JP4012383B2 - Method for producing modified α-glucosidase and oligosaccharide - Google Patents

Description

【図面の簡単な説明】
【図１】反応液を薄層クロマトグラフィーで分析した結果を示す。
【図２】ＯＤＳカラムを用いてＨＰＬＣ分析を行った際の、クロマトグラムを示した。
【図３】ピーク１〜３を薄層クロマトグラフィーで分析した結果を示す。
【図４】ピーク１〜３を部分加水分解したものを薄層クロマトグラフィーで分析した結果を示す。
【図５】SPG (G2444C)の作成スキーム。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an α-glucosidase modified so as not to substantially exhibit hydrolysis activity, and a method for producing an oligosaccharide using the modified α-glucosidase.
[0002]
[Prior art]
α-Glucosidase is an enzyme that exists in nature from microorganisms to animals and plants, and is an enzyme that hydrolyzes the α-glucoside bond at the non-reducing end of a substrate to produce α-D-glucose.
The 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 .: 280, 309-316 ( 1991)). Chiba et al. Classify them into Family 1 and Family 2 by focusing on the substrate specificity of α-glucosidase (Chiba et al., Biosci. Biotech. Biochem .: 61, 1233-1239 (1997)).
[0003]
[Problems to be solved by the invention]
α-Glucosidase catalyzes not only a hydrolysis (hydrolysis) reaction but also a transglycosylation reaction, and is also used for enzyme synthesis of various oligosaccharides and glycosides. However, since the oligosaccharide produced by the transglycosylation reaction is hydrolyzed again as a substrate of α-glucosidase, the resulting oligosaccharide increases with the progress of the transglycosylation reaction, but takes a certain maximum value. It will decrease after a while. Therefore, even if it tried to synthesize | combine a certain oligosaccharide by transglycosylation reaction, about 30 to 50% was a limit. The above phenomenon is observed not only with α-glucosidase but also with various glycosidases including β-glucosidase when synthesizing oligosaccharides using a glycosyl transfer reaction.
[0004]
Under such circumstances, Mackenzie et al., Wang et al., For β-glucosidase produced by Agrobacterium sp. A modified enzyme in which glutamic acid 358 is substituted with alanine has been prepared, and the synthesis of oligosaccharides having β-glucoside bonds using α-glucosyl azide or α-glucosyl fluoride as a glucosyl donor has been reported (Mackenzie et al , J. Am. Chem. Soc .: 116, 11594-11595 (1994), Wang et al., J. Am. Chem. Soc .: 120, 5583-5584 (1998)).
Mayer et al. Prepared a modified enzyme in which glutamic acid 358 was substituted with serine, and synthesized α-glucosyl fluoride and α-galactosyl fluoride as glycosyl donors to synthesize oligosaccharides having β-glycoside bonds. (Mayer et al., FEBS Letters: 466, 40-44 (2000)). Moracci et al. Also showed similar results to Mackenzie et al. And Wang et al. With a β-glycosidase-modified enzyme (glutamate 387 → glycine) derived from Sulfolobus solfataricus and α- Reported using glucosyl azide and α-glucosyl fluoride.
[0005]
However, these enzymes are all enzymes that hydrolyze β-glycoside bonds, and such modified enzymes have not been known for enzymes that hydrolyze α-glycoside bonds. In addition, the glucosyl donors obtained by synthesizing oligosaccharides using the modified enzymes obtained by Mackenzie et al., Wang et al., And Moracci et al. Are of the α-glucoside type. The oligosaccharide produced was limited to oligosaccharides having a β-glucoside bond.
[0006]
Also, US Pat. No. 5,716,812 indicates that the production rate of these oligosaccharides can be 80% or more. 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 a disaccharide derivative, it is necessary to use a glycosyl donor that does not cause re-extension of sugar chains such as α-galactosyl fluoride. However, when a glycosyl donor that does not cause re-elongation is used, the resulting oligosaccharide becomes a hetero-oligosaccharide.
[0007]
Accordingly, an object of the present invention is to provide an enzyme that hydrolyzes an α-glycoside bond, and maintains a transfer reaction activity while reducing or substantially eliminating the hydrolysis (hydrolysis) reaction. There is. Furthermore, this invention is providing the manufacturing method of an oligosaccharide using such a modified enzyme with the high production rate of a disaccharide derivative.
[0008]
[Means for Solving the Problems]
Therefore, the present inventors have intensively studied α-glucosidase, and substituted amino acids containing dissociable carboxyl groups, which are usually essential for hydrolytic reactions, with amino acids containing no carboxyl groups in the side chains, and do not exhibit hydrolytic activity. The inventors have found an α-glucosidase modified in this way, and have found a method for producing an oligosaccharide using the modified α-glucosidase and β-glucosyl fluoride as a glucosyl donor, thereby completing the present invention.
Here, “not exhibiting hydrolytic activity” means that the amount of glucose produced is 1 / 10,000 or less compared to the amount of glucose produced by using the enzyme before modification with maltose, p-phenyl α-glucoside or the like as a substrate. Indicates that there is.
[0009]
  The present invention that achieves the object of the present invention is as follows.
[Claims1] Shown in SEQ ID NO: 2 in the sequence listingTaThe aspartic acid at position 481 in the amino acid sequence was replaced with glycine.Consists of amino acid sequenceα-Glucosidase.
[Claims2]The aspartic acid at position 481 in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing was substituted with glycine, and in addition to the above substitution,1 orseveralAmino acid sequence in which amino acids are substituted, deleted, inserted or addedCompared with α-glucosidase consisting of the amino acid sequence shown in SEQ ID NO: 2, the transfer reaction activity was maintained, and the hydrolysis activity was reduced to 1 / 10,000 or less.α-Glucosidase.
[Claims3Claim1 or 2A method for producing an oligosaccharide, comprising reacting a glycoside derivative with β-glucosyl fluoride in the presence of α-glucosidase described in 1.
[Claims4The glycoside derivative is pNP-β-glucoside, pNP-α-xyloside, pNP-α-mannoside, pNP-α-maltoside.3The manufacturing method as described in.
[Claims5The oligosaccharide is a disaccharide derivative.3 or 4The manufacturing method as described in.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
  Modified α-glucosidase of the present inventionIs an α-glucosidase having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, wherein 481st aspartic acid in the amino acid sequence isTo glycineIt is characterized by being replaced. The α-glucosidase having the amino acid sequence shown in SEQ ID NO: 2 in which the amino acid has not been modified is α-glucosidase derived from Schizosaccharomyces pombe. Then, the 481st aspartic acid in the amino acid sequence shown in SEQ ID NO: 2 is substituted with another amino acid. Amino acid substitution isMaintains the transfer activity,The hydrolysis activity is performed so as to be reduced to 1 / 10,000 or less as compared with α-glucosidase having the amino acid sequence shown in SEQ ID NO: 2. Specifically, the 481st aspartic acid is substituted with an amino acid containing no carboxyl group in the side chain. And amino acids that do not contain a carboxyl group in the side chain are:Glycine.
[0011]
The α-glucosidase of the present invention is an α-glucosidase having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, wherein the aspartic acid at position 481 in the amino acid sequence is substituted with glycine.
[0012]
  Modified α-glucosidase of the present inventionIs an α-glucosidase derived from Schizosaccharomyces pombe, and an amino acid containing a dissociated carboxyl group among the active dissociating groups of the α-glucosidase,Maintains the transfer activity,It is characterized by substitution with an amino acid not containing a carboxyl group in the side chain so that the hydrolysis activity is reduced to 1 / 10,000 or less as compared with the wild-type enzyme.
[0013]
  Modified α-glucosidase of the present inventionThen, the basic α-glucosidase is not limited to the α-glucosidase having the amino acid sequence shown in SEQ ID NO: 2, and may be α-glucosidase derived from Schizosaccharomyces pombe. And the amino acid containing a dissociation-type carboxyl group among the active dissociation groups of (alpha) -glucosidase derived from Schizosaccharomyces pombe is substituted by the other amino acid which does not contain a carboxyl group in a 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. Since the α-glucosidase having the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing is also an α-glucosidase derived from Schizosaccharomyces pombe, the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing is used. Α-glucosidase derived from Schizosaccharomyces pombe other than α-glucosidase having high homology with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing is shown in SEQ ID NO: 2 in the sequence listing. The amino acid corresponding to the 481st aspartic acid in the amino acid sequence can be easily found.
[0014]
  Amino acid substitution isMaintains the transfer activity,The hydrolysis activity is reduced to 1 / 10,000 or less as compared with α-glucosidase derived from wild-type enzyme, ie, wild-type Schizosaccharomyces pombe. In addition, amino acids that do not contain a carboxyl group in the side chain areGlycine.
[0015]
The method for preparing 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 an α-glucosidase gene derived from Schizosaccharomyces pombe points to an amino acid residue having a target dissociable carboxyl group It can be prepared by substituting with other amino acids by mutation. The modified enzymes are prepared by culturing microorganisms and can be used as supernatants or crushed cells, but can be purified by various chromatographic methods as needed. it can.
[0016]
Chiba et al., Agric. Biol. Chem.: Α-glucosidase derived from Schizosaccharomyces pombe has already been reported for its purification method, enzymatic chemistry, and transglycosylation (Chiba et al., Agric. Biol. Chem. 29, 540-547 (1965), Chiba et al., Agric. Biol. Chem .: 30, 536-540 (1966)). The gene has also been obtained, and the active dissociation group has been identified (Journal of Japanese Society for Agricultural Chemistry, Vol. 72, 1998 Annual Meeting Abstracts p.134). Specific examples of the method for obtaining the α-glucosidase-modified enzyme derived from Schizosaccharomyces pombe are shown in Examples described later.
[0017]
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 for the purpose of elucidating the enzyme reaction mechanism. Regarding the preparation method, 2,3,4,6-tetraacetyl-D-glucosyl fluoride is prepared by reacting pentaacetyl D-glucose with anhydrous hydrogen fluoride, from which crystallization, silica gel column, TLC, etc. are prepared. And α-form and β-form are separated and prepared by deacetylation using sodium methoxide (Arch. Biolchem. Biophys .: 142, 382-393 (1971)), N-glucosyltriazole derivatives (Carbhydr. Res .: 327, 5-14 (2000)) and the like. The reagent is sold by Sigma-Aldrich Japan Co., Ltd.
[0018]
The glycoside derivative is a glucosyl receptor, and the glycoside derivative is not particularly limited. For example, any 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.
Examples of the glycoside derivative include pNP-β-glucoside, pNP-α-xyloside, pNP-α-mannoside, and pNP-α-maltoside other than the above.
[0019]
The reaction conditions can be as follows, for example.
(1) Range of molar ratio of glycoside derivative and β-glucosyl fluoride
The molar ratio of the glycoside derivative and β-glucosyl fluoride is not particularly limited, but a higher molar ratio of β-glucosyl fluoride, which is a glucosyl donor, is preferable, for example, 1: 3 to 1:10. The reaction may be performed at a molar ratio such as
[0020]
(2) Range of modified α-glucosidase amount for glycoside derivatives or β-glucosyl fluoride
The amount of the modified α-glucosidase added to the glycoside derivative or β-glucosyl fluoride is not particularly limited, and a desired reaction product can be obtained by adjusting the reaction conditions even in a reaction solution containing a very small amount of the modified glucosidase. Obtainable.
[0021]
(3) Concentration of glycoside derivative and β-glucosyl fluoride in the reaction solution
The concentration of the glycoside derivative and β-glucosyl fluoride in the reaction solution is not particularly limited as long as the glycoside derivative and β-glucosyl fluoride are dissolved.
[0022]
(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) Product purification method
As a purification method of the product, conventionally known carbohydrate purification methods such as chromatography such as gel filtration, ion exchange and adsorption, and precipitation using organic solvents and salts can be used.
[0023]
【Example】
EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.
[0024]
Example 1: Preparation of modified enzyme
α-Glucosidase activity measurement method
The α-glucosidase activity was measured by the following method. That is, 200 μl of 0.1 M acetate buffer (pH 4.5) and 100 μl of enzyme solution dissolved in 50 mM acetate buffer (pH 4.5), 0.05% Triton X-100 were added to an ice-cooled test tube, After 3 minutes, the reaction was initiated by adding 200 μl of 0.5% maltose. 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 was developed with a Glucostat reagent (glucose AR-II color former, manufactured by Wako Pure Chemical Industries, Ltd.), and the amount of glucose was measured by measuring absorption at a wavelength of 505 nm. Quantified. The enzyme activity that hydrolyzes 1 μmol of substrate per minute is defined as 1 unit of enzyme.
[0025]
SPG (G2444C) Create
SPG (G2444C) was prepared according to the scheme shown in FIG.
When constructing a mutant enzyme expression vector, the operation of returning the DNA fragment into which the mutation has been introduced to the vector is complicated, so the 2444th of the α-glucosidase cDNA derived from Schizosaccharomyces pombe shown in SEQ ID NO: 1 A BamHI recognition site was introduced by a megaprimer PCR method by converting the base from a silent mutation (469Ser TCG (TCC) guanine to cytosine) with no amino acid substitution.
That is, as a first PCR, 10 ng of a plasmid obtained by subcloning a DNA fragment of PstI (2546 nt) from EcoRV (1224 nt) of cDNA of Schizosaccharomyces pombe into Bluescript II KS (Stratagene) 1 unit of polymerase (Toyobo, KOD DNA polymerase), 20 pmol of Bam (5'-ct ttt gga tcg aat ggt act gt, sense primer) and M13 reverse universal primer for each primer. 20 cycles of reaction at 98 ° C. for 10 seconds at −53 ° C., for 2 seconds at −74 ° C. for 30 seconds were carried out using the buffer attached. The PCR product was purified using a QIAquick PCR purification kit (QIAGEN) to obtain a megaprimer.
[0026]
Second PCR consists of 1 unit of polymerase (Toyobo, KOD DNA polymerase), 10 ng of EcoRV to PstI DNA fragment subcloned as template into Bluescript II KS (Stratagene) as a megaprimer Five cycles of the reaction at 94 ° C. for 1 minute to 74 ° C. for 2 minutes were performed using 9 μl of the fast PCR reaction solution and the buffer attached to the polymerase. Primer M13 as an antisense primer at 74 ° C? 20 pmol of 20 universal primer was added, and 20 cycles of reaction at 94 ° C. for 30 seconds at −55 ° C. and 30 seconds at −74 ° C. for 1 minute were performed. As a result, introduction of mutation in the obtained amplified fragment, that is, introduction of the BamHI site was confirmed by sequencing. The introduced fragment of the BamHI site was converted back to α-glucosidase cDNA derived from Schizosaccharomyces pombe with BglII (1415 nt) and PstI, and this was named SPG (G2444C). It was confirmed by the maltase activity of the crude extract of the expression system in Schizosaccharomyces pombe that there was no effect on the expression due to silent mutation (SPG / pYES transformant 2.6 U / mg, SPG (G2444C) / pYES transformant 2.5 U / mg).
[0027]
D481G Creation of mutant enzyme
The D481G mutant enzyme links part of the SPG (G2444C) DNA fragment (BamHI, 2439 nt? EcoRI, 3315 nt) to the BamHI and EcoRI sites of the Vector Bluescript II SK (Stratagene). Plasmid BamHI? It was prepared by the megaprimer method using EcoRI / SK as a template.
That is, plasmid BamHI as fast PCR? EcoRI / SK 10ng as a template, polymerase 1 unit (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 aaa t) Using 20 pmol of the buffer attached to the polymerase, 20 cycles of reaction at 98 ° C for 10 seconds at -55 ° C for 2 seconds at -74 ° C for 30 seconds were performed. . Primer D481G is designed to be antisense so that the 481st aspartic acid of SEQ ID NO: 2 (primary sequence) is converted to glycine and the 2479th adenine of SEQ ID NO: 1 (cDNA base sequence) is converted to guanine. did. The obtained 173 bp amplified fragment was purified using a QIAquick PCR purification kit (QIAGEN). The purified amplified fragment matches the position of the primer predicted from SEQ ID NO: 1 (cDNA base sequence), and the base sequence converts the 481st aspartic acid of SEQ ID NO: 2 (primary sequence) to glycine. Since it was converted, it was set as a mega primer.
[0028]
Second PCR is 1 unit of polymerase (Toyobo, KOD DNA polymerase), BamHI as a template? EcoRI / SK (10 ng), 9 μl of the first PCR reaction solution as a mega primer, and 5 cycles of reaction at 94 ° C. for 1 minute to −74 ° C. for 2 minutes using the buffer attached to the polymerase. Primer M13 as an antisense primer at 74 ° C? 20 pmol of 20 universal primer (gta aaa cga cgg cca gt) was added, and 20 cycles of reaction at 94 ° C. for 30 seconds at −55 ° C. and 30 seconds at −74 ° C. for 1 minute were performed. As a result, the obtained amplified fragment of 1084 bp coincided with the positional relationship of the primer predicted from SEQ ID NO: 1 (cDNA base sequence), and the base sequence was the 481st aspartic acid of SEQ ID NO: 2 (primary sequence). Was identified as a cDNA fragment of α-glucosidase derived from a mutant form of Schizosaccharomyces pombe. BamHI of the cDNA portion of plasmid SPG / pPICZ in which this cDNA fragment and the cDNA of α-glucosidase derived from wild-type Schizosaccharomyces pombe are linked downstream of the AOX1 promoter of vector pPICZA (manufactured by Invitrogen) By replacing the EcoRI part, the D481G mutant enzyme expression vector SPG (D481G) / pPICZ was obtained.
[0029]
Transformant culture and enzyme induction
Plasmid SPG / pPICZ in which cDNA fragment of wild type α-glucosidase is ligated downstream of AOX1 promoter of expression vector pPICZA (Invitrogen) and plasmid SPG (D481G) / pPICZ in which cDNA fragment of mutant α-glucosidase is ligated Was introduced into Pichia pastoris strain GS115 by electroporation, and transformants wt SPG / PP and d481g SPG / PP were obtained using zeocin resistance as a selection marker, respectively.
[0030]
Seeds of each transformant were 2 ml of BMGY medium (1% Bacto yeast extract (Difco), 2% Bacto peptone (Difco)), 100 mM. Calcium phosphate buffer pH 6.0, 1.34% Yeast nitrogen base (Difco), 0.00004% biotin, 1% glycerol] was inoculated into yeast and cultured by shaking at 30 ° C. for 20 hours. After inoculating 800 μl of this inoculum into 800 ml of BMGY medium, shaking culture was performed at 30 ° C for 24 hours, and when the turbidity (600 nm) reached 8.8, 1,500 xg at 25 ° C for 5 minutes Bacterial cells were collected by centrifugation, and a portion of 1 L BMMY induction medium [1% Bacto yeast extract, 2% polypeptone, 100 mM calcium phosphate buffer pH 6.0, 1.34% yeast nitrogen It was transferred to a base (yeast nitrogen base, 0.00004% biotin, 1% methanol) to induce the recombinant enzyme. The cell turbidity (600 nm) immediately after replanting in the BMMY induction medium was 3.5. Methanol was added to the medium to a final concentration of 0.5% every 24 hours, and induction culture was performed for 102 hours. Thereafter, the culture supernatant was recovered by centrifugation at 5,500 × g for 5 minutes at 4 ° C. The bacterial turbidity (600 nm) at this time was 15.8. The activity of the culture supernatant of the transformant wt SPG / PP was 900 U with a total activity of 11.8 U / mg, whereas the culture supernatant of the transformant d481g SPG / PP showed activity. I couldn't. The protein was quantified by the Bradford method.
[0031]
Enzyme purification method
To 925 ml of the culture supernatant of the transformants wt SPG / PP and d481g SPG / PP, 1,850 ml of ethanol cooled in advance to −20 ° C. was gradually added with stirring to perform ethanol precipitation. The resulting precipitate was recovered by centrifuging at 15,000 xg for 30 minutes at 4 ° C. Add about 100 ml of lysis buffer (500 mM acetate buffer (pH 4.5)) and stir overnight to precipitate. Was dissolved. After dissolution, the supernatant was recovered by centrifugation at 15,000 × g for 30 minutes at 4 ° C. About 30 ml of lysis buffer was added to the remaining precipitate and stirred again, and centrifuged in the same manner to recover the supernatant. The obtained two ethanol precipitation lysates were each dialyzed against 20 mM acetate buffer (pH 4.5). In the lysate collected from the wild-type enzyme, 560 units of activity were recovered, 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 it was confirmed that the 180 kDa protein predicted by SDS-PAGE was present, and the protein amount was about 12 mg. These were used as crude enzyme solutions.
[0032]
The obtained wild-type crude enzyme solution was subjected to adsorption on a DEAE-Sepharose CL-6B column (φ1.8 × 30 cm, 76 ml) equilibrated with 20 mM acetate buffer (pH 4.5). Anion exchange column chromatography was performed by elution with a 0-1 M NaCl linear gradient. 21 ml of the active fraction was collected, concentrated to 2 ml, and dialyzed against 50 mM acetate buffer (pH 4.5) containing 50 mM NaCl. Next, gel filtration column chromatography was performed using the concentrated and dialyzed enzyme solution on a Sepharose 6B column (φ 1.6 x 102 cm, 204 ml) equilibrated with 50 mM acetate buffer (pH 4.5) containing 50 mM NaCl. It was. The obtained 20 ml active fraction was collected and dialyzed against 20 mM acetate buffer (pH 4.5). The dialyzed enzyme solution is adsorbed on a DEAE-Sepharose CL-6B column (φ 1.4 x 9 cm, 14 ml) equilibrated with 20 mM acetate buffer (pH 4.5), and a linear NaCl concentration of 0 to 0.6 M Anion exchange column chromatography was performed again by elution with a gradient. At this time, the protein elution pattern was obtained as two continuous peaks. It was confirmed by SDS-PAGE that the active fraction was contained in the first peak and that the latter half contained contaminating proteins. In order to remove contaminant proteins derived from the latter half of the peak, 3 ml of the first half from the top of the active fraction was collected using specific activity as an indicator. This purified enzyme fraction recovered 204 units of activity, with a specific activity of 104 U / mg and a recovery rate of 23%.
The mutant crude enzyme solution was purified by column chromatography similar to the wild-type enzyme, and 3 mg was recovered. However, since there was no enzyme activity, the purification index was determined by SDS-PAGE.
For purification of any enzyme, proteins were quantified by the UV method unless otherwise specified.
[0033]
Example 2: Preparation of oligosaccharide
0.1 ml of 1M acetate buffer (pH 4.5), 0.5 ml of the modified enzyme prepared in Example 1 of 260 μM, 1 ml of 10 mM pNP-α-glucoside, 55 mM β-glucosyl fluoride (manufactured by Sigma-Aldrich Japan) ) 0.9 ml was mixed and reacted at 30 ° C. for 2-20 hours.
The results of analyzing this sample by thin layer chromatography are shown in FIG. The thin layer plate uses silica gel 60 (manufactured by Merck), β-glucosyl fluoride using glucose and maltooligosaccharide, pNP-glucoside and pNP-maltoside as standard samples, and further a glucosyl donor, the above reaction system After removing the modified enzyme, the reaction solution was added and developed twice with a developing solvent of 1-butanol: 2-propanol: water = 10: 5: 4 (v / v / v), then anise Color was developed using an aldehyde reagent. As a result, a spot of the reaction product was observed in the reaction solution at substantially the same position as pNP-maltoside. Therefore, this reaction solution was analyzed by HPLC using an ODS column (YMC-Pack ODS-AP AP-303). Absorption at 303 nm was measured using a UV detector at a column temperature of 50 ° C. and a flow rate of 1 ml / min using 9% acetonitrile as the mobile phase. The results are shown in FIG. Three peaks were observed in the reaction solution, and peaks were set to 1 to 3 in the order of elution.
[0034]
Oligosaccharide analysis
The peaks 1 to 3 observed in the above HPLC analysis were collected and analyzed by thin layer chromatography. The results are shown in FIG. The thin layer plate was silica gel 60 (Merck), glucose and maltooligosaccharide, pNP-glucoside, pNP-maltoside and pNP-maltotrioside were attached as standard samples, and peaks 1 to 3 were attached as samples. After developing twice with a developing solvent of: 2-propanol: water = 10: 5: 4 (v / v / v), color was developed using an anisaldehyde reagent. As a result, peak 3 showed the same mobility as pNP-glucoside used as the glucosyl receptor, and peak 2 showed almost the same mobility as pNP-maltoside. None of the peaks 1 showed mobility consistent with the standard sample.
[0035]
The results obtained by adding 0.1% TFA to the samples of peaks 1 to 3 and treating them at 100 ° C. for 3 hours and analyzing the partially hydrolyzed samples by thin layer chromatography in the same manner as above are shown in FIG. Indicated. The thin layer plate uses silica gel 60 (Merck), glucose and maltooligosaccharide, isomaltose, pNP-glucoside, pNP-maltoside and pNP-maltotrioside as standard samples, and peaks 1 to 3 are partially hydrolyzed. Then, the mixture was added with a developing solvent of 2-propanol: 1-butanol: water = 12: 3: 4 (v / v / v), and color was developed using an anisaldehyde reagent. From the results, peak 1 was identified as pNP-isomaltoside, peak 2 as pNP-maltoside, and peak 3 as unreacted pNP-glucoside.
From the above structural analysis, the ratio of the ultraviolet absorption and the molecular weight of peaks 1 to 3 and the HPLC analysis of Example 2 were used to determine the production amount of the oligosaccharide product. As a result, the production amount was 70%.
[0036]
FIG. 1 shows an analysis of a reaction solution by thin layer chromatography. 1 is a standard sample of glucose and maltooligosaccharide, 2 is a reaction solution, 3 is a solution obtained by removing the modified enzyme from the reaction system, 4 is The β-glucosyl fluoride used as a glucosyl donor was analyzed by attaching 5 standard samples of pNP-glucoside and pNP-maltoside.
FIG. 2 shows a chromatogram when HPLC analysis was performed using an ODS column.
In FIG. 3, peaks 1 to 3 are analyzed by thin layer chromatography. 1 is a standard sample of glucose and maltooligosaccharide, 2 is peak 1, 3 is peak 2, 4 is peak 3, Are analyzed by attaching standard samples of pNP-glucoside, pNP-maltoside and pNP-maltotrioside.
FIG. 4 shows a result of partial hydrolysis of peaks 1 to 3 analyzed by thin layer chromatography. 1 is glucose, a standard sample of maltooligosaccharide, 2 is isomaltose, and 3 is partial hydrolysis of peak 1. 4 is a partial hydrolyzate of peak 2, 5 is a partial hydrolyzate of peak 3, 6 is a partial hydrolyzate of pNP-glucoside, 7 is a partial hydrolyzate of pNP-maltoside, 8 is a partial hydrolyzate of pNP-maltotrioside, and 9 is a standard sample of pNP-glucoside and pNP-maltoside pNP-maltotrioside.
[0037]
【The invention's effect】
As described above, it has been clarified that an oligosaccharide can be synthesized using β-glucosyl fluoride as a glucosyl donor using the modified enzyme of the present invention, and this modified enzyme does not exhibit hydrolytic activity. The resulting oligosaccharide is accumulated in the reaction system without being hydrolyzed. Synthesizing oligosaccharides or glycosides by such a reaction has high industrial utility value.
[Sequence Listing]

[Brief description of the 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 analyzing peaks 1 to 3 by thin layer chromatography.
FIG. 4 shows the result of analysis of partially hydrolyzed peaks 1 to 3 by thin layer chromatography.
FIG. 5 is a creation scheme of SPG (G2444C).

Claims (5)

Alpha-glucosidase which consists of an amino acid sequence which substituted the 481st aspartic acid of the amino acid sequence shown by sequence number 2 of the sequence table by glycine. Replacing the sequence 481 th aspartic acid number 2 amino acid sequence shown in the sequence listing glycine, in addition to said substitution, the other one or several amino acids are substituted, deleted, inserted or added Α-glucosidase consisting of an amino acid sequence and having the amino acid sequence shown in SEQ ID NO: 2 maintains the transfer reaction activity, and the hydrolysis activity is reduced to 1 / 10,000 or less. Glucosidase. A method for producing an oligosaccharide, comprising reacting a glycoside derivative with β-glucosyl fluoride in the presence of α-glucosidase according to claim 1. The production method according to claim 3, wherein the glycoside derivative is pNP-β-glucoside, pNP-α-xyloside, pNP-α-mannoside, or pNP-α-maltoside. The production method according to claim 3 or 4, wherein the oligosaccharide is a disaccharide derivative.

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