# Patent

## Title
Modified alpha-glucosidase and applications of same

## Abstract

The object is to provide an α-glucosidase in which a transglycosylation activity predominates, and use thereof, and the like. A modified α-glucosidase consisting of an amino acid sequence in which one or two or more of the amino acid(s) is selected from a group of specific amino acids.

## Background
This application is a divisional application of U.S. application Ser. No. 14/005,076, filed Sep. 13, 2013 which claims the right of priority under 35 U.S.C. §119 based on Japanese Patent Application No. 2011-057386 filed Mar. 16, 2011.


TECHNICAL FIELD
The present invention relates to a modified α-glucosidase. Specifically, the present invention relates to a modified α-glucosidase, a method of designing and a method of preparing the modified α-glucosidase, and use of the modified α-glucosidase. The present application claims priority based on Japanese Patent Application No. 2011-057386 filed on Mar. 16, 2011, and the whole content of the patent application is incorporated herein by reference.
BACKGROUND ART
Glycoside hydrolases are enzymes that cleave ester bonds, and many glycoside hydrolases that concurrently have a transglycosylation activity have also been reported. α-Glucosidase, which can be said to be a typical glycoside hydrolase, generally has a hydrolysis activity (hydrolysis reaction) and a transglycosylation activity (condensation reaction) in combination. α-Glucosidase is widely present in nature, from microorganisms to higher animals and higher plants. α-Glucosidase is the most useful enzyme in industries, and is utilized for the decomposition, synthesis and the like of saccharides.
Due to advances in technology, it has become possible to vary (improve) the characteristics of enzymes. Various attempts for modification have also been made for α-glucosidase (for example, see Patent Documents 1 to 3).
PRIOR ART DOCUMENT
Patent Literature
Patent Document 1: Japanese Unexamined Patent Publication No. 2003-88365
Patent Document 2: Japanese Unexamined Patent Publication No. 2005-253302
Patent Document 3: Japanese Unexamined Patent Publication No. 2009-22204
Patent Document 4: Japanese Unexamined Patent Publication No. 2001-046096
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
Since either the hydrolysis activity or transglycosylation activity is generally used in the case where α-glucosidase is used in industrial use, it is preferable that the other activity that is not used is low. In the case where α-glucosidase is used for the production of an oligosaccharide, transglycosylation activity of the enzyme could be decreased by increasing the hydrolysis activity. Therefore, the object of the present invention is to provide an α-glucosidase in which the transglycosylation activity predominates, which is suitable for the production of oligosaccharides. Furthermore, another object of the present invention is to provide the use thereof, and the like.
Means for Solving the Problem
The present inventors attempted to modify the structure of existing α-glucosidase at the molecular level for the purpose of designing a novel enzyme having an improved oligosaccharide production ability. Specifically, they conducted computer simulation and alignment comparison with four kinds of homologues by utilizing a result of an X-ray crystal structure analysis of α-glucosidase derived from human small intestine (Human Maltase-glucoamylase). As a result, three amino acids that constitute the substrate pockets of the enzyme were identified. Subsequently, amino acids that correspond to the amino acids were specified on the amino acid sequence of Aspergillus niger α-glucosidase, and modification was introduced into the those amino acids. As a result of an investigation on the characteristics of the obtained modified forms, it was confirmed that the characteristics of the enzyme (specifically the balance between the hydrolysis activity and transglycosylation activity) had significantly changed. Among the modified forms, those not showing a hydrolysis activity against α-1,4 glucosidic linkages and α-1,6 glucosidic linkages, those having a remarkably decreased hydrolysis activity against α-1,6 glucosidic linkages, those having a significantly decreased hydrolysis activity against α-1,3 glucosidic linkages and α-1,6 glucosidic linkages, and the like were observed. It is noteworthy that the wild-type enzymes catalyze formation of α-1,6 glucosidic linkages, mainly, whereas the modified forms (type W343M and type W343M/S496T) catalyze formation of α-1,4 glucosidic linkages, mainly. These facts mean that the above-mentioned amino acid positions that were successfully identified are effective as positions to which mutation is to be added, in the case where structural modification is performed for the purpose of obtaining an α-glucosidase that is suitable for the production of an oligosaccharide, and the like (i.e., an α-glucosidase in which the transglycosylation activity predominates). Furthermore, it can be said that the amino acid positions are especially effective as positions for mutation for acquiring an α-glucosidase having a unique characteristic that it shows a transglycosylation activity to form α-1,4 glucosidic linkages. On the other hand, modified α-glucosidases that are successfully obtained themselves are of great value in industry.
The present inventors further advanced their investigation and decided to consider annotated amino acids by a novel procedure. Specifically, first, steric structure models were constructed for Aspergillus niger α-glucosidase and Aspergillus nidulans α-glucosidase by using another computer simulation software. Subsequently, a docking simulation analysis was performed to thereby predict substrate pockets. Furthermore, based on the results, the amino acids to be an active center were searched, and the positions in each steric structure model were specified. Next, the amino acid sequences were compared between Aspergillus niger α-glucosidase and Aspergillus nidulans α-glucosidase, and the amino acids that were mismatched among the amino acids of the substrate pockets were specified as amino acids that are suitable for introducing modification. Subsequently, in order to confirm whether or not these amino acids are suitable for introducing mutation (in other words, whether or not these amino acids are beneficial for structural modification), mutation was actually introduced into two amino acids to give modified forms. As a result of the investigation on the characteristics of the obtained modified forms, a modified form showing a transglycosylation activity to form α-1,6 glucosidic linkages and a modified form showing a transglycosylation activity to form α-1,4 glucosidic linkages were observed. This fact supports that the amino acid positions that were successfully identified are effective for structural modification. On the other hand, the modified α-glucosidases that were successfully obtained themselves are of great value in industry.
On the other hand, as a result of further research, it was confirmed that the modified forms that were successfully obtained are useful for the generation or production of oligosaccharides. It is noteworthy that the modified form showing a transglycosylation activity to form α-1,6 glucosidic linkages was able to form oligosaccharides also from substrates having α1,4 glucosidic linkages by using the modified form in combination with a wild type α-glucosidase.
Meanwhile, it is often experienced that it is highly possible that an additive or synergistic effect is generated by combining effective two amino acid substitutions. Therefore, it can be said that the three mutation positions that were successfully identified are effective for changing the characteristics (especially for improving a transglycosylation activity), not only alone but also for the combinations thereof. Actually, the transglycosylation activity was also improved in the case where amino acid substitutions at two positions were combined (Examples mentioned below).
Furthermore, considering the technical common knowledge that enzymes of the same kind are highly similar in structures (primary structure and steric structure) and that it is highly possible that similar mutation generates a similar effect, it can be said that the mutation technique found by the present inventors can be applied to not only the α-glucosidase derived from Aspergillus niger as shown in the following Examples, but also α-glucosidases derived from other organisms.
The present invention shown below is based on the above-mentioned accomplishment and observation.
[1] A modified α-glucosidase consisting of an amino acid sequence in which one or two or more of the amino acid(s) selected from the group consisting of the following (1) to (14) has/have been substituted by other amino acid in the amino acid sequence of α-glucosidase:
(1) an amino acid corresponding to the amino acid at the position 385 of the amino acid sequence of SEQ ID NO. 1;
(2) an amino acid corresponding to the amino acid at the position 491 of the amino acid sequence of SEQ ID NO. 1;
(3) an amino acid corresponding to the amino acid at the position 535 of the amino acid sequence of SEQ ID NO. 1;
(4) an amino acid corresponding to the amino acid at the position 450 of the amino acid sequence of SEQ ID NO. 1;
(5) an amino acid corresponding to the amino acid at the position 534 of the amino acid sequence of SEQ ID NO. 1;
(6) an amino acid corresponding to the amino acid at the position 537 of the amino acid sequence of SEQ ID NO. 1;
(7) an amino acid corresponding to the amino acid at the position 538 of the amino acid sequence of SEQ ID NO. 1;
(8) an amino acid corresponding to the amino acid at the position 554 of the amino acid sequence of SEQ ID NO. 1;
(9) an amino acid corresponding to the amino acid at the position 556 of the amino acid sequence of SEQ ID NO. 1;
(10) an amino acid corresponding to the amino acid at the position 579 of the amino acid sequence of SEQ ID NO. 2;
(11) an amino acid corresponding to the amino acid at the position 585 of the amino acid sequence of SEQ ID NO. 2;
(12) an amino acid corresponding to the amino acid at the position 630 of the amino acid sequence of SEQ ID NO. 1;
(13) an amino acid corresponding to the amino acid at the position 683 of the amino acid sequence of SEQ ID NO. 1; and
(14) an amino acid corresponding to the amino acid at the position 689 of the amino acid sequence of SEQ ID NO. 1.
[2] The modified α-glucosidase according to [1], wherein the α-glucosidase is Human Maltase-glucoamylase, Aspergillus niger alpha-glucosidase, Human Neutral alpha-glucosidase C, Mouse Lysosomal alpha-glucosidase, Yeast GLU2A, Aspergillus nidulans Alpha-glucosidase AgdA, Aspergillus nidulans Alpha-glucosidase AgdB, Mucor javanicus alpha-glucosidase, Aspergillus oryzae alpha-glucosidase, Mortierella alliacea alpha-glucosidase, Schizosaccharomyces pombe alpha-glucosidase, Debaryomyces occidentalis alpha-glucosidase, Hordeum vulgare subsp. vulgare alpha-glucosidase, Arabidopsis thaliana alpha-glucosidase, Spinacia oleracea alpha-glucosidase, Beta vulgaris alpha-glucosidase or Solanum tuberosum alpha-glucosidase.
[3] The modified α-glucosidase according to [1], wherein the amino acid sequence of the α-glucosidase is the amino acid sequence of SEQ ID NO. 2, and
the amino acid of (1) is the amino acid at the position 343 of the amino acid sequence, the amino acid of (2) is the amino acid at the position 452 of the amino acid sequence, the amino acid of (3) is the amino acid at the position 496 of the amino acid sequence, the amino acid of (4) is the amino acid at the position 410 of the amino acid sequence, the amino acid of (5) is the amino acid at the position 495 of the amino acid sequence, the amino acid of (6) is the amino acid at the position 498 of the amino acid sequence, the amino acid of (7) is the amino acid at the position 499 of the amino acid sequence, the amino acid of (8) is the amino acid at the position 531 of the amino acid sequence, the amino acid of (9) is the amino acid at the position 533 of the amino acid sequence, the amino acid of (12) is the amino acid at the position 662 of the amino acid sequence, the amino acid of (13) is the amino acid at the position 715 of the amino acid sequence, and the amino acid of (14) is the amino acid at the position 721 of the amino acid sequence.
[4] The modified α-glucosidase according to [3], wherein the amino acid to be substituted is the amino acid of (1), and the amino acid after the substitution is cysteine, aspartic acid, methionine, histidine, alanine, phenylalanine, glycine, threonine, glutamic acid, valine, glutamine, asparagine or isoleucine.
[5] The modified α-glucosidase according to [3], wherein the amino acid to be substituted is the amino acid of (2), and the amino acid after the substitution is glycine, aspartic acid or glutamic acid.
[6] The modified α-glucosidase according to [3], wherein the amino acid to be substituted is the amino acid of (3), and the amino acid after the substitution is asparagine, glutamine or valine.
[7] The modified α-glucosidase according to [3], wherein the amino acids to be substituted are the amino acid of (1) and the amino acid of (2), and
the amino acids after the substitution are aspartic acid for the amino acid of (1) and alanine or glycine for the amino acid of (2).
[8] The modified α-glucosidase according to [3], wherein the amino acids to be substituted are the amino acid of (1) and the amino acid of (3), and
the amino acids after the substitution are aspartic acid or methionine for the amino acid of (1) and isoleucine, arginine, cysteine or threonine for the amino acid of (3).
[9] The modified α-glucosidase according to [3], wherein the amino acid to be substituted is the amino acid of (5), and the amino acid after the substitution is glycine, proline or valine.
[10] The modified α-glucosidase according to [3], wherein the amino acid to be substituted is the amino acid of (6), and the amino acid after the substitution is leucine or serine.
[11] The modified α-glucosidase according to [1], which is consisting of any of the amino acid sequences of SEQ ID Nos. 18 to 42 and 77 to 78.
[12] The modified α-glucosidase according to [1], which is consisting of any of the amino acid sequences of SEQ ID Nos. 74 to 76.
[13] A gene coding for the modified α-glucosidase according to any one of [1] to [12].
[14] The gene according to [13], which comprises any of base sequences of SEQ ID Nos. 43 to 67 and 79 to 83.
[15] A recombinant DNA comprising the gene according to [13] or [14].
[16] A microorganism having the recombinant DNA according to [15].
[17] An enzyme agent comprising the modified α-glucosidase according to any one of [1] to [12].
[18] A method for producing an oligosaccharide, comprising reacting the modified α-glucosidase according to any one of [1] to [8], [10] and [11] with an oligosaccharide or polysaccharide of di- or more saccharides having α-1,4 glucosidic linkage.
[19] A method for producing an oligosaccharide, comprising reacting the modified α-glucosidase according to [9] or [12] with an oligosaccharide or polysaccharide of di- or more saccharides having α-1,6 glucosidic linkage.
[20] The method for producing an oligosaccharide according to [18] or [19], comprising using a wild type enzyme in combination.
[21] A method for producing an oligosaccharide, comprising reacting the modified α-glucosidase according to [9] or [12] and a wild type enzyme with an oligosaccharide or polysaccharide of di- or more saccharides having α-1,4 glucosidic linkage(s).
[22] A pharmaceutical composition, a quasi-drug composition, a cosmetic composition, a food composition or a feed composition, comprising the modified α-glucosidase according to any one of [1] to [12] or the enzyme agent according to [17].
[23] A method for designing a modified α-glucosidase, comprising the following steps (i) and (ii):
(i) a step of identifying one or two or more amino acid(s) selected from the group consisting of the following (1) to (14) in the amino acid sequence of α-glucosidase as an enzyme to be mutated:
(1) an amino acid corresponding to the amino acid at the position 385 of the amino acid sequence of SEQ ID NO. 1;
(2) an amino acid corresponding to the amino acid at the position 491 of the amino acid sequence of SEQ ID NO. 1;
(3) an amino acid corresponding to the amino acid at the position 535 of the amino acid sequence of SEQ ID NO. 1;
(4) an amino acid corresponding to the amino acid at the position 450 of the amino acid sequence of SEQ ID NO. 1;
(5) an amino acid corresponding to the amino acid at the position 534 of the amino acid sequence of SEQ ID NO. 1;
(6) an amino acid corresponding to the amino acid at the position 537 of the amino acid sequence of SEQ ID NO. 1;
(7) an amino acid corresponding to the amino acid at the position 538 of the amino acid sequence of SEQ ID NO. 1;
(8) an amino acid corresponding to the amino acid at the position 554 of the amino acid sequence of SEQ ID NO. 1;
(9) an amino acid corresponding to the amino acid at the position 556 of the amino acid sequence of SEQ ID NO. 1;
(10) an amino acid corresponding to the amino acid at the position 579 of the amino acid sequence of SEQ ID NO. 2;
(11) an amino acid corresponding to the amino acid at the position 585 of the amino acid sequence of SEQ ID NO. 2;
(12) an amino acid corresponding to the amino acid at the position 630 of the amino acid sequence of SEQ ID NO. 1;
(13) an amino acid corresponding to the amino acid at the position 683 of the amino acid sequence of SEQ ID NO. 1; and
(14) an amino acid corresponding to the amino acid at the position 689 of the amino acid sequence of SEQ ID NO. 1.
(ii) a step of constructing an amino acid sequence in which the amino acid sequence(s) identified in the step (i) has/have been substituted by other amino acid, based on the amino acid sequence of the enzyme to be mutated.
[24] The designing method according to [23], wherein the α-glucosidase is Human Maltase-glucoamylase, Aspergillus niger alpha-glucosidase, Human Neutral alpha-glucosidase C, Mouse Lysosomal alpha-glucosidase, Yeast GLU2A, Aspergillus nidulans Alpha-glucosidase AgdA, Aspergillus nidulans Alpha-glucosidase AgdB, Mucor javanicus alpha-glucosidase, Aspergillus oryzae alpha-glucosidase, Mortierella alliacea alpha-glucosidase, Schizosaccharomyces pombe alpha-glucosidase, Debaryomyces occidentalis alpha-glucosidase, Hordeum vulgare subsp. vulgare alpha-glucosidase, Arabidopsis thaliana alpha-glucosidase, Spinacia oleracea alpha-glucosidase, Beta vulgaris alpha-glucosidase or Solanum tuberosum alpha-glucosidase.
[25] The designing method according to [23], wherein the α-glucosidase comprises any of the amino acid sequences of SEQ ID Nos. 1 to 17.
[26] The designing method according to [23], wherein the α-glucosidase is consisting of the amino acid sequence of SEQ ID NO. 2, and the amino acid(s) that is/are substituted in the step (i) is/are one amino acid selected from (1) to (14), two amino acids selected from (1) to (14) or three amino acids selected from (1) to (14).
[27] A method for preparing a modified α-glucosidase, comprising the following steps (I) to (III):
(I) a step of preparing a nucleic acid that codes for any of the amino acid sequences of SEQ ID Nos. 18 to 42 and 74 to 78 or an amino acid sequence constituted by the designing method according to any one of [23] to [26];
(II) a step of expressing the nucleic acid, and
(III) a step of collecting an expression product.
[28] An α-glucosidase having a transglycosylation activity to form α-1,4 or α-1,6 glucosidic linkages.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows positions of annotated amino acids in human α-glucosidase (A), comparison of alignments (B), and the positions of the amino acids corresponding to the annotated amino acids in an amino acid sequence of Aspergillus niger (C).
FIG. 2 shows a scheme of a modified TG (transglucosidase) screening system using yeast.
FIG. 3 compares oligosaccharide synthesis activities by HPLC. The oligosaccharide synthesis activities of a WT (wild type) (A) and modified TGs (W343M (B), V452G (C) and S496V (D)) were compared.
FIG. 4 compares synthesis amounts of the oligosaccharides of tri- or more saccharides.
FIG. 5 compares the synthesis amounts of the oligosaccharides of tri- or more saccharides (upper) and compares the synthesis amounts of the oligosaccharides of tetra- or more saccharides (lower). The amounts of the generated oligosaccharides were compared among the modified TGs having different amino acids after substitution (indicated below the graph).
FIG. 6 compares the synthesis amounts of the oligosaccharides of tri- or more saccharides (left) and compares the synthesis amounts of the oligosaccharides of tetra- or more saccharides (right). The amounts of the generated oligosaccharides were compared among the modified TGs having different amino acids after substitution (indicated below the graph).
FIG. 7 compares substrate specificities relating to hydrolysis activities.
FIG. 8 shows the hydrolysis activities of the modified TGs by substitution of two amino acids.
FIG. 9 shows the oligosaccharide synthesis activities of the modified TGs by substitution of two amino acids. The oligosaccharide synthesis activities of the WT (wild type) (A) and modified TGs (W343M (B), W343M/S496T (C) and W343D/V452G (D)) were compared.
FIG. 10 compares the synthesis amounts of the oligosaccharides of tri- or more saccharides (left) and the comparison of the synthesis amounts of the oligosaccharides of tetra- or more saccharides (right). The amounts of the generated oligosaccharides were compared among the modified TGs having different amino acids after substitution (indicated below the graph).
FIG. 11 shows the substrate specificities of the modified TGs by substitution of two amino acids.
FIG. 12 compares the oligosaccharides that are formed when maltopentaose is used as a start substrate.
FIG. 13 compares the oligosaccharides that are formed when a mixture of maltose and isomaltose is used as the start substrate.
FIG. 14 are computer-simulated steric structure models of α-glucosidase. Left: Aspergillus niger TG, right: Aspergillus nidulans TG
FIG. 15 shows the scheme for considering annotated amino acids based on computer simulation.
FIG. 16 compares the oligosaccharide synthesis activities of the modified TGs (S495X). Maltose was used as the substrate. The synthesis amount of the oligosaccharides of tri- or more saccharides (upper) and the synthesis amount of the oligosaccharides of tetra- or more saccharides (lower) were compared.
FIG. 17 compares the oligosaccharide synthesis activities of the modified TGs (S495X). Isomaltose was used as the substrate. The synthesis amount of the oligosaccharides of tri- or more saccharides (upper) and the synthesis amount of the oligosaccharides of tetra- or more saccharides (lower) were compared.
FIG. 18 shows the hydrolysis activities of the modified TGs (S495X).
FIG. 19 compares the oligosaccharide synthesis activities of the modified TGs. Maltopentaose was used as the substrate. (a) A HPLC chart of the WT (wild type). (b) The HPLC chart of the modified TG (S495V). (c) A ratio of the generated oligosaccharides.
FIG. 20 compares the oligosaccharide synthesis activities of the modified TGs (C498X). Maltose was used as the substrate.
FIG. 21 shows the hydrolysis activities of the modified TGs (C498X).
FIG. 22 compares the oligosaccharide-synthesis ability. (a) The case where TG is not added, (b) the case where TG of the WT (wild type) is added, (c) the case where the modified TG (W343M) was added, and (d) comparison of an area surface areas of the tri- or more saccharides.
FIG. 23 is the HPLC chart of the oligosaccharides formed from porridge (at 90 minutes after the initiation of the reaction).
FIG. 24 compares the oligosaccharide synthesis activities. The synthesis amount of the oligosaccharides of tri- or more saccharides (upper) and the synthesis amount of the oligosaccharides of tetra- or more saccharides (lower) were compared in the case where the modified TG (S495P) was used alone and the cases where the wild type was used in combination. Maltose was used as the substrate.
FIG. 25 is the HPLC chart of the oligosaccharides formed from maltose (substrate) (at 48 hours after the initiation of the reaction).
FIG. 26 compares the oligosaccharide-synthesis ability. (a) The case where the WT (wild type) TG is added, (b) the case where the modified TG (S495P) is added, (c) the case where the WT (wild type) and the modified TG (S495P) are used in combination, and (d) comparison of the area surface areas of the tri- or more saccharides.
FIG. 27 is the HPLC chart of the oligosaccharides formed from porridge (at 90 minutes after the initiation of the reaction).



DESCRIPTION OF EMBODIMENTS
For convenience in explanation, a part of the terms used with respect to the present invention will be defined below.
TERMS
α-Glucosidase is also referred to as transglucosidase. In the present specification, the term “α-glucosidase” and the term “transglucosidase” are interchangeably used. The term “modified α-glucosidase” is an enzyme that is obtained by modifying or mutating “a base α-glucosidase” by the means disclosed by the present specification. In the present specification, the term “modified α-glucosidase”, the term “modified α-glucosidase”, the term “modified enzyme” and the term “modified enzyme” are interchangeably used. The base α-glucosidase is typically a wild type α-glucosidase. However, this does not interfere with the application of α-glucosidase that has already undergone an artificial operation to the present invention as the “base α-glucosidase”. The “base α-glucosidase” is also referred to as “α-glucosidase to be mutated” or “enzyme to be mutated” in the present specification. In the present specification, an amino acid on a mutation introduction point is expressed by a combination of a single letter that represents the kind of the amino acid and a number that represents the position of the amino acid. For example, when the tryptophan at the position 343 is a mutation introduction point, the amino acid is expressed as “W343”.
(Modified α-Glucosidase)
The first aspect of the present invention relates to a modified α-glucosidase (modified enzyme). The modified enzyme of the present invention has an amino acid sequence in which one or two or more of the amino acid(s) selected from the group consisting of the following (1) to (14) has/have been substituted by other amino acid(s) in the amino acid sequence of an enzyme to be mutated.
(1) an amino acid corresponding to the amino acid at the position 385 of the amino acid sequence of SEQ ID NO. 1
(2) an amino acid corresponding to the amino acid at the position 491 of the amino acid sequence of SEQ ID NO. 1
(3) an amino acid corresponding to the amino acid at the position 535 of the amino acid sequence of SEQ ID NO. 1
(4) an amino acid corresponding to the amino acid at the position 450 of the amino acid sequence of SEQ ID NO. 1
(5) an amino acid corresponding to the amino acid at the position 534 of the amino acid sequence of SEQ ID NO. 1
(6) an amino acid corresponding to the amino acid at the position 537 of the amino acid sequence of SEQ ID NO. 1
(7) an amino acid corresponding to the amino acid at the position 538 of the amino acid sequence of SEQ ID NO. 1
(8) an amino acid corresponding to the amino acid at the position 554 of the amino acid sequence of SEQ ID NO. 1
(9) an amino acid corresponding to the amino acid at the position 556 of the amino acid sequence of SEQ ID NO. 1
(10) an amino acid corresponding to the amino acid at the position 579 of the amino acid sequence of SEQ ID NO. 2
(11) an amino acid corresponding to the amino acid at the position 585 of the amino acid sequence of SEQ ID NO. 2
(12) an amino acid corresponding to the amino acid at the position 630 of the amino acid sequence of SEQ ID NO. 1
(13) an amino acid corresponding to the amino acid at the position 683 of the amino acid sequence of SEQ ID NO. 1
(14) an amino acid corresponding to the amino acid at the position 689 of the amino acid sequence of SEQ ID NO. 1
As shown in Examples mentioned below, the amino acid at the position 385, the amino acid at the position 491 and the amino acid at the position 535 mentioned above are amino acids that were expected to be important for the functions of enzymes as a result of making full use of the steric structure analysis and prediction of functions of Human Maltase-glucoamylase (SEQ ID NO. 1), as well as techniques such as alignment comparison with homologues, and the like. In one embodiment of the present invention, the structure of the enzyme is modified by mutating amino acids that correspond to these amino acids to thereby change the characteristics of α-glucosidase. The “characteristics” herein are a transglycosylation activity and a hydrolysis activity. In the modified enzyme of the present invention, the balance between the transglycosylation activity and hydrolysis activity has shifted to the direction in which the transglycosylation activity predominates, on the basis of the balance before the modification. Typically, as a result of the modification, improvement of the transglycosylation activity or lowering of the hydrolysis activity, or both generate(s), and thus the enzyme enables effective transglycosylation.
On the other hand, the amino acids shown in the above-mentioned (4) to (14) are amino acids that were expected to be effective for structure modification as a result of making use of the comparison of the steric structures and the comparison of the amino acid sequences in Aspergillus niger alpha-glucosidase and Aspergillus nidulans alpha-glucosidase. In another embodiment of the present invention, the structure of the enzyme is modified by mutating amino acids that correspond to these amino acids to thereby change the characteristics of α-glucosidase.
Herein, the term “corresponding” when used for an amino acid residue in the present specification means contributing equally to exhibition of functions among proteins (enzymes) being compared. For example, when an amino acid sequence for comparison to the base amino acid sequence (that is, the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2) is aligned while considering partial homology of the primary structure (that is, an amino acid sequence) so that the most appropriate comparison can be achieved (in this event, the alignment may be optimized by introducing gaps if necessary), an amino acid located at a position corresponding to a specific amino acid in the base amino acid sequence can be specified as a “corresponding amino acid”. The “corresponding amino acid” can also be specified by comparison between conformations (three-dimensional structures) in place of or in addition to the comparison between primary structures. Utilization of conformational information can give highly credible comparison results. In this case, a technique of performing an alignment with comparing atomic coordinates of conformations of a plurality of enzymes can be adopted. Conformational information of an enzyme to be mutated is available from, for example, the Protein Data Bank.
One example of a method for determination of a protein conformation by the X-ray crystal structure analysis will be shown below.
(1) A protein is crystallized. Crystallization is essential to determine a conformation, and in addition, crystallization is industrially useful as a purification method of a protein at high purity and a stable preservation method of a protein at high density. In this case, it is preferable that a protein to which a substrate or its analogous compound as a ligand is bound is used for crystallization.
(2) The prepared crystal is irradiated with X-ray to collect diffraction data. There are many cases that a protein crystal is damaged due to X-ray irradiation and the diffraction ability is deteriorated. In such cases, a low-temperature measurement technique of rapidly cooling the crystal to about −173° C. and collecting diffraction data in the state has recently prevailed. In addition, ultimately, synchrotron orbit radiation having high luminance is utilized to collect high resolution data that is used for structural determination.
(3) In addition to the diffraction data, phase information is necessary in order to perform the crystal structure analysis. When a crystal structure of an analogous protein to a desired protein is unknown, it is impossible to determine the structure in a molecular substitution method, and a phase problem has to be solved by a heavy-atom isomorphous replacement method. The heavy-atom isomorphous replacement method is a method in which a metallic atom having a high atomic number such as mercury or platinum is introduced into a crystal and contribution of a large X-ray scattering ability of such a metallic atom to X ray diffraction data is utilized to collect phase information. The determined phase is possibly improved by smoothing an electron density of a solvent region in the crystal. Since a water molecule in the solvent region has large fluctuation, the electron density is hardly observed, and thus adjusting the electron density in this region to close to 0 makes it possible to approach the real electron density, which results in improving a phase. When plural molecules are contained in an asymmetrical unit, equation of electron densities of these molecules makes it possible to more significantly improve a phase. A model of a protein is fit to an electron density map calculated using the phase improved as described above. This process is performed on computer graphics using a program such as QUANTA made by MSI Co. (USA). After the process, structure refinement is performed using a program such as X-PLOR made by MSI Co. to complete the structure analysis. When a crystal structure of an analogous protein to a desired protein is known, it can be determined in a molecular substitution method using the atomic coordinate of the known protein. Molecular substitution and structure refinement can be performed using a program such as CNS_SOLVE ver.11.
Examples of the enzyme to be mutated in the present invention are Human Maltase-glucoamylase, Aspergillus niger alpha-glucosidase, Human Neutral alpha-glucosidase C, Mouse Lysosomal alpha-glucosidase, Yeast GLU2A, Aspergillus nidulans Alpha-glucosidase AgdA, Aspergillus nidulans Alpha-glucosidase AgdB, Mucor javanicus alpha-glucosidase, Aspergillus oryzae alpha-glucosidase, Mortierella alliacea alpha-glucosidase, Schizosaccharomyces pombe alpha-glucosidase, Debaryomyces occidentalis alpha-glucosidase, Hordeum vulgare subsp. vulgare alpha-glucosidase, Arabidopsis thaliana alpha-glucosidase, Spinacia oleracea alpha-glucosidase, Beta vulgaris alpha-glucosidase and Solanum tuberosum alpha-glucosidase. The amino acid sequences of these α-glucosidases, which are registered with public database, are shown below.
Human Maltase-glucoamylase: SEQ ID NO. 1
Aspergillus niger alpha-glucosidase: SEQ ID NO. 2
Human Neutral alpha-glucosidase C: SEQ ID NO. 3
Mouse Lysosomal alpha-glucosidase: SEQ ID NO. 4
Yeast GLU2A: SEQ ID NO. 5
Aspergillus nidulans Alpha-glucosidase AgdA: SEQ ID NO. 6
Aspergillus nidulans Alpha-glucosidase AgdB: SEQ ID NO. 7
Mucor javanicus alpha-glucosidase: SEQ ID NO. 8
Aspergillus oryzae alpha-glucosidase: SEQ ID NO. 9
Mortierella alliacea alpha-glucosidase: SEQ ID NO. 10
Schizosaccharomyces pombe alpha-glucosidase: SEQ ID NO. 11
Debaryomyces occidentalis alpha-glucosidase: SEQ ID NO. 12
Hordeum vulgare subsp. vulgare alpha-glucosidase: SEQ ID NO. 13
Arabidopsis thaliana alpha-glucosidase: SEQ ID NO. 14
Spinacia oleracea alpha-glucosidase: SEQ ID NO. 15
Beta vulgaris alpha-glucosidase: SEQ ID NO. 16
Solanum tuberosum alpha-glucosidase: SEQ ID NO. 17
Here, when the α-glucosidase derived from Aspergillus niger having the amino acid sequence of SEQ ID NO. 2 is an enzyme to be mutated, the amino acid of the above-mentioned (1) is the amino acid at the position 343 of SEQ ID NO. 2, the amino acid of the above-mentioned (2) is the amino acid at the position 452 of SEQ ID NO. 2, the amino acid of the above-mentioned (3) is the amino acid at the position 496 of SEQ ID NO. 2, the amino acid of the above-mentioned (4) is the amino acid at the position 410 of SEQ ID NO. 2, the amino acid of the above-mentioned (5) is the amino acid at the position 495 of SEQ ID NO. 2, the amino acid of the above-mentioned (6) is the amino acid at the position 498 of SEQ ID NO. 2, the amino acid of the above-mentioned (7) is the amino acid at the position 499 of SEQ ID NO. 2, the amino acid of the above-mentioned (8) is the amino acid at the position 531 of SEQ ID NO. 2, the amino acid of the above-mentioned (9) is the amino acid at the position 533 of SEQ ID NO. 2, the amino acid of the above-mentioned (10) is the amino acid at the position 579 of SEQ ID NO. 2, the amino acid of the above-mentioned (11) is the amino acid at the position 585 of SEQ ID NO. 2, the amino acid of the above-mentioned (12) is the amino acid at the position 662 of SEQ ID NO. 2, the amino acid of the above-mentioned (13) is the amino acid at the position 715 of SEQ ID NO. 2, and the amino acid of the above-mentioned (14) is the amino acid at the position 721 of SEQ ID NO. 2.
A kind of an amino acid after substitution is not particularly limited. Thus, it may be “conservative amino acid substitution” or “non-conservative amino acid substitution”. The “conservative amino acid substitution” herein refers to substituting a certain amino acid residue with an amino acid residue having a side chain with the same characteristics. Amino acid residues are classified into some families according to their side chains, such as basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., asparaginic acid, and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). The conservative amino acid substitution is typically substitution between amino acid residues in the same family.
When the α-glucosidase derived from Aspergillus niger having the amino acid sequence of SEQ ID NO. 2 is an enzyme to be mutated, examples of the amino acids after the substitution are cysteine, aspartic acid, methionine, histidine, alanine, phenylalanine, glycine, threonine, glutamic acid, valine, glutamine, asparagine or isoleucine for the amino acid of (1); glycine, aspartic acid, glutamic acid or alanine for the amino acid of (2); valine, asparagine, glutamine, isoleucine, arginine, cysteine or threonine for the amino acid of (3); glycine, proline or valine for the amino acid of (5); and leucine or serine for the amino acid of (6).
Two or more amino acids among the above-mentioned amino acids (1) to (14) may be substituted. As combinations of the amino acids to be substituted, a combination of (1) and (2) and a combination of (1) and (3) can be exemplified. These combinations correspond to the modified enzymes that were obtained in the Examples mentioned below.
Here, examples of the amino acid sequences of the modified enzymes are shown in SEQ ID Nos. 18 to 42 and 74 to 78. These sequences are the amino acid sequences of the modified enzymes obtained by conducting substitution of one amino acid (on any of the amino acids of (1) to (3), (5) and (6)), substitution of two amino acids (on the amino acids of (1) and (2), the amino acids of (1) and (3), or the amino acids of (2) and (3)) or substitution of three amino acids (on the amino acids of (1), (2) and (3)) on Aspergillus niger α-glucosidase. The corresponding relations of the sequence identification numbers and the amino acid substitutions are as follows. In the amino acid sequence of Aspergillus niger α-glucosidase (SEQ ID NO. 2), the tryptophan at the position 343 (W343) corresponds to the amino acid of (1), the valine at the position 452 (V452) corresponds to the amino acid of (2), the serine at the position 496 (S496) corresponds to the amino acid of (3), the isoleucine at the position 410 (I410) corresponds to the amino acid of (4), the valine at the position 499 (V499) corresponds to the amino acid of (7), the glutamic acid at the position 531 (E531) corresponds to the amino acid of (8), the phenylalanine at the position 533 (F533) corresponds to the amino acid of (9), the histidine at the position 579 (H579) corresponds to the amino acid of (10), the asparagine at the position 585 (N585) corresponds to the amino acid of (11), the tyrosine at the position 662 (Y662) corresponds to the amino acid of (12), the tyrosine at the position 715 (Y715) corresponds to the amino acid of (13), and the leucine at the position 721 (L721) corresponds to the amino acid of (14). Furthermore, in the amino acid sequence of Aspergillus nidulans α-glucosidase AgdB (SEQ ID NO. 7), the tyrosine at the position 296 (Y296) corresponds to the amino acid of (1), the asparagine at the position 456 (N456) corresponds to the amino acid of (3), the methionine at the position 363 (M363) corresponds to the amino acid of (4), the alanine at the position 455 (A455) corresponds to the amino acid of (5), the tyrosine at the position 458 (Y458) corresponds to the amino acid of (6), the asparagine at the position 459 (N459) corresponds to the amino acid of (7), the aspartic acid at the position 488 (D488) corresponds to the amino acid of (8), the leucine at the position 490 (L490) corresponds to the amino acid of (9), the arginine at the position 565 (R565) corresponds to the amino acid of (10), the glutamine at the position 571 (Q571) corresponds to the amino acid of (11), the isoleucine at the position 646 (I646) corresponds to the amino acid of (12), the phenylalanine at the position 700 (F700) corresponds to the amino acid of (13), and the isoleucine at the position 706 (I706) corresponds to the amino acid of (14).
Amino acid sequence: substitution of amino acid
A. SEQ ID NO. 18: W343C
B. SEQ ID NO. 19: W343D
C. SEQ ID NO. 20: W343M
D. SEQ ID NO. 21: W343H
E. SEQ ID NO. 22: W343A
F. SEQ ID NO. 23: W343F
G. SEQ ID NO. 24: W343G
H. SEQ ID NO. 25: W343T
I. SEQ ID NO. 26: W343E
J. SEQ ID NO. 27: W343V
K. SEQ ID NO. 28: W343Q
L. SEQ ID NO. 29: W343N
M. SEQ ID NO. 30: W343I
N. SEQ ID NO. 31: V452G
O. SEQ ID NO. 32: V452D
P. SEQ ID NO. 33: V452E
Q. SEQ ID NO. 34: S496V
R. SEQ ID NO. 35: S496N
S. SEQ ID NO. 36: S496Q
T. SEQ ID NO. 37: W343D and V452A
U. SEQ ID NO. 38: W343D and V452G
V. SEQ ID NO. 39: W343D and S496I
W. SEQ ID NO. 40: W343D and S496R
X. SEQ ID NO. 41: W343M and S496C
Y. SEQ ID NO. 42: W343M and S496T
a. SEQ ID NO. 74: S495G
b. SEQ ID NO. 75: S495P
c. SEQ ID NO. 76: S495V
d. SEQ ID NO. 77: C498L
e. SEQ ID NO. 78: C498S
In light of the experimental results shown in the following Examples (confirmation of the effect of amino acid substitution), the modified α-glucosidases of A to C, O and Q to S are highly useful in that they are specifically excellent in transglycosylation activity. Furthermore, V and W are highly useful in that they have a quite low hydrolysis activity. On the other hand, U, X and Y are highly useful in that they have a quite low decomposition activity against α-1,6 glucosidic linkages. Y is highly useful also in that it is excellent in transglycosylation activity. Furthermore, a to c are characterized in that they show a transglycosylation activity to form α-1,6 glucosidic linkages. d and e are excellent in transglycosylation activity to form α-1,4 glucosidic linkages.
Generally, when a part of an amino acid sequence of a certain protein is modified, the modified protein may have the equal function to that of the protein before the modification. That is to say, the modification of the amino acid sequence may not have a substantial effect on the function of the protein, so that the function of the protein may be maintained before and after the modification. When this technical common sense is considered, an enzyme that has a recognizable slight difference in the amino acid sequence (provided that the difference occurs in sites other than the sites in which the above substitutions have been made) and has Nos.ubstantially recognizable difference in the function can be regarded as an enzyme that is substantially the same as the above modified enzyme in comparison with the modified enzyme comprising the amino acid sequence in which one or more amino acids selected from the group consisting of the above (1) to (14) have been substituted with another amino acid. The term “slight difference in the amino acid sequence” as used herein typically means that the amino acid sequence is mutated (changed) by the deletion or substitution of one to several amino acids (the upper limit: e.g. 3, 5, 7, or 10 amino acids) constituting the amino acid sequence, or by the addition, insertion, or combination thereof, of one to several amino acids (the upper limit: e.g. 3, 5, 7, or 10 amino acids). The identity (%) of between the amino acid sequence in the “substantially the same enzyme” and the amino acid sequence of the above modified enzyme as a standard is preferably 90% or more, more preferably 95% or more, yet more preferably 98% or more, and most preferably 99% or more. In addition, the difference in the amino acid sequence may occur in a plurality of positions. The “slight difference in the amino acid sequences” is preferably generated by a conservative amino acid substitution.
(Nucleic Acid Coding for Modified α Glucosidase, Etc.)
The second aspect of the present invention provides a nucleic acid relating to the modified enzyme of the invention. That is, provided are a gene coding for the modified enzyme, a nucleic acid that can be used as a probe for identifying a nucleic acid coding for the modified enzyme, and a nucleic acid that can be used as a primer for amplifying or mutating a nucleic acid coding for the modified enzyme.
The gene coding for a modified enzyme is typically used in preparation of the modified enzyme. According to a genetic engineering procedure using the gene coding for a modified enzyme, a modified enzyme in a more homogeneous state can be obtained. Further, the method can be a preferable method also in the case of preparing a large amount of a modified enzyme. Note that uses of the gene coding for a modified enzyme are not limited to preparation of a modified enzyme. For example, the nucleic acid can also be used as a tool for an experiment intended for clarification of action mechanisms of a modified enzyme or a tool for designing or preparing a further mutant of an enzyme.
The “gene coding for a modified enzyme” herein refers to a nucleic acid capable of obtaining the modified enzyme when it is expressed, and includes, as a matter of course of a nucleic acid having a nucleotide sequence corresponding to the amino acid sequence of the modified enzyme, also a nucleic acid obtained by adding a sequence that does not code for an amino acid sequence to such a nucleic acid. Degeneracy of a codon is also considered.
Examples of sequences (nucleotide sequences) of genes coding for the modified enzyme are shown in SEQ ID Nos.: 43 to 67 and 79 to 83. These sequences are genes coding for the modified enzyme obtained by specific amino acids substitution to Aspergillus niger a glucosidase. The relationship between a SEQ ID NO and an amino acid substitution is as follows.
nucleotide sequence: amino acid substitution
A. SEQ ID NO 43: W343C
B. SEQ ID NO 44: W343D
C. SEQ ID NO 45: W343M
D. SEQ ID NO 46: W343H
E. SEQ ID NO 47: W343A
F. SEQ ID NO 48: W343F
G. SEQ ID NO 49: W343G
H. SEQ ID NO 50: W343T
I. SEQ ID NO 51: W343E
J. SEQ ID NO 52: W343V
K. SEQ ID NO 53: W343Q
L. SEQ ID NO 54: W343N
M. SEQ ID NO 55: W343I
N. SEQ ID NO 56: V452G
O. SEQ ID NO 57: V452D
P. SEQ ID NO 58: V452E
Q. SEQ ID NO 59: S496V
R. SEQ ID NO 60: S496N
S. SEQ ID NO 61: S496Q
T. SEQ ID NO 62: W343D and V452A
U. SEQ ID NO 63: W343D and V452G
V. SEQ ID NO 64: W343D and S496I
W. SEQ ID NO 65: W343D and S496R
X. SEQ ID NO 66: W343M and S496C
Y. SEQ ID NO 67: W343M and S496T
a. SEQ ID NO 79: S495G
b. SEQ ID NO 80: S495P
c. SEQ ID NO 81: S495V
d. SEQ ID NO 82: C498L
e. SEQ ID NO 83: C498S
The nucleic acid of the present invention can be prepared in an isolated state by use of a standard genetic engineering technique, molecular biological technique, biochemical technique, and the like in reference to the present specification or the sequence information disclosed in the appended sequence listing.
Another embodiment of the present invention provides a nucleic acid different in a nucleotide sequence in a part (hereinafter also referred to as a “homologous nucleic acid”, and a nucleotide sequence defining a homologous nucleic acid is also referred to as a “homologous nucleotide sequence”) as compared to the nucleotide sequence of the gene coding for the modified enzyme of the invention, although functions of a protein coded by the nucleic acid are equal. An example of the homologous nucleic acid includes a DNA composed of a nucleotide sequence containing substitution, deletion, insertion, addition or inversion of 1 to several nucleotides on the basis of the nucleotide sequence of the nucleic acid coding for the modified enzyme of the present invention and coding for a protein having enzyme activity characteristic to the modified enzyme (i.e. glycosyltransferase activity). Substitution or deletion of bases may occur in a plurality of sites. The “plurality” herein depends on positions or kinds of amino acid residues in a conformation of a protein coded by the nucleic acid but means, for example, 2 to 40 bases, preferably 2 to 20 bases, and more preferably 2 to 10 bases.
Such a homologous nucleic acid as described above can be obtained by, for example, a restriction enzyme treatment, a treatment with exonuclease, DNA ligase, etc., and introduction of mutation by a site-directed mutagenesis method (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York), and a random mutagenesis method (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York). The homologous nucleic acid can be obtained also in other methods such as exposure to ultraviolet radiation.
Another embodiment of the present invention relates to a nucleic acid having the complementary nucleotide sequence to the nucleotide sequence of the gene coding for the modified enzyme of the invention. Another embodiment of the present invention provides a nucleic acid having a nucleotide sequence with an identity of at least about 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% to the nucleotide sequence of the gene coding for the modified enzyme of the invention or the complementary nucleotide sequence
Another embodiment of the present invention relates to a nucleic acid having a nucleotide sequence hybridizing to the complementary nucleotide sequence to the nucleotide sequence of the gene coding for the modified enzyme of the invention or its homologous nucleotide sequence under stringent conditions. The “stringent conditions” herein refer to conditions wherein a so-called specific hybrid is formed and a nonspecific hybrid is not formed. Such stringent conditions are known by a person skilled in the art and can be set in reference to, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) and Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). Examples of the stringent conditions include conditions of using a hybridization liquid (50% formamide, 10×SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), a 5×Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg/ml of modified salmon sperm DNA, and a 50 mM phosphate buffer (pH7.5)) and incubating at about 42° C. to about 50° C., thereafter washing with 0.1×SSC and 0.1% SDS at about 65° C. to about 70° C. Examples of more preferable stringent conditions include conditions of using 50% formamide, 5×SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), a 1×Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg/ml of modified salmon sperm DNA, and a 50 mM phosphate buffer (pH 7.5) as a hybridization liquid.
Another embodiment of the present invention provides a nucleic acid (nucleic acid fragment) having a part of the nucleotide sequence of the gene coding for the modified enzyme of the invention or the complementary nucleotide sequence. Such a nucleic acid fragment can be used in detection, identification, and/or amplification of a nucleic acid having the nucleotide sequence of the gene coding for the modified enzyme of the present invention. For example, the nucleic acid fragment is designed so as to at least contain a part being hybridized to a sequential nucleotide moiety (for example, about 10 to about 100 nucleotides length, preferably about 20 to about 100 bases length, more preferably about 30 to about 100 bases length) in the nucleotide sequence of the gene coding for the modified enzyme of the invention. When used as a probe, the nucleic acid fragment can be labeled. Examples such as fluorescent substances, enzymes, and radioactive isotopes can be used for the labeling.
Another aspect of the present invention relates to a recombinant DNA containing the gene of the present invention (the gene coding for a modified enzyme). The recombinant DNA of the invention is provided in, for example, a form of a vector. The term “vector” in the present specification refers to a nucleic acid molecule that can transfer a nucleic acid inserted in the vector to a target such as a cell.
A suitable vector is selected according to its intended use (cloning, expression of a protein) and in consideration of a kind of a host cell. Examples include a M13 phage or an altered form thereof, a λ phage or an altered form thereof, and pBR322 or an altered form thereof (e.g., pB325, pAT153, pUC8), etc. as a vector having Escherichia coli as a host, pYepSec1, pMFa, and pYES2 as a vector having a yeast as a host, pAc, pVL, etc. as a vector having an insect cell as a host, and pCDM8, pMT2PC, etc. as a vector having a mammal cell as a host.
The vector of the present invention is preferably an expression vector. The “expression vector” refers to a vector capable of introducing a nucleic acid inserted in the expression vector into a target cell (host cell) and expressing it in the cell. The expression vector generally contains a promoter sequence necessary for expression of a nucleic acid inserted, an enhancer sequence for promoting expression, and the like. An expression vector containing a selective marker can also be used. When such an expression vector is used, presence or absence (and its degree) of introduction of the expression vector can be confirmed using a selective marker.
Insertion of the nucleic acid of the present invention into the vector, insertion of a selective marker gene (if necessary), insertion of a promoter (if necessary), and the like can be performed by using a standard recombinant DNA technique (for example, a known method of using a restriction enzyme and a DNA ligase, which can be referred in Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York).
For the host cell, microorganisms such as Escherichia coli and budding yeasts (Saccharomyces cerevisiae) are preferably used from the viewpoint of easiness of handling, and host cells capable of duplicating a recombinant DNA and expressing a gene of a modified enzyme can be used. Examples of Escherichia coli include Escherichia coli BL21(DE3)pLysS in the case of using a T7 promoter, and Escherichia coli JM109 in other cases. Examples of budding yeasts include budding yeast SHY2, AH22, or INVSc1 (Invitrogen Ltd.).
Another aspect of the present invention relates to a microorganism having the recombinant DNA of the invention (that is, a transformant). The microorganism of the invention can be obtained by transfection or transformation using the vector of the invention described above. The transfection or transformation can be performed in, for example, the calcium chloride method (J. Mol. Biol., 53, 159 (1970)), the Hanahan method (J. Mol. Biol., 166, 557 (1983)), the SEM method (Gene, 96, 23 (1990)), a method by Chung, et al. (Proc. Natl. Acad. Sci. U.S.A. 86, 2172 (1989)), the calcium phosphate coprecipitation method, the electroporation method (Potter, H. et al., Proc. Natl. Acad. Sci. U.S.A. 81, 7161-7165 (1984)), and the lipofectin method (Feigner, P. L. et al., Proc. Natl. Acad. Sci. U.S.A. 84, 7413-7417 (1984)). Note that the microorganism of the present invention can be used in producing the modified enzyme of the present invention (see the section of the preparation method of modified α glucosidase described later).
(Enzyme Agent Containing Modified α-Glucosidase)
The modified enzyme of the present invention is provided, for example, in the form of an enzyme agent. The enzyme agent may contain an excipient, a buffer agent, a suspending agent, a stabilizer, a preservative, an antiseptic, saline and the like besides the active ingredient (the modified enzyme of the present invention). As the excipient, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, white soft sugar, glycerol and the like can be used. As the buffer agent, phosphates, citrates, acetates and the like can be used. As the stabilizer, propylene glycol, ascorbic acid and the like can be used. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben and the like can be used. As the antiseptic, ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
(Use of Modified α-Glucosidase)
A further aspect of the present invention relates to use of the modified enzyme. As one of the use, a method for producing an oligosaccharide is provided. In the first embodiment of the method for producing an oligosaccharide of the present invention, in order to utilize the feature of the modified enzyme of the present invention of high specificity against α-1,4 glucosidic linkages, an oligosaccharide or polysaccharide of di- or more saccharides having α-1,4 glucosidic linkage(s) is used as a substrate, and the modified enzyme of the present invention is reacted with the substrate. In this embodiment, enzymes having high specificity against α-1,4 glucosidic linkages such as modified enzymes W343M, W343M/S496T, C498L and C498S shown in the following Examples, and the like are used. Examples of the substrate are maltose, maltotriose, panose, pullulan, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose, dextrin, starches (potato starch, sweet potato starch, corn starch, wheat starch, tapioca starch and the like), and the like. A mixture of two or more oligosaccharides or polysaccharides may be the substrate. According to the production method of the present invention, an oligosaccharide having low-branched chains can be produced in a specific and efficient manner. The reaction conditions may follow those generally adopted in the generation of an oligosaccharide using α-glucosidase.
In the second embodiment of the method for producing an oligosaccharide of the present invention, modified enzymes that show a transglycosylation activity to form α-1,6 glucosidic linkages (for example, S495G, S495P and S495V shown in the following Examples) are used. Examples of the substrate in this case are isomaltose, isomaltotriose, panose, isopanose, starches (potato starch, sweet potato starch, corn starch, wheat starch, tapioca starch and the like). According to this embodiment, typically, an oligosaccharide having much branched chains can be produced in a specific and efficient manner.
In order to increase the generation amount of the intended oligosaccharide (an oligosaccharide having low-branched chains in the case of the first embodiment, or typically an oligosaccharide having much branched chains in the case of the second embodiment), a wild type enzyme may be used in addition to the modified enzyme. The wild type enzyme is not specifically limited as long as it is suitable for the generation of the intended oligosaccharide. For example, a wild type enzyme that corresponds to the modified enzyme to be used (i.e., an enzyme that has undergone modification for obtaining the modified enzyme) can be adopted. In contrast to this example, a modified enzyme and wild type enzyme whose origins are different can be used in combination. As an example, an enzyme obtained by modifying Aspergillus niger α-glucosidase (modified enzyme) and Aspergillus nidulans wild type α-glucosidase AgdB are used in combination.
Here, as shown in the following Examples, it has become clear that an oligosaccharide can be formed from a substrate having α-1,4 glucosidic linkages when a modified enzyme that shows a transglycosylation activity to form α-1,6 glucosidic linkages and a wild type enzyme in combination. Therefore, in a further embodiment of the present invention, a modified enzyme that shows a transglycosylation activity to form α-1,6 glucosidic linkages (for example, S495G, S495P and S495V shown in the following Examples) is reacted in combination with a wild type enzyme to give an oligosaccharide.
Due to the transglycosylation activity of the modified enzyme of the present invention, an effect of preventing aging of starches, an effect of improving flavors of foods, an effect of regulating the intestines and the like can be expected. Therefore, examples of the use of the modified enzyme of the present invention may include prevention of the aging of starches in foods, improvement of the flavors of foods and regulation of the function of the intestines. Assuming application to these uses, the present invention provides compositions containing the modified enzyme (a pharmaceutical composition, a quasi-drug composition, a cosmetic composition, a food composition or a feed composition) as a further aspect.
The pharmaceutical composition or quasi-drug composition of the present invention can be formulated according to a conventional method. In the case of the formulation, other components that are acceptable in formulation (for example, a carrier, an excipient, a disintegrating agent, a buffer agent, an emulsifying agent, a suspending agent, a soothing agent, a stabilizer, a preservative, an antiseptic, saline and the like) can be incorporated. As the excipient, lactose, starch, sorbitol, D-mannitol, white soft sugar and the like can be used. As the disintegrating agent, starch, carboxymethyl cellulose, calcium carbonate and the like can be used. As the buffer agent, phosphates, citrates, acetates and the like can be used. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glycerin monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, ascorbic acid and the like can be used. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben and the like can be used. As the antiseptic, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
The dosage form in the case of the formulation is also not specifically limited, and the pharmaceutical composition or quasi-drug composition of the present invention can be provided, for example, as a tablet, a powder, a fine granule, a granular agent, a capsule agent, a syrup agent, an injection agent, an external preparation and a suppository, and the like.
The pharmaceutical composition of the present invention contains an active ingredient (modified enzyme) in an amount necessary to obtain the expected therapeutic effect or prophylactic effect (i.e., a therapeutically effective amount). Similarly, the quasi-drug composition of the present invention contains an active ingredient in an amount necessary to obtain the expected improvement effect, prophylactic effect and the like. Although the amount of the active ingredient contained in the pharmaceutical composition or quasi-drug composition of the present invention generally differs depending on the dosage form or form, the amount of the active ingredient is set within the range of, for example, about 0.1% by weight to about 95% by weight so that a desired dose can be achieved.
The pharmaceutical composition or quasi-drug composition of the present invention is applied to a subject in an oral or parenteral manner (intravenous, intraarterial, subdermal, intramuscular, or interperitoneal injection, transdermal, pernasal, transmucosal, application, or the like) depending on the dosage form and form thereof. The “subject” as used herein is not specifically limited, and includes humans and mammals other than humans. (Pet animals, farm animals, and experimental animals are included. Specific examples are mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, cats, chickens, quails and the like.) In a preferable embodiment, the subject to be applied is a human.
The doses and use amounts of the pharmaceutical composition and quasi-drug composition of the present invention are preset so that expected effects would be obtained. In the setting of an effective dose, the symptom, age, sex, body weight and the like of a subject to be applied are considered. Any person skilled in the art can set a suitable dose with consideration for these matters. As an administration schedule, for example, once to several times a day, once every second day, or once every third day can be adopted. In the preparation of the administration schedule, the symptom of the subject, the duration of the effect of the active ingredient, and the like can be considered.
The cosmetic composition of the present invention can be obtained by incorporating the modified enzyme, and components and substrates that are generally used in cosmetics (for example, various fats and oils, mineral oils, vaseline, squalane, lanolin, beeswax, modified alcohols, dextrin palmitate, glycerin, fatty acid esters of glycerin, ethylene glycol, paraben, camphor, menthol, various vitamins, zinc oxide, titanium oxide, benzoic acid, edetic acid, chamomile oil, carrageenan, chitin powder, chitosan, perfume materials, coloring materials and the like). As the forms of the cosmetic composition, an emulsion liquid for face or body, a skin lotion, a cream, a lotion, an essence, an oil, a pack, a sheet, a cleanser and the like can be exemplified. The addition amount of the modified enzyme in the cosmetic composition is not specifically limited. For example, the modified enzyme can be added so as to be 0.01% by weight to 10% by weight.
On the other hand, examples of the “food composition” in the present invention may include general foods (cooked rice, bread, cereal, vegetables, edible meat, various processed foods, sweets, milk, soft drink, alcohol beverages and the like), nutritional supplements (supplements, nutrition-supplement drinks and the like) and food additives. In the cases of the nutritional supplement or food additive, it can be provided in the form of a powder, a granule, a tablet, a paste, a liquid or the like.
Examples of the “feed composition” of the present invention are pet foods (feeds for dogs, cats, birds, fishes, reptiles, amphibians, rodents and the like) and feedstuffs (feeds for farm animals, poultries, cultured fishes and the like).
(Method for Designing Modified α-Glucosidase)
A further aspect of the present invention relates to a method of designing the modified enzyme. In the designing method of the present invention, the following steps (i) and (ii) are carried out.
Step (i): one or two or more amino acid(s) selected from the group consisting of the following (1) to (14) is/are identified in the amino acid sequence of α-glucosidase as an enzyme to be mutated:
(1) an amino acid corresponding to the amino acid at the position 385 of the amino acid sequence of SEQ ID NO. 1
(2) an amino acid corresponding to the amino acid at the position 491 of the amino acid sequence of SEQ ID NO. 1
(3) an amino acid corresponding to the amino acid at the position 535 of the amino acid sequence of SEQ ID NO. 1
(4) an amino acid corresponding to the amino acid at the position 450 of the amino acid sequence of SEQ ID NO. 1
(5) an amino acid corresponding to the amino acid at the position 534 of the amino acid sequence of SEQ ID NO. 1
(6) an amino acid corresponding to the amino acid at the position 537 of the amino acid sequence of SEQ ID NO. 1
(7) an amino acid corresponding to the amino acid at the position 538 of the amino acid sequence of SEQ ID NO. 1
(8) an amino acid corresponding to the amino acid at the position 554 of the amino acid sequence of SEQ ID NO. 1
(9) an amino acid corresponding to the amino acid at the position 556 of the amino acid sequence of SEQ ID NO. 1
(10) an amino acid corresponding to the amino acid at the position 579 of the amino acid sequence of SEQ ID NO. 2
(11) an amino acid corresponding to the amino acid at the position 585 of the amino acid sequence of SEQ ID NO. 2
(12) an amino acid corresponding to the amino acid at the position 630 of the amino acid sequence of SEQ ID NO. 1
(13) an amino acid corresponding to the amino acid at the position 683 of the amino acid sequence of SEQ ID NO. 1
(14) an amino acid corresponding to the amino acid at the position 689 of the amino acid sequence of SEQ ID NO. 1
The amino acids to be substituted (1) to (14) are amino acids that have been identified as amino acids that are important for the characteristics of enzymes. Therefore, it can be highly expected that change in the characteristics of α-glucosidase is generated if these amino acids are substituted. The characteristics for which change is especially expected are a transglycosylation activity and a hydrolysis activity. The designing method of the present invention is especially useful as a means for designing an enzyme in which the balance of these two activities has been changed. For example, the designing method of the present invention can be utilized for the purpose of decreasing or eliminating a hydrolysis activity and increasing a transglycosylation activity.
The enzyme to be mutated in the designing method of the invention is α-glucosidase. The enzyme to be mutated is typically a wild type enzyme (an enzyme found in nature). However, this does not interfere use of an enzyme that has already undergone any mutation or modification as an enzyme to be mutated. Examples of the enzyme to be mutated are Human Maltase-glucoamylase, Aspergillus niger alpha-glucosidase, Human Neutral alpha-glucosidase C, Mouse Lysosomal alpha-glucosidase, Yeast GLU2A, Aspergillus nidulans Alpha-glucosidase AgdA, Aspergillus nidulans Alpha-glucosidase AgdB, Mucor javanicus alpha-glucosidase, Aspergillus oryzae alpha-glucosidase, Mortierella alliacea alpha-glucosidase, Schizosaccharomyces pombe alpha-glucosidase, Debaryomyces occidentalis alpha-glucosidase, Hordeum vulgare subsp. vulgare alpha-glucosidase, Arabidopsis thaliana alpha-glucosidase, Spinacia oleracea alpha-glucosidase, Beta vulgaris alpha-glucosidase and Solanum tuberosum alpha-glucosidase. As mentioned above, the amino acid sequences of these α-glucosidases are registered with public database (SEQ ID Nos. 1 to 17). In a preferable embodiment, an enzyme consisting of any of these amino acid sequences is an enzyme to be mutated.
In the present invention, the following step (ii) is conducted after the step (i).
Step (ii): an amino acid sequence in which the amino acid sequence(s) identified in the step (i) has/have been substituted by other amino acid is constructed based on the amino acid sequence of the enzyme to be mutated.
The kind(s) of the amino acid(s) after the substitution is/are not specifically limited. Therefore, either conservative amino acid substitution or non-conservative amino acid substitution is acceptable.
EXAMPLES
Preparation Method of Modified α Glucosidase
A further aspect of the present invention relates to a preparation method of a modified enzyme. In one embodiment of the preparation method of a modified enzyme of the present invention, the modified enzyme that the present inventors succeeded in obtaining is prepared in a genetic engineering technique. In the case of this embodiment, a nucleic acid coding for any one of the amino acid sequences of SEQ ID Nos.: 18 to 42 and 74 to 78, is prepared (step (I)). Herein, “a nucleic acid coding for a specific amino acid sequence” is a nucleic acid capable of obtaining a polypeptide having the amino acid sequence in the case of being expressed, and as a matter of course of a nucleic acid having a nucleotide sequence corresponding to the amino acid sequence, may be a nucleic acid added with an extra sequence (may be a sequence coding for an amino acid sequence or a sequence not coding for an amino acid sequence). Degeneracy of a codon is also considered. “A nucleic acid coding for any one of the amino acid sequences of SEQ ID Nos.: 18 to 42 and 74 to 78” can be prepared into a state of being isolated by using a standard genetic engineering technique, molecular biological technique, biochemical technique, and the like in reference to sequence information disclosed in the present specification or the appended sequence listing. Herein, all of the amino acid sequences of SEQ ID Nos.: 18 to 42 and 74 to 78 are obtained by mutation to the amino acid sequence of the Aspergillus niger-derived a glucosidase. Therefore, a nucleic acid (gene) coding for any one of the amino acid sequences of SEQ ID Nos.: 7 to 10 can be obtained also by adding necessary mutation to the gene coding for the Aspergillus niger-derived a glucosidase (SEQ ID NO: 73). A large number of methods for site-directed mutagenesis have been known in the present technical field (for example, see Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York), and among those methods, a suitable method can be selected to be used. A method of saturation mutagenesis can be adopted as the method of site-directed mutagenesis. The method of saturation mutagenesis is a “semi-rational, semi-random” technique of assuming a position which relates to a desired function based on a conformation of a protein and introducing amino acid saturation (J. Mol. Biol. 331, 585-592 (2003)). For example, use of a kit such as KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.), Quick change (Stratagene Corporation) and Overlap extension PCR (Nucleic Acid Res. 16, 7351-7367 (1988)) makes it possible to introduce position specific amino acid saturation. A Taq polymerase and the like can be used for a DNA polymerase used in PCR. Provided that a DNA polymerase having high precision such as KOD-PLUS-(TOYOBO CO., LTD.) or Pfu turbo (Stratagene Corporation) is preferably used.
In another embodiment of the present invention, a modified enzyme is prepared based on an amino acid sequence that is designed by the designing method of the present invention. In the case of this embodiment, a nucleic acid coding for an amino acid sequence constructed by the designing method of the present invention is prepared in the step (i). For example, based on the amino acid sequence constructed by the designing method of the present invention, necessary mutation (that is, substitution of an amino acid in a specific position in a protein that is an expressed product) is added to a gene coding for a modified enzyme and a nucleic acid (gene) coding for the modified enzyme is obtained.
Following the step (I), the prepared nucleic acid is expressed (step (II)). For example, firstly, an expression vector inserted with the above described nucleic acid is prepared and a host cell is transformed using this constructed vector. The “expression vector” refers to a vector that can introduce a nucleic acid inserted therein into a desired cell (host cell) and is capable of being expressed in the cell. The expression vector generally contains a promoter sequence that is necessary for expression of an inserted nucleic acid, an enhancer sequence that promotes expression, and the like. An expression vector containing a selection marker can also be used. When such an expression vector is used, presence or absence (and its degree) of the expression vector can be confirmed by using a selection marker.
Then, a transformant is cultured under the condition of producing a modified enzyme that is an expressed product. Culture of the transformant may follow a general method. An assimilable carbon compound may be used as a carbon source used for a medium, and examples such as glucose, sucrose, lactose, maltose, molasses, and pyruvic acid are used. An available nitrogen compound may be used as a nitrogen source, and examples such as peptone, meat extract, yeast extract, casein hydrolysate, and soybean bran alkali extract are used. Other than those substances, phosphate, carbonate, sulfate, salts of magnesium, calcium, potassium, iron, manganese and zinc, specific amino acids, specific vitamins, and the like are used according to necessity.
On the other hand, a culture temperature can be set within the range from 30 to 40° C. (preferably at around 37° C.). A culture time can be set by considering growing characteristics of a transformant to be cultured and production characteristics of a mutant-type enzyme. A pH of a medium is set within the range wherein a transformant grows and an enzyme is produced. The pH of a medium is preferably set at about 6.0 to 9.0 (preferably at around pH 7.0).
Subsequently, the expressed product (modified enzyme) is recovered (step (III)). A culture liquid containing fungas bodies after culture may be used as an enzyme solution directly or after undergoing condensation, removal of impurities, or the like, but the expressed product is generally once recovered from the culture liquid or fungas bodies. When the expressed product is a secretion type protein, it can be recovered from the culture liquid, and in other cases, the expressed product can be recovered from cells. In the case of recovering from the culture liquid, for example, an undissolved substance is removed by filtration and centrifugation on a culture supernatant, and then, a purified product of a modified enzyme can be obtained by separation and purification in combination of vacuum concentration, membrane concentration, salting out using ammonium sulfate or sodium sulfate, fractional precipitation by methanol, ethanol, or acetone, dialysis, heating treatment, isoelectric treatment, various kinds of chromatography such as gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography (for example, gel filtration with Sephadex gel (GE Healthcare Life Sciences), etc., DEAE sepharose CL-6B (GE Healthcare Life Sciences), octyl sepharose CL-6B (GE Healthcare Life Sciences), CM sepharose CL-6B (GE Healthcare Life Sciences)). On the other hand, in the case of recovering the expressed product from cells, a culture liquid is subjected to filtration, centrifugation, or the like, to thus obtain the cells, then the cells are crushed by a mechanical method such as a pressure treatment and an ultrasonic treatment, or an enzymatic method with a lysozyme or the like, thereafter carrying out separation and purification in the same manner as described above, and a purified product of a modified enzyme can be thus obtained.
The purified enzyme obtained as described above can be provided after being powdered, for example, by freeze dry, vacuum dry, or spray dry. In this time, the purified enzyme may be previously dissolved in a phosphoric acid buffer solution, a triethanol amine buffer solution, a tris-hydrochloric acid buffer solution, or a GOOD buffer solution. Preferably, a phosphoric acid buffer solution and a triethanol amine buffer solution can be used. Note that, for the GOOD buffer solution herein, PIPES, MES or MOPS is exemplified.
Generally, genetic expression and recovery of the expressed product (modified enzyme) are carried our using an appropriate host-vector system as described above, but a cell-free synthesis system may also be employed. Herein, the “cell-free synthesis system (cell-free transcription system, cell-free transcription/translation system)” refers to in vitro synthesis of mRNA or a protein from a nucleic acid (DNA or mRNA) being a template, which codes for the mRNA or the protein, using a ribosome, a transcription/translation factor derived from living cells (alternately, obtained in a genetic engineering technique) or the like, not using living cells. In the cell-free synthesis system, a cell extraction obtained from a cell disruptor that is purified according to necessity is generally used. The cell extraction generally includes ribosome necessary for protein synthesis and various factors such as an initiation factor, and various enzymes such as tRNA. When a protein is synthesized, this cell extraction is added with other substances necessary for protein synthesis, such as various amino acids, energy sources (e.g., ATP and GTP), and creatine phosphate. As a matter of course, ribosome and various factors and/or various enzymes, and the like, which are separately prepared, may be supplemented if necessary in the protein synthesis.
Development of a transcription/translation system reconstructing various molecules (factors) necessary for protein synthesis has also been reported (Shimizu, Y. et al.: Nature Biotech., 19, 751-755, 2001). In this synthesis system, a gene of 31 kinds of factors composed of 3 kinds of initiation factors constituting a protein synthesis system of bacteria, 3 kinds of elongation factors, 4 kinds of factors associated with termination, 20 kinds of aminoacyl tRNA synthesis enzymes that make each amino acid combine with tRNA, and a methionyl tRNA formyl transfer enzyme is amplified from an Escherichia coli genome, and a protein synthesis system is reconstructed in vitro using them. Such a reconstructed synthesis system may be used in the present invention.
The term “cell-free transcription/translation system” is interchangeably used with a cell-free protein synthesis system, an in vitro translation system or an in vitro transcription/translation system. In the in vitro translation system, RNA is used as a template to synthesize a protein. Any of RNA, mRNA, an in vitro transcribed product, or the like is used as the template RNA. On the other hand, in the in vitro transcription/translation system, DNA is used as a template. The template DNA should include in a ribosome bonding region, and preferably contains a suitable terminator sequence. In addition, in the in vitro transcription/translation system, a condition of adding factors necessary for each reaction is established so that a transcription reaction and a translation reaction proceed sequentially.
A. Acquirement of Modified α-Glucosidase 1
Using α-glucosidase (also referred to as transglucosidase. Hereinafter referred to as “TG”.) as a model, a technique for directing a hydrolysis reaction and a dehydration condensation reaction in one direction (One direction technology) was studied by using protein engineering. TG is an enzyme used for the production of oligosaccharides and has an activity to hydrolyze glucosidic linkages and also has a transglycosylation activity, and thus forms oligosaccharides from maltose. The study proceeded in an effort to develop a TG that conducts transglycosylation more efficiently.
(1) Study of Annotated Amino Acids Using Computer Program
Computer study was carried out aiming at designing a novel enzyme having improved oligosaccharide-producing ability by the structure modification of TG at the molecular level. Using α-glucosidase derived from human small intestine (SEQ ID NO. 1), for which an X-ray crystal structure analysis was reported, annotated amino acids were determined by computer simulation based on the steric structure of TG and functional expectation based on evolutionary findings. Specifically, amino acids that are expected to be annotated amino acids in human α-glucosidase were calculated by using HotSpot Wizard program (Non-patent Document: Pavelka et al., Nucleic Acids Res. 37 W376-83. (2009)), and the three amino acids (the amino acid at the position 385: Y385, the amino acid at the position 491: V491, the amino acid at the position 535: N535) were selected. Since all of the amino acids are present on the regions that contact with a substrate, it can be expected that the amino acids are involved in the activity of the enzymes (FIG. 1, A). Subsequently, five kinds of α-glucosidase homologues were selected, and the sequences in the vicinity of the above-mentioned annotated amino acids were subjected to alignment comparison by using CLUSTALW. Since the homology of the sequences in the vicinity was high and highly conserved in all of Y385, V491 and N535, it was able to be expected that the regions are regions that are important for the functions of the enzyme. The amino acids corresponding to these annotated amino acids were identified by a Aspergillus niger TG (Asp. Niger) (FIG. 1, C). The amino acid corresponding to Y385 is W343, the amino acid corresponding to V491 is V452, and the amino acid corresponding to N535 is 5496. A primer for introducing mutation into these amino acids was designed, and mutation was introduced by an inverse PCR method using a random primer. Transformation into Escherichia coli was conducted, and a sequence analysis was conducted after miniprep to confirm introduction of mutation.
(2) Development of Screening System
The plasmid prepared was transformed into yeast INVsc1 strain and cultured. The culture supernatant was collected by centrifugation, and the secreted enzyme was used as modified TG. The broth was concentrated and desalted by ultrafiltration using a spin column. Consequently, an enzyme sample concentrated to approximately 10 to 50-fold was obtained.
Using maltose, which is an enzyme substrate, the hydrolysis activity was measured. The substrate was adjusted so as to have a final concentration of 1%, and the glucose formed by hydrolysis was reacted with an aminoantipyrine-phenol color developing liquid containing glucose oxidase (GO) and peroxidase (PO) to give a color and quantified by measuring OD500. A calibration curve (standard) was prepared by a glucose standard preparation, and the amount of the generated glucose was calculated from the absorbance.
In order to examine the oligosaccharide synthesis activities, the generated oligosaccharide was analyzed by HPLC. Using a 50% maltose aqueous solution as a substrate, the generated oligosaccharide was determined. The conditions for HPLC were as follows.
Column: TSKGEL Amido-80 (Tosoh Corporation)
Solvent: MeCN/H2O=2/1
Detection: RI
The area surface areas of HPLC were represented as a graph to thereby estimate the respective saccharide numbers of the generated oligosaccharides. The scheme of the above-mentioned screening method is shown in FIG. 2.
(3) Evaluation of Modified TG by Substitution of One Amino Acid
Random mutation was introduced by a PCR method into the position corresponding to the above-mentioned mutation introduction point (Y385, V491, N535), i.e., W343, V452 or 5496, in Aspergillus niger α-glucosidase (SEQ ID NO. 2) (substitution of one amino acid). After the introduction of mutation, sequencing was conducted to thereby identify the introduced point mutation. The obtained three modified TGs (W343M, V452G and S496V) were analyzed. In addition, a preliminary experiment was conducted to confirm that a protein derived from the plasmid was expressed in the culture supernatant, and that the enzyme had a hydrolysis activity.
First, the oligosaccharide synthesis activities were measured by using a high-concentration maltose solution as a substrate. A typical HPLC pattern as the result thereof is shown in FIG. 3. In a sample in which a wild type (WT) TG was expressed, the peak of the substrate maltose decreased, and the peaks of glucose, isomaltose, panose and isomaltotriose were seen (FIG. 3, A). Furthermore, a bipartite peak was seen in the area of the tetrasaccharide (FIG. 3, A).
On the other hand, as compared to the WT, in the modified TGs, the peak of the substrate maltose decreased, and peaks of glucose, maltotriose and panose were seen (FIG. 3, B to D). Furthermore, gathered peaks were seen in the area of the tetrasaccharide (FIG. 3, B to D). In the oligosaccharides formed by the modified TGs, unlike the WT, the generation amount of the isomalto-based oligosaccharide significantly decreased.
Subsequently, the oligosaccharide synthesis activities of the modified TGs were compared to that of the WT by quantifying the area surface areas of HPLC. The graph in FIG. 4 shows the total of the area surface areas of tri- or more saccharides. Oligosaccharides of tri- or more saccharides were formed also in the WT, but the area surface areas of the formed oligosaccharides were larger than those of the WT in all of the modified TGs (increased by approximately 20%).
For the purpose of investigating the relationship between the kinds of the amino acids after substitution and the transglycosylation activity, various modified TGs were prepared with respect to the amino acid W343 corresponding to the mutation introduction point (385Y), and the oligosaccharide synthesizing abilities were compared. As a result, in the cases where the amino acid after the substitution was cysteine (W343C), aspartic acid (W343D), methionine (W343M), histidine (W343H), alanine (W343A), phenylalanine (W343F), glycine (W343G), threonine (W343T), glutamic acid (W343E), valine (W343V), glutamine (W343Q), asparagine (W343N) or isoleucine (W343I), the amount of the oligosaccharides of tri- or more saccharides formed by the modified TG increased more than that in the WT (FIG. 5, upper). Similar comparison was made for the oligosaccharides of tetra- or more saccharides, and consequently found that the modified TG had more excellent synthesis ability than that of the WT in the cases where the amino acid after the substitution was methionine (W343M), aspartic acid (W343D), cysteine (W343C), phenylalanine (W343F), glutamine (W343Q), histidine (W343H), threonine (W343T), glutamic acid (W343E), glycine (W343G), asparagine (W343N), valine (W343V) or alanine (W343A) (FIG. 5, lower). It can be evaluated that the modified TGs that were in the upper level for either of the synthesized amount of the oligosaccharides of tri- or more saccharides and the synthesized amount of the oligosaccharides of tetra- or more saccharides, i.e., W343C, W343D and W343M, are especially excellent in oligosaccharide synthesis ability. Furthermore, it is suggested that W343M, W343D, W343C, W343F, W343Q and W343H, in which the synthesized amount of the oligosaccharides of tetra- or more saccharides is significantly higher than that in the WT, are advantageous for the synthesis of oligosaccharides having longer chains.
The transglycosylation activities of the modified TGs were compared to that of the WT by quantifying the area surface areas of HPLC. Various modified TGs were prepared with respect to the amino acid V452, and the oligosaccharide synthesis abilities were compared. As a result, in the cases where the amino acid after the substitution was glycine, aspartic acid or glutamic acid, the amount of the oligosaccharides of tri- or more saccharides increased more than that in the WT (FIG. 6). Furthermore, the oligosaccharides of tetra- or more saccharides were similarly compared, and consequently found that the modified TG in which the amino acid after the substitution was aspartic acid (V452D) was superior to the WT in synthesis ability (FIG. 6). Similarly, various modified TGs were prepared with respect to the amino acid S496, and the oligosaccharide synthesis abilities were compared. As a result, in the cases where the amino acid after the substitution was valine, asparagine or glutamine, the modified TGs were superior to the WT in synthesis ability in both cases of the oligosaccharides of tri- or more saccharides and the oligosaccharides of tetra- or more saccharides (FIG. 6).
As mentioned above, it was found that the generation amount of the isomalto-based oligosaccharide was greatly decreased in the modified TGs. Therefore, it was thought that the reactivity against glucosidic linkages should change, and thus the hydrolysis activities against various glucosidic linkages were investigated. As enzyme substrates, aqueous solutions of maltose (α-1,4) and isomaltose (α-1,6), which are disaccharides, were prepared. The WT showed substrate affinity for both α-1,4 glucosidic linkages and α-1,6 glucosidic linkages (FIG. 7). In contrast, in the modified TGs, the hydrolysis activity against α-1,4 glucosidic linkages was approximately the same as that of the wild type, whereas the hydrolysis activity against α-1,6 glucosidic linkages decreased to one-fifth or less (FIG. 7).
(4) Evaluation of Modified TG by Substitution of Two Amino Acids
Modified TGs in which mutation had been introduced into two amino acids were prepared by introducing further mutation (either of the two amino acids present on the surface of the tunnel structure (V452 and S496)) in the modified TG in which the tryptophan at the position 343 had been substituted. The hydrolysis activities of the obtained modified TGs were measured. As shown in FIG. 8, the activity was not able to be detected at all for W343D/S496I (the W at the position 343 was substituted by D and the S at the position 496 was substituted by I) and W343D/S496R (the W at the position 343 was substituted by D and the S at the position 496 was substituted by R). On the other hand, the hydrolysis activity of isomaltose greatly decreased (approximately 1/20) in the three kinds of modified TGs: W343D/V452G (the W at the position 343 was substituted by D and the V at the position 452 was substituted by G), W343M/S496C (the W at the position 343 was substituted by M and the S at the position 496 was substituted by C) and W343M/S496T (the W at the position 343 was substituted by M and the S at the position 496 was substituted by T).
Subsequently, the oligosaccharide synthesis activities were measured. In the samples in which the wild type TG was expressed, the peaks of panose and isomaltotriose were seen with respect to trisaccharides (FIG. 9, A). On the other hand, in W343M, the peaks of maltotriose and panose were seen with respect to the trisaccharides as in the previous results (FIG. 9, B). In W343M/S496T and W343D/V452G, which are TGs in which two amino acids have been substituted, only one peak was seen with respect to a trisaccharide, and the peak was anticipated to be of maltotriose (FIG. 9, the arrows in C and D). Furthermore, the peak seen in HPLC was only one also in the region of four saccharides, and this resulted in an analysis pattern greatly different from the analysis pattern of the wild type (FIGS. 9, C and D). This peak for the tetrasaccharide is a peak that is seen in only TGs in which two amino acids have been substituted, and shows that the generated oligosaccharide is different from the wild type.
The transglycosylation activities of the modified TGs were compared to that of the WT by quantifying the area surface areas of HPLC. In all of the TGs in which two amino acids had been substituted, the area surface areas of the generated oligosaccharides were larger than that in the WT, and the synthesis amount of the oligosaccharide of tri- or more saccharides increased by 20 to 50% (FIG. 10). Furthermore, in W343M/S496T, the synthesis amount of the oligosaccharides of tetra- or more saccharides was significantly larger than that of the WT, and increased by about 70% (FIG. 10).
On the other hand, the hydrolysis activities against glucosidic linkages of the modified TGs were studied. In W343M/S496T, the hydrolysis activities against maltose and maltotriose were each 50%, and the hydrolysis activity against isomaltose was 10% or less (FIG. 11). The hydrolysis activity against kojibiose was approximately 40%, and the hydrolysis activity against nigerose decreased to approximately 10% (FIG. 11). It is suggested that the hydrolysis activity decreased in the modified TGs in which two amino acids had been substituted, and that the hydrolysis activities against specifically α-1,6 glucosidic linkages and α-1,3 glucosidic linkages greatly decreased.
(5) Analysis of Oligosaccharides Formed by Modified TGs
According to the previous reports, when the number of saccharides in an initial substrate used for the synthesis of an oligosaccharide is increased, the ratio of the α-1,6 glucosidic linkages in the oligosaccharide formed by a TG increases. Therefore, oligosaccharide synthesis reactions were conducted by using maltopentaose, which is a pentasaccharide, as a substrate, and the formed oligosaccharides were compared between the wild type and modified TGs. First, as a standard, glucose (G=1), maltose (G=2), maltotriose (G=3) and maltopentaose (G=5) were measured, and the positions of the peaks were confirmed (FIG. 12). In the wild type TG, decomposition and production widely occurred from glucose to the areas of hepta- or more saccharides (FIG. 12). However, the ratio of penta- or less saccharides was large, and thus it was suggested that hydrolysis proceeded more than oligosaccharide synthesis did. On the other hand, in the modified TGs (W343M and W343M/S496T), saccharides that had been hydrolyzed to monosaccharide and disaccharide were decreased, and hexa- or more saccharides had been formed more (FIG. 12). In the areas indicated by the arrows in FIG. 12, the difference is specifically great between the WT and modified TGs.
Next, oligosaccharide synthesis was conducted in a mixed solution of maltose and isomaltose as a start substrate. Although the ratio of the generated oligosaccharides was not greatly changed, a difference appeared in the kinds of the generated trisaccharides. In the wild type, isomaltotriose, which has two α-1,6 glucosidic linkages, was formed much (FIG. 13). On the other hand, in the modified TGs (W343M and W343M/S496T), panose, which has an α-1,4 glucosidic linkage and an α-1,6 glucosidic linkage was formed most (FIG. 13, arrow). Furthermore, the isomaltose as a start substrate was decomposed little in the cases of modified TGs.
It was suggested by the above-mentioned results that the modified TGs specifically have a transglycosylation activity to form α-1,4 glucosidic linkages, in contrast to that the wild type TG mainly have a transglycosylation activity to form α-1,6 glucosidic linkages.
The amino acid sequences of the various modified TGs prepared in the investigation at this time are shown below.
SEQ ID NO. 18: W343C
SEQ ID NO. 19: W343D
SEQ ID NO. 20: W343M
SEQ ID NO. 21: W343H
SEQ ID NO. 22: W343A
SEQ ID NO. 23: W343F
SEQ ID NO. 24: W343G
SEQ ID NO. 25: W343T
SEQ ID NO. 26: W343E
SEQ ID NO. 27: W343V
SEQ ID NO. 28: W343Q
SEQ ID NO. 29: W343N
SEQ ID NO. 30: W343I
SEQ ID NO. 31: V452G
SEQ ID NO. 32: V452D
SEQ ID NO. 33: V452E
SEQ ID NO. 34: S496V
SEQ ID NO. 35: S496N
SEQ ID NO. 36: S496Q
SEQ ID NO. 37: W343D/V452A
SEQ ID NO. 38: W343D/V452G
SEQ ID NO. 39: W343D/S496I
SEQ ID NO. 40: W343D/S496R
SEQ ID NO. 41: W343M/S496C
SEQ ID NO. 42: W343M/S496T
B. Acquirement of Modified α-Glucosidase 2
(1) Study of Annotated Amino Acids Using Computer Software MOE
For the purpose of identifying annotated amino acids by a novel means, the annotated amino acids of TGs were studied by using computer software MOE (Chemical Computing Group). At this time, the annotated amino acids were selected by comparing the sequences of Aspergillus niger and Aspergillus nidulans. 
The amino acid sequences of the Aspergillus niger TG and Aspergillus nidulans TG (α-glucosidase B) were obtained from known data bank and read into MOE, and protein modeling was builted on a computer. Based on the respective amino acid sequences, sequences having high homology were searched on the database of MOE. As a result, the same sequence was hit in both cases, and human TG was selected. Subsequently, using the steric structures as templates, the respective proteins are modeled. The force field was “AMBER99”, five intermediate models were prepared and one model structure was finally prepared.
Subsequently, a docking simulation analysis was performed to thereby expect the substrate pockets. The force field was set to “MMFF99x”, and the pockets were searched by Site Finder of MOE. As a result, a plurality of candidate regions having possibility of becoming pockets in view of the steric structures were predicted. Therefore, amino acids that become the active center of TG reported in previous articles and the like were searched, and the position of each amino acid on a model was determined. Furthermore, the region containing a plurality of amino acids, which is an active center, was determined as a substrate pocket calculated by the simulation.
The steric structures that were successfully modeled are shown in FIG. 14. When the structures of Aspergillus niger TG and Aspergillus nidulans TG were compared, a significant difference was observed between the respective steric structures even though they were modeled based on the same steric structure of human TG When the simulated substrate pockets were compared, the substrate pocket of Aspergillus nidulans was smaller than that of Aspergillus niger. The substrate pocket was composed of 30 amino acids in Aspergillus niger, whereas the substrate pocket was composed of 22 amino acids in Aspergillus nidulans (FIG. 14).
Subsequently, the amino acid sequence of Aspergillus niger TG and the amino acid sequence of Aspergillus nidulans TG were aligned by CLASTAL-W. When the whole amino acid sequences were compared, the homology of these proteins was low, and the ratio at which the amino acids were identical was calculated as 35%. However, in the case where only the sequences of the substrate pockets were compared, the sequences had high homology, and 19 of 30 amino acids were identical in the substrate pocket of Aspergillus niger and 19 of 22 amino acids were identical in Aspergillus nidulans (FIG. 15). This result suggests that the amino acids relating to the activity are highly conserved as compared to the entire sequence. On the other hand, we predicted the amino acids that were not identical have low evolutionary conservation property, and thus it was expected that mutation can be introduced. Therefore, these amino acids were used as candidate annotated amino acids for introducing mutation (FIG. 15). Furthermore, since W343 and 5496 in which mutation had been already introduced were selected, it was thought that there is a correlation with simulation by Hot spots wizard using human TG that has been used in the past.
Primers were designed so as to actually introduce mutation into Aspergillus niger TG, and mutation was introduced by an inverse PCR method using a random primer. Transformation into Escherichia coli was conducted, and a sequence analysis was conducted after miniprep to confirm introduction of mutation. Furthermore, transformation into yeast was conducted, and a recombinant modified TG was expressed and analyzed. The analysis results for two amino acids: 5495 and C498 among the actual preparation are shown.
(2) Evaluation of S495-Modified TG
In Aspergillus niger TG, random mutation was introduced into the 495th serine (S495) by a PCR method (substitution of one amino acid). Sequence was conducted to identify the introduced point mutation, and the modified TG was analyzed.
These modified TGs were compared by measuring the oligosaccharide synthesis activities by using HPLC. A 50% maltose aqueous solution, an isomaltose aqueous solution, and an aqueous solution of maltopentaose, which is an oligosaccharide of a pentasaccharide, were used as substrates. The aqueous solutions were incubated at a reaction temperature of 50° C. for a predetermined time (48 hours for maltose and maltopentaose, and 96 hours for isomaltose).
The oligosaccharide synthesis activities of the modified TGs were compared by quantifying the area surface areas of HPLC. In the case where the substrate was maltose, the total of the area surface areas of the tri- or more saccharides and tetra- or more saccharides was larger than that of the WT in S495G but was conversely decreased in S495P and S495T (FIG. 16).
In the case where the substrate was isomaltose, the total of the area surface areas of the tri- or more saccharides was larger than that of the WT in S495G, S495P and S495V (FIG. 17). Furthermore, the total of the area surface areas of the tetra- or more saccharides was larger than that of the WT in S495G and S495P, and thus it was suggested that the oligosaccharide synthesis ability was improved (FIG. 17). When the peaks in the HPLC are observed, isomaltotriose increased in the trisaccharides, and thus it can be presumed that the modified TGs have a transglycosylation activity to form α-1,6 glucosidic linkages as in the WT. It was found from the above-mentioned results that the three kinds: S495G, S495P and S495V increase oligosaccharides with α-1,6 glucosidic linkages by 50% or more.
Therefore, it was thought that the reactivity against glucosidic linkages should differ in the modified TGs, and thus the hydrolysis activities against various glucosidic linkages were studied (FIG. 18). Using a maltose aqueous solution and an isomaltose aqueous solution as substrates, the amounts of the generated glucose were quantified to thereby evaluate the hydrolysis activities. It is shown that the WT has substrate affinity for both α-1,4 glucosidic linkages and α-1,6 glucosidic linkages against glucosidic linkages. On the other hand, the hydrolysis activity of maltose decreased more than in the WT whereas the hydrolysis activity of isomaltose relatively increased in S495A, S495G, S495P, S495T and S495V. As mentioned above, the modified TG in which 5495 had been substituted had increased transglycosylation activity to form α-1,6 glucosidic linkages.
Finally, oligosaccharide synthesis reactions were conducted by using maltopentaose, which is a pentasaccharide, as an initial substrate, and the generated oligosaccharides were compared between the wild type TG and modified TGs (FIG. 19). In the wild type TG, decomposition and generation occurred widely in the areas from glucose to hexa- or more saccharides. When a graph was drawn, the amount of monosaccharide was the largest, and hydrolysis proceeded during the reaction. Increase in the oligosaccharides was observed in all of the modified TGs. Specifically, in the modified TGs of S495P-type and S495V-type, the hydrolyzed saccharides were small, whereas hexa- or more saccharides were formed much (FIG. 19c).
As mentioned above, three kind of modified TGs having transglycosylation activity to form α-1,6 glucosidic linkages were successfully obtained. Furthermore, it was found that the oligosaccharides formed by the modified TGs are polymerized by a bonding manner including α1,6 glucosidic linkages to thereby increase the formation amounts of the oligosaccharides.
(3) Evaluation of C498-Modified TG
In Aspergillus niger TG, random mutation was introduced into the 498th cysteine (C498) by a PCR method (substitution of one amino acid). Sequence was conducted to identify the introduced point mutation, and the modified TG was analyzed.
These modified TGs were compared by measuring the oligosaccharide synthesis activities by using HPLC. Using a 60% maltose aqueous solution as a substrate, incubation was conducted at a reaction temperature of 50° C. for 48 hours.
The oligosaccharide synthesis activities of the modified TGs were compared by quantifying the area surface areas of HPLC (FIG. 20). When the totals of the area surface areas of tri- or more saccharides were compared, the area surface areas were larger in C498L and C498S than that of the WT. Furthermore, increase in the total of the area surface areas of tetra- or more saccharides was similarly observed in C498L and C498S as compared to the WT.
It was thought based on the results that the reactivity against glucosidic linkages could be changed in the modified TGs, and thus the hydrolysis activities were measured. Using a maltose aqueous solution and an isomaltose aqueous solution as substrates, the amounts of the generated glucose were quantified. As shown in FIG. 21, it was found that the hydrolysis activity of maltose was higher in C498L and C498S as compared to the WT. Furthermore, it is also understood that a significant change was observed in the ratio of the maltose hydrolysis activity and isomaltose hydrolysis activity and thus the specificity against α-1,4 glucosidic linkages was improved in these modified TGs.
As mentioned above, it was shown that the synthesis of oligosaccharides is increased in C498 by introducing mutation. Furthermore, two kinds of modified TGs (these show transglycosylation activity to form α-1,4 glucosidic linkages) in which the ability of synthesizing oligosaccharides had been enhanced were successfully obtained.
The amino acid sequences of the various modified TGs prepared in the investigation at this time are shown below.
SEQ ID NO. 74: S495G
SEQ ID NO. 75: S495P
SEQ ID NO. 76: S495V
SEQ ID NO. 77: C498L
SEQ ID NO. 78: C498S
C. Application of Modified TGs to Field of Foods
In order to evaluate the possibility of application of modified TGs to the field of health foods, an experiment using an artificial digestion model system was conducted. In this model system, how TGs act on oligosaccharides that are formed by decomposing porridge by amylase derived from saliva is evaluated. Amylase and TG were reacted with commercially available porridge at an acidic region being close to the pH of gastric juice (30 minutes to 2 hours, warmed at 37° C.), sampling was then conducted, and the enzymatic reaction was stopped. Furthermore, the generated oligosaccharides were analyzed by HPLC. The amount of the added amylase was 1.45 U/ml, which corresponds to the amount of the amylase in saliva. Furthermore, in light of the result of the preliminary research, the amount of TG was set to 20 μg/mL. The specific of the experimental protocol is shown below.
(Protocol)
(i) Porridge (Ajinomoto Co., Inc.) is crushed in a blender, and 1M NaOAc buffer (pH 5.0) is added in an amount of 1/20.
(ii) 340 μl of an enzyme solution is prepared (protein concentration: 20 μg/mL).
(iii) 0.65 g of the porridge of (i), 1.45 U of amylase and 340 μl of the enzyme solution are put into a glass test tube.
(iv) Incubation is conducted in a hot water bath at 37° C., and 200 μl is sampled into a micro test tube every 30 minutes.
(v) Boiling is conducted for 5 minutes to thereby stop the enzymatic reaction.
(vi) Centrifugation is conducted at 12,000 rpm, and the supernatant is transferred to a new tube.
(vii) 120 μl is sorted out and diluted to three-fold with MilliQ (registered trademark) water.
(viii) An HPLC analysis is conducted under the following conditions.
(Column Conditions)
Column; TSKGEL Amido-80
Solvent; MeCN/H2O=2/1
Detector; RI
Flow rate; 1 ml/min
The result of the analysis of the obtained oligosaccharides by HPLC is shown in FIG. 22. By adding amylase, 40 to 50% of the whole saccharides becomes maltose (FIG. 22 (a)). Furthermore, maltotriose, which is a trisaccharide, and maltotetraose, which is a tetrasaccharide, are formed. Glucose generates little, and decomposition does not proceed from disaccharides. In the case where a wild type (WT) TG was added, the generation amount of glucose increased over time, generation of isomaltose was observed from 60 minutes after the initiation of the reaction, and the oligosaccharides of tri- or more saccharides decreased over the passage of time (FIGS. 22 (b) and (d)). In the case where W343M, which is a modified TG having a transglycosylation activity to form α-1,4 glucosidic linkages is added, maltose is decomposed as in the case of the wild type TG and a transglycosylation reaction proceeds. At 30 minutes after the initiation of the reaction, the generated oligosaccharides are not significantly different from those in the wild type TG, and the peaks of maltotriose, maltose and glucose are observed (FIG. 22 (c)). On the other hand, W343M transfers maltose to oligosaccharides of tri- or more saccharides while suppressing the generation of glucose (FIG. 22 (c)). In contrast to the wild type TG, in the case where W343M is added, the decrease in the tri- or more oligosaccharides is small, and the amount of the finally generated oligosaccharides is dramatically large (FIG. 22 (d)). When the actual HPLC pattern (at 90 minutes after the initiation of the reaction) was seen, in the case where W343M was added, the peak of isomaltose was not observed, and the peaks of the oligosaccharides having larger sizes were seen (FIG. 23).
As mentioned above, when W343M, which is a modified TG, was used, the generation amount of isomaltose decreased as compared to the case where the wild type TG was used, whereas the generation amounts of the oligosaccharides with α-1,4 glucosidic linkages having long chain lengths such as maltotriose increased. When compared at 90 minutes after the initiation of the reaction, the generation amount of glucose in the modified TG decreased by 19.2%, and the generation amount of the oligosaccharides of tri- or more saccharides increased by 42.7%, as compared to that in the wild type TG Therefore, the usefulness of the modified TG, i.e., the modified TG is suitable for the generation of oligosaccharides even in an artificial digestion model system, was demonstrated.
D. Combination Use of Wild Type (WT) TG and Modified TG
(1) Synthesis of Oligosaccharides Using Maltose as Substrate
When a reaction for synthesizing oligosaccharides was attempted by using a modified TG having a transglycosylation activity to form α-1,6 glucosidic linkages and using maltose, which is a disaccharide with an α1,4 glucosidic linkage, as a substrate, the synthesis of oligosaccharides did not proceed. The reason therefor was considered that the modified TG having a transglycosylation activity to form α-1,6 glucosidic linkages has low reactivity against α-1,4 glucosidic linkages, and thus the reaction for synthesizing oligosaccharides does not sufficiently proceed even when maltose is used as a substrate. Therefore, it was thought that a reaction for synthesizing oligosaccharides could proceed even when maltose is used as a substrate, if an enzyme that supplies isomaltose is used in combination, and thus the synthesis of oligosaccharides was conducted by using a wild type TG having an enzyme activity to convert maltose to isomaltose and a modified TG (S495P) in combination. The generated oligosaccharides were analyzed by HPLC. In addition, a 50% maltose aqueous solution was used as a substrate, and the reaction was conducted at 50° C. for 48 hours.
The synthesis of oligosaccharides was conducted for the case of S495P alone and the cases where wild type and S495P were mixed (mixing ratio 2:1, 1:1 or 1:2), and the generated oligosaccharides were analyzed by HPLC. In the case of S495P alone, panose, which is a trisaccharide, is formed, whereas the generation of oligosaccharides of tetra- or more saccharides is very low. Namely, the reactions of the tetra- or more saccharides do not proceed (S495P in FIG. 24, and S495P in FIG. 25). On the other hand, in the case where the wild type and S495P were used in combination, both of the amount of the tri- or more oligosaccharides and the amount of the tetra- or more oligosaccharides increased in either mixing ratio (WT/S495P=2/1, WT/S495P=1/1 and WT/S495P=1/2 in FIG. 24). Of particular note is that most of the generated oligosaccharides was panose in the case of S495P alone, whereas in the case where the wild type and S495P were used in combination, generation of panose and isomaltotriose was observed (FIG. 25, the peaks indicated by the arrows), and the generation of oligosaccharides of tetra- or more saccharides was further observed. As mentioned above, combination use of the wild type and the modified TG having a transglycosylation activity to form α-1,6 glucosidic linkages was effective, and it was possible to generate oligosaccharides also from a saccharide with an α-1,4 glucosidic linkage on which a reaction does not proceed by single use of the modified TG having an α-1,6 transglycosylation activity.
(2) Application of Modified TG to Field of Foods
For a modified TG having a transglycosylation activity to form α-1,6 glucosidic linkages, an experiment using an artificial digestion model system was conducted. The protocol is the same as in the case of the above-mentioned C.
In the case where only amylase is added to porridge, 40 to 50% of the generated saccharides is maltose. Furthermore, maltotriose, which is a trisaccharide, and maltotetraose, which is a tetrasaccharide, are formed. Since all of these are saccharides with α-1,4 glucosidic linkages, it was thought that the reaction does not proceed by single use of a modified α-glucosidase having a transglycosylation activity to form α-1,4 glucosidic linkages. Therefore, similarly to (1) of D, synthesis of oligosaccharides was attempted by using a wild type TG and a modified TG (S495P) in combination.
First, in the case where amylase and the wild type were added to porridge, the reaction proceeded from after 30 minutes, and the generation amount of glucose gradually increased (FIG. 26 (a)). Furthermore, isomaltose was formed from 60 minutes after the initiation of the reaction, and the tri- or more oligosaccharides gradually decreased. On the other hand, in the case where amylase and S495P were added to porridge, only generation of saccharides by amylase occurred, and even at 120 minutes after the initiation of the reaction, most of the saccharides remained as maltose, maltotriose and maltotetraose (FIG. 26 (b)). In contrast, in the case where three kinds of enzymes: amylase, a wild type and S495P were used in combination (the mixing ratio of the wild type and S495P was 1:2), the decomposition of maltose occurred and a transglycosylation reaction proceeded. After 60 minutes after the initiation of the reaction, oligosaccharides of tri- or more saccharides increased as compared to that in the wild type alone (FIG. 26 (d)). In the case where three kinds of enzymes were used in combination, decrease in the maltose and increase in the ratio of the oligosaccharides of tri- or more saccharides were observed (FIG. 26 (c)), and thus it is thought that the synthesis of the oligosaccharides proceeded. When the actual HPLC pattern is seen, in the case where the three kinds of enzymes were used in combination, the peaks of the maltose and maltotriose generated by amylase decreased, whereas the peaks of panose and oligosaccharides of tetra- or more saccharides were observed (the arrows at the lower right of FIG. 27). Furthermore, it was found that the oligosaccharides of tri- or more saccharides decreased little as compared to the case where only the wild type was used in combination with amylase (FIG. 27, lower left), and thus it can be said that combination use of three kinds of enzymes (amylase, wild type and S495P) is effective for the generation of oligosaccharides.
Accordingly, it was shown that combination use of a wild type and a modified TG having a transglycosylation activity to form α-1,4 glucosidic linkages is extremely effective for generating oligosaccharides.
INDUSTRIAL APPLICABILITY
The modified α-glucosidase of the present invention has a high transglycosylation activity. By utilizing this property, utilization in the production (synthesis) of oligosaccharides is expected. Furthermore, the designing method of the present invention is useful as a means for enhancing the transglycosylation activities of wild type enzymes and the like, and thus can be used for the modification of various α-glucosidases.
This invention is not limited at all by the above-mentioned embodiments for carrying out the invention and the explanations in the Examples. Various modified embodiments are also encompassed in this invention within a scope that does not deviate from the recitation of the claims and can be easily conceived by a person skilled in the art. All of the contents of the articles, patent publications and patent gazettes that are clearly indicated in the present specification are incorporated herein by reference.

## Claims

1. A modified α-glucosidase which (a) consists of an amino acid sequence which is identical to SEQ ID NO: 2 except for a substitution at a position corresponding to position 495 of SEQ ID NO: 2, or (b) comprises an amino acid sequence which has 95% or more identity with SEQ ID NO: 2 and comprises a substitution at a position corresponding to position 495 of SEQ ID NO: 2,
wherein the amino acid at position 495 is substituted by a alanine, glycine, proline, threonine or valine, and
wherein the modified α-glucosidase has increased transglycosylation activity to form α-1,6 glucosidic linkages compared to the α-glucosidase of SEQ ID NO: 2.

2. A method for producing an oligosaccharide, comprising reacting the modified α-glucosidase of claim 1 with an oligosaccharide or polysaccharide having an α-1,4 glucosidic linkage.

3. A method for producing an oligosaccharide, comprising reacting the modified α-glucosidase of claim 1 with an oligosaccharide or polysaccharide having α-1,6 glucosidic linkage.

4. The modified α-glucosidase of claim 1, wherein said modified α-glucosidase comprises an amino acid sequence which has 95% or more identity with SEQ ID NO: 2 and comprises a substitution at a position corresponding to position 495 of SEQ ID NO: 2, wherein the amino acid at position 495 is substituted by a alanine, glycine, proline, threonine or valine, wherein the modified α-glucosidase has increased transglycosylation activity to form α-1,6 glucosidic linkages compared to the α-glucosidase of SEQ ID NO: 2, and wherein said modified α-glucosidase further comprises one or more amino acid substitutions at an amino acid selected from the group consisting of:
(i) an amino acid corresponding to the amino acid at position 343 of SEQ ID NO: 2;
(ii) an amino acid corresponding to the amino acid at position 452 of SEQ ID NO: 2;
(iii) an amino acid corresponding to the amino acid at position 496 of SEQ ID NO: 2;
(iv) an amino acid corresponding to the amino acid at position 410 of SEQ ID NO: 2;
(v) an amino acid corresponding to the amino acid at position 498 of SEQ ID NO: 2;
(vi) an amino acid corresponding to the amino acid at position 499 of SEQ ID NO: 2;
(vii) an amino acid corresponding to the amino acid at position 531 of SEQ ID NO: 2;
(viii) an amino acid corresponding to the amino acid at position 533 of SEQ ID NO: 2;
(ix) an amino acid corresponding to the amino acid at position 579 of SEQ ID NO: 2;
(x) an amino acid corresponding to the amino acid at position 585 of SEQ ID NO: 2;
(xi) an amino acid corresponding to the amino acid at position 662 of SEQ ID NO: 2;
(xii) an amino acid corresponding to the amino acid at position 715 of SEQ ID NO: 2; and
(xiii) an amino acid corresponding to the amino acid at position 721 of SEQ ID NO: 2.