JP2001321178A - HEAT-RESISTANT alpha-GALACTOSIDASE - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an α-galactosidase gene of a new gene, cloned from a chromosomal DNA of the 8084 strain, capable of providing a gene recombinant obtained by using Escherichia coli as a host, and capable of enabling an expression enzyme to be efficiently produced because the gene recombinant obtained by using the Escherichia coli as the host can express a heat-resistant α- galactosidase in high activities compared to the 8084 strain, and capable of enabling raffinose to be mass-synthesized and produced by utilizing the expression enzyme because the enzyme can synthesize the raffinose from sucrose and galactose. SOLUTION: This gene is the new α-galactosidase gene having heat resistance.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to α-galactosidase, and more particularly, it is excellent in thermostability and cleaves α-galactoside bond. The present invention relates to a conventionally unknown novel enzyme having new and useful characteristics such as catalyzing a reaction for synthesizing a compound.

Further, Absidi successfully analyzed by the present invention
The α-galactosidase gene of a corymbifera IFO 8084 (hereinafter sometimes referred to as strain 8084) is novel,
By linking the present structural gene to an expression plasmid vector and introducing the obtained recombinant plasmid into a system using Escherichia coli as a host, α-galactosidase can be efficiently expressed in large amounts, and the present enzyme is used. It has become possible to supply large quantities to the industrial field at low cost. The filamentous fungus Absidia corymbifer according to the present invention
The novel thermostable α-galactosidase gene obtained from a), the recombinant plasmid obtained by ligating the present structural gene to an expression plasmid vector, and the transformant obtained by introducing this into a host microorganism are all unknown hitherto. It is.

According to the present invention, industrial production of this novel thermostable α-galactosidase has been made possible. The present invention is effective for the production of raffinose as a raw material, particularly for industrial production.

[0004]

2. Description of the Related Art In recent years, as eating habits have diversified, lifestyle-related diseases and the like have been regarded as a serious problem, and consumers have become increasingly aware of foods and food materials. Under such circumstances, excessive intake of sugar has been taken up, and development and production of oligosaccharides as new sweeteners with low calories and physiological functions have become important, and their research has been diverse. .

[0005] From such a viewpoint, the inventors paid attention to raffinose. Raffinose is a trisaccharide oligosaccharide having a structure in which D-galactose is α1-6 bonded to a sucrose molecule. In the natural world, sugar beet (sugar beet), which is a raw material for sugar, as well as seeds of legumes such as soybeans, sugarcane, honey, cabbage, yeast, potatoes, grapes, wheat, and corn are widely distributed. It is used not only as a sweetener, but also as a stabilizer for frozen storage of bovine semen, and is also incorporated in a transport solution for human organ transplantation used worldwide. On the other hand, as research on human intestinal bacteria progresses, as a factor for growing bifidobacteria, a useful bacterium in the intestine,
Indigestible oligosaccharides have attracted attention, and clinical studies have revealed that raffinose has an excellent growth effect on bifidobacteria.In 1993, it was approved as a food material for specified health uses specified by the Ministry of Health and Welfare. I have. In addition, clinical clinical research has revealed that oral administration of raffinose may be effective in some cases of atopic dermatitis, which is one of the allergic conditions. I have.

[0006] Raffinose is industrially recovered as a by-product during the production of beet sugar. Raffinose in beets is considered as a bioprotective component against freezing because it increases with decreasing temperature before and after harvest in autumn. However, the content of raffinose in beet is only about 0.1% of the rhizome weight at the maximum, and the production amount is greatly related to the production amount of sugar, but there is a limit. In the future, as described above, the physiological function of raffinose will be clarified, and an increase in demand will be indispensable. Therefore, not only extraction and production from plants but also synthetic products from inexpensive raw materials will be required.

Prior to the present invention, the present inventors utilized the α-galactosidase activity of strain 8084 to efficiently decompose raffinose in order to decompose raffinose, which is a component that hindered the crystallization of sugar in the sugar production process. A decomposition method has been established. This α-galactosidase is theoretically capable of synthesizing raffinose using sucrose and galactose as raw materials as a reverse reaction.

[0008]

As described above, α-galactosidase is used in the sugar industry and the like for the purpose of decomposing raffinose which hinders the crystallization of sugar, utilizing its original property of decomposing α-galactoside. In addition to this, it can be used for the production of raffinose using sucrose and galactose as raw materials according to the reverse reaction.

In the sugar industry, a large amount of α-galactosidase is required to decompose raffinose, and a large amount of α-galactosidase is required for the synthesis of raffinose using a reverse reaction.
-Galactosidase is required. That is, as mentioned above, the production of raffinose by the present enzyme uses not the original forward reaction but the reverse reaction, so that the efficiency is extremely deteriorated when the amount of the enzyme is small or the reaction conditions are not optimal. there is a possibility. In the present invention, in order to supply a sufficient amount of α-galactosidase to this raffinose production method, efficient enzyme production was captured at the gene level and succeeded. The present invention has been accomplished for the purpose of mass-producing enzymes by introducing a genetic engineering technique.

[0010]

Means for Solving the Problems The present invention has been made to achieve the above-mentioned object, and aims to create a target microorganism and enzyme by a genetic engineering technique.

[0011] Then, the present inventors, chromosome D of 8084 strain
Cloning the α-galactosidase gene from NA,
Elucidation of the primary sequence of the structural gene and DNA containing it
The fragment was ligated to an expression plasmid vector, Escherichia coli (hereinafter, also referred to as Escherichia coli) BL21 (DE3) strain was transformed, the cells were cultured, and α-galactosidase was expressed in large amounts. . The obtained α-galactosidase not only has excellent α-galactoside resolution, but also can efficiently synthesize raffinose by the reverse reaction, and
It has activity even at an extremely high temperature of ℃, and has the characteristics of having extremely high heat resistance.
This enzyme was identified as a novel enzyme that was previously unknown. Hereinafter, the present invention will be described in detail.

In order to clone the α-galactosidase gene of the 8084 strain, the internal amino acid sequence of the α-galactosidase protein purified by enzyme was first determined. The 8084 strain was cultured, and the expressed α-galactosidase was purified to single electrophoretically by ammonium sulfate fractionation and various column chromatography.
This purified α-galactosidase protein was cleaved with a protease, the resulting peptide chains were separated, and the N-terminal amino acid sequence of each was decoded using a peptide sequencer. Oligonucleotide primers were synthesized based on the amino acid sequence thus obtained.

Separately, the chromosomal DNA of the 8084 strain prepared as a template was used as a template, mixed with the oligo DNA previously synthesized as a primer, and the α-galactosidase gene was partially amplified by PCR to obtain a probe.

Then, a chromosome is extracted from the 8084 strain,
A cDNA was synthesized from the obtained chromosome, the synthesized cDNA was inserted into a multiple cloning site of a plasmid vector, and a host such as Escherichia coli was transformed using the plasmid vector group to construct a cDNA library. From the E. coli colonies of the gene library thus prepared, positive clones were selected by colony hybridization. Plasmid DNA is extracted from the positive clone strain, and the DNA of the region that may contain the desired α-galactosidase gene
The NA base sequence was decoded. As a result, the entire DNA base sequence of the structural gene of the α-galactosidase gene was determined. This nucleotide sequence is shown in SEQ ID NO: 2 in the sequence listing (FIG. 2), and this sequence was a novel gene which was hitherto unknown.
Also, from this DNA base sequence, the α-
The amino acid sequence of galactosidase is inferred to be SEQ ID NO: 1 (FIG. 1).

Then, a fragment containing the structural gene of α-galactosidase completely is inserted into an Escherichia coli expression plasmid vector,
For example, a new recombinant plasmid was constructed by inserting and ligating the pET32-EK / LIC plasmid vector (Takara Shuzo) downstream of the thioredoxin sequence. In this plasmid vector, a promoter capable of efficiently expressing a gene linked as a foreign gene in Escherichia coli has been introduced, and the recombinant plasmid is transformed into Escherichia coli and induced to culture, whereby a large amount of α-galactosidase can be expressed. Enabled.

The α-galactosidase according to the present invention has the following physicochemical properties and not only has a high ability to degrade α-galactoside as a normal reaction, but also has a property of synthesizing raffinose from sucrose and galactose as a reverse reaction. It has excellent characteristics, such as having an excellent temperature range of 50 to 70 ° C. and extremely high resistance to heat. Such α-galactosidase is a novel enzyme which has never been seen in the past and is unknown in the past.

(1) Action The present enzyme generally performs a reaction as defined for α-galactosidase (cleaving an α-galactoside bond at the non-reducing end of a sugar chain, having an exoglycosidase activity) as a positive reaction. In particular, it has the substrate specificity described in (2). As a reverse reaction, a reaction for synthesizing raffinose is performed using sucrose and galactose as substrates. The test methods for substrate specificity and enzyme titer are described in (4).
Detailed reaction conditions for the reverse reaction will be described later.

(2) Substrate Specificity Table 1 shows the relative activity of each substrate when the activity of the present enzyme for ONPα-galactoside is defined as 100% (substrate specificity of a positive reaction). As a result, this enzyme is extremely ON
Pα-galactoside had high substrate specificity.

[0019]

(3) Optimum pH and stable pH range FIG. 3 shows the effect of the present enzyme on pH. Stability studies were performed at 4 ° C. for 24 hours in a buffer at each pH.
After the reaction, the solution was replaced with a buffer solution of pH 5.5, and the enzyme activity of the forward reaction was measured according to a standard method using ONPα-galactoside as a substrate. As a result, the optimal pH of the enzyme
Was 5.5 and stable at pH 5.0 to 11.0.

(4) Method for measuring titer The activity of the present enzyme was measured using ONPG (o-nitrophenyl α-D-
(Galactopyranoside) method. The enzymatic reaction was performed using a 0.1 M sodium phosphate buffer (pH 5.5).
Medium at 40 ° C. 0.1 M in 0.1 ml of enzyme solution
The reaction is started by adding 0.2 ml of sodium phosphate buffer (pH 5.5) and 0.2 ml of 20 mM ONPG, and stopped by adding 5 ml of 0.1 M sodium carbonate solution 10 minutes later. I let it. Generated o
-The amount of nitrophenol was measured at an absorbance of 415 nm. o-nitrophenyl β-D-galactopyranoside,
The same test was performed using o-nitrophenyl β-D-glucopyranoside as a substrate, and the amount of o-nitrophenol produced was quantified.

When raffinose, melibiose, sucrose, lactose and maltose were used as substrates, the test was carried out in the same manner except that the reaction was stopped by keeping the reaction in boiling water for 5 minutes. Then, when using raffinose as a substrate,
Galactose was quantified using a galactose UV test, and when melibiose, sucrose, lactose and maltose were used as substrates, glucose was quantified by the glucostat method. Here, one unit of the enzyme was defined as an amount of the enzyme that produced 1 μmol of a reaction product per minute under the present enzyme reaction conditions.

(5) Optimum temperature and stable temperature range FIG. 4 shows the effect of the present enzyme on the temperature. To examine the stability, the enzyme solution was kept at each temperature for 20 minutes, then quickly cooled to 4 ° C., and the enzyme activity of the positive reaction was measured according to a standard method using ONPα-galactoside as a substrate. As a result, the optimum temperature was 60 ° C., and the temperature was stable up to 60 ° C. As is clear from the drawing, the present enzyme is extremely characteristic in that it has extremely high thermostability.

(6) Conditions for deactivation by pH, temperature, etc. Although no particular details have been examined, the relative activity decreases under the conditions of pH and temperature described in (3) and (5), especially stability. Above this range is considered to be a condition for deactivation. Further, disappearance of the enzyme activity after keeping the enzyme solution in boiling water for 5 minutes has been confirmed.

(7) Purification method The expression system of the present Escherichia coli BL21 (DE3) -pET32Trx / galα includes His.T derived from plasmid vector pET32-Ek / LIC.
It contains an ag sequence and is designed so that the expressed α-galactosidase can be purified from the cell-free extract by affinity chromatography in one step.
The His-Tag sequence is a sequence in which about 6 to 10 histidine residues are arranged and has a binding ability with a metal ion.
For this reason, the expression product is converted to His • Tag R
A single protein can be purified with high efficiency by chelation chromatography using esin. The technique is a known fact, and it followed the purification manual of Novagent. As a result, α-galactosidase was singly purified from a cell-free extract of E. coli BL21 (DE3) -pET32Trx / galα with a high recovery of 80% or more.

(8) Molecular Weight and Method of Measuring Molecular Weight The molecular weight of the expressed α-galactosidase protein was determined by
Laemmli (Laemmli: Laemmli:
Nature (London), 1970, 227, 680-68.
It was performed by SDS-PAGE according to the method of 5). The gel concentration was 7.5%. Coomassi for protein staining
e Brilliant Blue was used. As a result, the molecular weight was measured to be about 82,000 Da. This value is similar to the molecular weight of 82,712 Da calculated from the amino acid sequence deduced from the DNA base sequence of the gene, and is reliable.

The novel thermostable 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 is exemplified as an example. . Regarding the production method, a novel transformant containing the gene (FER)
MBP-7140 (deposited internationally with Life Science Research Laboratories) by IPTG induction culture or by normal cultivation without induction, the novel thermostable α- according to the present invention.
Since galactosidase can be obtained and one example of its novel amino acid sequence has been identified (SEQ ID NO: 1),
The present enzyme can also be obtained by synthesis.

The present enzyme can efficiently decompose α-galactoside bond, which is a normal reaction, and can efficiently decompose, for example, melibiose and raffinose, and very efficiently perform raffinose synthesis, which is a reverse reaction. be able to. Raffinose synthesis can be performed very efficiently by incubating with the novel thermostable α-galactosidase according to the present invention in the presence of sucrose, galactose (and / or UDP-galactose).

Raffinose can be synthesized, for example, as follows. For example, as the present enzyme, in addition to a purified product, it is naturally possible to use a crude product, and at least one of a culture of a transformant, a culture solution, a cell-free extract, and a concentrate thereof is appropriately used. Can be used. The higher the substrate concentration of sucrose and galactose, the better. Each is preferably at least 20%, preferably at least 25%. As a preferred example, sucrose 40-80% (preferably 50-70%), galactose 5-5 For example, 45% (preferably 15 to 35%) is exemplified.

The reaction pH is preferably on the neutral to alkaline side than 6.0, and the reaction temperature is preferably high enough not to cause a positive reaction. Specifically, the reaction temperature may be set at about 70 ° C., although the stability of the enzyme is slightly lowered, preferably 50 ° C. or higher, more preferably 60 ° C. or higher.

In this case, since the present enzyme has the characteristic of having excellent heat resistance, it is epoch-making that raffinose can be efficiently synthesized for the first time. In addition, since the enzyme is active even at high temperatures,
The reaction system can be maintained at a high temperature, the reaction system is not contaminated by various bacteria, and is particularly suitable for industrial practice. In addition, the enzyme solution and other reaction solutions can be sterilized at high temperature, and the present invention is also excellent in this respect.

The reaction conditions can be appropriately selected without departing from the above-mentioned range, unless the reaction rate, yield and the like are taken into account. Hereinafter, the present invention will be described more specifically with reference to examples.

[Example 1] (1) Enzymatic purification of α-galactosidase of strain 8084 Unless otherwise specified, the enzyme was purified at 4 ° C and the buffer was 10 mM sodium phosphate buffer, pH 7.0 (hereinafter referred to as “pH 7.0”). Buffer).

The 8084 strain was cultured according to the method of Honjo et al. (Honjo et al., Journal of Sugar Refining Technology, 1985, 35, 67-73). The cultured cells were suspended in a 6-fold amount of glycine-sodium hydroxide buffer, pH 8.7, and autolyzed at 50 ° C for 24 hours. Thereafter, cell residues were removed by centrifugation, and the resulting supernatant was used as a crude enzyme solution. The wet cells cultured in this manner were also used in (3).

The crude enzyme solution was stirred for 5 hours.
Ammonium sulfate was added so as to be 0% saturated and left for 1 hour. The protein salted out by this operation was recovered as a precipitate by centrifugation. This precipitate was dissolved in a small amount of buffer and dialyzed against the same buffer overnight.

The crude enzyme solution was used as a carrier and subjected to ion exchange column chromatography (2.7 × 45 cm) using DEAE Sephacel equilibrated with a buffer solution. Elution of the adsorbed protein was performed by linearly changing the concentration of sodium chloride contained in the buffer solution from 0 to 0.5M. The α-galactosidase active fraction was measured according to a standard method, and the active fraction was concentrated with a centrifugal ultrafiltration membrane.

This crude enzyme solution was used as a carrier and subjected to gel filtration column chromatography (2.5 × 50 cm) using Sephacryl S-300 equilibrated with a buffer solution. The active fraction was concentrated with a centrifugal ultrafiltration membrane. The crude enzyme solution was used as a carrier and subjected to DEAE-Sepharose ion exchange column chromatography (1.8 × 13 cm) equilibrated with a buffer solution. Elution of the adsorbed protein was performed by linearly changing the concentration of sodium chloride contained in the buffer solution from 0 to 0.3M. The active fraction was concentrated with a centrifugal ultrafiltration membrane.

The purity of the protein was determined by Laemmli (Laemmli:
Nature (London), 1970, 227 , 680-685)
And SDS-PAGE. The gel concentration was 7.5%. Coomassie for protein staining
Briliant Blue was used. By the above operation, α of 8084 strains
-The galactosidase protein could be purified to a single electrophoretically. The recovery was 36% and the specific activity was 216.2.
U / ml.

(2) Internal amino acid sequence of purified α-galactosidase and synthesis of oligonucleotide primer

The purified enzyme preparation prepared in (1) and lysyl endopeptidase are mixed in a 0.1 M Tris-HCl buffer (pH 9.0) and reacted at 37 ° C. for 24 hours to convert the polypeptide chain. Decomposed. The degraded peptide fragment after the reaction was separated by SDS-PAGE, the gel after electrophoresis was transferred to a PVDF membrane, the transferred peptide chain was cut out, and each N-terminal amino acid sequence was subjected to a gas-phase protein sequencer. Decrypted. As a result, four internal amino acid sequences could be decoded. These sequences were all included in SEQ ID NO: 1 (FIG. 1).

Based on the determined amino acid sequences, two types of synthetic oligonucleotide primers shown in SEQ ID NOs: 3 and 4 (FIGS. 5 and 6) were prepared. That is, a site containing a large number of amino acid sequences with low degeneracy was selected, and a DNA database, DNA Information Stock Center, DISC (http:
//www.dna.affrc.go.jp/,National Institute of Agrob
The codon usage of the filamentous fungus Absidia, registered in Iological Resources, Tsukuba) was taken into account. Inosine was also used.

The sense primers and antisense primers used are shown in SEQ ID NOs: 3 and 4 (FIGS. 5 and 6). In the sequence, v indicates A or G, y indicates T or C, and n indicates I (inosine).

(3) Preparation of chromosomal DNA of strain 8084 The basic techniques of gene cloning experiments frequently used in the following experiments are known in the art, and unless otherwise specified, the method of Sambrook et al. (Sambrook et al .: Molecular・ Cloning A Laboratory Manual,
Second Edition, 1989).

About 2 of the wet cells of the 8084 strain prepared in (1)
g of TEN buffer (10 mM Tris-HCl, 1 mM ED)
TA, 0.1 M NaCl, pH 8.0) and washed well. The cells were collected by centrifugation, and the washing operation was repeated three times. Thereafter, the washed cells were frozen at −80 ° C. and freeze-dried. Thereafter, the dried cells were ground in a mortar to obtain a powder. Lysozyme and N-acetylmuramide were respectively added to this powder, and 37
Incubated at 24 ° C. for 24 hours. Minus 8 for processed material
After freezing at 0 ° C for 1 hour, a TEN buffer was added, the mixture was heated to 37 ° C, SDS was added to 3.3% (w / v), and the mixture was further incubated at 37 ° C for 3 hours to break cells. This solution was treated with phenol and precipitated with ethanol. The precipitated nucleic acid was wound around a glass rod and collected. 70% of this nucleic acid
After washing with ethanol, drying, re-dissolving in TE, RNase treatment, phenol treatment, phenol-chloroform treatment, ethanol precipitation, recovery, and chromosome D
NA. By this operation, about 10 mg of chromosomal DNA
Could be prepared.

(4) Probe synthesis and confirmation The primer prepared in (2) and the chromosomal DNA prepared in (3) were mixed, and Taq DNA polymerase and Dig were mixed.
PCR was performed in a system using a fluorescent dye of -11-dUTP (total volume was 50 microliters, the number of cycles was 35, one cycle was 98 ° C for 1 minute, and 68 ° C-3
Minutes), and the amplified fluorescently labeled DNA fragment of about 180 base pairs was used as a probe.

The prepared probe was subjected to formamide-formaldehyde agarose gel electrophoresis to separate mRNA extracted from the 8084 strain by the method described in (5), followed by blotting on a nylon membrane by the capillary method, followed by Northern blotting. Analysis confirmed that it could be used for experiments. The conditions for hybridization with the probe were as follows: hybridization solution (6 × SSC (1 × SSC)
Are 0.15M NaCl, 0.015M trisodium citrate), 3% blocking reagent, 0.1% SDS, 1%
Overnight at 68 ° C. in 0 mM EDTA, 150 microgram / ml salmon sperm DNA).

(5) Construction of cDNA Gene Library of strain 8084 The strain 8084 was cultured, total RNA was extracted from the cells, and stored at minus 80 ° C. After purifying the polyA mRNA using a simple column, cDNA was synthesized. The synthesized cDNA was inserted into the multiple cloning site of the pSPORT II plasmid vector. This plasmid vector group was transformed into E. coli 5α strain by electroporation to construct a cDNA library. Transformants were LB agar medium containing 150 microgram / ml ampicillin (10 g / L polypeptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, p.
H7.0).

(6) Obtaining the α-galactosidase gene of strain 8084 and its primary sequence The DNA of the E. coli colony of the gene library prepared in (5) was blotted on a nylon membrane. (4)
Using the Dig-labeled probe prepared in (4), (4)
Positive clones were screened by colony hybridization under the same conditions as in the above hybridization. For screening, two-stage selection was carried out. As a final selection, Escherichia coli was colonized into a single colony, plasmid DNA was extracted, and fragment Southern hybridization was performed. Those which showed a strong signal were defined as positive clones.

[0049] DN of the region that may contain the target gene
The fragment A was subcloned diligently, and the nucleotide sequence of the DNA was determined. A series of operations were commissioned to Takara Shuzo Co., Ltd. Gene Analysis Center.

The DNA sequence analysis was performed using GENETYX
-MAC was used. Various information regarding DNA sequencing was obtained from the network service of the DNA Information Stock Center, DISC.

As a result, 5 ′ shown in SEQ ID NO: 2 (FIG. 2)
A 2,190 base pair α-galactosidase structural gene having a DNA base sequence from the end was decoded. This sequence was a novel gene that had not been found so far. Also, 8084 deduced from this DNA base sequence
The α-galactosidase produced by the strain was composed of 729 amino acids and had an amino acid sequence from the N-terminus as shown in SEQ ID NO: 1 (FIG. 1).

(7) Construction and Transformation of α-Glucosidase Gene Expression Plasmid Vector The cloned α-galactosidase DNA fragment was
Restriction enzyme (EcoR) from which the structural gene can be completely extracted
I and NotI), blunt-ended, and a region of about 2.2 kilobase pairs between the cleavage sites is pET32-BK / LIC.
A new plasmid vector pET32Trx / ga was ligated downstream of the thioredoxin sequence of the plasmid vector.
Iα was constructed. This plasmid vector efficiently transcribes the gene linked as a foreign gene in E. coli,
A translatable T7Lac promoter has been introduced, and α-galactosidase can be expressed and produced with high efficiency by the culture method shown in (8).

The recombinant plasmid vector was transformed into competent cells of Escherichia coli BL21 (DB3) by the heat shock method to create a recombinant microorganism. This microorganism is Escherichia coli BL21 (DE3) -pET32Trx / gal
α, and the Institute of Biotechnology and Industrial Technology
Deposited internationally as ERM BP-7140.

(8) Culture of transformant and expression of α-galactosidase E. coli BL21 (DE3) -pET32Trx / galα prepared in (7)
5 ml containing 50 microgram / ml ampicillin
LB medium at 37 ° C until mid-logarithmic growth. This bacterial solution was treated with isopropyl-β- so that the final concentration was 1 mM.
100 ml of the same medium containing D-thiogalactopyranoside (hereinafter abbreviated as IPTG) was inoculated and cultured with shaking at 37 ° C. After the culture, E. coli was recovered by centrifugation.
Cells are 10 mM sodium phosphate buffer (pH 6.0)
, And then resuspended in the same buffer and crushed with an ultrasonic crusher. The crushed liquid was centrifuged, and the supernatant was used as a cell-free extract.

Escherichia coli BL21 (DE3) -pET32T thus prepared
When the α-galactosidase activity of the cell-free extract of rx / galα was measured according to a standard method, 18.7 units / culture solution (m
l). The 8084 strain is 0.449 even under optimal conditions
Since only .alpha.-galactosidase can be produced per unit / culture solution (ml), the genetically modified product according to the present invention is the original strain 8
084 higher productivity. The activity comparison is shown in Table 2.

(Table 2) Table 2: Comparison of α-galactosidase production by transformants酵素 Enzyme activity (unit / ml culture) Microorganism ──────────────────────── Control IPTG no addition IPTG addition (Induction for 6 hours ) (3 hour induction) (6 hour induction) ──────────────────────────────────── E.coli BL21 (DE3) / 0.19 ^{* 1} 14.1 15.5 18.7 pET32Trx / galα strain A. corybifera IFO8084 strain 0.45 ^{* 2} − − − ──────────────────────── ──────────── * 1: pET32-EK / LIC transformed. * 2: Cultured for 9 days.

The α-galactosidase expressed from the cell-free extract can be completely purified electrophoretically by a single operation using affinity chromatography using histidine Tag derived from a plasmid vector.
The expressed α-galactosidase can be supplied completely and inexpensively with a recovery rate of 92%.

(9) Production of raffinose using expressed α-galactosidase Dissolve in 10 mM phosphate buffer (pH 7.0) so that the final concentration of sucrose is 600 g / L and the final concentration of galactose is 250 g / L. The liquid thus obtained was used as a substrate solution. This substrate solution and the purified recombinant recombinant expression α-prepared in (8) were used.
Mix with galactosidase solution (100 U) and mix
The enzyme reaction was performed at ℃. After completion of the reaction, the reaction was stopped by maintaining the mixture in boiling water for 5 minutes. Raffinose synthesized by this reaction was measured in a system using HPLC. The column was Shodex SUGARKS801 (8.0 × 3).
00 mm), water was used as the eluent, and RI was used as the detector.

As a result, as shown in Table 3, the yield of raffinose was 10% of the galactose used. That is, 0.14 mol / L (25 g / L) of raffinose was synthesized. When galactose was changed to UDP-galactose and used as a substrate, the raffinose yield increased to 45%. That is, 0.63 mol / L (1
13 g / L) of raffinose was synthesized.

(Table 3) Table 3: Production of raffinose by expressed α-galactosidase Substrate conditions Yield (%) ^{* 1} ３０ 30% sucrose + 25% galactose 3 60% Sucrose + 25% galactose 10 30% sucrose + 25% UDP-galactose 38 60% sucrose + 25% UDP-galactose 45─────────────────────────── * * 1: Yield based on molar amount of substrate galactose Amount of enzyme used: 100 U Reaction conditions: 70 ° C, 72 hours

In this example, as described above, the novel transformant Escherichia coli BL21 (DE-3) -pET32Trx
/ galα was induction-cultured with IPTG to prepare a cell-free extract. Under the main culture conditions, α-galactosidase can be produced at 18.7 units / ml of the culture solution. This enzyme can be purified by affinity chromatography.However, considering the industrial production of raffinose, the cost is involved, so there seems to be no problem in terms of raffinose production efficiency. The cell-free extract was used as it was.

The total volume of the reaction solution was set to 500 ml, and a 10 mM phosphate buffer (p) was added so that the final concentration of sucrose was 600 g / L and the concentration of galactose was 250 g / L.
H7.0) was used as a substrate solution. This substrate solution and the prepared α-galactosidase solution (for 100 units) were mixed, and an enzyme reaction was performed at 70 ° C. After completion of the reaction, the reaction was stopped by maintaining the mixture in boiling water for 5 minutes.

The optimal pH of the enzyme in the forward reaction was 5.5, but in the reverse reaction of raffinose synthesis, it was 7.0. The pH was set at 7.0.

Further, the efficiency of the raffinose synthesis reaction is higher at the highest possible temperature (the normal reaction is carried out at a normal reaction temperature of around 40 ° C.), so that the stability of the enzyme deteriorates. The reaction temperature was set at 70 ° C.

The raffinose synthesis reaction did not proceed unless the substrate concentrations of sucrose and galactose were 25% or more. Therefore, the stability of the enzyme deteriorates,
Sucrose 60% and galactose 25% were set.

[0066]

The heat-stable α-galactosidase (as well as the enzyme gene) is a novel substance which has hitherto been unknown. This enzyme not only has the effect of efficiently decomposing galactoside bonds, but also has been able to synthesize raffinose in large amounts from sucrose and galactose by utilizing the reverse reaction. Although this reverse reaction needs to be performed under high temperature conditions, the novel enzyme according to the present invention has an outstanding feature of having excellent heat resistance, and therefore, it is possible to efficiently perform this reverse reaction. It became possible for the first time.

Further, according to the present invention, the gene encoding thermostable α-galactosidase was successfully cloned, and its nucleotide sequence was revealed, and the amino acid sequence of the enzyme was also revealed. In addition, the cloned gene was successfully inserted into a vector to transform a host. Therefore, by culturing the resulting transformant without induction or induction, heat-resistant α-
Galactosidase can be produced in significant quantities.

[0068]

[Sequence List] SEQUENCE LISTING <110> Nippon Tensaiseito Kabushiki kaisha <120> Novel Thermotolerant α-Galactosidase <130> 6300 <141> 2000-5-15 <160> 4 <210> 1 <211> 729 <212> PRT < 213> Absidia corymbifera <400> 1 Ala Leu Asp Val Gly Ile His Lys His Pro Ser Phe Glu Phr Trp Phe 16 MET Val Thr Lys Lys Ser Thr Tyr Val Val Gly Ala Thr Ala Asp Gly 32 Tyr Val Ile Asn Leu His Trp Gly Lys Arg Leu Thr Gln Leu Asp Asp 48 Leu Asn Ala Thr Val Pro Leu Ser Asp Gln Ser Gln Asn Pro Pro Ile 64 Ser Tyr Ala MET Glu Glu Leu Pro Ala Phe Gly Gly Leu Arg Tyr Arg 80 Asp Asn Val Leu Lys Val Asp Leu Pro Asp Gly Thr Arg Glu Leu Asn 96 Leu Leu Tyr Ser Gly Ala Lys Ser Lys Asp Asp Thr Leu Leu Asp Ile 112 Glu Leu Ser Asp Gly Asn Arg Thr Asp Phe Lys Val Thr Leu His Tyr 128 Glu Leu Asp Thr Glu Asn Asp MET Ile Arg Arg Ser Tyr Thr Val Glu 144 Asn Gly Leu Lys Gly Arg Val Asn Leu Asp Leu Ala Leu Ser Ala Ala 160 Trp His Pro Pro Thr Ala Leu Asp Val Asp Glu Lys Arg Glu Leu Leu 176 T hr Leu Ala Gly Glu Trp Asn Asn Glu Ala Gln Val Gln Ser Thr Glu 192 Leu Lys Pro Gly Ile Ala His Thr Ile Gln Thr Pro Lys Gly Phe Thr 208 Asp His Val Ser Tyr Pro Tyr Phe Ala Ile Arg Gln Val Pro Ser Glu 224 Val Asn Pro Gly Thr Glu Val Tyr Phe Gly Thr Leu Ala Trp Gly Gly 240 Ser Trp Glu Ile Thr Ala His Thr Asn Thr Tyr Gly Tyr Thr Arg Ile 256 Thr Gly Gly MET His His Leu Asp Phe Gly Trp Thr Leu Glu Pro Gly 272 Glu Ser Phe Thr Thr Pro Val Phe Ile Ala Gly Phe Thr Asp Glu Gly 288 Leu Pro Gly Ala Arg Arg Arg Leu Pro Arg His Ala Arg Lys Tyr Gln 304 Gln Leu Ser Leu Gln Thr Gln Lys Asn Asp Ser Leu Tyr His Pro Val 320 Leu Tyr Asn Ser Trp Glu Ala Val Thr Phe Asp Val Thr Phe Asp Lys 336 Gln Val Ala Leu Ala Glu Lys Ala Ala Pro Leu Gly Val Glu Leu Phe 352 Val Ile Asp Asp Gly Trp Phe Gly Asp Arg Asn Asn Asp Ser Ala Gly 368 Leu Gly Asp Trp Tyr Pro Asn Lys Glu Lys Phe Pro Asn Gly Leu Lys 384 Pro Leu Ala Asp His Val His Asp Leu Gly MET Gln Phe Gly Val Trp 400 Phe Glu Pro Glu Ser Val Asn Pro Asn Ser Asn Leu T yr Arg Glu His 416 Pro Asp Trp Val Leu Tyr Tyr Asp Gly Val Pro Arg Tyr Glu Ala Arg 432 Asn Gln Leu Leu Leu Asn Leu Gly Leu Pro Glu Val Gln Asp Tyr Ile 448 Tyr Asp Arg Val Ser Ser Ile Ile Glu Glu Asn Asp Ile Asp Tyr Ile 464 Lys Trp Asp MET Asn Arg Pro Tyr Gln Gly Val Thr MET His His Tyr 480 Asp Arg Asn Pro Arg Glu Ala Trp Val Leu Ile Ala Arg Gly Tyr Gln 496 Asn Leu Leu Ala Lys Leu Lys Lys Arg Phe Pro Asn Leu Trp Ile Glu 512 Ser Cys Ala Ser Gly Gly Gly Arg MET Asp Leu Ser Val Leu Glu His 528 Ala Asp Gln Val Trp Thr Ser Asp Asn Thr Arg Pro Asp Ala Arg Leu 544 Lys Ile Gln Tyr Gly Ala Ser Leu Phe Leu Pro Pro Arg Ala MET Tyr 560 Gly Trp Val Thr Glu Ser Gly Asp Asp Asn Asn Val His Ile Pro Leu 576 Ser Phe Arg Phe His Thr Ser Phe MET Gly Gly Leu Gly Ile Gly Ala 592 Asn Leu Asn Lys Tyr Ser Asp Asn Asp MET Lys Glu Ser Thr Ala Trp 608 Ile Ala Leu Tyr Lys Lys Leu Arg Pro Val Ile Gln Asn Gly Asp Leu 624 Asp Trp Leu Val Pro Pro Ser Lys Val Gly Glu MET Ile Ala Val Ser 640 Gln Thr Thr Ser Lys Asp Gln G ln Glu Ala Val Ile Leu Ala Phe Arg 656 Ile Ser Ser Pro Phe Ser Ala Pro MET Asn Pro Val Arg Pro Arg Phe 672 Leu Lys Asp Asp Val Val Tyr Arg Val Gln Ile Trp Leu Gln Asp Pro 688 His Lys Val Ala Glu Glu Tyr Asp MET Ser Gly Ala Leu Leu MET His 704 Lys Gly Ile Ser Leu Asp Gly Leu Asn Arg Ala MET Phe Thr Ser Ala 720 Val Val Tyr Val Lys Gln Ser Tyr Asp <210> 2 <211> 2190 <212> DNA < 213> Absidia corymbifera <400> 2 10 20 30 40 50 60 gcgttggatg tggggattca taagcatccc tctttcgaga catggttcat ggtgacaaag 70 80 90 100 110 120 aagtctacat acgtggttgg tgctacagca gatggatatg ttatcaacct tcactgggcacat gatca gagcaca tca tca tca tca tca tca tca tca tca tca tca t tactcaacct tcact aatccaccta ttagctatgc catggaagaa ttaccagctt ttggtggact 250 actgca ctatgaactg gacactgaga acgatatgat acgtcgatcg 430 440 450 460 470 480 tatactgttg agaatggact gaagggtcgt gtcaaccttg acttggcatt gtcagctgca 490 500 510 520 530 540 tggcatccac caacagcact tgacgttgac gagaaacgag agttactgac attggcaggc 550 560 570 580 590 600 gaatggaaca atgaagcaca agtacaatca accgagttga agcctggcat agcccatacg 610 620 630 640 650 660 atccagacac ccaaaggatt tactgatcat gtgtcatatc catactttgc catcagacaa 670 680 690 700 710 720 gtaccaagtg aagtgaatcc tggcactgaa gtttactttg gtactcttgc atggggaggt 730 740 750 760 770 780 agttgggaga ttacagcaca taccaacaca tatggctata cccgtatcac tggtggcatg 790 800 810 820 830 840 catcatcttg attttggatg gacacttgaa ccaggagaat cattcacaac accagtgttt 850 860 870 880 890 900 attgctggat tcacagatga aggcttacca ggcgcacgta gacgcttacc acgtcatgct 910 920 930 940 950 960 cgaaagtatc agcaattgag cctgcagaca caaaagaatg acagtcttta tcatcctgtt 970 980 990 1000 1010 1020 ctttacaact cttgggaagc tgttactttt gatgtc10t10gct 10ctgct 10g10gct 10g ctg ga ccact tggtgttgaa ttgtttgtta ttgatgatgg ctggtttggg 1090 1100 1110 1120 1130 1140 gatcgtaaca acgactctgc aggattagga gattggtatc ccaacaagga aaagttccca 1150 1160 1170 1180 1190 1200 aatggtctca agccccttgc agaccatgtg catgatctcg gcatgcagtt tggcgtgtgg 1210 1220 1230 1240 1250 1260 tttgaacccg agtcggtcaa tcccaattcc aatctttatc gtgaacatcc tgattgggtg 1270 1280 1290 1300 1310 1320 ctttactatg acggtgtccc aagatacgaa gcacgtaatc agctgttact caaccttgga 1330 1340 1350 1360 1370 1380 ttaccagaag tgcaagatta catttatgat cgagtgagca gtatcattga agagaatgat 1390 1400 1410 1420 1430 1440 attgactata tcaagtggga tatgaatcga ccctatcaag gcgttactat gcatcattat 1450 1460 1470 1480 1490 1500 gatagaaatc ctcgagaagc atgggtgctg attgcgcgtg gatatcaaaa tttgctagcc 1510 1520 1530 1540 1550 1560 aagctcaaga aacgatttcc caatctgtgg attgaaagct gtgctagtgg tggtggacgt 1570 1580 1590 1600 1610 1620 atggatctca gtgtgcttga acatgctgat caagtttgga ccagtgacaa tacaagacct 1630 1640 1650 1660 1670 1680 gatgctcgtc ttaagattca atatggtgct tcattgttag ag gccatgtat 1690 1700 1710 1720 1730 1740 ggttgggtga ctgaaagtgg ggatgataac aatgtgcaca tacctttatc cttccggttc 1750 1760 1770 1780 1790 1800 cacacatctt tcatgggtgg ccttggtatt ggtgccaact tgaacaagta ctcagacaat 1810 1820 1830 1840 1850 1860 gatatgaaag agagcacagc ttggattgca ttgtataaga agcttcgacc agtgattcaa 1870 1880 1890 1900 1910 1920 aatggtgatc tcgattggtt agtgccacct tcaaaggttg gtgaaatgat tgctgtatcc 1930 1940 1950 1960 1970 1980 cagacaacat caaaggatca acaagaagca gtgatcttgg cattccgaat cagcagccct 1990 2000 2010 2020 2030 2040 ttttcagctc caatgaatcc agtacgacca cgttttctca aggatgatgt agtatatcgg 2050 2060 2070 2080 2090 2100 gttcaaatct ggttacagga tccacacaag gttgctgaag aatacgacat gtctggtgct 2110 2120 2130 2140 2150 2160 ttactcatgc acaagggaat tagtcttgat ggattgaata gagccatgtt tactagtgca 2170 2180 2190 gttgtttatg tgaaagagtc atacgattaa <210> 3 <211> 28 <212> DNA <213> Artificial sequence <400> 3 garytnttyg tnatngayga yggntggt <210> 4 <211> 29 <212> DNA <213> Artificial se quence <400> 4 ttnggrttna cngaytcngg ytcraacca

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of thermostable α-galactosidase derived from 8084 strain.

FIG. 2 shows the nucleotide sequence of a thermostable α-galactosidase gene DNA derived from 8084 strain.

FIG. 3 shows the effect of expressed α-galactosidase on pH.

FIG. 4 shows the effect of expressed α-galactosidase on temperature.

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

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

Claims (6)

[Claims] 1. Heat-resistant α- having the following physicochemical properties:
Galactosidase. (1) Action The present enzyme performs a reaction as defined in α-galactosidase (having an exoglycosidase activity that cleaves an α-galactoside bond at the non-reducing end of a sugar chain) as a positive reaction. As a reverse reaction, a reaction for synthesizing raffinose is performed using sucrose and galactose as substrates. (2) Substrate specificity (substrate specificity of forward reaction) ONPα-galactoside is degraded well. Decomposes melibiose moderately or more. Decomposes raffinose a little.
ONPβ-galactoside, lactose, maltose, O
The ability to decompose NPβ-glucoside and sucrose is weak. (3) Optimum pH and stable pH range Optimum pH: 5.5 Stable pH range: 5 to 11 (4) Optimum temperature and stable temperature range Optimum temperature: 60 ° C The enzyme is stable up to 60 ° C. And has very high heat resistance. 2. A thermostable α-galactosidase represented by the amino acid sequence of SEQ ID NO: 1. 3. A thermostable α-galactosidase corresponding to the thermostable α-galactosidase gene represented by the nucleotide sequence of SEQ ID NO: 2. 4. The heat-resistant α- according to any one of claims 1 to 3, wherein the enzyme is derived from the genus Absidia.
Galactosidase. 5. The thermostable α-galactosidase according to claim 4, wherein the genus Absididia is Absidia corymbifera. 6. A transformant transformed with a plasmid containing the DNA of the thermostable α-galactosidase gene represented by the nucleotide sequence of SEQ ID NO: 2 is cultured, and the thermostable α-galactosidase is collected from the culture. A method for producing a thermostable α-galactosidase having the following physicochemical properties: (1) Action The present enzyme performs a reaction as defined in α-galactosidase (having an exoglycosidase activity that cleaves an α-galactoside bond at the non-reducing end of a sugar chain) as a positive reaction. As a reverse reaction, a reaction for synthesizing raffinose is performed using sucrose and galactose as substrates. (2) Substrate specificity (substrate specificity of forward reaction) ONPα-galactoside is degraded well. Decomposes melibiose moderately or more. Decomposes raffinose a little.
ONPβ-galactoside, lactose, maltose, O
The ability to decompose NPβ-glucoside and sucrose is weak. (3) Optimum pH and stable pH range Optimum pH: 5.5 Stable pH range: 5 to 11 (4) Optimum temperature and stable temperature range Optimum temperature: 60 ° C The enzyme is stable up to 60 ° C. And has very high heat resistance.