Maltotriose-to-amylase

Abstract

The present invention provides a novel enzyme for producing maltotriose. A maltotriose-producing amylase comprising a polypeptide represented by any one of the following: a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 3; a polypeptide which has a maltotriose-producing ability and in which one or more amino acids are substituted, added, inserted or deleted in each of the amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 3; the polypeptide has a sequence identity of 70% or more with respect to each of the amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 3, and has a maltotriose-producing ability.

Description

[0001] This application is a divisional application based on the Chinese invention patent application with application number 201980070365.2, application date October 28, 2019, and invention name “Maltotriose-generating amylase”. Technical Field

[0002] The present invention relates to a maltotriose generating amylase. Background Art

[0003] Maltotriose is a sugar obtained by partially decomposing starch using enzymes or acids, in which three glucose molecules are bonded via α-1,4-glucosidic bonds. The sweetness intensity of maltotriose is about 17% of that of granulated sugar, but it has a mellow sweetness, so it is used as a low-sweetener for food. In addition, maltotriose is useful as an anti-desiccant for food, an anti-crystallization agent for granulated sugar, and an anti-aging agent for starch because of its excellent hygroscopicity and water retention. In addition, maltotriose has excellent thermal stability compared to glucose and maltose, so it is also useful as a carbohydrate material used in food processing. Maltotriose can be obtained from the by-products when maltose is manufactured. However, from the viewpoint of stable industrial supply, it is preferred to manufacture by chemically decomposing starch. As enzymes for generating maltotriose from starch, N-A468 enzyme (Non-patent Document 1) derived from Streptomyces griseus and amylase G3 (Non-patent Document 2) derived from Bacillus subtilis are known. N-A468 enzyme is classified in the same way as β-amylase, and decomposes starch using maltotriose units, and amylase G3 is classified in the same way as α-amylase, and produces starch degradation products containing maltotriose as the main component. Prior art literature Non-patent literature

[0004] Non-patent document 1: Starch Science, Vol. 23, No. 3, pp. 175-181 (1979) Non-patent document 2: Agricultural and Biological Chemistry 1985 Volume 49 Issue 4 Pages 1091-1097 Summary of the invention Problems to be solved by the invention

[0005] However, these enzymes have low production efficiency from microorganisms and still have problems from the viewpoint of stable industrial supply. Therefore, it is desired to obtain a new enzyme that produces maltotriose.

[0006] An object of the present invention is to provide a novel enzyme for producing maltotriose. Technical solutions to solve problems

[0007] The present inventors have conducted intensive research and found that maltotriose-producing amylase (hereinafter referred to as "maltotriose-producing amylase") is derived from bacteria of the genus Cellulosimicrobium. The present invention has been completed based on these findings. That is, the present invention provides the invention of the following disclosed aspects.

[0008] Item 1. A maltotriose-producing amylase comprising a polypeptide represented by any one of the following (1-1) to (1-3), (2-1) to (2-3), and (3-1) to (3-3): (1-1) a polypeptide comprising the amino acid sequence represented by SEQ 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 represented by SEQ ID NO: 1, (1-3) a polypeptide having a sequence identity of 70% or more to the amino acid sequence represented by SEQ ID NO: 1 and having the ability to produce maltotriose, (2-1) a polypeptide comprising the amino acid sequence represented by SEQ 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 represented by SEQ ID NO: 2, (2-3) a polypeptide having a sequence identity of 70% or more to the amino acid sequence represented by SEQ ID NO: 2 and having the ability to produce maltotriose, (3-1) a polypeptide comprising the amino acid sequence represented by SEQ 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 represented by SEQ ID NO: 3, (3-3) A polypeptide having a sequence identity of 70% or more to the amino acid sequence represented by SEQ ID NO: 3 and having the ability to produce maltotriose. Item 2. A DNA encoding the maltotriose-producing amylase according to Item 1. Item 3. A recombinant vector comprising the DNA described in Item 2. Item 4. A transformant obtained by transforming a host using the DNA described in Item 2 or the recombinant vector described in Item 3. Item 5. A method for producing the maltotriose-producing amylase according to Item 1, comprising the step of culturing the transformant according to Item 4. Item 6. An enzyme preparation comprising the maltotriose-producing amylase according to Item 1. Item 7. A method for producing maltotriose, comprising the step of producing maltotriose from starch using the maltotriose-producing amylase according to Item 1.

[0009] According to the present invention, a novel enzyme for producing maltotriose can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The results of purification by SDS-PAGE of the α-amylase having maltotriose-producing activity obtained in Test Example 1 and obtained from bacteria belonging to the genus Cellulosimicrobium are shown. DETAILED DESCRIPTION

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

[0012] In the present specification, the left end of the amino acid sequence is the N-terminus, and the right end is the C-terminus.

[0013] In this specification, "non-polar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine and tryptophan. "Non-charged 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] 1. Maltotriose produces amylase The maltotriose-producing amylase of the present invention is derived from bacteria of the genus Cellulosimicrobium.

[0015] The first embodiment of the maltotriose-producing amylase of the present invention is a polypeptide represented by any one of the following (1-1) to (1-3).

[0016] (1-1) A polypeptide comprising the amino acid sequence represented by SEQ ID NO:1. (1-2) A polypeptide having the ability to produce maltotriose, in which one or more amino acids are substituted, added, inserted or deleted in the amino acid sequence represented by SEQ ID NO: 1. (1-3) A polypeptide having a sequence identity of 70% or more to the amino acid sequence represented by SEQ ID NO: 1 and having the ability to produce maltotriose.

[0017] The polypeptides represented by (1-1) to (1-3) above have α-amylase activity that cleaves α-1,4-bonds of starch and has activity that produces maltotriose.

[0018] The amino acid changes introduced into the polypeptide of (1-2) above may include only one change among substitution, addition, insertion and deletion (e.g., substitution), or may include two or more changes (e.g., substitution and insertion). In the polypeptide of (1-2) above, the number of amino acids to be substituted, added, inserted or deleted may be one or more, 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] In addition, in the above-mentioned polypeptide (1-3), the sequence identity relative to the amino acid sequence represented by sequence number 1 can be 70% or more, preferably 80% or more, 85% or more, 90% or more, further preferably 95% or more, 97% or more, 98% or more, and particularly preferably 99% or more.

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

[0021] In the polypeptides of (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 represented by SEQ ID NO: 1 are considered to contribute to the activity, and therefore, it is preferred that no substitution or deletion is introduced into these positions. The amino acids at positions 107 and 166 are estimated to be the Ca binding site, and the amino acids at positions 196, 223, and 284 are estimated to be the active center, and therefore, it is particularly preferred that no substitution or deletion is introduced into these positions.

[0022] When amino acid substitutions are introduced into the polypeptides of (1-2) and (1-3) above, conservative substitutions can be cited as the form of amino acid substitutions. That is, in the polypeptides of (1-2) and (1-3), as examples of amino acid substitutions introduced relative to the amino acid sequence represented by SEQ ID NO: 1, if the amino acid before substitution is a non-polar amino acid, it is substituted with another non-polar amino acid; if the amino acid before substitution is a non-charged amino acid, it is substituted with another non-charged amino acid; if the amino acid before substitution is an acidic amino acid, it is substituted with another acidic amino acid; and if the amino acid before substitution is a basic amino acid, it is substituted with another basic amino acid.

[0023] In the polypeptides of (1-2) and (1-3) above, "having maltotriose-producing ability" means: having activity to the extent that it can function as a maltotriose-producing amylase. Specifically, it means that the peak detection of maltotriose can be confirmed in the "<Method for determining the activity of maltotriose-producing amylase>" described later. Preferably, it means: the maltotriose-producing ability confirmed for starch is the same as or higher than the maltotriose-producing ability of the polypeptide based on (1-1) above (for example, the peak intensity ratio of maltotriose detected by the "<Method for determining the activity of maltotriose-producing amylase>" is 70% or more of that of the polypeptide of (1-1) above).

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

[0025] The second embodiment of the maltotriose-producing amylase of the present invention is a polypeptide represented by any one of the following (2-1) to (2-3).

[0026] (2-1) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2. (2-2) A polypeptide having the ability to produce maltotriose, in which one or more amino acids are substituted, added, inserted or deleted in the amino acid sequence represented by SEQ ID NO: 2. (2-3) A polypeptide having a sequence identity of 70% or more to the amino acid sequence represented by SEQ ID NO: 2 and having the ability to produce maltotriose.

[0027] The polypeptide of (2-1) above includes the polypeptide of (1-1) of the above-mentioned 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 represented by sequence number 2 correspond to the polypeptide of (1-1) above. In addition, positions 582 to 674 of the amino acid sequence represented by sequence number 2 are considered to correspond to the CBM region. It is believed that the CBM region causes the maltotriose-producing amylase to be locally present on the surface of the substrate (starch) to increase the efficiency of hydrolysis. The CBM region can be directly or indirectly bonded to the C-terminal side of the polypeptide represented by any one of (1-1) to (1-3) of the above-mentioned first embodiment. It should be noted that indirect bonding refers to: for example, bonding via other amino acid sequences such as a linker sequence containing 1 to 250, preferably 10 to 220 amino acids.

[0028] The amino acid changes introduced into the polypeptide of (2-2) above may contain only one change (e.g., substitution) among substitution, addition, insertion and deletion, or may contain two or more changes (e.g., substitution and insertion). In the region corresponding to the CBM region of (2-2) above, the number of amino acids replaced, added, inserted or deleted may be one or more, 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] In addition, in the above-mentioned polypeptide (2-3), the sequence identity relative to the amino acid sequence represented by sequence number 2 can be 70% or more, preferably 80% or more, 85% or more, 90% or more, further preferably 95% or more, 97% or more, 98% or more, and particularly preferably 99% or more.

[0030] Here, in the polypeptide of (2-3) above, the sequence identity relative to the amino acid sequence represented by sequence number 2 is the sequence identity calculated by comparison with the amino acid sequence represented by sequence number 2, and the "sequence identity" is the same as the sequence identity in (1-3) in the above-mentioned first embodiment.

[0031] In the above-mentioned polypeptides (2-2) and (2-3), it is preferred that the positions of the amino acids in sequence number 2 at which substitution or deletion is not introduced are not introduced. As the positions of the amino acids in sequence number 1 at which substitution or deletion is preferably not introduced in the polypeptides (1-2) and (1-3) of the above-mentioned first embodiment, as shown above, 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 represented by sequence number 2 contribute to the activity, and therefore, it is preferred that substitution or deletion is not introduced in these positions. The amino acids at positions 107 and 166 are estimated as Ca bonding sites, and the amino acids at positions 196, 223, and 284 are estimated as active centers, and therefore, it is particularly preferred that no substitution or deletion is introduced in these sites. In addition, regarding the CBM region, it is believed that the amino acids at positions 608, 639, 651, 652, and 656 contribute to bonding with starch, and therefore, when it is desired to make the maltotriose generating amylase localized on the surface of the substrate (starch), it is preferred that no substitution or deletion is introduced in these sites.

[0032] When amino acid substitution is introduced into the polypeptides of (2-2) and (2-3) above, conservative substitution can be cited as the mode of amino acid substitution. Conservative substitution is the same as the conservative substitution as the mode of amino acid substitution when amino acid substitution is introduced into the polypeptides of (1-2) and (1-3) in the first embodiment above.

[0033] In the polypeptides of (2-2) and (2-3) above, "having maltotriose-producing ability" means: having activity to the extent that it can function as a maltotriose-producing amylase. Specifically, it means that the peak detection of maltotriose can be confirmed using the "<Method for determining the activity of maltotriose-producing amylase>" described later. Preferably, it means that the maltotriose-producing ability confirmed for starch is the same as or higher than the maltotriose-producing ability of the polypeptide based on (2-1) above (for example, the peak intensity ratio of maltotriose detected by the "<Method for determining the activity of maltotriose-producing amylase>" is 70% or more of that of the polypeptide of (2-1) above).

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

[0035] The third embodiment of the maltotriose-producing amylase of the present invention is a polypeptide represented by any one of the following (3-1) to (3-3).

[0036] (3-1) A polypeptide comprising the amino acid sequence represented by SEQ ID NO:3. (3-2) A polypeptide having the ability to produce maltotriose, in which one or more amino acids are substituted, added, inserted or deleted in the amino acid sequence represented by SEQ ID NO: 3. (3-3) A polypeptide having a sequence identity of 70% or more to the amino acid sequence represented by SEQ ID NO: 3 and having the ability to produce maltotriose.

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

[0038] The amino acid changes introduced into the polypeptide of (3-2) above may contain only one change among substitution, addition, insertion and deletion (e.g., substitution), or may contain two or more changes (e.g., substitution and insertion). In the polypeptide of (3-2) above, the number of amino acids to be substituted, added, inserted or deleted may be one or more, 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] In addition, in the above-mentioned polypeptide (3-3), the sequence identity relative to the amino acid sequence represented by sequence number 3 can be 70% or more, preferably 80% or more, 85% or more, 90% or more, further preferably 95% or more, 97% or more, 98% or more, and particularly preferably 99% or more.

[0040] Here, in the polypeptide of the above (3-3), the sequence identity relative to the amino acid sequence represented by sequence number 3 is the sequence identity calculated by comparison with the amino acid sequence represented by sequence number 3, and the "sequence identity" is the same as the sequence identity in (1-3) of the above-mentioned first embodiment.

[0041] When amino acid substitution is introduced into the polypeptides of (3-2) and (3-3) above, conservative substitution can be cited as the form of amino acid substitution. Conservative substitution is the same as the conservative substitution as the form of amino acid substitution when amino acid substitution is introduced into the polypeptides of (1-2) and (1-3) in the first embodiment above.

[0042] In the above-mentioned polypeptides (3-2) and (3-3), it is preferred that the position of the amino acid in sequence number 3 not be introduced into which substitution or deletion is made, as shown above, as the position of the amino acid in sequence number 1 not be introduced into which substitution or deletion is made in the above-mentioned polypeptides (1-2) and (1-3) of the first embodiment.

[0043] In the polypeptides of (3-2) and (3-3) above, "having maltotriose-producing ability" means having activity to the extent that it can function as a maltotriose-producing amylase. Specifically, it means that the peak detection of maltotriose can be confirmed using the "<Method for determining the activity of maltotriose-producing amylase>" described later. Preferably, it means that the maltotriose-producing ability confirmed for starch is the same as or higher than the maltotriose-producing ability of the polypeptide based on (3-1) above (for example, the peak intensity ratio of maltotriose detected by the "<Method for determining the activity of maltotriose-producing amylase>" is 70% or more of that of the polypeptide of (3-1) above).

[0044] The molecular weight of the maltotriose-producing amylase comprising the polypeptide of (3-1) of the third embodiment is about 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 containing 200 μL of a substrate solution (1% (w / v) soluble starch solution) and 50 μL of an enzyme solution was reacted by shaking at 1000 rpm at 50°C for 48 hours, and then boiled at 100°C for 5 minutes to stop the reaction. The reaction solution after the reaction was stopped was diluted 30 times with purified water and filtered with a 0.45 μm filter to obtain an analysis sample. The maltotriose-generating amylase activity was determined by performing HLPC analysis on the analysis sample and detecting the peak of maltotriose.

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

[0047] In addition, when introducing a specific mutation into a specific site of a base sequence, the mutation introduction method is well known, and for example, a site-specific mutation introduction method of DNA can be used. As a specific method for converting bases in DNA, for example, a commercially available kit can be used.

[0048] The base sequence of the DNA obtained by introducing mutations into the base sequence can be confirmed using a DNA sequencer. Once the base sequence is determined, the DNA encoding the above-mentioned maltotriose-producing amylase can be obtained by PCR using a probe obtained by chemical synthesis or cloning as a template, or by hybridization using a DNA fragment having the base sequence as a probe.

[0049] In addition, a mutant of the DNA encoding the maltotriose-producing amylase and a DNA having the same function as before the mutation can be synthesized by site-specific mutagenesis, etc. It should be noted that when introducing a mutation into the DNA encoding the maltotriose-producing amylase, it can be carried out by a known method such as the Kunkel method, the Gapped duplex method, and the megaprimer PCR method.

[0050] A person 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] The first embodiment of the DNA of the present invention is a DNA encoding the polypeptides of (1-1) to (1-3) above. For example, the DNA encoding the polypeptide of (1-1) above includes a DNA comprising the base sequence represented by SEQ ID NO: 4.

[0052] In addition, the first embodiment of the DNA of the present invention includes: a DNA encoding the polypeptides of (1-1) to (1-3) above and containing a base sequence complementary to the DNA containing the base sequence represented by SEQ ID NO: 4, and a DNA that hybridizes under stringent conditions.

[0053] In addition, the first embodiment of the DNA of the present invention also includes a DNA encoding the polypeptides of (1-1) to (1-3) above and having an identity of 70% or more to the DNA comprising the base sequence represented by SEQ ID NO: 4. The above-mentioned 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.

[0054] The second embodiment of the DNA of the present invention is a DNA encoding any one of the polypeptides (2-1) to (2-3) above. For example, as a DNA encoding the polypeptide of (2-1) above, a DNA comprising the base sequence represented by SEQ ID NO: 5 can be cited.

[0055] In addition, the second embodiment of the DNA of the present invention includes: a DNA encoding the polypeptides of (2-1) to (2-3) above and containing a base sequence complementary to the DNA containing the base sequence represented by sequence number 5, and a DNA that hybridizes under stringent conditions.

[0056] Furthermore, the second embodiment of the DNA of the present invention also includes a DNA encoding the polypeptides of (2-1) to (2-3) above and having a homology of 70% or more to the DNA comprising the base sequence represented by SEQ ID NO: 5. The above 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] The third embodiment of the DNA of the present invention is a DNA encoding any one of the polypeptides (3-1) to (3-3) above. For example, as a DNA encoding the polypeptide of (3-1) above, there is a DNA comprising the base sequence represented by SEQ ID NO: 6.

[0058] In addition, the third embodiment of the DNA of the present invention includes: a DNA encoding the polypeptides of (3-1) to (3-3) above and containing a base sequence complementary to the DNA containing the base sequence represented by SEQ ID NO: 6, and a DNA that hybridizes under stringent conditions.

[0059] Furthermore, the third embodiment of the DNA of the present invention also includes a DNA encoding the polypeptides of (3-1) to (3-3) above and having a homology of 70% or more to the DNA comprising the base sequence represented by SEQ ID NO: 6. The above 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.

[0060] Here, "stringent conditions" refers to incubation at 50°C to 65°C for 4 hours to overnight in 6×SSC (1×SSC contains 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% SDS, 5×Denhartz's [Denhartz's, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll400] and 100μg / ml salmon sperm DNA.

[0061] Hybridization under stringent conditions is specifically performed by the following method. That is, a nylon membrane is prepared on which a DNA library or cDNA library is immobilized, and the nylon membrane is blocked at 65°C in a prehybridization solution containing 6×SSC, 0.5% SDS, 5×Denhartz's, and 100 μg / ml salmon sperm DNA.32 Each probe labeled with P was incubated overnight at 65° C. The nylon membrane was washed in 6×SSC at room temperature for 10 minutes, in 2×SSC containing 0.1% SDS at room temperature for 10 minutes, and in 0.2×SSC containing 0.1% SDS at 45° C. for 30 minutes, and then DNA specifically hybridized with the probe was detected by autoradiography.

[0062] In addition, the "homology" of DNA refers to the identity value obtained by bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, 247-250, 1999) of BLAST PACKAGE [sgi32bit edition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]. The parameters can be set as Gapinsertion Cost value: 11 and Gap extension Cost value: 1.

[0063] The DNA of the present invention is preferably a DNA whose codon usage frequency is optimized in the host. For example, when Escherichia coli is used as the host, it is preferably a DNA whose codon usage frequency is optimized in Escherichia coli.

[0064] 3. Recombinant vector The recombinant vector of the present invention comprises a 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 comprises control factors such as promoters operably linked to the DNA of the present invention. As control factors, representatively, promoters can be cited, but further transcription elements such as enhancers, CCAAT boxes, TATA boxes, and SPI sites can be included as needed. In addition, operably linked refers to that various control factors such as promoters and enhancers regulating the DNA of the present invention are linked to the DNA of the present invention in an operable state in a host cell.

[0066] As the expression vector, preferably a phage, plasmid, or an expression vector constructed by a virus for gene recombination purposes that can autonomously proliferate in the host. The above-mentioned expression vector is well known, for example, as commercially available expression vectors, pQE vectors (QIAGEN Co., Ltd.), pDR540, pRIT2T (GE Healthcare Sciences Co., Ltd.), pET vectors (Merck Co., Ltd.), etc. can be cited. The expression vector can be selected and used in appropriate combinations with host cells. For example, when Escherichia coli is used as the host cell, a combination of pET vectors and BL21 (DE3) Escherichia coli strains, or a combination of pDR540 vectors and JM109 Escherichia coli strains, etc. are preferably cited.

[0067] 4. Transformants The transformant of the present invention is obtained by transforming a host with the DNA of the present invention or the recombinant vector of the present invention.

[0068] As the host used in the preparation of the transformant, there is no particular limitation as long as the gene can be introduced and can proliferate autonomously and express the gene of the present invention. As preferred examples, for example, bacteria belonging to the genus Escherichia such as Escherichia coli, the genus Bacillus subtilis such as Bacillus subtilis, the genus Pseudomonas such as Pseudomonas putida, etc.; actinomycetes, etc.; yeast, etc.; filamentous fungi, etc., in addition, animal cells, insect cells, plants, etc., can be mentioned. Among them, Escherichia coli is particularly preferred. In addition, as a host, it can also be the source bacteria of the maltotriose-producing amylase of the present invention, that is, Cellulosimicrobium.

[0069] The transformant of the present invention can be obtained by introducing the above-mentioned DNA of the present invention or the above-mentioned 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 can be introduced into the host. In addition, the position of the introduced DNA is not particularly limited as long as the target gene can be expressed, and it can be on a plasmid or on a genome. As a specific method for introducing the DNA of the present invention or the recombinant vector of the present invention, for example, a recombinant vector method and a genome editing method can be cited.

[0070] The conditions for introducing the DNA of the present invention or the recombinant vector of the present invention into the host can be appropriately set according to the type of the introduction method and the host. If the host is a bacterium, for example, a method using competent cells based on calcium ion treatment and electroporation can be enumerated. If the host is a yeast, for example, electroporation (electroporation), spheroplast method and lithium acetate method can be enumerated. If the host is an animal cell, for example, electroporation, calcium phosphate method and lipofection method can be enumerated. If the host is an insect cell, for example, calcium phosphate method, lipofection method and electroporation method can be enumerated. If the host is a plant, for example, electroporation, Agrobacterium transformation, particle gun method and PEG method can be enumerated.

[0071] 5. Method for producing maltotriose-generating amylase The maltotriose-producing amylase of the present invention can be produced by culturing the transformant of the present invention. In addition, the maltotriose-producing amylase of the present invention can also be produced by culturing the producing bacterium, Cellulosimicrobacterium sp. (without transformation). When the producing bacterium, Cellulosimicrobacterium sp. (without transformation), is cultured, the maltotriose-producing amylase of the present invention is produced by being treated in a state where any one of the polypeptides (1-1) to (1 to 3) (polypeptides of the first embodiment), any one of the polypeptides (2-1) to (2 to 3) (polypeptides of the second embodiment), and any one of the polypeptides (3-1) to (3 to 3) (polypeptides of the third embodiment) are mixed. On the other hand, when the transformant of the present invention is used, the introduced DNA allows the host to express only a gene encoding any one of the polypeptides of the first embodiment, the polypeptide of the second embodiment, and the polypeptide of the third embodiment. Therefore, it is possible to produce maltotriose-producing amylase containing any one of the polypeptides of the first embodiment, the polypeptide of the second embodiment, and the polypeptide of the third embodiment alone, or to produce it in a state where the polypeptides of the first embodiment, the polypeptide of the second embodiment, and the polypeptide of the third embodiment are mixed.

[0072] The culture conditions of the transformant of the present invention can be appropriately set in consideration of the nutritional physiological properties of the host, and liquid culture is preferred. In addition, in the case of industrial production, aeration and stirring culture is preferred.

[0073] The transformant of the present invention is cultured, and the culture supernatant or bacterial cells are recovered from the culture solution by centrifugation or the like. If the maltotriose-producing amylase of the present invention is accumulated in the bacterial cells, the bacterial cells are treated by a mechanical method such as ultrasonic wave or French press or by a lysozyme such as lysozyme, and if necessary, dissolved using an enzyme such as a protease or a surfactant such as sodium dodecyl sulfate (SDS), to obtain a water-soluble fraction containing the maltotriose-producing amylase of the present invention.

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

[0075] The culture solution or water-soluble fraction containing the maltotriose-producing amylase of the present invention obtained as described above may be directly subjected to purification treatment, or may be subjected to purification treatment after concentrating the polypeptide of the present invention in the culture solution or water-soluble fraction.

[0076] The concentration can be performed, for example, by reduced pressure concentration, membrane concentration, salting-out treatment, or a separate precipitation method using a hydrophilic organic solvent (eg, methanol, ethanol, and acetone).

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

[0078] The maltotriose-producing amylase of the present invention thus purified can be powdered by freeze drying, vacuum drying, spray drying, etc. as necessary.

[0079] 6. Enzyme preparations The enzyme preparation of the present invention contains the above-mentioned maltotriose generating amylase of the present invention as an active ingredient. The maltotriose generating amylase contained in the enzyme preparation may contain only a maltotriose generating amylase containing any one of the polypeptides (1-1) to (1 to 3), or only a maltotriose generating amylase containing any one of the polypeptides (2-1) to (2-3), or only a maltotriose generating amylase containing any one of the polypeptides (3-1) to (3-3). It may also contain multiple maltotriose generating amylases mentioned above. The type and content ratio of the maltotriose generating amylase contained can be appropriately selected according to the substrate.

[0080] The enzyme preparation of the present invention may contain an additive selected from excipients, buffers, suspending agents, stabilizers, preservatives, preservatives, and saline solutions in addition to the maltotriose-producing amylase of the present invention. As excipients, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, white sugar, glycerol, etc. may be mentioned. As buffers, phosphates, citrates, acetates, etc. may be used. As stabilizers, propylene glycol, ascorbic acid, etc. may be mentioned. As preservatives, sodium chloride, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methyl parahydroxybenzoate, etc. may be mentioned. As preservatives, sodium chloride, ethanol, benzalkonium chloride, p-hydroxybenzoic acid, chlorobutanol, etc. may be mentioned.

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

[0082] The enzyme agent of the present invention may contain other enzymes. As other enzymes, for example, amylase (α-amylase, β-amylase, glucoamylase), glucosidase (α-glucosidase, β-glucosidase), β-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, glucanase, transglutaminase, protein deamidase, pullulanase, etc. can be mentioned.

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

[0084] 7. Method for producing maltotriose The method for producing maltotriose of the present invention comprises a step of producing maltotriose from starch using the maltotriose-producing amylase of the present invention.

[0085] As starch, there can be mentioned amylose, amylopectin, glycogen and starch; and starch partial degradation products such as amylodextrin, maltodextrin, maltooligosaccharide, etc. obtained by partially hydrolyzing these substances by amylase or acid. As the starch partial degradation products obtained by decomposition using amylase, for example, amylase such as α-amylase (EC 3.2.1.1), maltopentaose generating amylase, maltohexaose generating amylase (EC 3.2.1.98) can be used to decompose amylose, amylopectin, glycogen, starch, etc. and obtain partial degradation products. In addition, when preparing partial degradation products, starch pruning enzymes such as pullulanase (EC3.2.1.41) and isoamylase (EC 3.2.1.68) can be allowed to act.

[0086] Examples of the starch include starch derived from corn, wheat, rice, potato, sweet potato, cassava, etc. These starches can be used in the form of a liquefied starch solution obtained by gelatinization and liquefaction.

[0087] When the maltotriose generating amylase of the present invention is allowed to act on starch, the concentration of the starch solution is not particularly limited, and from an industrial point of view, a concentration of 10% (w / v) or more can be cited. The reaction temperature is not particularly limited as long as it is a temperature at which the reaction is carried out, and specifically, about 65°C or less can be cited, preferably 45 to 60°C. The reaction pH can be cited as usually 5 to 9, preferably 5.5 to 7.5. The amount of the enzyme used can be appropriately selected according to the speed of the target enzyme reaction.

[0088] In addition, during the maltotriose production reaction, other enzymes may be used simultaneously to increase the maltotriose content in the saccharified liquid. For example, by using a starch debranching enzyme such as pullulanase or isoamylase in combination, the maltotriose content in the saccharified liquid can be increased.

[0089] The reaction solution obtained by the above reaction can be used directly as a sugar solution containing maltotriose, but it is preferred to further purify the sugar solution containing maltotriose. As a purification method, a common method for purifying sugar can be appropriately adopted, for example, decolorization using activated carbon, decolorization using H + Type, OH - One or more purification methods include desalting with an ion exchange resin, ion exchange column chromatography, activated carbon column chromatography, fractionation using column chromatography such as silica gel column chromatography, separation using an organic solvent such as alcohol and acetone, and separation using a membrane with appropriate separation performance.

[0090] As a method for obtaining a highly pure maltotriose-containing saccharide, ion exchange column chromatography can be cited. Specifically, by column chromatography using a strongly acidic cation exchange resin, contaminating saccharides can be removed to produce maltotriose or saccharides containing the same with an increased content of the target product.

[0091] The saccharide containing maltotriose or the saccharide with increased maltotriose content thus obtained can be concentrated to form a syrupy product, and the syrupy product can also be dried and pulverized to form a powdery product.

[0092] Maltotriose or saccharides containing the same obtained by the production method of the present invention can be used in the form of syrup or powder as a sweetener, taste improver, quality improver, stabilizer, etc., and as an additive to various compositions such as food, drink, snacks, feed, bait, cosmetics, quasi-drugs, and pharmaceuticals. Example

[0093] Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention should not be construed as being limited to the following Examples.

[0094] As shown below, α-amylase was obtained from the candidate producing bacteria, and the maltotriose-producing activity of the obtained α-amylase was confirmed. In addition, the candidate producing bacteria that produced the α-amylase confirmed to have the maltotriose-producing activity was determined. In addition, the α-amylase confirmed to have the maltotriose-producing activity was subjected to amino acid sequence analysis and base sequence determination.

[0095] [Test Example 1: Obtaining α-amylase from candidate producing bacteria] [1] Cultivation of candidate production bacteria A liquid culture medium having the following composition was placed in a conical flask and sterilized at 121°C for 20 minutes using an autoclave. A candidate producer bacterium was inoculated into the liquid culture medium and cultured at 30°C for 3 days.

[0096] [Table 1] 0 Culture medium composition

[0097] [Table 2] ○Cultivation conditions

[0098] [2] Enzyme purification The enzyme produced by the candidate producing bacteria is purified by bacterial cell separation, ultrafiltration, ammonium sulfate fractionation, dialysis, the first DEAE-Sepharose treatment, the second DEAE-Sepharose treatment, the first Sephadex G-150 treatment and the second Sephadex G-150 treatment.

[0099] The bacterial cell separation was performed by centrifuging the culture supernatant (7000 rpm, 5 min). Ultrafiltration was performed by using an ultrafiltration membrane (AIV membrane, Asahi Kasei) on the centrifuged supernatant. The 0-40 fractions were obtained by ammonium sulfate fractionation. Dialysis was performed using Tris hydrochloric acid buffer (pH 7.0). In the first DEAE-Sepharose treatment, a DEAE-sepharose column containing 10 -3 2.5×10 ML-cysteine -3 The amylase fraction was collected and concentrated using a membrane filter and then re-fractionated using DEAE-Sepharose. In the second DEAE-Sepharose treatment, the same elution operation as the first DEAE-Sepharose treatment was performed. In the first Sephadex G-150 treatment, a Sephadex G-150 column containing 10 -3 2.5×10 M L-cysteine -3 The amylase fraction was collected by equilibration and washing with MTris hydrochloric acid buffer (pH 7.0), eluted with KCl (0-1.0 M gradient), concentrated with a membrane filter, and re-fractionated with a Sephadex G-150 column. In the second Sephadex G-150, the same operation as the first Sephadex G-150 was performed.

[0100] The amylase fraction is a fraction in which the α-amylase activity was confirmed using an α-amylase assay kit (manufactured by Kikkoman Chemiphar) according to the instructions of the kit.

[0101] The obtained fractions were developed by SDS-PAGE. In SDS-PAGE, 10 μL of the obtained fractions were 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 electrophoresis samples, and after boiling at 99.9°C for 10 minutes, 10 μL of the electrophoresis samples were applied to the electrophoresis gel [SDS gel; SuperSep TM Ace 15% (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)] was used for electrophoresis. The electrophoresis gel was transferred to a PVDF membrane [Trans-Blot Turbo TMThe membrane was stained with a staining solution [0.1% (w / v) CBB R-250 in a 50% (v / v) methanol aqueous solution] and destained with a 50% (v / v) methanol aqueous solution.

[0102] The results of SDS gel staining are shown in Figure 1 . Figure 1 In the figure, the rightmost lane is the molecular marker. Figure 1 As shown, it is suggested that the obtained enzyme has three forms after treatment. The molecular weights of the enzymes in each form are: the molecular weight indicated by arrow 1 is 55 kDa, the molecular weight indicated by arrow 2 is 70 kDa, and the molecular weight indicated by arrow 3 is 77 kDa. In addition, regarding the enzyme with a molecular weight of 55 kDa indicated by arrow 1, it was confirmed that it was purified purely. Figure 1 The 55 kDa purified fraction was assayed for α-amylase activity using the α-amylase assay kit described above.

[0103] [Test Example 2: Confirmation of Maltotriose Production Activity] right Figure 1 The 55 kDa purified fraction was further confirmed for maltotriose production activity as follows.

[0104] [1] Preparation of substrate solution 1 g of soluble starch was suspended in about 60 mL of purified water and heated at 100°C for 5 minutes to dissolve the starch. After cooling, the suspension was made up 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 the substrate solution and 50 μL of the enzyme solution to be analyzed were mixed to prepare 250 μL of a reaction solution.

[0106] [3] Preparation of samples for analysis The reaction solution was reacted at 50°C and 1000 rpm for 48 hours and then boiled at 100°C for 5 minutes to stop the reaction. The reaction solution after the reaction was stopped was diluted 30 times with purified water and filtered with a 0.45 μm filter to obtain a filtrate as an analysis sample.

[0107] The sample was transferred to a glass vial for analysis and subjected to HPLC analysis under the following conditions.

[0108] [Table 3] ○HPLC analysis

[0109] As a result of HPLC analysis, a peak of maltotriose was detected as a product. That is, the maltotriose-producing activity of the 55 kDa purified fraction was confirmed.

[0110] Likewise, Figure 1 Maltotriose-producing activity was confirmed in the 70 kDa purified fraction and the 77 kDa purified fraction.

[0111] [Test Example 3: Identification of a strain producing α-amylase having maltotriose-producing activity] The candidate α-amylase producer confirmed to have maltotriose-producing activity was subjected to 16S rRNA analysis. As a result, the candidate α-amylase producer was identified as belonging to the genus Cellulosimicrobacterium sp.

[0112] [Test Example 4: Amino Acid Sequence Analysis and Base Sequence Determination of α-Amylase Having Maltotriose-Producing Activity] [1] Analysis of N-terminal amino acid sequence and internal amino acid sequence The three bands (55 kDa, 70 kDa, and 77 kDa) obtained in Item [2] of Test Example 1 were cut out and analyzed by a protein sequencer. As a result, the N-terminal amino acid sequence of each band was determined. After each band was further treated with trypsin, the internal amino acid sequence was determined by analysis using LC-MSMS.

[0113] [2] Determination of base sequence [2-1]PCR Primers were designed based on the specific N-terminal amino acid sequence and internal amino acid sequence. PCR amplification was performed using 20 μL / tube of PCR reaction solution. The composition of the PCR reaction solution and the PCR conditions are shown below. Single amplification was confirmed by electrophoresis in 1% agarose gel.

[0114] [Table 4]

[0115] [2-2]TA-cloning A ligation reaction solution containing 2 μL of PCR product, 1 μL of T-Vecor pMD20, and 3 μL of Ligation Mix was used to carry out ligation reaction at 16°C for 30 minutes, and then transformed into 25 μL of E. coli BL21 (DE3). About 30 μL of the obtained transformation solution was applied to a liquid culture medium containing LB (Thermo Fisher Scientific) and Amp (final concentration 100 μg / mL) (hereinafter referred to as "LB+Amp liquid culture medium") plate, and cultured at 37°C, O / N.

[0116] [2-3] Plasmid extraction The transformant was cultured in LB+Amp liquid medium (2 mL) at 37°C, O / N. After the culture was completed, the bacteria were harvested and the plasmid was extracted by the Miniprep method.

[0117] [2-4] Sequence analysis The Sanger method was used for sequence analysis to determine the partial base sequence.

[0118] [2-5] Full-length base sequence analysis Colony hybridization was performed using a probe prepared based on the obtained partial base sequence, and the gene sequence of the obtained positive clone was analyzed to determine the target base sequence. As a result, the base sequence of sequence number 4 was obtained from the 55kDa band. The base sequence of sequence number 5 was obtained from the 70kDa band. The base sequence of sequence number 6 was obtained from the 77kDa band.

[0119] [3] Determination of amino acid sequence The amino acid sequence was determined from the above base sequence. The amino acid sequence encoded by the base sequence of sequence number 4 is sequence number 1, the amino acid sequence encoded by the base sequence of sequence number 5 is sequence number 2, and the amino acid sequence encoded by the base sequence of sequence number 6 is sequence number 3. That is, it can be seen that: through Test Example 1, maltotriose-producing amylases of sequence numbers 1, 2, and 3 can be obtained.

[0120] [Test Example 5: Recombinant production of maltotriose-producing amylase] [1] Construction of expression vector [1-1]PCR To amplify the amylase gene, the primer represented by sequence number 7 was designed as the forward primer, and the primer represented by sequence number 8 was designed as the reverse primer. PCR amplification was performed using 50 μL / tube of PCR reaction solution. The composition of the PCR reaction solution and the PCR conditions are shown below. Electrophoresis was performed on 1% agarose gel to confirm single amplification.

[0121] [Table 5]

[0122] [1-2. Connection and conversion] A ligation solution (5 μL) containing a mixture of PCR product and pUBCM21 (a shuttle vector of pUC19 and pUB110) was used to perform a ligation reaction at 16°C for 30 minutes, followed by transformation into 50 μL of E. coli DH5α. Approximately 55 μL of the resulting transformation solution was applied to an LB+Amp (100 μg / mL) plate and cultured at 37°C, O / N. The composition of the ligation reaction solution is shown below. [Table 6] ○) Ligation reaction solution composition

[0123] [1-3. Plasmid extraction] Plasmid extraction was performed by the same method as in 2-3 of Test Example 4 to obtain plasmid pUBCM21-amy.

[0124] [2] Transformation of Bacillus subtilis The obtained vector pUBCM21-amy was used to transform the amyE-deficient strain derived from Bacillus subtilis 168. The transformation method was carried out according to the instruction manual of B. subtilis Secretory Protein Expression System (manufactured by TaKaRa).

[0125] [3] Production of maltotriose-generating amylase [3-1] Cultivation of transformants The transformant was inoculated into a liquid medium containing LB (Thermo Fisher Scientific) and kanamycin (20 μg / mL) and cultured with shaking at 37° C. for 4 days. After the start of the culture, 0.5 mL of each sample was collected every day and centrifuged at 15,000 rpm for 5 minutes at 4° C. to recover the supernatant to obtain maltotriose-producing amylase.

[0126] [3-2] Confirmation of α-amylase productivity The recovered supernatant (maltotriose-generating amylase fraction) was subjected to activity assay and SDS-PAGE to confirm the α-amylase productivity. The activity assay was performed using an α-amylase assay kit (Kikkoman Chemiphar) according to the instructions of the kit. In addition, SDS-PAGE was performed in the same manner as that of Test Example 1.

[0127] The result of the activity assay is: compared with the negative control, i.e., pUBCM21 (empty vector introduced strain), it can be confirmed that the maltotriose generating amylase fraction has significant activity. In addition, the result of SDS-PAGE is: by optimizing the codon in Bacillus subtilis, three bands derived from α-amylase can be confirmed. The three bands are estimated to be bands of amylases of sequence numbers 1, 2, and 3 by their respective molecular weights. That is, through this test example, it is believed that maltotriose generating amylases of sequence numbers 1, 2, and 3 can be obtained.

[0128] Sequence numbers 7 and 8 are primers.

Claims

1. A maltotriose-generating amylase, characterized in that A polypeptide comprising any one of the following (1-1), (1-3), (2-1), (2-3), (3-1), and (3-3): (1-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, (1-3) a polypeptide having a sequence identity of 80% or more to the amino acid sequence represented by SEQ ID NO: 1 and having the ability to produce maltotriose, (2-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, (2-3) a polypeptide having a sequence identity of 80% or more to the amino acid sequence represented by SEQ ID NO: 2 and having the ability to produce maltotriose, (3-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3, (3-3) A polypeptide having a sequence identity of 80% or more to the amino acid sequence represented by SEQ ID NO: 3 and having the ability to produce maltotriose.

2. A DNA, characterized in that: Encodes the maltotriose-producing amylase of claim 1.

3. A recombinant vector, characterized in that: Containing the DNA according to claim 2.

4. A transformant, characterized in that The invention is obtained by transforming a host using the DNA according to claim 2 or the recombinant vector according to claim 3.

5. A method for producing a syrup or powder containing maltotriose, characterized in that: Including the following processes: Using the maltotriose-generating amylase according to claim 1 to generate maltotriose from starchy substances, thereby obtaining a sugar solution containing maltotriose; refining the sugar solution containing maltotriose to obtain sugar with increased maltotriose content; and The saccharide with increased maltotriose content is concentrated or dried to obtain the maltotriose-containing syrup or powder.

6. Use of the maltotriose-containing syrup or powder obtained by the production method according to claim 5 as a sweetener, taste improver, quality improver or stabilizer added to food, drink, snacks, feed, bait, cosmetics, quasi-drugs or pharmaceuticals.

Images