Maltotriose-producing amylase

Abstract

To provide novel enzymes that produce maltotriose.SOLUTION: Disclosed is a maltotriose-producing amylase consisting of any of the following polypeptides: polypeptides consisting of any of amino acid sequences of SEQ ID NOs:1-3; polypeptides consisting of any of amino acid sequences of SEQ ID NOs:1-3 with one or more amino acid substitution, addition, insertion or deletion and capable of maltotriose-production; and polypeptides having 70% or more sequence identity to any of amino acid sequences of SEQ ID NOs:1-3 and capable of maltotriose-production.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to maltotriose-producing amylase. [Background technology]

[0002] Maltotriose is a sugar obtained by partially decomposing starch with enzymes or acids, in which three glucose molecules are linked by an α-1,4-glucosidic bond. While its sweetness is about 17% of that of sugar, its mild sweetness makes it suitable for use as a low-sweetener in food. Furthermore, due to its excellent hygroscopic and water-retaining properties, maltotriose is useful as a food desiccant, a sugar crystal precipitation inhibitor, and a starch retrogradation inhibitor. Additionally, because maltotriose has superior thermal stability compared to glucose and maltose, it is also useful as a carbohydrate source in food processing.

[0003] Maltotriose can be obtained as a by-product of maltose production. However, from the perspective of stable industrial supply, it is desirable to produce it by enzymatically decomposing starch. Known enzymes that produce maltotriose from starch include the N-A468 enzyme from Streptomyces griseus (Non-Patent Literature 1) and amylase G3 from Bacillus subtilis (Non-Patent Literature 2). The N-A468 enzyme is classified similarly to β-amylase and decomposes starch in maltotriose units, while amylase G3 is classified similarly to α-amylase and produces starch hydrolysates mainly composed of maltotriose. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Starch Science, Vol. 26, No. 3, pp. 175-181 (1979) [Non-Patent Document 2] Agricultural and Biological Chemistry 1985 Volume 4 [Overview of the project] [Problems that the invention aims to solve]

[0005] However, these enzymes have low production efficiency from microorganisms, and there are still challenges in terms of stable industrial supply. Therefore, the acquisition of new enzymes that produce maltotriose is desirable.

[0006] The object of the present invention is to provide a novel enzyme that produces maltotriose. [Means for solving the problem]

[0007] As a result of diligent research, the inventors have discovered a maltotriose-producing amylase (hereinafter referred to as "maltotriose-producing amylase") derived from bacteria of the genus Cellulosimicrobium. The present invention is completed based on these findings. That is, the present invention provides the invention in the following embodiments.

[0008] Item 1. Maltotriose-producing amylases comprising polypeptides shown in any of the following (1-1)~(1-3), (2-1)~(2-3), and (3-1)~(3-3): (1-1) A polypeptide consisting of the amino acid sequence shown in Sequence ID No. 1, (1-2) A polypeptide having the ability to produce maltotriose, wherein one or more amino acids are substituted, added, inserted, or deleted in the amino acid sequence shown in Sequence ID No. 1. (1-3) A polypeptide having sequence identity of 70% or more to the amino acid sequence shown in Sequence ID No. 1, and having the ability to produce maltotriose. (2-1) A polypeptide consisting of the amino acid sequence shown in Sequence ID No. 2, (2-2) A polypeptide having the ability to produce maltotriose, wherein one or more amino acids are substituted, added, inserted, or deleted in the amino acid sequence shown in Sequence ID No. 2. (2-3) A polypeptide having a sequence identity of 70% or more with respect to the amino acid sequence shown in Sequence ID No. 2, and having the ability to produce maltotriose. (3-1) A polypeptide consisting of the amino acid sequence shown in Sequence ID No. 3, (3-2) A polypeptide having the ability to produce maltotriose, wherein one or more amino acids are substituted, added, inserted, or deleted in the amino acid sequence shown in Sequence ID No. 3. (3-3) A polypeptide having a sequence identity of 70% or more with respect to the amino acid sequence shown in SEQ ID NO: 3, and possessing the ability to produce maltotriose. Item 2. The DNA encoding the maltotriose-producing amylase described in Item 1. Item 3. A recombinant vector containing the DNA described in Item 2. Item 4. A transformant obtained by transforming a host with the DNA described in Item 2 or the recombinant vector described in Item 3. Item 5. A method for producing maltotriose-producing amylase according to Item 1, comprising the step of culturing the transformant described in Item 4. Item 6. An enzyme preparation containing the maltotriose-producing amylase described in Item 1. Item 7. A method for producing maltotriose, comprising the step of producing maltotriose from starch using the maltotriose-producing amylase described in Item 1. [Effects of the Invention]

[0009] The present invention provides a novel enzyme that produces maltotriose. [Brief explanation of the drawing]

[0010] [Figure 1] The results of SDS-PAGE purification of α-amylase with maltotriose-producing activity obtained from bacteria of the genus Cellulosimicrobium, as obtained in Test Example 1, are shown. [Modes for carrying out the invention]

[0011] Hereinafter, the present invention will be described in detail. In addition to the sequence listing, the 20 types of amino acid residues in the amino acid sequence may be represented by one-letter abbreviations. That is, glycine (Gly) is represented by G, alanine (Ala) is represented by A, valine (Val) is represented by V, leucine (Leu) is represented by L, isoleucine (Ile) is represented by I, phenylalanine (Phe) is represented by F, tyrosine (Tyr) is represented by Y, tryptophan (Trp) is represented by W, serine (Ser) is represented by S, threonine (Thr) is represented by T, cysteine (Cys) is represented by C, methionine (Met) is represented by M, aspartic acid (Asp) is represented by D, glutamic acid (Glu) is represented by E, asparagine (Asn) is represented by N, glutamine (Gln) is represented by Q, lysine (Lys) is represented by K, arginine (Arg) is represented by R, histidine (His) is represented by H, and proline (Pro) is represented by P.

[0012] In addition, in this specification, the amino acid sequence to be displayed has the N-terminus at the left end and the C-terminus at the right end.

[0013] In this specification, "nonpolar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. "uncharged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. "acidic amino acids" include aspartic acid and glutamic acid. "basic amino acids" include lysine, arginine, and histidine.

[0014] <0000​​​​​​​​​​​​(1-2) A polypeptide having the ability to produce maltotriose, wherein one or more amino acids are substituted, added, inserted, or deleted in the amino acid sequence shown in Sequence ID No. 1. (1-3) A polypeptide having a sequence identity of 70% or more with respect to the amino acid sequence shown in SEQ ID NO: 1, and possessing the ability to produce maltotriose.

[0017] The polypeptides described in (1-1) to (1-3) above have α-amylase activity that cleaves the α-1,4-bond of starch and also have the activity to produce maltotriose.

[0018] The amino acid modifications introduced into the polypeptide (1-2) above may include only one type of modification (e.g., substitution) from among substitution, addition, insertion, and deletion, or may include two or more types of modifications (e.g., substitution and insertion). In the polypeptide (1-2) above, the number of amino acids that are substituted, added, inserted, or deleted may be one, more than one, or several, for example, 1 to 10, preferably 1 to 8, 1 to 6, 1 to 5, or 1 to 4, more preferably 1 to 3, and particularly preferably 1 or 2 or 1.

[0019] Furthermore, in the polypeptides described in (1-3) above, the sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 should be 70% or more, but preferably 80% or more, 85% or more, 90% or more, more preferably 95% or more, 97% or more, 98% or more, and especially preferably 99% or more.

[0020] Here, in the polypeptides described in (1-3) above, the sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 is the sequence identity calculated by comparing it with the amino acid sequence shown in SEQ ID NO: 1. Furthermore, "sequence identity" refers to the value of amino acid sequence identity obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247-250, 1999) from BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)]. The parameters should be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

[0021] In the polypeptides described in (1-2) and (1-3) above, the amino acids at positions 196, 223, 284, 107, 166, 118, 119, 122, 123, 158, 161, 214, 215, 216, 217, 247, 250, 252, 253, 278, 283, 336, and 342 in the amino acid sequence shown in Sequence ID No. 1 are thought to contribute to activity, and therefore it is desirable not to introduce substitutions or deletions at these sites. The amino acids at positions 107 and 166 are presumed to be Ca binding sites, and the amino acids at positions 196, 223, and 284 are presumed to be active sites, so it is particularly desirable not to introduce substitutions or deletions at these sites.

[0022] When amino acid substitutions are introduced into the polypeptides of (1-2) and (1-3) above, one type of amino acid substitution is a conservative substitution. Specifically, in the polypeptides of (1-2) and (1-3), examples of amino acid substitutions introduced into the amino acid sequence shown in SEQ ID NO: 1 include substitution with another nonpolar amino acid if the amino acid before substitution is a nonpolar amino acid, substitution with another noncharged amino acid if the amino acid before substitution is an uncharged amino acid, substitution with another acidic amino acid if the amino acid before substitution is an acidic amino acid, and substitution with another basic amino acid if the amino acid before substitution is a basic amino acid.

[0023] In the polypeptides described in (1-2) and (1-3) above, "having maltotriose-producing ability" means having sufficient activity to function as a maltotriose-producing amylase. Specifically, it means that the detection of a maltotriose peak can be confirmed using the "<Method for Measuring the Activity of Maltotriose-Producing Amylase>" described later. Preferably, the maltotriose-producing ability observed in relation to starch is equivalent to or higher than that of the polypeptide described in (1-1) above (for example, the peak intensity ratio of maltotriose detected by the "<Method for Measuring the Activity of Maltotriose-Producing Amylase>" is 70% or higher than that of the polypeptide described in (1-1) above).

[0024] The molecular weight of the maltotriose-producing amylase consisting of polypeptide (1-1) of the first embodiment is approximately 55 kDa.

[0025] A second embodiment of the maltotriose-producing amylase of the present invention is a polypeptide shown in any of the following (2-1) to (2-3).

[0026] (2-1) A polypeptide consisting of the amino acid sequence shown in Sequence ID No. 2. (2-2) A polypeptide having the ability to produce maltotriose, wherein one or more amino acids are substituted, added, inserted, or deleted in the amino acid sequence shown in Sequence ID No. 2. (2-3) A polypeptide having a sequence identity of 70% or more with respect to the amino acid sequence shown in Sequence ID No. 2, and possessing the ability to produce maltotriose.

[0027] The polypeptide (2-1) described above includes the polypeptide (1-1) of the first embodiment, and further has a carbohydrate-binding module (CBM region) on its C-terminal side. Positions 1 to 533 of the amino acid sequence shown in SEQ ID NO: 2 correspond to the polypeptide (1-1). Positions 582 to 674 of the amino acid sequence shown in SEQ ID NO: 2 are considered to correspond to the CBM region. The CBM region is thought to localize maltotriose-producing amylase on the surface of the substrate (starch) to improve the efficiency of hydrolysis. The CBM region may be directly or indirectly bound to the C-terminal side of any polypeptide shown in (1-1) to (1-3) of the first embodiment. Indirect binding means binding via another amino acid sequence, such as a linker sequence consisting of 1 to 250, preferably 10 to 220, amino acids.

[0028] The amino acid modification introduced into the polypeptide described in (2-2) above may include only one type of modification (e.g., substitution) from among substitution, addition, insertion, and deletion, or it may include two or more types of modifications (e.g., substitution and insertion). In the region corresponding to the CBM region described in (2-2) above, the number of amino acids that are substituted, added, inserted, or deleted may be one, more than one, or several, for example, 1 to 10, preferably 1 to 8, 1 to 6, 1 to 5, or 1 to 4, more preferably 1 to 3, and particularly preferably 1 or 2 or 1.

[0029] Furthermore, in the polypeptides described in (2-3) above, the sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 2 should be 70% or more, but preferably 80% or more, 85% or more, 90% or more, more preferably 95% or more, 97% or more, 98% or more, and especially preferably 99% or more.

[0030] Here, in the polypeptide of (2-3) above, the sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 2 is the sequence identity calculated by comparing it with the amino acid sequence shown in SEQ ID NO: 2, and "sequence identity" is the same as the sequence identity in (1-3) of the first embodiment.

[0031] In the polypeptides of (2-2) and (2-3) described above, the amino acid positions in SEQ ID NO: 2 where substitution or deletion is not desirable are the same as the amino acid positions in SEQ ID NO: 1 in the polypeptides of (1-2) and (1-3) of the first embodiment where substitution or deletion is not desirable. Specifically, the amino acids at positions 196, 223, 284, 107, 166, 118, 119, 122, 123, 158, 161, 214, 215, 216, 217, 247, 250, 252, 253, 278, 283, 336, and 342 in the amino acid sequence shown in SEQ ID NO: 2 are considered to contribute to activity, and therefore it is desirable not to introduce substitution or deletion at these sites. The amino acids at positions 107 and 166 are presumed to be Ca binding sites, and the amino acids at positions 196, 223, and 284 are presumed to be active sites; therefore, it is particularly desirable not to introduce substitutions or deletions in these sites. Furthermore, regarding the CBM region, the amino acids at positions 608, 639, 651, 652, and 656 are thought to contribute to starch binding; therefore, if the goal is to localize maltotriose-producing amylase to the surface of the substrate (starch), it is desirable not to introduce substitutions or deletions in these sites.

[0032] When amino acid substitutions are introduced into the polypeptides of (2-2) and (2-3) above, conservative substitutions can be considered as forms of amino acid substitution. The conservative substitutions are the same as the conservative substitutions in the first embodiment when amino acid substitutions are introduced into the polypeptides of (1-2) and (1-3) above.

[0033] In the polypeptides described in (2-2) and (2-3) above, "having maltotriose-producing ability" means having sufficient activity to function as a maltotriose-producing amylase. Specifically, it means that the maltotriose peak can be detected using the "<Method for Measuring the Activity of Maltotriose-Producing Amylase>" described later. Preferably, the maltotriose-producing ability observed in relation to starch is equivalent to or higher than that of the polypeptide described in (2-1) above (for example, the peak intensity ratio of maltotriose detected by the "<Method for Measuring the Activity of Maltotriose-Producing Amylase>" is 70% or higher than that of the polypeptide described in (2-1) above).

[0034] The molecular weight of the maltotriose-producing amylase consisting of the polypeptide (2-1) of the second embodiment is approximately 70 kDa.

[0035] A third embodiment of the maltotriose-producing amylase of the present invention is a polypeptide shown in any of the following (3-1) to (3-3).

[0036] (3-1) A polypeptide consisting of the amino acid sequence shown in Sequence ID No. 3. (3-2) A polypeptide having the ability to produce maltotriose, wherein one or more amino acids are substituted, added, inserted, or deleted in the amino acid sequence shown in Sequence ID No. 3. (3-3) A polypeptide having a sequence identity of 70% or more with respect to the amino acid sequence shown in Sequence ID No. 3, and also having the ability to produce maltotriose.

[0037] The polypeptide (3-1) also includes the polypeptide (1-1) of the first embodiment. More specifically, the polypeptide (3-1) includes the polypeptide (2-1) of the second embodiment and further has a pro sequence at its N-terminus. Positions 54 to 586 of Sequence ID No. 3 correspond to the polypeptide (1-1) of the first embodiment, and positions 54 to 728 correspond to the polypeptide (2-1) of the second embodiment.

[0038] The amino acid modification introduced into the polypeptide (3-2) above may consist of only one type of modification (e.g., substitution) from among substitution, addition, insertion, and deletion, or it may consist of two or more types of modifications (e.g., substitution and insertion). In the polypeptide (3-2) above, the number of amino acids that are substituted, added, inserted, or deleted may be one, more than one, or several, for example, 1 to 10, preferably 1 to 8, 1 to 6, 1 to 5, or 1 to 4, more preferably 1 to 3, and particularly preferably 1 or 2 or 1.

[0039] Furthermore, in the polypeptide described in (3-3) above, the sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 3 should be 70% or more, but preferably 80% or more, 85% or more, 90% or more, more preferably 95% or more, 97% or more, 98% or more, and especially preferably 99% or more.

[0040] Here, in the polypeptide of (3-3) above, the sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 3 is the sequence identity calculated by comparing it with the amino acid sequence shown in SEQ ID NO: 3, and "sequence identity" is the same as the sequence identity in (1-3) of the first embodiment.

[0041] When amino acid substitutions are introduced into the polypeptides of (3-2) and (3-3) above, conservative substitutions can be considered as forms of amino acid substitution. The conservative substitutions are the same as the conservative substitutions in the first embodiment when amino acid substitutions are introduced into the polypeptides of (1-2) and (1-3) above.

[0042] In the polypeptides of (3-2) and (3-3) described above, the amino acid positions in SEQ ID NO: 3, where substitution or deletion is not desirable, are as previously described, as the amino acid positions in SEQ ID NO: 1, where substitution or deletion is not desirable in the polypeptides of (1-2) and (1-3) of the first embodiment.

[0043] In the polypeptides described in (3-2) and (3-3) above, "having maltotriose-producing ability" means having sufficient activity to function as a maltotriose-producing amylase. Specifically, it means that the maltotriose peak can be detected using the "<Method for Measuring the Activity of Maltotriose-Producing Amylase>" described later. Preferably, the maltotriose-producing ability observed in relation to starch is equivalent to or higher than that of the polypeptide described in (3-1) above (for example, the peak intensity ratio of maltotriose detected by the "<Method for Measuring the Activity of Maltotriose-Producing Amylase>" is 70% or higher than that of the polypeptide described in (3-1) above).

[0044] The molecular weight of the maltotriose-producing amylase consisting of the polypeptide of the third embodiment (3-1) is approximately 77 kDa.

[0045] The enzymatic activity of the maltotriose-producing amylase of the present invention can be measured by the method shown below. <Method for measuring the activity of maltotriose-producing amylase> A reaction solution is prepared by mixing 200 μL of substrate solution (1% (w / v) soluble starch solution) and 50 μL of enzyme solution. The mixture is shaken at 1000 rpm at 50°C for 48 hours, and then the reaction is stopped by boiling at 100°C for 5 minutes. The reaction solution is diluted 30-fold with purified water and filtered through a 0.45 μm filter to obtain an analytical sample. The maltotriose-producing amylase activity is measured by detecting the maltotriose peak in the analytical sample using HLPC analysis.

[0046] 2. DNA The DNA of the present invention is DNA encoding the maltotriose-producing amylase described above. The DNA of the present invention can be obtained, for example, by using DNA encoding any of the polypeptides (3-1) to (3-3) above, derived from bacteria of the genus Cellulosimicrobium, as a template, and obtaining at least the region encoding any of the polypeptides (1-1) to (1-3) above by PCR or the like. Furthermore, the DNA encoding the maltotriose-producing amylase of the present invention can also be artificially synthesized by gene synthesis methods.

[0047] Furthermore, when introducing specific mutations to specific sites in a base sequence, the methods for introducing mutations are publicly known, and for example, site-directed mutagenesis of DNA can be used. As for specific methods for converting bases in DNA, for example, commercially available kits can be used.

[0048] DNA into which mutations have been introduced into the base sequence can be sequenced using a DNA sequencer. Once the base sequence is determined, the DNA encoding the maltotriose-producing amylase can then be obtained by chemical synthesis, PCR using a cloned probe as a template, or hybridization using a DNA fragment containing the sequence as a probe.

[0049] Furthermore, mutant versions of the DNA encoding the maltotriose-producing amylase that have the same function as the pre-mutation DNA can be synthesized by site-directed mutagenesis or the like. The mutation can be introduced into the DNA encoding the maltotriose-producing amylase using known methods such as the Kunkel method, gapped duplex method, or megaprimer PCR method.

[0050] Those skilled in the art can appropriately design the base sequence of the DNA encoding the maltotriose-producing amylase of the present invention according to the amino acid sequence of the maltotriose-producing amylase of the present invention.

[0051] A first embodiment of the DNA of the present invention is DNA encoding the polypeptides (1-1) to (1-3) described above. For example, an example of DNA encoding the polypeptide (1-1) is DNA consisting of the base sequence shown in Sequence ID No. 4.

[0052] Furthermore, the first embodiment of the DNA of the present invention includes DNA that encodes the polypeptides (1-1) to (1-3) and contains a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 4, and that hybridizes under stringent conditions.

[0053] Furthermore, the first embodiment of the DNA of the present invention also includes DNA that encodes the polypeptides (1-1) to (1-3) and has 70% or more homology to the DNA consisting of the base sequence shown in SEQ ID NO: 4. Preferably, the homology is 80% or more, 90% or more, more preferably 95% or more, 97% or more, 98% or more, and particularly preferably 99% or more.

[0054] A second embodiment of the DNA of the present invention is DNA encoding any of the polypeptides described in (2-1) to (2-3) above. For example, an example of DNA encoding the polypeptide described in (2-1) is DNA consisting of the base sequence shown in Sequence ID No. 5.

[0055] Furthermore, a second embodiment of the DNA of the present invention includes DNA that hybridizes under stringent conditions with DNA that encodes the polypeptides of (2-1) to (2-3) and has a base sequence complementary to the DNA consisting of the base sequence shown in Sequence ID No. 5.

[0056] Furthermore, the second embodiment of the DNA of the present invention also includes DNA that encodes the polypeptides of (2-1) to (2-3) and has 70% or more homology to the DNA consisting of the base sequence shown in Sequence ID No. 5. The homology is preferably 80% or more, 90% or more, more preferably 95% or more, 97% or more, 98% or more, and particularly preferably 99% or more.

[0057] A third embodiment of the DNA of the present invention is DNA encoding any of the polypeptides described in (3-1) to (3-3) above. For example, an example of DNA encoding the polypeptide described in (3-1) is DNA consisting of the base sequence shown in SEQ ID NO: 6.

[0058] Furthermore, a third embodiment of the DNA of the present invention includes DNA that encodes the polypeptides of (3-1) to (3-3) above and contains a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 6, and that hybridizes under stringent conditions.

[0059] Furthermore, a third embodiment of the DNA of the present invention also includes DNA that encodes the polypeptides of (3-1) to (3-3) and has 70% or more homology to the DNA consisting of the base sequence shown in SEQ ID NO: 6. Preferably, the homology is 80% or more, 90% or more, more preferably 95% or more, 97% or more, 98% or more, and particularly preferably 99% or more.

[0060] Here, "stringent conditions" refers to conditions in which a sample is incubated at 50°C to 65°C for 4 hours to overnight in a 6x SSC (1x SSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% SDS, 5x Denhartz's (0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficol 400), and 100 μg / ml salmon sperm DNA.

[0061] Hybridization under stringent conditions is specifically performed by the following method: A nylon membrane immobilizing a DNA library or cDNA library is prepared, and the nylon membrane is blocked at 65°C in a pre-hybridization solution containing 6×SSC, 0.5% SDS, 5×Denharts, and 100 μg / ml salmon sperm DNA. Then, 32Each probe labeled with P is added and incubated overnight at 65°C. After washing this nylon membrane in 6×SSC for 10 minutes at room temperature, in 2×SSC containing 0.1% SDS for 10 minutes at room temperature, and in 0.2×SSC containing 0.1% SDS for 30 minutes at 45°C, autoradiography can be performed to detect DNA that specifically hybridizes with the probe.

[0062] Furthermore, DNA "homology" refers to the identity value obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol.174, 247-250, 1999) in BLAST PACKAGE [sgi32 bitedition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]. The parameters should be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

[0063] The DNA of the present invention is preferably one whose codon usage frequency is optimized for the host. For example, if E. coli is used as the host, DNA with codon usage frequency optimized for E. coli is preferable.

[0064] 3. Recombinant vectors The recombinant vector of the present invention contains DNA encoding the maltotriose-producing amylase of the present invention. The recombinant vector of the present invention can be obtained by inserting the DNA of the present invention into an expression vector.

[0065] The recombinant vector of the present invention includes regulatory factors such as promoters operably linked to the DNA of the present invention. Typical regulatory factors include promoters, but may also include enhancers, CCAAT boxes, TATA boxes, SPI sites, and other transcription elements as needed. Operable linkage means that the DNA of the present invention is linked to various regulatory factors such as promoters and enhancers that regulate the DNA of the present invention in a manner that allows it to function within a host cell.

[0066] Preferred expression vectors are those constructed for genetic recombination from phages, plasmids, or viruses that can autonomously proliferate within a host. Such expression vectors are well known, and commercially available examples include pQE vectors (Qiagen Co., Ltd.), pDR540, pRIT2T (GE Healthcare Biosciences Co., Ltd.), and pET vectors (Merck KGaA). The expression vector can be used in any combination with an appropriate host cell. For example, when using Escherichia coli as the host cell, a combination of a pET vector and the BL21(DE3) strain of Escherichia coli, or a combination of a pDR540 vector and the JM109 strain of Escherichia coli, are preferred.

[0067] 4. Transformed organism The transformant of the present invention is obtained by transforming a host using the DNA of the present invention described above or the recombinant vector of the present invention described above.

[0068] The host used in the production of the transformant is not particularly limited as long as it is capable of gene introduction, autonomous proliferation, and expression of the gene traits of the present invention. For example, bacteria belonging to the Escherichia genus such as Escherichia coli, the Bacillus genus such as Bacillus subtilis, the Pseudomonas genus such as Pseudomonas putida, etc., actinomycetes, yeasts, filamentous fungi, etc., are preferred examples, but animal cells, insect cells, plants, etc., may also be used. Among these, Escherichia coli is particularly preferred. The host may also be a Cellulosimicrobium genus, which is the source of the maltotriose-producing amylase of the present invention.

[0069] A transformant of the present invention can be obtained by introducing the DNA of the present invention or the recombinant vector of the present invention into a host. The method of introducing the DNA of the present invention or the recombinant vector of the present invention is not particularly limited as long as the target gene is introduced into the host. Furthermore, the site on which the DNA is introduced is not particularly limited as long as the target gene can be expressed, and may be on a plasmid or on the genome. Specific methods for introducing the DNA of the present invention or the recombinant vector of the present invention include, for example, recombinant vector methods and genome editing methods.

[0070] The conditions for introducing the DNA or recombinant vector of the present invention into a host can be appropriately set depending on the introduction method and the type of host. If the host is a bacterium, for example, methods using competent cells treated with calcium ions and electroporation can be used. If the host is a yeast, for example, electroporation, spheroplast, and lithium acetate can be used. If the host is an animal cell, for example, electroporation, calcium phosphate, and lipofection can be used. If the host is an insect cell, for example, calcium phosphate, lipofection, and electroporation can be used. If the host is a plant, for example, electroporation, Agrobacterium, particle gun, and PEG can be used.

[0071] 5. Method for producing maltotriose-producing amylase The maltotriose-producing amylase of the present invention can be produced by culturing the transformant of the present invention. Alternatively, the maltotriose-producing amylase of the present invention can also be produced by culturing the production bacterium, Cellulosimicrobacterium sp. (untransformed). When the production bacterium, Cellulosimicrobacterium sp. (untransformed), is cultured, the maltotriose-producing amylase of the present invention is produced in a mixture of polypeptides (1-1) to (1 to 3) (polypeptide of the first embodiment), polypeptides (2-1) to (2 to 3) (polypeptide of the second embodiment), and polypeptides (3-1) to (3 to 3) (polypeptide of the third embodiment). On the other hand, when using the transformant of the present invention, the introduced DNA makes it possible to express in the host only the gene encoding one of the polypeptides of the first embodiment, the second embodiment, or the third embodiment. Therefore, it is possible to produce a maltotriose-producing amylase consisting of one of the polypeptides of the first embodiment, the second embodiment, or the third embodiment alone, or to produce it in a mixture of the polypeptides of the first embodiment, the second embodiment, and the third embodiment.

[0072] The culture conditions for the transformants of the present invention can be appropriately set considering the nutritional and physiological properties of the host, but liquid culture is preferred. Furthermore, in the case of industrial production, aerated and agitated culture is preferred.

[0073] The transformant of the present invention is cultured, and the culture supernatant or bacterial cells are recovered from the culture medium by methods such as centrifugation. If the maltotriose-producing amylase of the present invention is accumulated in the bacterial cells, the bacterial cells are treated with mechanical methods such as ultrasound or French press, or with lytic enzymes such as lysozyme, and solubilized by using enzymes such as proteases or surfactants such as sodium dodecyl sulfate (SDS) as needed to obtain a water-soluble fraction containing the maltotriose-producing amylase of the present invention.

[0074] Furthermore, by selecting an appropriate expression vector and host, the expressed maltotriose-producing amylase of the present invention can be secreted into the culture medium.

[0075] The culture medium or water-soluble fraction containing the maltotriose-producing amylase of the present invention obtained as described above may be subjected to purification treatment as is, or the polypeptide of the present invention in the culture medium or water-soluble fraction may be concentrated before being subjected to purification treatment.

[0076] Concentration can be carried out, for example, by vacuum concentration, membrane concentration, salting-out treatment, or fractional precipitation using hydrophilic organic solvents (e.g., methanol, ethanol, and acetone).

[0077] The purification of the maltotriose-producing amylase of the present invention can be carried out by appropriately combining methods such as gel filtration, hydrophobic chromatography, ion exchange chromatography, and affinity chromatography.

[0078] The maltotriose-producing amylase of the present invention, purified in this manner, may be powdered by freeze-drying, vacuum drying, spray drying, or the like, if necessary.

[0079] 6. Enzyme preparations The enzyme preparation of the present invention contains the maltotriose-producing amylase of the present invention as an active ingredient. The maltotriose-producing amylase contained in the enzyme preparation may consist only of maltotriose-producing amylase composed of any polypeptide from (1-1) to (1 to 3), or only of maltotriose-producing amylase composed of any polypeptide from (2-1) to (2-3), or only of maltotriose-producing amylase composed of any polypeptide from (3-1) to (3-3). Multiple of the above maltotriose-producing amylases may be contained. The type and content ratio of the maltotriose-producing amylases included can be appropriately selected depending on the substrate.

[0080] The enzyme preparation of the present invention may contain, in addition to the maltotriose-producing amylase of the present invention described above, additives selected from the group consisting of excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, and physiological saline. Examples of excipients include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, and glycerol. Examples of buffers include phosphates, citrates, and acetates. Examples of stabilizers include propylene glycol and ascorbic acid. Examples of preservatives include sodium chloride, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of antiseptics include sodium chloride, ethanol, benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol.

[0081] The content of maltotriose-producing amylase in the enzyme preparation of the present invention is appropriately set within a range in which the effect of maltotriose-producing amylase is exerted.

[0082] The enzyme preparation of the present invention may contain other enzymes. Examples of other enzymes include amylase (α-amylase, β-amylase, glucoamylase), glucosidase (α-glucosidase, β-glucosidase), galactosidase (α-galactosidase, β-galactosidase), protease (acid protease, neutral protease, alkaline protease), peptidase (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acid phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, dehydrogenase, glutaminase, pectinase, catalase, dextranase, transglutaminase, protein deamide enzyme, pullulanase, and the like.

[0083] The form of the enzyme preparation of the present invention is not particularly limited, but examples include liquid, powder, granules, etc. The enzyme preparation of the present invention can be prepared by generally known methods.

[0084] 7. Method for producing maltotriose The present invention provides a method for producing maltotriose, which includes a step of producing maltotriose from starch using the maltotriose-producing amylase of the present invention described above.

[0085] Examples of starches include amylose, amylopectin, glycogen, and starch; as well as partially hydrolyzed starch products such as amylodextrin, maltodextrin, and maltooligosaccharides obtained by partially hydrolyzing them with amylase or acid. Examples of partially hydrolyzed starch products obtained by hydrolyzing amylase, such as α-amylase (EC 3.2.1.1), maltopentaose-producing amylase, and maltohexaose-producing amylase (EC 3.2.1.98), include partially hydrolyzed products obtained by hydrolyzing amylose, amylopectin, glycogen, and starch. Furthermore, when preparing the partially hydrolyzed products, starch branching enzymes such as pullulanase (EC 3.2.1.41) and isoamylase (EC 3.2.1.68) may be used.

[0086] Examples of starches include those derived from corn, wheat, rice, potatoes, sweet potatoes, and tapioca. These starches can be used in the form of liquefied starch solutions obtained by gelatinization and liquefaction.

[0087] When the maltotriose-producing amylase of the present invention is reacted with starch, the concentration of the starch solution is not particularly limited, but from an industrial standpoint, a concentration of 10% (w / v) or higher is preferred. The reaction temperature is not particularly limited as long as the reaction proceeds at that temperature, but specifically, up to about 65°C, preferably 45-60°C. The reaction pH is usually 5-9, preferably 5.5-7.5. The amount of enzyme used can be appropriately selected depending on the rate at which the desired enzymatic reaction proceeds.

[0088] Furthermore, during this maltotriose production reaction, other enzymes may be used simultaneously to increase the maltotriose content in the saccharified solution. For example, the maltotriose content of the saccharified solution can be increased by using starch debranching enzymes such as pullulanase and isoamylase in combination.

[0089] The reaction solution obtained by the above reaction may be used as is as a maltotriose-containing sugar solution, but preferably, the maltotriose-containing sugar solution is further purified. For the purification method, any conventional method used for sugar purification may be used, for example, decolorization with activated carbon, H + Type, OH - Examples of purification methods include desalting with ion exchange resins, fractionation by column chromatography such as ion exchange column chromatography, activated carbon column chromatography, and silica gel column chromatography, separation with organic solvents such as alcohol and acetone, and separation with membranes having appropriate separation performance.

[0090] One method for obtaining high-purity maltotriose-containing carbohydrates is ion-exchange column chromatography. Specifically, by removing contaminating sugars using column chromatography with a strongly acidic cation exchange resin, maltotriose or carbohydrates containing it with an increased content of the target substance can be produced.

[0091] The maltotriose-containing carbohydrate obtained in this way, or a carbohydrate with an increased maltotriose content, can be concentrated to produce a syrup-like product. This syrup-like product can be further dried and pulverized to produce a powder-like product.

[0092] Maltotriose or carbohydrates containing the same obtained by the manufacturing method of the present invention can be used in the form of syrup or powder as an additive to various compositions such as food and beverages, treats, animal feed, cosmetics, quasi-drugs, and pharmaceuticals, as a sweetener, flavor enhancer, quality enhancer, stabilizer, etc. [Examples]

[0093] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0094] As described below, α-amylase was obtained from candidate production bacteria, and its maltotriose production activity was confirmed. Furthermore, the candidate production bacteria that produced the α-amylase exhibiting maltotriose production activity were identified. In addition, amino acid sequence analysis and base sequence determination were performed on the α-amylase exhibiting maltotriose production activity.

[0095] [Test Example 1: Acquisition of α-amylase from candidate production bacteria] [1] Culture of candidate bacteria for production A liquid culture medium with the following composition was placed in an Erlenmeyer flask and sterilized in an autoclave at 121°C for 20 minutes. Candidate bacteria were inoculated into the liquid culture medium and incubated at 30°C for 3 days.

[0096] [Table 1]

[0097] [Table 2]

[0098] [2] Purification of enzymes The enzymes produced by candidate enzyme-producing bacteria were purified by cell isolation, ultrafiltration, ammonium sulfate fractionation, dialysis, first DEAE-Sepharose treatment, second DEAE-Sepharose treatment, first Sephadex G-150 treatment, and second Sephadex G-150 treatment.

[0099] Cell isolation was performed by centrifugation (7,000 rpm, 5 min) of the culture supernatant. Ultrafiltration was performed on the centrifugation supernatant using an ultrafiltration membrane (AIV membrane, Asahi Kasei). Fractions 0-40 were obtained by ammonium sulfate fractionation. Dialysis was performed using Tris-HCl buffer (pH 7.0). In the first DEAE-Sepharose treatment, a DEAE-sepharose column was used for 10 -3 M L-cysteine ​​2.5 × 10 -3 The samples were equilibrated and washed with M Tris-HCl buffer (pH 7.0), eluted with KCl (0-1.0 M gradient), the amylase fraction was collected, concentrated using a membrane filter, and then re-fractionated using DEAE-Sepharose. The second DEAE-Sepharose treatment followed the same procedure as the first DEAE-Sepharose treatment. In the first Sephadex G-150 treatment, a Sephadex G-150 column was used, and 10 -3 M L-cysteine ​​2.5 × 10 -3 The samples were equilibrated and washed with M Tris-HCl buffer (pH 7.0), eluted with KCl (0-1.0 M gradient), the amylase fraction was collected, concentrated using a membrane filter, and then re-fractionated using a Sephadex G-150 column. The same procedure as the first Sephadex G-150 column was followed for the second Sephadex G-150 column.

[0100] The amylase fraction is the fraction in which the α-amylase activity was confirmed using an α-amylase measurement kit (manufactured by Kikkoman Biochemifa Co., Ltd.) according to the protocol of the kit.

[0101] The obtained fraction was developed by SDS-PAGE. In SDS-PAGE, 10 μL of the obtained fraction was diluted with 5 μL of SDS sample buffer [Tris-HCL buffer (pH 6.8) 0.125 M, SDS 4% (w / v), sucrose 10% (w / v), BPB (bromophenol blue) 0.01% (w / v), DTT (dithiothreitol) 0.2 M] to prepare an electrophoresis sample, which was boiled at 99.9 °C for 10 minutes. Then, 10 μL of the electrophoresis sample was applied to an electrophoresis gel [SDS gel; SuperSep TM Ace 15% (manufactured by Fujifilm Wako Pure Chemical Corporation)] and electrophoresed. The electrophoresis gel was transferred to a PVDF membrane [Trans-Blot Turbo TM Mini PVDF Transfer Pack (manufactured by BIO-RAD)], and the transfer membrane was stained with a staining solution [containing 0.1% (w / v) CBB R-250 in 50% (v / v) aqueous methanol solution] and decolorized with 50% (v / v) aqueous methanol solution.

[0102] The staining results of the SDS gel are shown in Fig. 1. In Fig. 1, the rightmost lane is the molecular marker. As shown in Fig. 1, it was suggested that the obtained enzyme takes three forms by processing. The molecular weights of the enzymes in each form were 55 kDa indicated by arrow 1, 70 kDa indicated by arrow 2, and 77 kDa indicated by arrow 3, respectively. Also, it was confirmed that the enzyme with a molecular weight of 55 kDa indicated by arrow 1 was purified alone. Regarding the 55 kDa purified fraction in Fig. 1, the α-amylase activity was confirmed using the above-mentioned α-amylase measurement kit.

[0103] [Test Example 2: Confirmation of Maltotriose Production Activity] Regarding the 55 kDa purified fraction in Fig. 1, the maltotriose production activity was further confirmed as follows.

[0104] [1] Preparation of Substrate Solution 1 g of soluble starch was suspended in approximately 60 mL of purified water and heated at 100°C for 5 minutes to dissolve. After cooling, the solution was diluted to 100 mL with purified water to obtain a substrate solution containing 1% (w / v) soluble starch.

[0105] [2] Preparation of reaction solution 200 μL of substrate solution and 50 μL of the enzyme solution to be analyzed were mixed to prepare 250 μL of reaction solution.

[0106] [3] Preparation of samples for analysis The reaction solution was reacted at 50°C for 48 hours with shaking at 1000 rpm, and then the reaction was stopped by boiling at 100°C for 5 minutes. The stopped reaction solution was diluted 30-fold with purified water and filtered through a 0.45 μm filter to obtain the filtrate as an analytical sample.

[0107] The sample was transferred to an analytical sample vial and analyzed by HPLC under the following conditions.

[0108] [Table 3]

[0109] HPLC analysis revealed a peak for maltotriose as a product. This confirms the maltotriose-producing activity of the 55 kDa purified fraction.

[0110] Similarly, maltotriose production activity was confirmed in the 70kDa and 77kDa purified fractions shown in Figure 1.

[0111] [Test Example 3: Identification of bacteria producing α-amylase with maltotriose-producing activity] Candidate α-amylase-producing bacteria that demonstrated maltotriose production activity were subjected to 16S rRNA analysis. As a result, the candidate bacteria were identified as Cellulosimicrobacterium sp.

[0112] [Test Example 4: Amino acid sequence analysis and base sequence determination of α-amylase with maltotriose-producing activity] [1] Analysis of the N-terminal amino acid sequence and internal amino acid sequence The three bands (55kDa, 70kDa, and 77kDa) obtained in item [2] of Test Example 1 were extracted and analyzed using a protein sequencer. As a result, the N-terminal amino acid sequence of each band was identified. Furthermore, the internal amino acid sequence was identified by analyzing each band after trypsin treatment using LC-MSMS.

[0113] [2] Determination of the base sequence [2-1]PCR Primers were designed based on the identified N-terminal and internal amino acid sequences. PCR amplification was performed using 20 μL / tube of PCR reaction mixture. The composition of the PCR reaction mixture and the PCR conditions are as follows. Electrophoresis was performed on a 1% agarose gel to confirm single amplification.

[0114] [Table 4]

[0115] [2-2] TA-cloning Ligation was performed at 16°C for 30 minutes using a ligation reaction mixture prepared by mixing 2 μL of PCR product, 1 μL of T-Vecor pMD20, and 3 μL of Ligation Mix. Subsequently, 25 μL of E. coli BL21 (DE3) was transformed. Approximately 30 μL of the resulting transformation solution was spread onto a liquid medium containing LB (Thermo Fisher Scientific) and Amp (final concentration 100 μg / mL) (hereinafter referred to as "LB+Amp liquid medium") and incubated at 37°C over-the-air.

[0116] [2-3] Plasmid extraction Transformants were cultured in LB+Amp liquid medium (2 mL) at 37°C over-the-air. After culturing was complete, the cells were harvested and plasmids were extracted using the Miniprep method.

[0117] [2-4] Sequence analysis Sequencing analysis was performed using the Sanger method to determine the partial base sequence.

[0118] [2-5] Full-length nucleotide sequence analysis Colony hybridization was performed using probes created based on the obtained partial nucleotide sequences. The gene sequences of the resulting positive clones were analyzed to determine the target nucleotide sequences. As a result, the nucleotide sequence for Sequence ID No. 4 was obtained from the 55 kDa band. The nucleotide sequence for Sequence ID No. 5 was obtained from the 70 kDa band. The nucleotide sequence for Sequence ID No. 6 was obtained from the 77 kDa band.

[0119] [3] Determination of amino acid sequence The amino acid sequences were determined from the above nucleotide sequences. The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4 was SEQ ID NO: 1, the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 5 was SEQ ID NO: 2, and the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 6 was SEQ ID NO: 3. In other words, it was found that maltotriose-producing amylases of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 were obtained by Experiment 1.

[0120] [Test Example 5: Recombinant Production of Maltotriose-Producing Amylase] [1] Construction of the expression vector [1-1]PCR To amplify the amylase gene, we designed the primer shown in SEQ ID NO: 7 as a forward primer and the primer shown in SEQ ID NO: 8 as a reverse primer. PCR amplification was performed using 50 μL / tube of PCR reaction mixture. The composition of the PCR reaction mixture and the PCR conditions are as follows. Electrophoresis was performed on a 1% agarose gel to confirm single amplification.

[0121] [Table 5]

[0122] [1-2. Ligation and Transformation] A ligation reaction was carried out at 16°C for 30 minutes using a ligation solution (5 μL) containing the PCR product and pUBCM21 (a shuttle vector for pUC19 and pUB110), followed by transformation of 50 μL of E. coli DH5α. Approximately 55 μL of the resulting transformation solution was spread onto an LB+Amp (100 μg / mL) plate and incubated at 37°C over-the-air. The composition of the ligation reaction solution is as follows: [Table 6]

[0123] [1-3. Plasmid Extraction] Plasmid extraction was performed using the same method as in Test Example 4, Sections 2-3, and plasmid pUBCM21-amy was obtained.

[0124] [2] Transformation of Bacillus subtilis The obtained vector pUBCM21-amy was used to transform amyE-deficient strains derived from Bacillus subtilis strain 168. The transformation method followed the protocol of the B. subtilis Secretory Protein Expression System (TaKaRa).

[0125] [3] Production of maltotriose-producing amylase [3-1] Culture of transformed strains Transformed strains were inoculated into liquid medium containing LB (Thermo Fisher Scientific) and kanamycin (20 μg / mL) and cultured with shaking at 37°C for 4 days. 0.5 mL samples were taken daily after the start of culture, and the supernatant was collected by centrifugation at 15,000 rpm at 4°C for 5 minutes to obtain maltotriose-producing amylase.

[0126] [3-2] Confirmation of α-amylase productivity The recovered supernatant (maltotriose-producing amylase fraction) was subjected to activity testing and SDS-PAGE to confirm α-amylase productivity. Activity testing was performed using an α-amylase measurement kit (Kikkoman Biochemifa Co., Ltd.) according to the kit's protocol. SDS-PAGE was performed in the same manner as in Test Example 1.

[0127] Activity measurements revealed significant activity in the maltotriose-producing amylase fraction compared to the negative control, pUBCM21 (empty vector-introduced strain). Furthermore, SDS-PAGE analysis, by optimizing the codons for Bacillus subtilis, identified three bands derived from α-amylase. Based on their molecular weights, these three bands were estimated to belong to the amylases of SEQ ID NOs: 1, 2, and 3. Therefore, this study is considered to have yielded the maltotriose-producing amylases of SEQ ID NOs: 1, 2, and 3. [Sequence Listing Free Text]

[0128] Sequence IDs 7 and 8 are primers.

Claims

1. An enzyme preparation for producing maltotriose from starch, comprising any of the polypeptides shown in (1-1) to (1-3) below as an active ingredient: (1-1) Polypeptide consisting of the amino acid sequence shown in Sequence ID No. 1, (1-2) A polypeptide having the ability to produce maltotriose, wherein the amino acid sequence shown in Sequence ID No. 1 has 1 to 10 amino acids substituted, added, inserted, or deleted. (1-3) A polypeptide having a sequence identity of 90% or more with respect to the amino acid sequence shown in SEQ ID NO: 1, and having the ability to produce maltotriose.

2. A method for producing maltotriose, comprising the step of producing maltotriose from starch using a maltotriose-producing amylase consisting of any of the polypeptides shown in (1-1) to (1-3) below: (1-1) Polypeptide consisting of the amino acid sequence shown in Sequence ID No. 1, (1-2) A polypeptide having the ability to produce maltotriose, wherein the amino acid sequence shown in Sequence ID No. 1 has 1 to 10 amino acids substituted, added, inserted, or deleted. (1-3) A polypeptide having a sequence identity of 90% or more with respect to the amino acid sequence shown in SEQ ID NO: 1, and having the ability to produce maltotriose.

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