala Leu Gly Gly Ile Glu Cys Pro Asp Leu Phe Glu MET Thr 230 235 240 Ala Gly Asp Gly Thr Arg His Trp Val Phe Gly Ala Ser MET Asp 245 250 255 Ala Tyr Ser Ile Gly Leu Pro MET Thr Phe Ala Tyr Trp Thr Gly 260 265 270 Ser Trp Asn Gly Thr Ala Phe Ile Ala Asp Asn Leu Thr Pro Gln 275 280 285 Trp Leu Asp Trp Gly Trp Asp Trp Tyr Ala Ala Val Thr Trp Pro 290 295 300 Ala Val Glu Ala Pro Glu Thr Lys Arg Leu Ala Thr Ala Trp MET 305 310 315 Asn Asn Trp Lys Tyr Ala Ala Arg Asn Val Pro Thr Asp Ala Ser 320 325 330 Asp Gly Tyr Asn Gly Gln Asn Ser Ile ThrArg Glu Leu Arg Leu 335 340 345 Glu Arg Gln Ser Gly Gly Trp Tyr Thr Leu Leu Ser Thr Pro Val 350 355 360 Pro Ala Leu Ser Asn Tyr Ala Thr Ser Ser Thr Thr Leu Pro Asp 365 370 375 Arg Thr Val Asn Gly Ser Phe Val Leu Pro Trp Ser Gly Arg Ala 380 385 390 Tyr Glu Leu Glu Leu Asp Ile Ser Trp Asp Thr Ala Ala Asn Val 395 400 405 Gly Val Ser Val Gly Arg Ser Ser Asp Gly Ser Arg His Thr Asn 410 415 420 Ile Gly Lys Tyr Gly Asp Glu Leu Tyr Val Asp Arg Ala Ser Ser 425 430 435 Glu Gln Ser Gly Tyr Ala Leu Ala Pro Tyr Thr Arg Ala Ala Ala 440 445 450 Pro Ile Asp Ala Asn Ala Arg Ser Val His Leu Arg Ile Phe Val 455 460 465 Asp Thr Gln Ser Val Glu Val Phe Val Asn Ser Gly His Thr Val 470 475 480 Val Ser Gln Gln Val His Phe Ala Ala Gly Asp Thr Gly Ile Ser 485 490 495 Leu Tyr Ala Asp Gly Gly Pro Ala Asn Phe Thr Gly Ile Thr Ile 500 505 510 Arg Glu Phe Gly Asn Pro Ile 515 <210> 4 <211> 1752 <212> DNA <213> Arthrobacter nicotinovorans <400> 4 cccgggacct ttcccgacgg tctcacacac ccatcaccat gacgtttggc gtgtcgtcgc60 gcc cgccgaa cactgagagg aaacgaatcg atgacgtatg acatctctcg ccgcactgcc 120 ctgcaaggtg caggggttgg tgccttggca cttttcatga gcaatgccat tcccgtggcc 180 gcccacgccc aggcatccct ccgggcgatc taccacatga ccccgccctc gggctggcta 240 tgtgatccgc agcgacccgt acatacaaac ggcgcctacc agctctacta cctccactcc 300 ggccagaaca acggaccggg cggatgggac cacgcgacca ccggcgacgg agtgtcttac 360 acccaccatg gagtggtgat gccaatgcaa ccggacttcc ccgtgtggtc gggatcggca 420 gtagtggaca ccgccaacac cgccggcttc ggcgccggcg cagtcatcgc gctcgccacc 480 caacccaccg acggaaaatt ccaggaacag tacctttact ggtccacgga tggcggctac 540 tccttcaccg cattgcctga cccggtcatt gtgaacactg atggacggac ggccaccacc 600 cccgccgagg tggagaacgc agaatggttc cgcgacccga aaattcactg ggacgcgacg 660 cgcaacgagt gggtctgtgt catcggcagg gcccgctacg ctgccttcta cacctctccc 720 aacctgcggg attggcaatg gaagtccaac ttcgactacc ccaaccacgc cctcggcggt 780 atcgaatgcc cggatctgtt cgaaatgacc gcaggagacg gaacccggca ctgggtgttc 840 ggggcgagca tggacgccta cagcatcggc ttgcccatga cctttgccta ctggacaggt 900 tcatggaacg gcacagcatt c atcgccgac aacctcacac cacagtggct tgactgggga 960 tgggactggt acgccgccgt gacctggccg gccgtggaag cacctgagac caagcggctt 1020 gccacagcgt ggatgaacaa ctggaaatat gccgcccgca acgtgcccac ggacgcgtcc 1080 gatggctata acgggcaaaa ttccatcacg cgcgagctca ggctcgagcg ccaatcgggc 1140 ggctggtaca ccttgctcag cacgcccgtt ccggcgcttt cgaactatgc cacctccagc 1200 accacccttc cggaccgcac agtcaacggc agtttcgtac ttccgtggag cggccgggcg 1260 tatgaactgg aactcgatat ttcatgggac acggcagcga acgtgggagt ctcggtgggc 1320 cgctcgtccg atggcagccg ccatacgaac atcggcaaat acggtgacga gttgtacgtc 1380 gatcgcgcat cctcggagca aagcggttat gcgctggcac cctacacccg cgccgccgcg 1440 cccatcgatg cgaacgccag atccgtccac ctgcgcatct ttgtagacac ccaaagtgtt 1500 gaggtgttcg taaattccgg gcacacggtg gtttcgcagc aggtgcactt cgcggccggg 1560 gacacgggga tctccctcta tgcggacggc ggtccggcca acttcaccgg catcaccatc 1620 cgcgagttcg ggaaccccat ctaagcctgc gtcccacggc tggaaaggac gacggcgacg 1680 ctgcagcagg cgggtgcgtc gccgtcgttc ttccgggtcc gttgggcagt tcgcatctga 1740 caagcacccg gg <210 > 5 <211> 27 <212> DNA <213> Artificial sequence <400> 5 ccgcccatat gcaggcatcc ctccggg 27 <210> 6 <211> 27 <212> DNA <213> Artificial sequence <400> 6 gggacggatc cttagatggg gttcccg 27

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of LFTase according to the present invention.

FIG. 2 shows the nucleotide sequence of the LFTase gene DNA according to the present invention.

FIG. 3 shows the amino acid sequence of LFTase derived from GS-9.

FIG. 4 shows the nucleotide sequence of GS-9-derived LFTase gene DNA.

FIG. 5 shows the nucleotide sequence of a sense primer.

FIG. 6 shows the nucleotide sequence of an antisense primer.

FIG. 7 is a construction diagram of a large-scale expression system of LFTase.

FIG. 8 shows a flowchart of a method for mass production of DFA IV from sucrose.

FIG. 9 shows Levan synthesis by a fermenter of Serratia levanicum in Example 1. The viscosity is an actually measured value of a B-type viscometer (No. 2 rotation axis, 20 ° C.).

FIG. 10: H before and after desalination of a DFA IV-containing solution
It is a PLC chromatogram.

FIG. 11 is a purification diagram of DFA IV.

FIG. 12 is an HPLC chromatogram of a crude DFA IV solution. The vertical axis represents mV, and the horizontal axis represents retention time (minutes).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12N 9/10 C12R 1:19) C12R 1:19) 1:06) (C12N 15/09 C12N 15 / 00 A C12R 1:06) C12R 1:06) (72) Inventor Hiroaki Sakurai 13th west of Inadacho Minami 9 Line, Obihiro City, Hokkaido Japan Beet Sugar Manufacturing Co., Ltd. (72) Inventor Koji Sayama 9 Inadamachi Minami 9 Obihiro City, Hokkaido 13 Line West Nippon Sugar Beet Sugar Co., Ltd. (72) Inventor Tsutomu Arizuka 13 Nishi Line 9 Inadacho Minami Line, Obihiro City, Hokkaido Inside Japan Sugar Beet Sugar Co., Ltd. (72) Inventor Fumio Tomita Kita, Sapporo, Hokkaido 9-chome, Kita-ku, Ku-ku, Hokkaido University, Faculty of Agriculture (72) Inventor Gyzo Asano, 9-chome, Kita 9-Jo Nishi, Kita-ku, Sapporo, Hokkaido Who Atsushi Yokota Sapporo, Hokkaido Kita-ku, Kita 9 Jo Nishi 9-chome, Hokkaido University Faculty of Agriculture in the F-term (reference) 4B024 AA05 BA80 CA04 DA06 GA11 GA19 HA01 4B050 CC01 CC03 DD02 LL02 4B064 AF03 CA19 CB07 CC24 CD19 CE10 CE11 DA10

Claims (9)

[Claims] 1. Lefructose dianhydride IV (DFA) by reacting levan fructotransferase (LFTase) having the following physicochemical properties with levan
IV). A method for producing DFA IV. (1) Action Decomposes levan, a polyfructan having a β-2,6 fructoside bond, to give difructose dianhydride I
V (DFA IV). (2) Substrate specificity It acts on levan and levan oligosaccharides having a chain length of 3 to 7. (3) Optimum pH and stable pH range Optimum pH: 6.0 Stable pH range: 4.0 to 12.0 (4) Optimum temperature and stable temperature range Optimum temperature: 50 ° C Stable temperature range: the present enzyme Was stable up to 40 ° C. (5) Molecular weight Molecular weight: about 50,000 Da (SDS-PAGE, gel temperature 7.5%) (6) Inducibility of enzyme Levan is not required for inducible expression of this enzyme. 2. L represented by the amino acid sequence of SEQ ID NO: 1.
A method for producing DFA IV, wherein FTase is allowed to act on levan to produce DFA IV. 3. An LFT represented by the nucleotide sequence of SEQ ID NO: 2.
LFTa having an amino acid sequence corresponding to the ase gene
A method for producing DFA IV, wherein se is allowed to act on levan to produce DFA IV. 4. An LFT represented by the nucleotide sequence of SEQ ID NO: 2.
A transformant obtained by transforming with a plasmid containing the DNA of the ase gene is cultured, and L-derived from the resulting culture is cultured.
The method for producing DFAIV according to claim 1, wherein FTase is used. 5. A transformant, Escherichia coli BL21 (DE
3) -pET / LTFsa (FERM P-1789)
The method according to claim 4, wherein 6) is used.
Manufacturing method of FA IV. 6. A DFA IV fraction is purified by at least one of a treatment for assimilating and / or removing sugar in an enzyme reaction solution, a desalting treatment, and a chromatographic separation treatment,
The method according to any one of claims 1 to 5, wherein the collection is performed.
The method described in the section. 7. The method according to claim 6, wherein sugar in the enzyme reaction solution is assimilated and / or removed by aeration culture treatment with yeast. 8. The method according to claim 6, wherein desalting treatment is performed using a strongly basic ion exchange resin and a strongly acidic ion exchange resin, or a strongly basic ion exchange resin. 9. A carrier for chromatographic separation,
The method according to any one of claims 6 to 8, wherein a strongly acidic ion exchange resin of Na type or Ca type is used.