JP4236949B2 - Method for selecting β-fructofuranosidase used for producing crystalline 1-kestose - Google Patents

Description

【００４３】
さらに、これらの株の培養上清をFFase粗酵素液として、FFase活性測定とスクロースを基質とした酵素反応を行い、反応液の糖組成をＨＰＬＣ（カラム：リクロスフェア１００ＮＨ_２、溶離液：７２％アセトニトリル、流速：１ｍｌ／分、検出器：示差屈折計）で分析した。FFase活性は、Agric. Biol. Chem., 53, 667-673 (1989)に記載の方法に従って測定した。また、酵素反応は、培養上清中に４８重量％のスクロースを添加し、この反応液を、４０℃、ｐＨ７でインキュベートすることにより行なった。
【００４４】
上記表１に記載の株はいずれも、FFase活性を有していることが確認された。さらに、スクロースを基質とした酵素反応により得られる反応液中の糖組成を下記の表２に示す。ここで、各株についてのデータは、それぞれ１−ケストース（ＧＦ_２）の量が最大となる時点での反応液の糖組成を示す。
【００４５】
【表２】

【００４６】
１−ケストースの製造に用いるFFaseは、１−ケストースの含有量が多く、他の糖類、特にニストースおよびフラクトシルニストースの含有量が少ない酵素反応産物を与えるものであることが好ましいと考えられる。このような観点から表１および表２に記載のFFaseを評価すると、（21-36）株、（22-95）株および（64-60）株では、上記両培地における濁度の比が野生型よりも大きく、１−ケストースの含有量が多く、ニストースの生成が抑制されている。すなわち、これらの株により生産されるFFaseは、１−ケストースの製造に有利な反応特性を有するといえる。
【００４７】
一方で、（M-2）株、（64-81）株および（51-24）株では、上記両培地における濁度の比が野生型と同程度で、１−ケストースおよびニストースの含有量に変化は見られない。
【００４８】
【配列表】

[0001]
BACKGROUND OF THE INVENTION
Field of Invention
The present invention relates to a method for selecting β-fructofuranosidase with modified reaction characteristics.
[0002]
Background art
Fructooligosaccharides are oligosaccharides in which one or more fructose is added to sucrose in a chain, and the binding mode is β-type for the carbon at the 1st position in the fructose part of sucrose and the carbon at the 2nd position of the fructose to be added. Bond (β2 → 1 bond), and this is repeated. Schematic representation of the fructooligosaccharide binding mode is as follows: (Fru) β2- {1 (Fru) β2-} nα1 (Glc) (where Fru is a D-fructose residue, Glc is a D-glucose residue, n Represents the number of fructose residues added to sucrose). Fructooligosaccharides have been shown to have various physiological functions such as difficult caries, growth promotion of bifidobacteria, metabolism improvement of lipids such as cholesterol, and immunoregulation, and are extremely useful industrially as functional food materials. It is. Fructooligosaccharides are widely distributed in plants in nature, and are known to be contained in, for example, asparagus, onion, juniper and honey.
[0003]
In recent years, a technique for producing a large amount of fructooligosaccharides from sucrose using a transfer reaction of β-fructofuranosidase derived from microorganisms has been established, and fructooligosaccharides are industrially produced. The fungal β-fructofuranosidase used in this industrial production is characterized by high transfer activity and low isomer formation, while only producing 1-kestose. Plant-derived sucrose: Sucrose fructosyltransferase (SST) is not as high in oligosaccharide production selectivity and can only produce a mixture of oligosaccharides with a degree of polymerization of 2-6. For this reason, fructooligosaccharides are used as liquids (syrups) or powders, and since these are non-crystalline mixtures, they are highly hygroscopic compared to crystalline food materials such as sugar (sucrose). There were problems such as poor processability.
[0004]
On the other hand, by combining column chromatography, crystallization, and the like, a crystalline fructooligosaccharide having a single oligosaccharide component as a main component, such as 1-kestose (trisaccharide; GF₂), Nystose (tetrasaccharide; GF₃) Etc. can be obtained. In particular, crystalline 1-kestose has excellent physical properties and processability while retaining the physiological function of fructooligosaccharide (Patent Document 1: JP-A-9-65829, Patent Document 2: JP-A-6-31160). Issue gazette). However, the enzyme 1-kestose (trisaccharide; GF) used in the production of fructooligosaccharides₂) Is about 42%, and in the reaction product, the by-product nystose (tetrasaccharide; GF)₃) Is about 12% and the substrate sucrose is about 23%. Therefore, there is still a demand for β-fructofuranosidase capable of industrially and efficiently obtaining higher purity 1-kestose. That is, it is desired to obtain β-fructofuranosidase or a variant thereof suitable for production of crystalline fructo-oligosaccharide having high 1-kestose production selectivity and improved composition of oligosaccharide as a transfer reaction product. It can be said that.
[0005]
International Publication No. 97/34004 (Patent Document 3) discloses a β-fructofuranosidase mutant that selectively produces 1-kestose. On the other hand, as far as the present inventors know, a method for efficiently selecting β-fructofuranosidase or its mutant that selectively produces 1-kestose has not been reported.
[0006]
[Patent Document 1]
JP-A-9-65829
[Patent Document 2]
JP-A-6-31160
[Patent Document 3]
WO 97/34004 Pamphlet
[0007]
SUMMARY OF THE INVENTION
In order to produce a low hygroscopic crystalline fructooligosaccharide, 1-kestose (GF) by β-fructofuranosidase is used to reduce the chromatographic fraction of the enzyme reaction solution and to recycle the crystal mother liquor.₂) And the by-product nystose (GF)₃It is considered preferable that the amount of other fructooligosaccharides produced is low.
[0008]
The present inventors introduced a candidate β-fructofuranosidase or a mutant gene thereof into a host cell, and transformed the transformant into a medium containing sucrose as the sole carbon source and 1-kestose as the sole. It is found that the β-fructofuranosidase or a variant thereof satisfies the above conditions when the growth rate in the former is higher than the growth rate in the latter when cultured separately in a carbon source medium. It was. The present invention is based on such knowledge.
[0009]
Therefore, according to the present invention, a method for determining whether or not β-fructofuranosidase or a variant thereof is advantageous for the production of 1-kestose, as well as a β-fructofuranosidase mutation advantageous for the production of 1-kestose. And a method for producing a gene encoding the same.
[0010]
And the determination method by this invention is a method of determining whether (beta) -fructofuranosidase or its variant is advantageous for manufacture of 1-kestose, Comprising:
(A) transforming a host cell with a vector containing a gene encoding β-fructofuranosidase or a variant thereof in an expressible form;
(B) a step of culturing the transformant obtained in step (a) in a medium containing only sucrose as a carbon source, and measuring the growth rate of the transformant;
(C) a step of culturing the transformant obtained in step (a) in a medium containing only 1-kestose as a carbon source and measuring the growth rate of the transformant; and (d) 1 as a carbon source. -Β-fructofuranosidase produced by the transformant or a mutation thereof when the growth rate in a medium containing only kestose is smaller than the growth rate in a medium containing only sucrose as a carbon source Determining the body as advantageous for the production of 1-kestose,
Is included.
[0011]
In addition, the method for producing a β-fructofuranosidase variant according to the present invention is a method for producing a β-fructofuranosidase variant advantageous for producing 1-kestose, which comprises the following steps:
(A) introducing a mutation into the β-fructofuranosidase gene and preparing a gene encoding a β-fructofuranosidase mutant;
(B) transforming a host cell with a vector containing the gene obtained in step (a) in an expressible form;
(C) a step of culturing the transformant obtained in step (b) in a medium containing only sucrose as a carbon source and measuring the growth rate of the transformant;
(D) a step of culturing the transformant obtained in step (b) in a medium containing only 1-kestose as a carbon source and measuring the growth rate of the transformant; and (e) 1 as a carbon source. A step of selecting a β-fructofuranosidase mutant produced by a transformant having a growth rate in a medium containing only kestose, which is smaller than a growth rate in a medium containing only sucrose as a carbon source;
Is included.
[0012]
Furthermore, the method for producing a β-fructofuranosidase mutant gene according to the present invention is a method for producing a β-fructofuranosidase mutant gene advantageous for the production of 1-kestose, which comprises the following steps:
(A) introducing a mutation into the β-fructofuranosidase gene and preparing a gene encoding a β-fructofuranosidase mutant;
(B) transforming a host cell with a vector containing the gene obtained in step (a) in an expressible form;
(C) a step of culturing the transformant obtained in step (b) in a medium containing only sucrose as a carbon source and measuring the growth rate of the transformant;
(D) a step of culturing the transformant obtained in step (b) in a medium containing only 1-kestose as a carbon source and measuring the growth rate of the transformant; and (e) 1 as a carbon source. A step of selecting a β-fructofuranosidase mutant gene contained in a transformant showing a growth rate in a medium containing only kestose, which is smaller than a growth rate in a medium containing only sucrose as a carbon source;
Is included.
[0013]
The β-fructofuranosidase obtained by the present invention and variants thereof have a reaction characteristic that 1-kestose is selectively produced and the amount of other oligosaccharides produced is low. By using such β-fructofuranosidase, a fructooligosaccharide having a high 1-kestose content can be obtained, so that crystalline 1-kestose can be easily produced.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
As used herein, “β-fructofuranosidase” refers to the ability to hydrolyze β-D-fructofuranoside to release fructose and transfer the β-fructofuranosyl group to other sugars. An enzyme classified as EC 3.2.1.26 that has the ability, and is obtained from a natural organism. Furthermore, “β-fructofuranosidase” includes naturally occurring β-fructofuranosidase mutants without artificial mutagenesis. In the present specification, the term “mutant” used for β-fructofuranosidase means an amino acid mutation artificially introduced into β-fructofuranosidase.
[0015]
1. Preparation of genes for β-fructofuranosidase or its variants
In the method according to the present invention, a candidate β-fructofuranosidase or a mutant gene thereof is first prepared.
[0016]
β-fructofuranosidase may be any derived from a natural organism. For example, a known β-fructofuranosidase may be used, or a newly discovered one may be used. Such a β-fructofuranosidase gene can be obtained by methods known to those skilled in the art.
[0017]
The β-fructofuranosidase mutant is obtained by artificially introducing an amino acid mutation into β-fructofuranosidase, and the gene is obtained by introducing the mutation into the β-fructofuranosidase gene. be able to. The β-fructofuranosidase gene to be mutated may be derived from any organism, but is preferably a fungal β-fructofuranosidase gene, more preferably Aspergillus spp., Penicillium Β-fructofuranosidase gene derived from a fungus belonging to the genus, scoprariopsis, fusarium, or aureobasidium, more preferably a β-fructofuranosidase gene derived from a fungus belonging to the genus Aspergillus .
[0018]
The mutation as described above may be a mutation designated in advance or may be a random (random) mutation. Predesignated mutations can be introduced by methods known to those skilled in the art, such as the Gapped duplex method (Methods in Enzymology, 154, 350, 1987), the Kunkel method (Methods in Enzymology, 154, 367, 1987), and the like. it can.
[0019]
According to a preferred embodiment of the present invention, the mutation is a random mutation. Introduction of random mutations into the β-fructofuranosidase gene can be performed by methods known to those skilled in the art, for example, methods using mutagens, radiation, PCR methods and the like.
[0020]
For example, random mutagenesis using a mutagen can be performed by, for example, transferring a target microorganism having a β-fructofuranosidase gene to a base analog such as 5-bromouracil or 5-chlorouracil, or a base such as nitrous acid. This can be done by exposure to chemicals such as oxidative deamination inducers, hydroxyamines that react with cytosine guanine, alkylating reagents such as N-methyl-N′-nitro-N-nitrosoguanidine.
[0021]
The introduction of random mutation using the PCR method is, for example, under conditions that reduce fidelity of the DNA polymerase replication reaction in the amplification reaction of the gene using the DNA fragment containing the target β-fructofuranosidase gene as a template. PCR can be carried out by causing a replication error in the amplified DNA sequence. Such a method is generally known as an error-prone PCR method. Conditions for reducing the fidelity of the DNA polymerase replication reaction include high pH, high magnesium ion concentration, changes in each concentration of the four types of deoxyribonucleic acid (dNTP) serving as a substrate, or A DNA polymerase with low fidelity may be used.
[0022]
2. Determination of whether β-fructofuranosidase or a variant thereof is advantageous for the production of 1-kestose
A host cell is transformed with a prepared vector containing a gene encoding β-fructofuranosidase or a variant thereof, and the resulting transformant contains only sucrose as a carbon source. The growth rate of the transformant was measured by separately culturing in a medium (hereinafter referred to as “sucrose medium”) and a medium containing only 1-kestose as a carbon source (hereinafter referred to as “1-kestose medium”). By comparing the growth rate, it can be determined whether the β-fructofuranosidase or a variant thereof is advantageous for producing 1-kestose.
[0023]
In the comparison of the growth rate, β-fructofuranosidase produced by a transformant having a growth rate in the 1-kestose medium lower than that in the sucrose medium or a mutant thereof is 1 -Determined to be advantageous for the production of kestose. Moreover, the one where the difference of said reaction rate is larger is preferable. Therefore, according to a preferred embodiment of the present invention, the growth rate on the sucrose medium is 1.5 times or more, more preferably 2 times or more, more preferably 2.5 times the growth rate on the 1-kestose medium. It is determined that β-fructofuranosidase produced by a transformant showing the above values or a variant thereof is advantageous for producing 1-kestose.
[0024]
The growth rate of the transformant can be measured by methods known to those skilled in the art, for example, colony size measurement, culture solution turbidity measurement, cell volume measurement, and cell component quantification. According to a preferred embodiment of the present invention, the growth rate of the transformant is measured using the turbidity of the culture solution as an index. Turbidity can be measured, for example, as the absorbance of the culture at 595 nm. The growth rates to be compared may be those measured simultaneously for both culture media, but preferably the average growth rate in a certain period after the start of culture (hereinafter referred to as “initial average growth rate”). The initial average growth rate is a value obtained by dividing various measured values after a certain period of time after the start of the culture by the time. Therefore, the comparison of the initial average growth rate can be performed by comparing the measured values themselves. it can. The elapsed time after the start of culture for measuring the initial average growth rate varies depending on the type of host cell used for transformation, and can be appropriately determined by those skilled in the art while monitoring the state of the culture. 72 hours, more preferably 10 to 30 hours, still more preferably about 20 hours.
[0025]
The host cell used for transformation is not particularly limited as long as it can assimilate D-glucose and / or D-fructose and allows expression of a foreign gene. More preferred are fungi such as yeast and mold. Particularly preferred host cells are those that do not assimilate sucrose, ie, sucrose non-assimilable host cells. Since the sucrose non-assimilating host cell does not produce an enzyme using sucrose as a substrate other than β-fructofuranosidase introduced by transformation or a mutant thereof, the above growth rates can be easily and clearly compared. . Examples of such sucrose non-assimilating host cells include those known to those skilled in the art, such as filamentous fungi described in WO 97/34004, several fungi belonging to the genus Trichoderma, and specific yeasts (Oda , Y. and Ouchi, K., Appl. Environ. Microbiol., 55, 1742-1747, 1989).
[0026]
The vector used for transformation is prepared so that the gene of the incorporated β-fructofuranosidase or a mutant thereof can be expressed in the host cell as described above. As such a vector, a viral vector, a plasmid vector, a cosmid vector or the like can be appropriately used depending on the type of host cell used. For example, when the host is Escherichia coli, a λ phage bacteriophage, pBR, pUC-type plasmids, etc. are used. When the host is Bacillus subtilis, a pUB-type plasmid is used. When the host is yeast, a plasmid vector such as YEp-type, YRp-type, YCp-type is used. it can.
[0027]
The above vector is a DNA sequence for efficiently expressing a gene of β-fructofuranosidase or a mutant thereof, such as a promoter, a transcription initiation signal, a ribosome binding site, a translation termination signal, a transcription termination signal, etc. It is preferable that a transcriptional control signal, a translational control signal, etc. are included in a functional form. Such DNA sequences can be appropriately selected and used by those skilled in the art.
[0028]
Said vector may contain the selection marker for selecting a transformant. As the selection marker, a drug resistance marker gene, an auxotrophic marker gene, or the like can be used as appropriate. For example, when the host is a bacterium, an ampicillin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, or the like is used as the host. In some cases, tryptophan synthesis gene (eg, TRP1), uracil synthesis gene (eg, URA3), leucine synthesis gene (eg, LEU2), etc. When the host is mold, hygromycin resistance gene (Hyg), bialaphos resistance gene (Bar) Nitrate reductase gene (niaD) etc. can be used.
[0029]
Many suitable combinations of host cells and vectors as described above are known, and those skilled in the art can appropriately select and use them. As a particularly preferred host cell and vector combination, for example, the sucrose non-assimilating yeast strain S. cerevisiae MS-161 described in Biosci. Biotechnol. Biochem., 65 (4), 766-773, 2001 is used. And expression vectors therefor.
[0030]
The medium used for culturing the transformant may be either a solid medium such as a plate medium or a liquid medium. However, it is preferable to select an appropriate medium according to the method for measuring the growth rate. For example, when measuring the growth rate by the size of the colony, a solid medium is appropriate, and the growth rate is determined by the turbidity of the culture solution. A liquid medium is suitable for measuring. The specific composition of the medium other than the carbon source is appropriately determined by those skilled in the art. The carbon source in the sucrose medium is only sucrose, and the content thereof is preferably 0.5 to 5% by weight, more preferably 1.5 to 3% by weight, and most preferably about 2%, based on the total weight of the medium. % By weight. Further, the carbon source in the 1-kestose medium is only 1-kestose, and the content thereof is the same as the sucrose concentration in the sucrose medium.
[0031]
Furthermore, other culture conditions for the transformant are appropriately determined by those skilled in the art depending on the type of host cell to be used and the like, and are not particularly limited. Preferred temperature conditions are 25 to 37 ° C., more preferably about 30. ° C.
[0032]
As described above, it can be determined whether or not β-fructofuranosidase or a mutant thereof is advantageous for the production of 1-kestose. Prior to the above-described determination method, preliminary selection of transformants is possible. May be performed. For example, when the vector used for transformation contains the above selection marker, a host cell (that is, a transformant) containing the vector can be selected using the selection marker. In addition, when sucrose-non-assimilating cells are used as host cells, the introduced β-fructofuranosidase or a variant thereof is expressed by culturing on a medium containing only sucrose as a carbon source. Can be selected, and at the same time, it can be confirmed that the introduced β-fructofuranosidase mutant has β-fructofuranosidase activity. Such preliminary selection is advantageous in reducing labor, for example, when the number of candidate β-fructofuranosidases or variants thereof is large.
[0033]
3.1 Recovery of β-fructofuranosidase or its variant or its gene determined to be advantageous for the production of kestose
Β-fructofuranosidase or a variant thereof determined to be advantageous for the production of 1-kestose by the above method can be recovered as follows. First, the above transformant is cultured under appropriate conditions, and a culture supernatant or cells are obtained from the obtained culture by a known method such as centrifugation. When the cells are obtained, suspend them in an appropriate buffer, crush the cells by freeze-thawing, sonication, grinding, etc., and centrifuge or filter the β-fructofuranosidase or A bacterial cell extract containing the mutant is obtained. Purification of the enzyme from the culture supernatant or bacterial cell extract can be performed by appropriately combining separation and purification methods known to those skilled in the art. Such separation / purification methods include, for example, a method utilizing a difference in heat resistance such as heat treatment, a method utilizing a difference in solubility such as salt precipitation and solvent precipitation, dialysis, ultrafiltration, and gel filtration. And methods utilizing molecular weight differences such as SDS-polyacrylamide gel electrophoresis, methods utilizing difference in charges such as ion exchange chromatography, methods utilizing specific affinity such as affinity chromatography, hydrophobicity Examples thereof include a method utilizing a difference in hydrophobicity such as chromatography and reverse phase chromatography, and a method utilizing a difference in isoelectric point such as isoelectric focusing.
[0034]
Moreover, the gene of β-fructofuranosidase or its variant determined to be advantageous for the production of 1-kestose by the above method is known to those skilled in the art from the vector in the transformant containing the gene. It can be recovered by the method.
[0035]
4.1 Utilization of β-fructofuranosidase or its variant or its gene determined to be advantageous for the production of 1-kestose
The β-fructofuranosidase determined to be advantageous for the production of 1-kestose by the above method or a variant thereof can be used for the production of 1-kestose using sucrose as a raw material. The enzyme reaction conditions in this case are the enzyme characteristics of the β-fructofuranosidase or its mutant, for example, sugar composition, specific activity, substrate specificity, optimum pH, optimum pH in the product during the enzyme reaction. It is appropriately set by those skilled in the art depending on temperature, pH stability, thermal stability, and the like. These enzyme characteristics can be confirmed by methods known to those skilled in the art. The β-fructofuranosidase or a mutant thereof may be used as a crude enzyme or as a purified enzyme. Moreover, you may use the said (beta) -fructofuranosidase or its variant fix | immobilized in the suitable support | carrier.
[0036]
For the β-fructofuranosidase gene or its mutant gene determined to be advantageous for the production of 1-kestose by the above method, a transformant using the gene should be prepared using an appropriate host-vector system. Can be used for the production of β-fructofuranosidase or a variant thereof. The production of the transformant is as described above. In particular, if sucrose non-assimilating cells are used as host cells, enzymes that produce sucrose as a substrate other than β-fructofuranosidase introduced by transformation or a variant thereof are not produced. In the state of crude enzyme, it can be used for the production of 1-kestose.
[0037]
【Example】
Specific examples of the present invention are shown below, but the scope of the present invention is not limited thereto.
[0038]
Example 1: β-fructofuranosidase ( FFase ) Introduction of random mutations into genes
Aspergillus niger used in fructooligosaccharide production (Aspergillus niger) A random mutation was introduced into the gene of β-fructofuranosidase (hereinafter referred to as “FFase”) derived from the ATCC20611 strain. This random mutation was introduced using a commercially available PCR mutagenesis kit (Gene Morph, Stratagene) as follows.
[0039]
The composition of the PCR reaction solution (50 μl) was as follows: template DNA containing FFase gene (1 μl), dNTP (40 mM, 1 μl), 10-fold concentration buffer (5 μl), forward primer: 5′-GCGAATTCATGAAGCTCACCACTACCA-3 ′ (SEQ ID NO: 1) and reverse primers: 5′-GCGGATCCCGGTCAATTTCTCT-3 ′ (SEQ ID NO: 2) (250 ng / ml, 0.5 μl, respectively), Mutazyme (2.5 U / μl, 1 μl), DMSO (5 μl), and sterile water ( 36 μl). The reaction conditions were as follows: pretreatment at 94 ° C. for 2 minutes, followed by 94 ° C. for 1 minute (denaturation step), 50 ° C. for 2 minutes (annealing step), and 72 ° C. for 2.5 minutes (extension step) Incubation was 30 cycles. Finally, incubation was performed at 72 ° C. for 3 minutes. The reaction product was extracted with phenol / chloroform / isoamyl alcohol (25: 24: 1) from the obtained reaction solution, and ethanol was added to the extract to precipitate. The precipitate was dissolved in TE buffer and then subjected to agarose electrophoresis, and a specifically amplified 1.9 kbp band was excised according to a conventional method to recover a DNA fragment. A plasmid in which a 1.9 kbp EcoRI-BamHI fragment was inserted into the EcoRI-BamHI site of an expression vector in yeast described in Biosci. Biotechnol. Biochem., 65 (4), 766-773, 2001 was used. The transformant was obtained by introducing into the oxidative yeast S. cerevisiae MS-161 strain (described in Biosci. Biotechnol. Biochem., 65 (4), 766-773, 2001) by the lithium acetate method. Individual transformants were replicated on SD-GF agar medium (0.67% yeast nitrogen base (w / o amino acids), 2% sucrose, 2% agar, 50 μg / ml L-Uracil) at 30 ° C. for 3 days. Transformants that grew well in the culture of were selected as strains retaining FFase activity, and a random mutation library was prepared.
[0040]
Example 2: Modification FFase Screening
From the random mutation library prepared in Example 1, FFase with modified reaction characteristics was selected as follows. That is, each strain was cultured at 30 ° C. in a 150 μl SD medium (0.67% yeast nitrogen base (w / o amino acids), 2% glucose, 50 μg / ml L-Uracil) using a 96-well microplate. Cultured overnight.
[0041]
Next, this preculture solution was mixed with SD-GF medium (0.67% yeast nitrogen base (w / o amino acids), 2% sucrose, 50 μg / ml L-Uracil) and SD-GF.₂4.5 μl of each medium was inoculated into 150 μl of two types of medium (0.67% yeast nitrogen base (w / o amino acids), 2% 1-kestose, 50 μg / ml L-Uracil) and cultured at 30 ° C. . The turbidity (absorbance at 595 nm) of the culture solution after 20 hours was measured, and (turbidity of the culture solution in SD-GF medium) / (SD-GF₂Table 1 shows the results of calculating the ratio of the turbidity of the culture solution in the medium.
[0042]
[Table 1]

[0043]
Furthermore, the culture supernatant of these strains was used as a crude FFase enzyme solution, FFase activity measurement and an enzyme reaction using sucrose as a substrate were performed, and the sugar composition of the reaction solution was determined by HPLC (column: recrosssphere 100NH).₂, Eluent: 72% acetonitrile, flow rate: 1 ml / min, detector: differential refractometer). FFase activity was measured according to the method described in Agric. Biol. Chem., 53, 667-673 (1989). The enzyme reaction was carried out by adding 48% by weight of sucrose to the culture supernatant and incubating the reaction solution at 40 ° C. and pH 7.
[0044]
All the strains listed in Table 1 above were confirmed to have FFase activity. Furthermore, the sugar composition in the reaction solution obtained by the enzyme reaction using sucrose as a substrate is shown in Table 2 below. Here, the data for each strain is 1-kestose (GF₂) Shows the sugar composition of the reaction solution at the time when the amount of) becomes the maximum.
[0045]
[Table 2]

[0046]
It is considered that the FFase used for the production of 1-kestose preferably provides an enzyme reaction product having a high content of 1-kestose and a low content of other saccharides, particularly nystose and fructosyl nystose. From this point of view, when the FFases described in Tables 1 and 2 were evaluated, the turbidity ratios in both media were wild in the (21-36), (22-95) and (64-60) strains. It is larger than the mold, has a high 1-kestose content, and suppresses the production of nystose. That is, FFase produced by these strains can be said to have advantageous reaction characteristics for the production of 1-kestose.
[0047]
On the other hand, in the (M-2) strain, (64-81) strain and (51-24) strain, the ratio of turbidity in the above two media is similar to that of the wild type, and the content of 1-kestose and nystose There is no change.
[0048]
[Sequence Listing]

Claims (9)

A method for determining whether β-fructofuranosidase or a variant thereof is advantageous for the production of 1-kestose, comprising the following steps:
(A) transforming a host cell with a vector containing a gene encoding β-fructofuranosidase or a variant thereof in an expressible form;
(B) a step of culturing the transformant obtained in step (a) in a medium containing only sucrose as a carbon source, and measuring the growth rate of the transformant;
(C) a step of culturing the transformant obtained in step (a) in a medium containing only 1-kestose as a carbon source and measuring the growth rate of the transformant; and (d) 1 as a carbon source. -Β-fructofuranosidase produced by the transformant or a mutation thereof when the growth rate in a medium containing only kestose is smaller than the growth rate in a medium containing only sucrose as a carbon source Determining the body as advantageous for the production of 1-kestose,
And the host cell is a sucrose non-assimilating yeast .   The method according to claim 1, wherein in step (b) and step (c), the growth rate of the transformant is measured by the turbidity of the culture solution in a liquid medium.   In the step (d), the growth rate measured by the turbidity of the culture solution in the medium containing only sucrose as the carbon source is the growth rate measured by the turbidity of the culture solution in the medium containing only 1-kestose as the carbon source. The β-fructofuranosidase produced by the transformant or a variant thereof is determined to be advantageous for the production of 1-kestose when the value is 1.5 times or more of The method described. A method for producing a β-fructofuranosidase variant advantageous for the production of 1-kestose, comprising the following steps:
(A) introducing a mutation into the β-fructofuranosidase gene and preparing a gene encoding a β-fructofuranosidase mutant;
(B) transforming a host cell with a vector containing the gene obtained in step (a) in an expressible form;
(C) a step of culturing the transformant obtained in step (b) in a medium containing only sucrose as a carbon source and measuring the growth rate of the transformant;
(D) a step of culturing the transformant obtained in step (b) in a medium containing only 1-kestose as a carbon source and measuring the growth rate of the transformant; and (e) 1 as a carbon source. A step of selecting a β-fructofuranosidase mutant produced by a transformant showing a growth rate in a medium containing only kestose, which is smaller than a growth rate in a medium containing only sucrose as a carbon source;
And the host cell is a sucrose non-assimilating yeast .   The method according to claim 4, wherein in step (c) and step (d), the growth rate of the transformant is measured by the turbidity of the culture solution in a liquid medium.   In step (e), the growth rate measured by the turbidity of the culture solution in a medium containing only sucrose as a carbon source is measured by the turbidity of the culture solution in a medium containing only 1-kestose as the carbon source. The method according to claim 4, wherein a β-fructofuranosidase mutant produced by a transformant exhibiting a value of 1.5 times or more of is selected. A method for producing a β-fructofuranosidase mutant gene advantageous for the production of 1-kestose, comprising the following steps:
(A) introducing a mutation into the β-fructofuranosidase gene and preparing a gene encoding a β-fructofuranosidase mutant;
(B) transforming a host cell with a vector containing the gene obtained in step (a) in an expressible form;
(C) a step of culturing the transformant obtained in step (b) in a medium containing only sucrose as a carbon source and measuring the growth rate of the transformant;
(D) a step of culturing the transformant obtained in step (b) in a medium containing only 1-kestose as a carbon source and measuring the growth rate of the transformant; and (e) 1 as a carbon source. A step of selecting a β-fructofuranosidase mutant gene contained in a transformant having a growth rate in a medium containing only kestose, which is smaller than a growth rate in a medium containing only sucrose as a carbon source;
And the host cell is a sucrose non-assimilating yeast .   The method according to claim 7, wherein in step (c) and step (d), the growth rate of the transformant is measured by the turbidity of the culture solution in a liquid medium.   In step (e), the growth rate measured by the turbidity of the culture solution in a medium containing only sucrose as a carbon source is measured by the turbidity of the culture solution in a medium containing only 1-kestose as the carbon source. The method according to claim 7, wherein a β-fructofuranosidase mutant gene contained in a transformant showing a value of 1.5 times or more is selected.