JP2003088365A - MODIFIED alpha-GLUCOSIDASE AND METHOD FOR PRODUCING OLIGOSACCHARIDE - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a modified α-glucosidase hydrolyzing α-glycoside bond, of which hydrolyzing activity is weakened or substantially extinguished, while maintaining its rearrangement activity, and a method for producing an oligosaccharide exhibiting a high disaccharide derivative yield by using the modified enzyme. SOLUTION: This α-glucosidase is derived from Schizosaccharmoyces pombe, and modified so as to decrease the hydrolyzing activity by <=1/10,000 as compared with that of the α-glucosidase by substituting the 481st asparagic acid in the amino acid sequence with e.g. glycine. The method for producing the oligosaccharide by performing a reaction of a glycoside derivative with a β- glucosyl fluoride in the presence of the α-glucosidase is also provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an α-glucosidase modified so as not to substantially exhibit hydrolytic activity, and a method for producing an oligosaccharide using the modified α-glucosidase.

[0002]

BACKGROUND ART α-Glucosidase is an enzyme widely existing in the natural world from microorganisms to animals and plants, and is an enzyme that hydrolyzes an α-glucoside bond at the non-reducing end of a substrate to produce α-D-glucose. . This α-glucosidase is classified into family 13 and family 31 according to the primary sequence of the enzyme by B. Henrissat.
(B. Henrissat, Biochem. J.: 28
0, 309-316 (1991)). Chiba et al. Have focused on the substrate specificity of α-glucosidase and classified it into family 1 and family 2 (Chiba et al., Biosci. Biotech.
Biochem .: 61, 1233-1239 (1997)).

[0003]

[Alpha] -Glucosidase catalyzes not only the hydrolysis (hydrolysis) reaction but also the transglycosylation reaction and is also used for the enzymatic synthesis of various oligosaccharides and glycosides. However, the oligosaccharide produced by the transglycosylation reaction becomes a substrate for α-glucosidase again and is hydrolyzed, so that the oligosaccharide produced increases with the progress of the transglycosylation reaction, but has a certain maximum value. Then decreases. Therefore, even if it is attempted to synthesize a certain oligosaccharide by a transglycosylation reaction, the limit is about 30 to 50%. The above phenomenon is observed not only in α-glucosidases but also in various glycosidases including β-glucosidases when synthesizing oligosaccharides using a transglycosylation reaction.

In this situation, Mackenzie (Macke
nzie) and Wang et al.
For β-glucosidase produced by (bacterium sp.), a modified enzyme was prepared by substituting alanine for glutamic acid 358, which is a dissociative carboxyl group as one of the active dissociative groups, to prepare α-glucosyl azide and α-glucosyl fluoride. As a glucosyl donor, we have reported the synthesis of oligosaccharides with β-glucosidic bonds (Mackenzie et a
l., J. Am. Chem. Soc .: 116, 11594-11595 (1994), W
ang et al., J. Am. Chem. Soc .: 120, 5583-5584 (19
98)). Mayer et al. Prepared a modified enzyme in which glutamic acid 358 was replaced with serine, and α-glucosyl fluoride and α-galactosyl fluoride were used as glycosyl donors to synthesize an oligosaccharide having a β-glycoside bond. (Mayer et al., FEBS Letters: 466,
40-44 (2000)). Morakci et al., Mackenzie
(Mackenzie) et al. (Wang) et al. With similar results, Sulfolobus solfataricus (Sulfolobus solfataricus)
Original β-glycosidase modifying enzyme (glutamic acid 3
87 → glycine) and α-glucosyl azide or α-glucosyl fluoride.

However, all of these enzymes are β
An enzyme that hydrolyzes a glycoside bond, and such an enzyme that hydrolyzes an α-glycoside bond has not been known. Also, Mackenzie
All of the glucosyl donors used in the synthesis of oligosaccharides using the modified enzymes obtained by Wara et al., And Moracci et al. Are α-glucoside type, and the oligosaccharides produced are Was limited to oligosaccharides with β-glucosidic bonds.

The production rate of these oligosaccharides is 80%.
What can be said is US Pat. No. 5,716,812.
No. However, the production rate shown here is a numerical value as an oligosaccharide mixture, and the production rate as a disaccharide derivative was only 54% at the maximum. On the other hand, in order to increase the production rate of the disaccharide derivative, it is necessary to use a glycosyl donor such as α-galactosyl fluoride that does not cause re-extension of the sugar chain. However, when a glycosyl donor that does not cause re-extension is used, the resulting oligosaccharide is a heterooligosaccharide.

Therefore, an object of the present invention is an enzyme which hydrolyzes an α-glycoside bond, and a modified enzyme which weakens or substantially eliminates a hydrolysis (hydrolysis) reaction while maintaining a transfer reaction activity. To provide. Further, the present invention is to provide a method for producing an oligosaccharide having a high production rate of a disaccharide derivative using such a modified enzyme.

[0008]

Therefore, the present inventors have
Diligently researching α-glucosidase, and finding an amino acid containing a dissociative carboxyl group, which is usually essential for the hydrolysis reaction,
Substitution with an amino acid that does not contain a carboxyl group in the side chain, found a modified α-glucosidase so as not to show hydrolytic activity, further oligosaccharides using the modified α-glucosidase and β-glucosyl fluoride as a glucosyl donor The present invention has been completed by discovering a manufacturing method of. Here, "does not show hydrolytic activity" means that the amount of glucose produced is 1 / 10,000 or less when the enzyme before modification is compared with the amount of glucose produced using maltose or p-phenyl α-glucoside as a substrate. Indicates that there is.

The present invention which achieves the above-mentioned object of the present invention is as follows. [Claim 1] An α-glucosidase having the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, wherein the 481st aspartic acid in the amino acid sequence is hydrolyzed with the amino acid sequence represented by SEQ ID NO: 2. Modified α-characterized by substituting with another amino acid so that it is reduced to 1 / 10,000 or less as compared with α-glucosidase having
Glucosidase. [Claim 2] The modified α-glucosidase according to claim 1, wherein the 481st aspartic acid is substituted with an amino acid having no carboxyl group in the side chain. [Claim 3] The modified α-glucosidase according to claim 2, wherein the amino acid containing no carboxyl group in the side chain is glycine or alanine. [Claim 4] An α-glucosidase having the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, wherein the 481st aspartic acid in the amino acid sequence is replaced with glycine. [Claim 5] The modified α-glucosidase according to claim 4, which has an amino acid sequence in which one or more amino acids are further substituted, deleted, inserted or added in the amino acid sequence. [Claim 6] Schizosaccha
romyces pombe) -derived α-glucosidase,
Among the active dissociative groups of the α-glucosidase, an amino acid containing a dissociative carboxyl group has an amino acid containing no carboxyl group in its side chain so that the hydrolysis activity is reduced to 1 / 10,000 or less as compared with the wild-type enzyme. A modified α-glucosidase, characterized in that [Claim 7] The modification according to claim 6, wherein the amino acid containing a dissociative carboxyl group among the active dissociative groups is an amino acid corresponding to the 481st aspartic acid in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. α-glucosidase. [8] The modified α-glucosidase according to [6] or [7], wherein the amino acid containing no carboxyl group in the side chain is glycine or alanine. [Claim 9] [alpha]-according to any one of claims 1-8.
A method for producing an oligosaccharide, which comprises reacting a glycoside derivative with β-glucosyl fluoride in the presence of glucosidase. [Claim 10] The glycoside derivative is pNP-β-glucoside, pNP-α-xyloside, pNP-α-mannoside, pN.
The method according to claim 9, which is P-α-maltoside. [Claim 11] The oligosaccharide is a disaccharide derivative.
Or the manufacturing method according to 10.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The modified α-glucosidase according to the first aspect of the present invention described in claim 1 is an α-glucosidase having the amino acid sequence represented by SEQ ID NO: 2 in the Sequence Listing, The aspartic acid at position 481 of the amino acid sequence was compared with that of α-glucosidase having the amino acid sequence shown in SEQ ID NO: 2 for hydrolyzing activity to be 1000.
It is characterized in that it was replaced with another amino acid so as to be reduced to 1/0 or less. The α-glucosidase having the amino acid sequence shown in SEQ ID NO: 2 in which the amino acid is not modified is a diazosaccharomyces pombe (Schizosaccharomyce pombe).
α-glucosidase derived from S. pombe). And
The aspartic acid at position 481 of the amino acid sequence shown in SEQ ID NO: 2 is replaced with another amino acid. The amino acid substitution is performed so that the hydrolysis activity is reduced to 1 / 10,000 or less as compared with α-glucosidase having the amino acid sequence shown in SEQ ID NO: 2. Specifically, 481
The second aspartic acid is replaced with an amino acid containing no carboxyl group in the side chain. Then, the amino acid having no carboxyl group in the side chain can be, for example, glycine or alanine.

The α-glucosidase of the present invention is an α-glucosidase having the amino acid sequence shown in SEQ ID NO: 2 of the Sequence Listing, wherein the 481st aspartic acid in the amino acid sequence is replaced with glycine. To do.

[0012] The modified α-glucosidase of the second aspect of the present invention according to claim 6 is an α-glucosidase derived from Schizosaccharomyces pombe, wherein among the active dissociative groups of the α-glucosidase, It is characterized in that an amino acid containing a dissociated carboxyl group is substituted with an amino acid containing no carboxyl group in its side chain so that the hydrolysis activity is reduced to 1 / 10,000 or less as compared with the wild-type enzyme.

In the modified α-glucosidase according to the second aspect of the present invention, the basic α-glucosidase is not limited to the α-glucosidase having the amino acid sequence shown in SEQ ID NO: 2, but it is not limited to Dizosaccharomyces pombe (Schizosaccharo).
Any α-glucosidase derived from myces pombe) may be used. And, Zizosaccharomyces pombe (Schizosaccha
Among the active dissociative groups of α-glucosidase derived from romyces pombe), an amino acid containing a dissociative carboxyl group is replaced with another amino acid having no carboxyl group in its side chain. The amino acid substituted with another amino acid is, for example, the amino acid corresponding to the 481st aspartic acid in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. The α-glucosidase having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing is also a dizosaccharomyces pombe (Schizosaccharomyces pombe).
Since it is an α-glucosidase derived from pombe), α-glucosidase having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing
Dizosaccharomyces pombe other than glucosidase (Schi
α-glucosidase derived from zosaccharomyces pombe) also has a high homology with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and corresponds to the 481st aspartic acid in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. The amino acid to be used can be easily found.

Amino acid substitution is carried out by using a wild-type enzyme having a hydrolytic activity, that is, wild-type dizosaccharomyces pombe (Sc
hizosaccharomyces pombe) -derived α-glucosidase is reduced to 1 / 10,000 or less. Further, the amino acid having no carboxyl group in the side chain can be, for example, glycine or alanine.

The modified enzyme of the present invention can be prepared by a conventional method. For example, an α-glucosidase gene having the amino acid sequence shown in SEQ ID NO: 2 or a dizosaccharomyces pombe (Schizosaccharomyce
It can be prepared by substituting the target amino acid residue having a dissociative carboxyl group with another amino acid by point mutation in the α-glucosidase gene. Modification enzymes, these modification enzymes,
It can be prepared by culturing a microorganism, and can be used as a supernatant or a crushed product of the cells, but if necessary, it can be purified by various chromatography and used.

Schizosaccha
The α-glucosidase derived from romyces pombe) has already been reported by Chiba et al. for its purification method, enzymatic chemical properties, and glycosyl transfer reaction (Chiba et al., Agric. B.
iol. Chem .: 29, 540-547 (1965), Chiba et al., Agr
ic. Biol. Chem .: 30, 536-540 (1966)). In addition, the gene has been obtained and the active dissociative group has been identified (Agricultural Chemical Society of Japan, Vol. 72, 1998 Annual Meeting Abstracts p.134). This Dizo Saccharomyces pombe (Schi
A specific example of the method for obtaining a modified enzyme of α-glucosidase derived from zosaccharomyces pombe) will be shown in Examples described later.

The present invention includes a method for producing an oligosaccharide, which comprises reacting a glycoside derivative with β-glucosyl fluoride in the presence of the modified α-glucosidase of the present invention. The β-glucosyl fluoride used in the above production method is a glucosyl donor and has been used so far for the purpose of elucidating the enzyme reaction mechanism. Regarding its preparation method, pentaacetyl D-glucose was reacted with anhydrous hydrogen fluoride to prepare 2,3,4,6-tetraacetyl-
D-glucosyl fluoride was prepared and crystallized from this.
A method in which α-form and β-form are separated using a silica gel column, TLC, etc., and deacetylated using sodium methoxide to prepare (Arch. Biolchem. Biophys .: 142, 3
82-393 (1971)) and a method using an N-glucosyltriazole derivative (Carbhydr. Res .: 327, 5-14 (2000)). Also, as a reagent, Sigma-Aldrich Japan
Sold by Co., Ltd.

The glycoside derivative is a glucosyl acceptor, and the glycoside derivative is not particularly limited. For example, a glycoside having a glycosyl residue on the non-reducing terminal side may be used, but a glycoside having an aglycone such as pNP-glycoside is more preferable. As the glycoside derivative, other than the above, for example, pNP-β-glucoside, pNP-α-xyloside, pNP-α-mannoside,
Mention may be made of pNP-α-maltoside.

The reaction conditions can be as follows, for example. (1) Range of molar ratio of glycoside derivative and β-glucosylfluoride The molar ratio of glycoside derivative and β-glucosylfluoride is not particularly limited, but the molar ratio of β-glucosylfluoride that is a glucosyl donor is not limited. The higher the ratio is, the better, for example, the reaction may be performed at a molar ratio of 1: 3 to 1:10.

(2) Range of modified α-glucosidase amount relative to glycoside derivative or β-glucosylfluoride The addition amount of the modified α-glucosidase relative to the glycoside derivative or β-glucosylfluoride is not particularly limited and is very small. A desired reaction product can be obtained by adjusting the reaction conditions even in the reaction solution containing the modified glucosidase of.

(3) β- and glycoside derivative in the reaction solution
Concentration of glucosylfluoride The concentration of the glycoside derivative and β-glucosylfluoride in the reaction solution is not particularly limited as long as the concentration of the glycoside derivative and β-glucosylfluoride is dissolved.

(4) Reaction temperature and time The reaction temperature is preferably 10 ° C to 60 ° C, more preferably 20 ° C to 30 ° C. The reaction time is not particularly limited. (5) Purification method of product As the purification method of the product, a conventionally known method for purifying sugars such as chromatography such as gel filtration, ion exchange, and adsorption, or a precipitation method using an organic solvent or salts is used. be able to.

[0023]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.

Example 1: Preparation of modified enzyme Method for measuring activity of α-glucosidase The activity of α-glucosidase was measured by the following method. That is, 0.1M acetate buffer (pH 4.5
) Add 200 μl and 100 μl of enzyme solution dissolved in 50 mM acetate buffer (pH 4.5), 0.05% Triton X-100, and add 3
After holding at 5 ° C for 3 minutes, 200 µl of 0.5% maltose was added to start the reaction. After reacting for a certain period of time, 1 ml of 2M Tris-HCl buffer (pH 7.0) was added to stop the reaction. Glucose released from the substrate maltose glucostat (Glucostat) reagent (glucose AR-II color developing agent, manufactured by Wako Pure Chemical Industries, Ltd.) After the color was developed, the glucose content was measured by measuring the absorption at a wavelength of 505 nm. It was quantified. In addition,
Enzyme activity that hydrolyzes 1 μmol of substrate per minute is defined as 1 unit of enzyme.

Preparation of SPG (G2444C) SPG (G2444C) was prepared according to the scheme shown in FIG.
Mutation was introduced when constructing a mutant enzyme expression vector
Since the operation of returning the DNA fragment to the vector is complicated, the dizosaccharomyces pombe (Schizosaccharo
A mutation was introduced at the BamHI recognition site by the megaprimer PCR method by converting the 2444th base of the α-glucosidase cDNA derived from (myces pombe) by a silent mutation (469Ser TCG (TCC) guanine to cytosine by a silent mutation that does not cause amino acid substitution. In other words, as a first PCR, the number of Diazosaccharomyces pombe (Schizosaccharomyces pombe)
Plasmid 10n obtained by subcloning the PstI (2546 nt) DNA fragment from the cDNA EcoRV (1224 nt) into Bluescript II KS (Stratagene).
1 unit of polymerase using g as a template (TOYOBO, KOD DN
A polymerase), Bam (5'-ct ttt gga
20 pmol each of tcg aat ggt act gt, sense primer) and M13 reverse universal primer
Using the buffer provided with the polymerase,
Reaction at 98 ℃, 10 seconds −53 ℃, 2 seconds −74 ℃, 30 seconds
Twenty cycles were performed. QIA Quick PCR for PCR products
Purification kit (QIAquick PCR purification kit)
(QIAGEN)) and used as a mega primer.

In the second PCR, 1 unit of polymerase (TOYOBO, KOD DNA polymerase) is used, and EcoRV to Ps is used as a template.
Bluescript II KS of tI DNA fragment
10 ng of the subcloned (Stratagene), 9 μl of the fast PCR reaction solution as a mega primer, and the buffer solution attached to the polymerase
Five cycles of reaction at 94 ° C for 1 minute and -74 ° C for 2 minutes were performed.
As an antisense primer at 74 ℃,
M13? Add 20 pmol of 20 universal primer and add 94
Reaction at -55 ° C for 30 seconds, -74 ° C for 30 seconds, 1 minute
0 cycles were performed. As a result, introduction of mutation of the obtained amplified fragment, that is, introduction of BamHI site was confirmed by sequence. BglHI (1415
nt) and PstI for Zizosaccharomyces pombe (Schizosacchar
The cDNA of α-glucosidase derived from omyces pombe) was reverted to SPG (G2444C). It was confirmed by the maltase activity of the crude extract of the expression system in Schizosaccharomyces pombe that the silent mutation had no effect on the expression (SPG / pYES transformant 2.6 U /
mg, SPG (G2444C) / pYES transformant 2.5 U / mg).

Preparation of D481G mutant enzyme The D481G mutant enzyme is a part of the DNA fragment of SPG (G2444C) (Ba
mHI, 2439 nt? EcoRI, 3315 nt) Vector Bluescript II SK (Stratagene (STRATAG
ENE)) BamHI and plasmid BamHI linked to EcoRI site
? It was prepared by the megaprimer method using EcoRI / SK as a template. That is, the plasmid BamHI is used as the first PCR.
? EcoRI / SK 10 ng as a template, 1 unit of polymerase (Toyobo, KOD DNA polymerase), sense primer M13 reverse universal primer (g gaa aca gct
atg acc atg) 20 pmol, antisense primer D481G
(5'-cgc aga acg agc tcg gtt cgt tca ttc cag tcc a
aat) 20 pmol of the buffer attached to the polymerase was used to perform 20 cycles of reaction at 98 ° C for 10 seconds at -55 ° C for 2 seconds at -74 ° C for 30 seconds. In addition, primer D481G
Is SEQ ID NO: 1 (cDNA nucleotide sequence) so that the 481st aspartic acid of SEQ ID NO: 2 (primary sequence) is converted to glycine.
Was designed to convert the adenine at position 2479 into guanine. The 173 bp amplified fragment obtained was Q
IA Quick PCR Purification Kit (QIAquick PCR purifica
purification kit (QIAGEN). The purified amplified fragment matches the positional relationship of the primer expected from SEQ ID NO: 1 (cDNA nucleotide sequence), and the nucleotide sequence converts the aspartic acid at position 481 of SEQ ID NO: 2 (primary sequence) into glycine. Since it was converted, it was used as a mega primer.

In the second PCR, 1 unit of polymerase (manufactured by Toyobo, KOD DNA polymerase) and BamHI?
10 ng of EcoRI / SK, Fast P as mega primer
Using the CR reaction solution (9 µl) and the buffer solution attached to the polymerase, 94 ° C, 1 minute-74 ° C, 2 minutes reaction was performed for 5 cycles. At 74 ° C, primer M13? 20 universal primer (gta aaa cga
cgg cca gt) 20 pmol, 94 ℃, 30 seconds at −55 ℃,
Twenty cycles of 1 minute reaction at −74 ° C. for 30 seconds were performed.
As a result, the obtained amplified fragment of 1084 bp was SEQ ID NO: 1 (c
DNA sequence) and the nucleotide sequence was confirmed to convert the aspartic acid at position 481 of SEQ ID NO: 2 (primary sequence) into glycine. It was identified as a cDNA fragment of α-glucosidase derived from Saccharomyces pombe. This cDNA fragment and the vector pPICZA (manufactured by Invitrogen) were placed downstream of the AOX1 promoter in the wild type Dizosaccharomyces pombe (Schiz
cDN of α-glucosidase from osaccharomyces pombe)
BamH of cDNA part of plasmid SPG / pPICZ with A ligation
By replacing the EcoRI part from I, a D481G mutant enzyme expression vector SPG (D481G) / pPICZ was obtained.

Culture of transformant and induction of enzyme Expression vector pPICZA (manufactured by Invitrogen)
Of the wild-type α-glucosidase downstream of the AOX1 promoter of
Plasmid SPG / pPICZ ligated with cDNA fragment and mutant α
-A plasmid SPG ligated with a glucosidase cDNA fragment
(D481G) / pPICZ to Pichia pastori (Pichia pastori
s) was introduced into GS115 strain by electroporation, and zeocin (zeoc
in) Resistance as a selection marker Transformants wt SP
G / PP and d481g SPG / PP were obtained.

The inoculum of each transformant was 2 ml of B
MGY medium [1% Bacto yeast extract]
(Difco), 2% Bacto pepton
e) (Difco), 100 mM calcium phosphate buffer
pH6.0, 1.34% Yeast nitrogen ba
se) (Difco), 0.00004% biotin, 1% glycerol], and cultured by shaking at 30 ° C. for 20 hours.
After inoculating 800 μl of this inoculum into 800 ml of BMGY medium,
After culturing with shaking for 24 hours, when the cell turbidity (600 nm) reached 8.8, the cells were collected by centrifugation at 25 ° C for 1,500 xg for 5 minutes, and a part of them was collected in 1 L of BMMY induction medium [1 % Bacto yeast extract, 2% polypeptone (pol
ypeptone), 100 mM calcium phosphate buffer pH 6.0, 1.3
4% yeast nitrogen base, 0.0000
4% biotin, 1% methanol] to induce the recombinant enzyme. The cell turbidity (600 nm) immediately after transplanting to the BMMY induction medium was 3.5. Final concentration 0.5 every 24 hours
Methanol was added to the medium so that the concentration became 100%, and induction culture was performed for 102 hours. Then, the culture supernatant was recovered by centrifugation at 4,500 xg for 5 minutes at 4 ° C. Cell turbidity at this time (600
nm) was 15.8. The activity of the transformant wt SPG / PP culture supernatant was 900 U in total activity and 11.8 U / mg in specific activity, whereas the activity was found in the culture supernatant of transformant d481g SPG / PP. I couldn't do it. The protein was quantified by the Bradford method.

Method for Purifying Enzyme 925 ml of the culture supernatant of transformants wt SPG / PP and d481 g SPG / PP was previously cooled to −20 ° C.
Ethanol precipitation was carried out by gradually adding ml of ethanol while stirring. The resulting precipitate was recovered by centrifugation at 15,000 xg for 30 minutes at 4 ° C, added with about 100 ml of lysis buffer (500 mM acetate buffer (pH 4.5)), and stirred overnight to allow precipitation. Was dissolved. After dissolution, the supernatant was recovered by centrifugation at 15,000 xg for 30 minutes at 4 ° C. About 30 ml of the lysis buffer was added again to the remaining precipitate, and the mixture was stirred and centrifuged in the same manner to collect the supernatant. Each of the obtained two ethanol precipitate dissolution solutions was dialyzed against a 20 mM acetate buffer solution (pH 4.5). 560 units of activity were recovered in the recovered lysate of the wild-type enzyme, and the specific activity was 53.8 units / mg. Protein quantification was performed by the Bradford method.
The activity of the mutant enzyme was not confirmed, but SDS
-It was confirmed by PAGE that the expected protein of 180 kDa was present, and the protein amount was about 12 mg. These were used as crude enzyme solutions.

The obtained wild-type crude enzyme solution was equilibrated with 20 mM acetate buffer (pH 4.5), and DEAE-Sepharose (Sepharose
ose) CL-6B column (φ 1.8 x 30 cm, 76 ml) for adsorption. Anion exchange column chromatography was performed by elution with a linear gradient of NaCl from 0 to 1 M. Collect 21 ml of the active fraction and concentrate to 2 ml.
It was dialyzed against 50 mM acetate buffer (pH 4.5) containing 0 mM NaCl. Next, 50 mM acetate buffer containing 50 mM NaCl (pH
4.5) equilibrated with Sepharose 6B column (φ 1.6 x 102 c
m, 204 ml) was provided with the concentrated and dialyzed enzyme solution and subjected to gel filtration column chromatography. The obtained 20 ml of active fraction was collected and dialyzed against 20 mM acetate buffer (pH 4.5). The dialyzed enzyme solution was added to 20 mM acetate buffer (pH
4.5) DEAE-Sepharose CL-6 equilibrated with
Adsorb to B column (φ 1.4 x 9 cm, 14 ml), 0 to 0.6
Anion exchange column chromatography was performed again by performing elution with a linear concentration gradient of M NaCl. At this time, the protein elution pattern was obtained as two consecutive peaks. The active fraction is contained in the first peak,
It was confirmed by SDS-PAGE that the peak in the latter half contained a contaminant protein. To remove contaminating proteins derived from the latter half of the peak, 3 ml was collected from the top of the active fraction using the specific activity as an index. This purified enzyme fraction recovered 204 units of activity,
The specific activity was 104 U / mg, and the recovery rate was 23%. The mutant crude enzyme solution was purified by the same column chromatography as the wild-type enzyme, and 3 mg was recovered. However, since there was no enzyme activity, purification was performed by SDS-PAGE. In the purification of any enzyme, protein was quantified by the UV method unless otherwise specified.

Example 2: Preparation of oligosaccharides 0.1 ml of 1M acetate buffer (pH 4.5), 260μ
M modified enzyme prepared in Example 1 of M 0.5 ml, 10 mM
1 ml of pNP-α-glucoside, 55 mM β-glucosyl fluoride (manufactured by Sigma-Aldrich Japan Co., Ltd.)
0.9 ml was mixed and reacted at 30 ° C. for 2 to 20 hours. The result of analyzing this sample by thin layer chromatography is shown in FIG. Silica gel 60 (manufactured by Merck) is used as the thin layer plate, β-glucosyl fluoride using glucose and maltooligosaccharides, pNP-glucoside and pNP-maltoside as standard samples, and β-glucosyl fluoride as the glucosyl donor, the above reaction system. The reaction solution was affixed together with the modified enzyme except that 1-butanol: 2-propanol: water = 10: 5:
After developing twice with a developing solvent of 4 (v / v / v), color was developed using an anisaldehyde reagent. As a result, a spot of the reaction product was observed in the reaction solution at a position almost similar to that of pNP-maltoside. Therefore, this reaction solution is OD
HPL using S column (YMC-Pack ODS-AP AP-303)
Analysis was performed at C. The mobile phase was 9% acetonitrile, and the absorption at 303 nm was measured by using a UV detector at a column temperature of 50 ° C. and a flow rate of 1 ml / min. The results are shown in Fig. 2. Three peaks were observed in the reaction solution, and the peaks were designated as peaks 1 to 3 in the order of elution.

Analysis of oligosaccharides Peaks 1 to 3 observed in the above HPLC analysis were collected and analyzed by thin layer chromatography.
It was shown to. Thin layer plate is silica gel 60 (Merck)
And glucose and maltooligosaccharides, pNP-glucoside, pNP-maltoside and pNP-maltotrioside as standard samples, peaks 1 to 3 were attached, and 1-butanol: 2-propanol: water = 10: 5: After developing twice with a developing solvent of 4 (v / v / v), color was developed using an anisaldehyde reagent. As a result, peak 3 was pNP- used as a glucosyl acceptor.
Glucoside and peak 2 showed almost the same mobility as pNP-maltoside. No peak 1 showed a mobility consistent with the standard sample.

0.1% TF was added to the samples of peaks 1 to 3.
A was added and treated at 100 ° C. for 3 hours, and the partially hydrolyzed sample was analyzed by thin layer chromatography in the same manner as above, and the results are shown in FIG. Silica gel 60 (manufactured by Merck) is used as the thin layer plate, and glucose and maltooligosaccharide, isomaltose, p are used as standard samples.
NP-glucoside, pNP-maltoside and pNP-
As a sample, maltotrioside was partially hydrolyzed from peaks 1 to 3, and 2-propanol: 1-
After addition with a developing solvent of butanol: water = 12: 3: 4 (v / v / v), color was developed using an anisaldehyde reagent. From the results, peak 1 was identified as pNP-isomaltoside, peak 2 was identified as pNP-maltoside, and peak 3 was identified as unreacted pNP-glucoside. The ratio of the ultraviolet absorption of peaks 1 to 3 and the molecular weight was determined from the above structural analysis, and the amount of oligosaccharide product produced was determined from the HPLC analysis of Example 2. As a result, the amount produced was 70%.

FIG. 1 shows an analysis of the reaction solution by thin layer chromatography. 1 is a standard sample of glucose and maltooligosaccharide, 2 is a reaction solution, and 3 is a solution obtained by removing the modifying enzyme from the reaction system. 4 is the β-glucosyl fluoride used as the glucosyl donor, and 5 is the analysis by attaching a standard sample of pNP-glucoside and pNP-maltoside. Figure 2 shows the HPL using an ODS column.
The chromatogram when C analysis was performed is shown. Figure 3
Is a thin layer chromatography analysis of peaks 1 to 3, in which 1 is a standard sample of maltooligosaccharide containing glucose, 2 is peak 1, 3 is peak 2, 4 is peak 3 and 5 is pNP. -A standard sample of glucoside, pNP-maltoside and pNP-maltotrioside was attached and analyzed. FIG. 4 shows an analysis of thin-layer chromatography of partially hydrolyzed peaks 1 to 3, wherein 1 is a standard sample of maltooligosaccharide for glucose, 2 is isomaltose, and 3 is partial hydrolysis of peak 1. Decomposition products, 4 is a partial hydrolysis product of peak 2, 5 is a partial hydrolysis product of peak 3, 6 is a partial hydrolysis product of pNP-glucoside, 7 is a partial hydrolysis product of pNP-maltoside, 8 is a partial hydrolyzate of pNP-maltotrioside, 9 is pNP-glucoside, pNP-maltoside p
This is an analysis in which a standard sample of NP-maltotrioside was attached.

[0037]

As described above, it was revealed that oligosaccharides can be synthesized by using β-glucosyl fluoride as a glucosyl donor using the modifying enzyme of the present invention, and this modifying enzyme shows hydrolytic activity. Because there is no
The synthesized oligosaccharide is accumulated in the reaction system without being hydrolyzed. The synthesis of oligosaccharides and glycosides by such a reaction has high industrial utility value.

[Sequence list]                         SEQUENCE LISTING <110> Nihon Shokuhin Kako Co., Ltd. <120> Modified α-glucosidase and a process for preparation oligo-sacch aride <130> A15089H <160> 5 <210> 1 <211> 4176 <212> DNA <213> Schizosaccharomyces pombe <400> 1 ataagtcaag tgaatacctc gcgccttttt ggttctaagt tttcatcaac ttttttgatc 60 cgaaatcttc cagtgtctgt actctatcgg taacacagtt gggtataact caatagtttt 120 catactgttt tctcctgtga ttacaccact ccctttatta aaaccaagca atatctgttc 180 cactagaaca gaagtgctct caaaccttcc acacctttaa tttattttgt taagttttga 240 ttctttgcta cttgtttttt tgtcctcccc ccacatccgc cttagtaagt ctccccacat 300 tgcaaaagaa acgagtcgat tgattccatc cccacgagaa aatcgatcca ttttccaacc 360 tatcgcccta ctttttttac aaaaaagtta ctaaacgttg ggtttgatta tctttttttt 420 tgcttttaga ttgattcgat tcgacttggc tttcaaaggc tttacccatc aaatcaatta 480 atcaaggctt ttcgcttcct cctcgttctt tgcaccttca agcgaacata cacttcgttt 540 tgcaaaaagt gctaagtaac acaagctaca aaagtttctc ccccactttt ataaggttcg 600 cttagccgct cttaattacg tcgttttgga cttgattggt taatcctacc tcttaccttt 660 ttttgggttt cgctcctttt taaaacgcgc gtttaaacaa accctttcaa gcttttcgtg 720 aaagagcttt tctttcctaa cttactatat atatacatac acacacacat atatatatat 780 ataacttttt atttatattg aaaaaaaaaa cttggacaag aaagtctaca attctattgt 840 tgtagttctt gcttaatttt ttgtctgtct cttaaaaccc tccttttctt tacgatttcg 900 cctctttaaa ccgcccttct attgccaaat tgcctatttg gttaatcttt tccattcttt 960 ttctctacga ttctttttag ttggtacttt gatttttctt aaaacttttt gatctctttt 1020 ttgttttttt tttaacgatg atgatttcta ctgcctacca atctctattt ttaactgctc 1080 tgttttcagc aatctcgatt gctgtcggta acgtctacca aactttaaat gtcattggtg 1140 atcgcaatgt cactatccct accaatggta tccctcaacg cttatccgta tatgacccat 1200 atcgcggtgt aaattgtcaa ggatatcaag ctgttaacat atctgagtca caaaatggcg 1260 ttactgccta tctcgcacta ctcggcgagc cttgctatgc ctatggtact gattacccat 1320 tgttgttcct caacgtcaca tatgaggaag ccgaccgagt tcatatatca atcaaagacg 1380 ctaataacac tcaattccaa tttaccagta ggaaagatct ttgggatgct cccttatatt 1440 caccttctta caataacaca aaccttctgt acaacttttc gtacaatgcc aatcctttcg 1500 aattttgggt tacacgtaag agcgatggtg aagttttatt tgatacacgc ggacagaaat 1560 tggttttcga agatcagtat attgagttaa ctactaatat ggttgaaaat tataatcttt 1620 atggtctcgc tgaaaccatc catggtttac gtctgggaaa taacttaacc cgtacctttt 1680 gggctaatga tgaagctagc cccgtggacc aaaacatgta cggaagtcat ccatactatt 1740 tagaacaaag atacaaggcg gatggtataa attcaacttt gaacgaaacc acttatactt 1800 cttcttctca tggtgttctt atgcttacag ctaatggaat ggatgttctt ttgcgccaag 1860 attatcttca gtatcgaatg atcggtggtg ttatcgacct ttttgtatac agtggtagca 1920 ctgagagtcc caaggagact gtcaagcaat tcgttcaatc cattggaaag cctgctatgc 1980 atcaatattg gacattgggt taccactcat gtcgttgggg ttacacaaat atcacagaaa 2040 tcatggacgt tcgtcaaaat tacattgatg cagacattcc agtggaaacc ttttggtctg 2100 atattgatta catggagaaa tatagagatt ttaccgttga ccctgtttct tattcaaagt 2160 cagatatgca aacatttttc agtgatttgg taagcaatca tcagcattac gttccaatca 2220 ttgatgctgc gatttatgcc gcaaacccct acaatcacac tgacgactct tattatccat 2280 actatgcagg cgttgaaaag gacattttct taaaaaatcc taatggaagt atctacattg 2340 gtgcggtttg gccaggattc actgctttcc ctgatttcac caatcccgat gtggttgact 2400 attggaaaga ctgtcttatc aaccttactt atgcttttgg atcgaatggt actgttccat 2460 tcagtggaat ttggactgat atgaacgaac cctcttcgtt ctgcgtgggc tcttgtggga 2520 gtgctatgat tgacttaaac cctgcagagc ccttggtcgg aatttcaaag cagtattcca 2580 tcccagaagg atttaacgtt tccaatgtga ctgagtatag ttctgcttac agtgcttcac 2640 ttagcaacta ctatgccact gcaacatcat cggtgttcca aattgtttca ccaactgcta 2700 ctccattagg tttgaagcca gattacaaca ttaactggcc cccttatgct attaacaatg 2760 aacaaggaaa tcatgatatt gccaatcaca ttgtaagccc caatgcgacc actcatgatg 2820 gaacccaacg ttacgatatt ttcaacatgt atggttatgg tgagacaaag gtctcatacg 2880 cagctctaac ccaaatttct cctaatgaac gaccctttat cttgagtcgt tctaccttct 2940 tgggatctgg agtctatggt gcacattggt tgggtgataa tcattctcta tggtctaaca 3000 tgttcttctc catttctgga atgatcgttt ttaacatgat gggtattcca atggtaggag 3060 ctgatgtttg tggtttcctt ggtgattcag atgaagaact ttgctctcgt tggatggcta 3120 tgggtgcttt ttcgccattc tatagaaatc ataacaacat ttaccaaatc tcacaagagc 3180 cctacacatg gtcttctgtt gctgaggcct cacgtcgtgc tatgtacatt cgttattctt 3240 tactccctta ctggtatact atcatggcta aggcatccca agatggcaca cctgccctac 3300 gtgctttgtt cgttgaattc cccaacgatc ctactctagc agacgttgac cgtcaattca 3360 tggtaggcga ctctctattg gtgacacctg tcttggagcc taatgttgaa tacgttcaag 3420 gtgttttccc tggtgacaac agcactgtat ggtatgactg gtacaaccac actgaaattg 3480 ttcgtcaata caacgaaaat gttactctgt acgctccttt ggaacacatc aatgtcgcta 3540 ttcgtggtgg tagtgttctt cccatgcaac aaccttcgct cactacttat gaaagtcgtc 3600 aaaatccatt caacctcctc gtcgctttgg atagagatgg ttctgctact ggtgagcttt 3660 accttgatga tggtgtttcc attgagctaa acgctacact ttccgttagc ttcacattta 3720 gtgatggtgt tttgagtgcc gttccaactg gcagctacga agttagccaa cccttggcca 3780 acgtaacgat ccttggtctc actgaatccc ctagttcaat caccttgaat ggacaaaacg 3840 tctcctcctt ccagtactct aacgatactg aggaattgct aattaccggt ctacagaaca 3900 ttacttcctc tggtgcattt gccaacagct ggaatcttac tttgtagtcg ctctcgcttc 3960 acccgatttt tcaccggcaa tccaggaacc gagcactaat taacagtcta gtttgttgca 4020 tctagcaact ctcgagatgc ttgtagtata tggaataaac ttacagtcat tgttagtcaa 4080 ttcaataaat aaactacttt ttctaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4140 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 4176 <210> 2 <211> 969 <212> amino acid <213> Schizosaccharomyces pombe <400> 2    Met Met Ile Ser Thr Ala Tyr Gln Ser Leu Phe Leu Thr Ala Leu Phe     1 5 10 15   Ser Ala Ile Ser Ile Ala Val Gly Asn Val Tyr Gln Thr Leu Asn Val                20 25 30   Ile Gly Asp Arg Asn Val Thr Ile Pro Thr Asn Gly Ile Pro Gln Arg            35 40 45   Leu Ser Val Tyr Asp Pro Tyr Arg Gly Val Asn Cys Gln Gly Tyr Gln        50 55 60   Ala Val Asn Ile Ser Glu Ser Gln Asn Gly Val Thr Ala Tyr Leu Ala    65 70 75 80   Leu Leu Gly Glu Pro Cys Tyr Ala Tyr Gly Thr Asp Tyr Pro Leu Leu                    85 90 95   Phe Leu Asn Val Thr Tyr Glu Glu Ala Asp Arg Val His Ile Ser Ile               100 105 110   Lys Asp Ala Asn Asn Thr Gln Phe Gln Phe Thr Ser Arg Lys Asp Leu           115 120 125   Trp Asp Ala Pro Leu Tyr Ser Pro Ser Tyr Asn Asn Thr Asn Leu Leu       130 135 140   Tyr Asn Phe Ser Tyr Asn Ala Asn Pro Phe Glu Phe Trp Val Thr Arg   145 150 155 160   Lys Ser Asp Gly Glu Val Leu Phe Asp Thr Arg Gly Gln Lys Leu Val                   165 170 175   Phe Glu Asp Gln Tyr Ile Glu Leu Thr Thr Asn Met Val Glu Asn Tyr               180 185 190   Asn Leu Tyr Gly Leu Ala Glu Thr Ile His Gly Leu Arg Leu Gly Asn           195 200 205   Asn Leu Thr Arg Thr Phe Trp Ala Asn Asp Glu Ala Ser Pro Val Asp       210 215 220   Gln Asn Met Tyr Gly Ser His Pro Tyr Tyr Leu Glu Gln Arg Tyr Lys   225 230 235 240   Ala Asp Gly Ile Asn Ser Thr Leu Asn Glu Thr Thr Tyr Thr Ser Ser                   245 250 255   Ser His Gly Val Leu Met Leu Thr Ala Asn Gly Met Asp Val Leu Leu               260 265 270   Arg Gln Asp Tyr Leu Gln Tyr Arg Met Ile Gly Gly Val Ile Asp Leu           275 280 285   Phe Val Tyr Ser Gly Ser Thr Glu Ser Pro Lys Glu Thr Val Lys Gln       290 295 300   Phe Val Gln Ser Ile Gly Lys Pro Ala Met His Gln Tyr Trp Thr Leu   305 310 315 320   Gly Tyr His Ser Cys Arg Trp Gly Tyr Thr Asn Ile Thr Glu Ile Met                   325 330 335   Asp Val Arg Gln Asn Tyr Ile Asp Ala Asp Ile Pro Val Glu Thr Phe               340 345 350   Trp Ser Asp Ile Asp Tyr Met Glu Lys Tyr Arg Asp Phe Thr Val Asp           355 360 365   Pro Val Ser Tyr Ser Lys Ser Asp Met Gln Thr Phe Phe Ser Asp Leu       370 375 380   Val Ser Asn His Gln His Tyr Val Pro Ile Ile Asp Ala Ala Ile Tyr   385 390 395 400   Ala Ala Asn Pro Tyr Asn His Thr Asp Asp Ser Tyr Tyr Pro Tyr Tyr                   405 410 415   Ala Gly Val Glu Lys Asp Ile Phe Leu Lys Asn Pro Asn Gly Ser Ile               420 425 430   Tyr Ile Gly Ala Val Trp Pro Gly Phe Thr Ala Phe Pro Asp Phe Thr           435 440 445   Asn Pro Asp Val Val Asp Tyr Trp Lys Asp Cys Leu Ile Asn Leu Thr       450 455 460   Tyr Ala Phe Gly Ser Asn Gly Thr Val Pro Phe Ser Gly Ile Trp Thr   465 470 475 480   Asp Met Asn Glu Pro Ser Ser Phe Cys Val Gly Ser Cys Gly Ser Ala                   485 490 495   Met Ile Asp Leu Asn Pro Ala Glu Pro Leu Val Gly Ile Ser Lys Gln               500 505 510   Tyr Ser Ile Pro Glu Gly Phe Asn Val Ser Asn Val Thr Glu Tyr Ser           515 520 525   Ser Ala Tyr Ser Ala Ser Leu Ser Asn Tyr Tyr Ala Thr Ala Thr Ser       530 535 540   Ser Val Phe Gln Ile Val Ser Pro Thr Ala Thr Pro Leu Gly Leu Lys   545 550 555 560   Pro Asp Tyr Asn Ile Asn Trp Pro Pro Tyr Ala Ile Asn Asn Glu Gln                   565 570 575   Gly Asn His Asp Ile Ala Asn His Ile Val Ser Pro Asn Ala Thr Thr               580 585 590   His Asp Gly Thr Gln Arg Tyr Asp Ile Phe Asn Met Tyr Gly Tyr Gly           595 600 605   Glu Thr Lys Val Ser Tyr Ala Ala Leu Thr Gln Ile Ser Pro Asn Glu       610 615 620   Arg Pro Phe Ile Leu Ser Arg Ser Thr Phe Leu Gly Ser Gly Val Tyr   625 630 635 640   Gly Ala His Trp Leu Gly Asp Asn His Ser Leu Trp Ser Asn Met Phe                   645 650 655   Phe Ser Ile Ser Gly Met Ile Val Phe Asn Met Met Gly Ile Pro Met               660 665 670   Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asp Ser Asp Glu Glu Leu           675 680 685   Cys Ser Arg Trp Met Ala Met Gly Ala Phe Ser Pro Phe Tyr Arg Asn       690 695 700   His Asn Asn Ile Tyr Gln Ile Ser Gln Glu Pro Tyr Thr Trp Ser Ser   705 710 715 720   Val Ala Glu Ala Ser Arg Arg Ala Met Tyr Ile Arg Tyr Ser Leu Leu                   725 730 735   Pro Tyr Trp Tyr Thr Ile Met Ala Lys Ala Ser Gln Asp Gly Thr Pro               740 745 750   Ala Leu Arg Ala Leu Phe Val Glu Phe Pro Asn Asp Pro Thr Leu Ala           755 760 765   Asp Val Asp Arg Gln Phe Met Val Gly Asp Ser Leu Leu Val Thr Pro       770 775 780   Val Leu Glu Pro Asn Val Glu Tyr Val Gln Gly Val Phe Pro Gly Asp   785 790 795 800   Asn Ser Thr Val Trp Tyr Asp Trp Tyr Asn His Thr Glu Ile Val Arg                   805 810 815   Gln Tyr Asn Glu Asn Val Thr Leu Tyr Ala Pro Leu Glu His Ile Asn               820 825 830   Val Ala Ile Arg Gly Gly Ser Val Leu Pro Met Gln Gln Pro Ser Leu           835 840 845   Thr Thr Tyr Glu Ser Arg Gln Asn Pro Phe Asn Leu Leu Val Ala Leu       850 855 860   Asp Arg Asp Gly Ser Ala Thr Gly Glu Leu Tyr Leu Asp Asp Gly Val   865 870 875 880   Ser Ile Glu Leu Asn Ala Thr Leu Ser Val Ser Phe Thr Phe Ser Asp                   885 890 895   Gly Val Leu Ser Ala Val Pro Thr Gly Ser Tyr Glu Val Ser Gln Pro               900 905 910   Leu Ala Asn Val Thr Ile Leu Gly Leu Thr Glu Ser Pro Ser Ser Ile           915 920 925   Thr Leu Asn Gly Gln Asn Val Ser Ser Phe Gln Tyr Ser Asn Asp Thr       930 935 940   Glu Glu Leu Leu Ile Thr Gly Leu Gln Asn Ile Thr Ser Ser Gly Ala   945 950 955 960   Phe Ala Asn Ser Trp Asn Leu Thr Leu                   965 <210> 3 <211> 22 <212> DNA <213> Artificial <400> 3 cttttggatc gaatggtact gt 22 <210> 4 <211> 19 <212> DNA <213> Artificial <400> 4 ggaaacagct atgaccatg 19 <210> 5 <211> 37 <212> DNA <213> Artificial <400> 5 cgcagaacga gctcggttcg ttcattccag tccaaat 37

[Brief description of drawings]

FIG. 1 shows the results of analysis of a reaction solution by thin layer chromatography.

FIG. 2 shows a chromatogram when HPLC analysis was performed using an ODS column.

FIG. 3 shows the results of analysis of peaks 1 to 3 by thin layer chromatography.

FIG. 4 shows the results of thin layer chromatography analysis of partially hydrolyzed peaks 1 to 3.

FIG. 5 is a scheme for creating SPG (G2444C).

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Haruhide Mori             Kitako 28, East 28, Higashi-ku, Sapporo, Hokkaido             Home 505-25 (72) Inventor Masayuki Okuyama             Hokkaido Kita-ku Kita-ku Kitajo Nishi 4-chome 10 Sawada             Building 303 (72) Inventor Takehiro Unno             703-25 Nakamaru, Fuji City, Shizuoka Prefecture (72) Inventor Ken Yamamoto             2954 Imaizumi, Fuji City, Shizuoka Prefecture (72) Inventor Mikio Yamamoto             110-23 Miyashita, Fuji City, Shizuoka Prefecture F-term (reference) 4B024 AA03 BA80 CA04 DA12 EA04                       FA02 GA11 HA01                 4B050 CC03 CC04 CC07 DD04 LL05                 4B064 AF03 CA06 CA19 CC24 DA10                       DA16

Claims (11)

[Claims] 1. An α-glucosidase having the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, wherein the amino acid sequence shown in SEQ ID NO: 2 has a hydrolysis activity of 481st aspartic acid in the amino acid sequence. Modified α-characterized by substituting with another amino acid so that it is reduced to 1 / 10,000 or less as compared with α-glucosidase having
Glucosidase. 2. The modified α-glucosidase according to claim 1, wherein the 481st aspartic acid is substituted with an amino acid having no carboxyl group in the side chain. 3. The modified α-glucosidase according to claim 2, wherein the amino acid having no carboxyl group in the side chain is glycine or alanine. 4. An α-glucosidase having the amino acid sequence shown in SEQ ID NO: 2 of the Sequence Listing, wherein the 481 th aspartic acid in the amino acid sequence is replaced with glycine. 5. The modified α-glucosidase according to claim 4, which has an amino acid sequence in which one or more amino acids are further substituted, deleted, inserted or added in the amino acid sequence. 6. A Schizosaccha pombe
romyces pombe) -derived α-glucosidase,
Among the active dissociative groups of the α-glucosidase, an amino acid containing a dissociative carboxyl group has an amino acid containing no carboxyl group in its side chain so that the hydrolysis activity is reduced to 1 / 10,000 or less as compared with the wild-type enzyme. A modified α-glucosidase, characterized in that 7. The modification according to claim 6, wherein the amino acid containing a dissociative carboxyl group among the active dissociative groups is an amino acid corresponding to the 481st aspartic acid in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. α-glucosidase. 8. The modified α-glucosidase according to claim 6 or 7, wherein the amino acid having no carboxyl group in the side chain is glycine or alanine. 9. α- according to any one of claims 1 to 8.
A method for producing an oligosaccharide, which comprises reacting a glycoside derivative with β-glucosyl fluoride in the presence of glucosidase. 10. The glycoside derivative is pNP-β-glucoside, pNP-α-xyloside, pNP-α-mannoside, pN.
The method according to claim 9, which is P-α-maltoside. 11. The oligosaccharide is a disaccharide derivative.
Or the manufacturing method according to 10.