A method for preparing linear malthexasaccharide

Abstract

This invention discloses a method for preparing linear maltohexasaccharides, belonging to the field of functional sugar production technology. This invention provides a comprehensive method for preparing linear maltohexasaccharides, utilizing a synergistic reaction of linear maltohexasaccharide-generating enzyme, debranching enzyme, and metal ions, followed by yeast fermentation and enzymatic hydrolysis to fully utilize the substrate, improve substrate conversion rate, and increase maltohexasaccharide content. The linear maltohexasaccharides prepared by this invention have a substrate conversion rate ≥99%, and the maltohexasaccharide content in the reaction solution after yeast fermentation is ≥70%. It has advantages such as low production cost, short processing time, simple operation steps, and high product quality, and has good prospects for industrial application.

Description

Technical Field

[0001] This invention relates to a method for preparing linear maltohexasaccharide, belonging to the field of functional sugar production technology. Background Technology

[0002] Linear malthexasaccharide is a linear oligosaccharide composed of six glucose molecules linked by α-1,4 glycosidic bonds, typically produced by enzymatic hydrolysis of amylase. Its poor digestibility makes it a high-quality prebiotic, promoting the growth of beneficial gut bacteria and improving digestive health. It also possesses low sweetness, moisturizing properties, and stability, making it widely used in functional foods such as sugar-free products, baking modifiers, pharmaceutical carriers such as immunomodulators, and cosmetic moisturizing ingredients.

[0003] Traditional methods for preparing linear maltohexasaccharides mainly rely on chemical acid hydrolysis or enzymatic hydrolysis of starch. However, acid hydrolysis has problems such as numerous byproducts and harsh reaction conditions. Conventional enzymatic hydrolysis, which utilizes maltohexasaccharide generating enzymes, produces a mixture of several maltooligosaccharides, resulting in problems such as low maltohexasaccharide content and low substrate conversion rate.

[0004] Low maltohexasose content in the product leads to high production costs, while low substrate conversion rate fails to fully utilize the substrate, resulting in low production efficiency, further increasing production costs and energy consumption, and to some extent limiting the industrial production and further development and application of maltohexasose.

[0005] Patent CN106755197A discloses a method for preparing linear maltohexasaccharide using linear maltodextrin-producing enzyme, but the proportion of linear maltohexasaccharide in the product is below 35%, and the use of maltodextrin as a substrate results in high production costs. Patent CN 20191050033A discloses a linear maltodextrin-producing enzyme mutant with improved maltohexasaccharide production capacity, but the proportion of linear maltohexasaccharide in the final product is 42.40%, which is still low, and the substrate conversion rate is less than 80%.

[0006] Therefore, there is a need for a method for preparing linear maltohexasaccharide that can improve substrate conversion rate and linear maltohexasaccharide content, which has extremely high application and economic value. Summary of the Invention

[0007] To address the shortcomings of existing linear maltohexasaccharide preparation processes, such as low substrate conversion rate, low maltohexasaccharide content in the product, high production cost, and low production efficiency, this invention provides a comprehensive method for preparing linear maltohexasaccharides. This method utilizes a linear maltohexasaccharide generating enzyme (G109D / K71R) in conjunction with a debranching enzyme and metal ions in a synergistic reaction, followed by yeast fermentation and enzymatic hydrolysis. This approach aims to fully utilize the substrate, improve substrate conversion rate, and increase maltohexasaccharide content. The linear maltohexasaccharide prepared by this invention exhibits a substrate conversion rate ≥99%, and the maltohexasaccharide content in the reaction solution after yeast fermentation is ≥70%. It offers advantages such as low production cost, short processing time, simple operation steps, and high product quality, demonstrating promising prospects for industrial application.

[0008] The first objective of this invention is to provide a method for preparing high-purity linear maltohexasaccharide solution, wherein a linear maltohexasaccharide generating enzyme mutant and metal ions are added to corn starch milk, followed by liquefaction, enzymatic hydrolysis, and enzyme inactivation to obtain a reaction solution, which is then filtered, decolorized, and desalted to obtain the linear maltohexasaccharide solution.

[0009] The amino acid sequences of the linear maltohexasaccharide synthase mutant are shown in SEQ ID NO.3–5.

[0010] The metal ion is any one or more of calcium ions, sodium ions, magnesium ions, and zinc ions.

[0011] In one embodiment, the calcium ions are CaCl2 or CaSO4.

[0012] In one embodiment, the linear maltohexasaccharide synthase mutant is G109D / K71R, with the amino acid sequence shown in SEQ ID NO.3.

[0013] In one embodiment, the proportion of linear maltohexaose in the sugar solution reaches more than 70%.

[0014] In one embodiment, the corn starch milk contains 5% to 30% corn starch by mass.

[0015] In one embodiment, corn starch milk is prepared by adding corn starch to hot water at 60°C to 65°C and keeping it at that temperature for 10 to 20 minutes.

[0016] In one embodiment, the pH of the corn starch milk is 4.5 to 7.5.

[0017] In one embodiment, the liquefaction conditions are liquefaction at 80–90°C for 15–30 minutes.

[0018] In one embodiment, the amount of linear maltohexasaccharide synthase mutant added is 20 U / g to 45 U / g of dry substrate (i.e., corn starch), and the enzymatic hydrolysis conditions are 60 to 70°C for 18 to 48 hours.

[0019] In one embodiment, the concentration of metal ions is 0.5 mM to 20 mM.

[0020] In one embodiment, the concentration of metal ions is 1 mM to 15 mM.

[0021] In one embodiment, the metal ion is a calcium ion with a concentration of 5 mM to 15 mM.

[0022] In one embodiment, the calcium ion concentration is 8–12 mM.

[0023] In one embodiment, a debranching enzyme may be added during enzymatic hydrolysis to synergistically hydrolyze the enzyme, with the amount of debranching enzyme added being 2 U / g to 10 U / g of dry substrate.

[0024] The debranching enzyme is any one or more of pullulanase, dextrin debranching enzyme, isoamylase, and oligosaccharide debranching enzyme.

[0025] In one embodiment, the debranching enzyme is pullulanase, and the amount added is 2-6 U / g dry substrate.

[0026] In one embodiment, active dry yeast can be added after enzyme inactivation for further fermentation;

[0027] The active dry yeast is any one or more of baker's yeast, brewer's yeast, and beer yeast.

[0028] In one embodiment, after enzyme inactivation, the temperature is lowered to 30-35°C and kept warm for 15-30 minutes (to prevent the yeast from dying due to high temperature).

[0029] In one embodiment, the amount of active dry yeast inoculated is 0.5% to 3.0% of the substrate mass, and the fermentation conditions are 30℃ to 37℃ for 2 to 12 hours.

[0030] In one embodiment, the amount of active dry yeast inoculated is 1.0% to 2.0% of the substrate mass, and the fermentation conditions are 30℃ to 37℃ for 2 to 6 hours.

[0031] In one embodiment, filtration is performed using a filter membrane; decolorization is performed using activated carbon; and desalination is performed using anion / cation exchange resins.

[0032] In one embodiment, the anion / cation exchange resin desalination is performed using a macroporous weakly basic anion exchange resin / macroporous strong acid styrene-type cation exchange resin.

[0033] The second objective of this invention is to provide a method for improving the purity of maltohexasose in corn starch enzymatic hydrolysate. A linear maltohexasose generating enzyme mutant of corn starch and metal ions are added to corn starch milk, followed by liquefaction, enzymatic hydrolysis, and enzyme inactivation to obtain a reaction solution containing maltohexasose.

[0034] The amino acid sequences of the linear maltohexasaccharide synthase mutant are shown in SEQ ID NO.3–5.

[0035] The metal ion is any one or more of calcium ions, sodium ions, magnesium ions, and zinc ions.

[0036] In one embodiment, the proportion of linear maltohexaose in the sugar solution reaches more than 70%.

[0037] In one embodiment, the corn starch milk contains 5% to 30% corn starch by mass.

[0038] In one embodiment, corn starch milk is prepared by adding corn starch to hot water at 60°C to 65°C and keeping it at that temperature for 10 to 20 minutes.

[0039] In one embodiment, the pH of the corn starch milk is 4.5 to 7.5.

[0040] In one embodiment, the liquefaction conditions are liquefaction at 80–90°C for 15–30 minutes.

[0041] In one embodiment, the amount of linear maltohexasaccharide synthase mutant added is 20 U / g to 45 U / g of dry substrate, and the enzymatic hydrolysis conditions are 60 to 70°C for 18 to 48 hours.

[0042] In one embodiment, the concentration of metal ions is 0.5 mM to 20 mM.

[0043] In one embodiment, the concentration of metal ions is 1 mM to 15 mM.

[0044] In one embodiment, the metal ion is a calcium ion with a concentration of 5 mM to 15 mM.

[0045] In one embodiment, the calcium ion concentration is 8–12 mM.

[0046] In one embodiment, the amount of linear maltohexasaccharide synthase mutant added is 20 U / g to 45 U / g of dry substrate, and the enzymatic hydrolysis conditions are 60 to 70°C for 18 to 48 hours.

[0047] In one embodiment, a debranching enzyme may be added during enzymatic hydrolysis to synergistically hydrolyze the enzyme, with the amount of debranching enzyme added being 2 U / g to 10 U / g of dry substrate.

[0048] The debranching enzyme is any one or more of pullulanase, dextrin debranching enzyme, isoamylase, and oligosaccharide debranching enzyme.

[0049] In one embodiment, the debranching enzyme is pullulanase, and the amount added is 2-6 U / g dry substrate.

[0050] In one embodiment, active dry yeast can be added after enzyme inactivation for further fermentation;

[0051] The active dry yeast is any one or more of baker's yeast, brewer's yeast, and beer yeast.

[0052] In one embodiment, the amount of active dry yeast inoculated is 0.5% to 3.0% of the substrate mass, and the fermentation conditions are 30℃ to 37℃ for 2 to 12 hours.

[0053] In one embodiment, the amount of active dry yeast inoculated is 1.0% to 2.0% of the substrate mass, and the fermentation conditions are 30℃ to 37℃ for 2 to 6 hours.

[0054] In one embodiment, filtration is performed using a filter membrane; decolorization is performed using activated carbon; and desalination is performed using anion / cation exchange resins.

[0055] A third object of the present invention is to provide the application of any of the above-described methods in the preparation of linear maltohexasaccharide.

[0056] The fourth objective of this invention is to provide a linear maltodextrin-producing enzyme mutant with enhanced maltodextrin-producing ability, wherein the linear maltodextrin-producing enzyme mutant has an amino acid mutation at position 71 based on the amino acid sequence shown in SEQ ID NO.1;

[0057] Among them, the lysine at position 71 is mutated to arginine, serine or valine;

[0058] When the lysine at position 71 is mutated to arginine, the amino acid sequence of the linear maltodextrin synthase mutant is shown in SEQ ID NO.3;

[0059] When the lysine at position 71 is mutated to serine, the amino acid sequence of the linear maltodextrin synthase mutant is shown in SEQ ID NO.4.

[0060] When the lysine at position 71 is mutated to valine, the amino acid sequence of the linear maltodextrin synthase mutant is shown in SEQ ID NO.5.

[0061] Beneficial effects of the present invention

[0062] (1) This invention utilizes linear maltohexasaccharide generating enzyme for a one-step liquefaction and saccharification reaction, without the need to add other enzymes for liquefaction and re-saccharification, and without the need to adjust the pH during the production process. The process is simple and convenient, and the production cost is low.

[0063] (2) In this invention, debranching enzyme is added during saccharification to carry out a synergistic reaction, and the substrate conversion rate reaches more than 99%, which is more than 46.36% higher than that without debranching enzyme (67.64%).

[0064] (3) In this invention, metal ions are added during the reaction to help the enzyme maintain good stability under long-term high temperature.

[0065] (4) The linear maltodextrose syrup produced by this invention has a high content of linear maltodextrose, and it still has good application effects even without subsequent impurity removal process.

[0066] (5) The linear maltodextrin syrup produced by this invention has a low content of macrodextrin, which greatly reduces the pressure of subsequent separation and purification. Through yeast fermentation, linear maltodextrin syrup with a purity of more than 70% can be obtained. Attached Figure Description

[0067] Figure 1 Analysis of the products of G109D, G109D / K71R, G109D / K71S and G109D / K71V applied to 20% (w / w) corn starch in Example 2; wherein G1 to G7 represent glucose, maltose, linear maltotriose, linear maltotetraose, linear maltopentose, linear maltohexaose and linear maltoheptaose, respectively;

[0068] Figure 2 The purity of linear maltohexasaccharide in Examples 2, 3, and 5; wherein G1 to G7 represent glucose, maltose, linear maltotriose, linear maltotetraose, linear maltopentose, linear maltohexasaccharide, and linear maltoheptasaccharide, respectively.

[0069] Figure 3 The yield of linear maltohexasaccharide in Examples 2, 3, and 4;

[0070] Figure 4 Comparison of sugar solution and fermentation broth after final decolorization and desalination in Example 6 (left is after decolorization and desalination, right is fermentation broth);

[0071] Figure 5 The results show the thermal stability of the mutant G109D / K71R at 60–80 °C. Detailed Implementation

[0072] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0073] Some definitions or terms involved in this invention:

[0074] Mutant: As used herein, when referring to variations of the invention, the terms "mutant," "peptide variant," "peptide," or "linear maltodextrin mutant" mean a polypeptide having the enzyme activity of a linear maltodextrin mutant and having altered (i.e., substitution, insertion, and / or deletion) positions at one or more sites based on the sequence such as SEQ ID NO. 1. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following an amino acid occupying a position. In describing variations of the invention, the nomenclature described below has been adapted for ease of reference.

[0075] Accepted IUPAC single-letter or three-letter amino acid abbreviations were adopted. Substitution: For amino acid substitutions, the following nomenclature was used: original amino acid, position, substituted amino acid. Therefore, the substitution of lysine at position 71 with arginine was represented as "K71R". Multiple mutations were separated by the symbol (" / "), for example, "G109D / K71R" represents the substitution of glycine (G) and lysine (K) at positions 109 and 71 with aspartic acid (D) and arginine (R), respectively.

[0076] Parental or parental linear maltodextrin generating enzyme: As used herein, the term "parental" linear maltodextrin generating enzyme refers to a linear maltodextrin generating enzyme modified to produce a mutant of the linear maltodextrin generating enzyme of the present invention. The term also refers to a polypeptide to which the mutant of the present invention is compared. The parent can be a naturally occurring (wild-type) polypeptide, or it can be, or even a variant thereof, prepared by any suitable means. For example, the parental protein can be a variant of a naturally occurring polypeptide whose amino acid sequence has been modified or altered. Thus, the parental linear maltodextrin generating enzyme may have one or more (or one or more) amino acid substitutions, deletions, and / or insertions. Thus, the parental linear maltodextrin generating enzyme can be a variant of the parental linear maltodextrin generating enzyme. The parent can also be an allelic variant, which is a polypeptide encoded by any of two or more alternative forms of a gene occupying the same chromosomal locus.

[0077] Wild-type enzyme: When referring to an amino acid or nucleic acid sequence, the term "wild-type" means that the amino acid or nucleic acid sequence is a naturally occurring or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences). When the parent enzyme is not a variant enzyme, the terms "wild-type enzyme" and "parent enzyme" are used interchangeably.

[0078] Expression: As used in this article, “expression” refers to any step involving the generation of variants, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0079] Expression vector: As used herein, the term “expression vector” refers to a linear or circular DNA molecule that contains a polynucleotide encoding a variant and is operatively linked to a control sequence that provides for its expression.

[0080] Host cell: The term "host cell" means any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "host cell" encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication, along with recombinant host cells, isolated host cells (e.g., isolated recombinant host cells), and heterologous host cells.

[0081] Recombination: When used to refer to cells, nucleic acids, proteins, or vectors, the term "recombination" means that the cell has been modified from its natural state. Thus, for example, recombinant cells express genes not found in the natural (non-recombinant) form of the cell, or express natural genes at different levels or under different conditions compared to those found in nature. The difference between recombinant nucleic acids and their natural sequences lies in the operative linking of one or more nucleotides and / or a foreign sequence (e.g., a foreign promoter in an expression vector). The difference between recombinant proteins and their natural sequences may lie in the fusion of one or more amino acids and / or a foreign sequence. A vector containing nucleic acids encoding a polypeptide is a recombinant vector. The term "recombination" is synonymous with "genetically modified" and "transgenic."

[0082] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0083] It should be noted that the abbreviations of amino acid residues in this article follow the general definition in this field, but in some instances, the full name is still indicated in parentheses after the abbreviation. In this article, "amino acid residue" and "amino acid" can be used interchangeably. The amino acid sequence in this article starts from the N-terminus.

[0084] In some alternative embodiments, a linear maltodextrin-producing enzyme mutant with enhanced maltohexasaccharide production capacity is provided, wherein the linear maltodextrin-producing enzyme mutant has an amino acid mutation at position 71 based on the amino acid sequence shown in SEQ ID NO.1.

[0085] In an optional embodiment, the amino acid sequence of the linear maltodextrin synthase is as shown in SEQ ID NO.1, and the nucleotide sequence of the linear maltodextrin synthase is as shown in SEQ ID NO.2; the linear maltodextrin synthase is a single mutant G109D of linear maltodextrin synthase derived from Bacillus stearothermophilus STB04.

[0086] In an optional embodiment, the linear maltodextrin-producing enzyme monomutant derived from Bacillus stearothermophilus STB04 is a wild-type enzyme in which glycine at position 109 is mutated to aspartic acid.

[0087] In an optional implementation, G109D is used as the parent for mutation.

[0088] In an alternative implementation, based on G109D as the parent, the lysine at position 71 is mutated to arginine, serine, or valine.

[0089] In an optional embodiment, when the lysine at position 71 is mutated to arginine, the amino acid sequence of the linear maltodextrin-producing enzyme mutant is shown in SEQ ID NO.3 and named G109D / K71R.

[0090] In an optional embodiment, when the lysine at position 71 is mutated to serine, the amino acid sequence of the linear maltodextrin-producing enzyme mutant is shown in SEQ ID NO.4 and named G109D / K71S.

[0091] In an optional embodiment, when the lysine at position 71 is mutated to valine, the amino acid sequence of the linear maltodextrin-producing enzyme mutant is shown in SEQ ID NO.5 and named G109D / K71V.

[0092] In an optional implementation, the mutant is constructed using a two-step PCR method, with the following steps:

[0093] The first step, PCR, uses the G109D plasmid pST-MFA / G109D as a template and F1 and R2 as upstream and downstream primers. This amplifies the PCR product L1. Similarly, using the G109D plasmid pST-MFA / G109D as a template and R1 and F2 as upstream and downstream primers, the PCR product L2 is amplified.

[0094] The second step, PCR, uses the PCR products L1 and L2 from the first step as templates and R1 and R2 as primers for amplification.

[0095] In an optional implementation, the first step of PCR amplification conditions are: pre-denaturation at 98°C for 3 min; followed by 34 cycles of denaturation at 98°C for 20 s, annealing at 60°C for 30 s, and extension at 68°C for 4 min; and finally incubation at 68°C for 10 min.

[0096] In an optional implementation, the second-step PCR amplification conditions are as follows: pre-denaturation at 98°C for 3 min; followed by 34 cycles of denaturation at 98°C for 20 s, annealing at 60°C for 30 s, and extension at 68°C for 8 min; and finally incubation at 68°C for 10 min.

[0097] In some optional embodiments, a method for preparing linear maltohexasaccharide is provided, wherein a linear maltohexasaccharide generating enzyme mutant and metal ions are added to corn starch milk, followed by liquefaction, enzymatic hydrolysis, and enzyme inactivation to obtain a reaction solution, and the reaction solution is filtered, decolorized, and desalted to obtain a linear maltohexasaccharide solution.

[0098] The amino acid sequences of the linear maltohexasaccharide synthase mutant are shown in SEQ ID NO.3–5.

[0099] The metal ion is any one or more of calcium ions, sodium ions, magnesium ions, and zinc ions.

[0100] In an optional embodiment, the pure enzyme activities of the linear maltodextrin generating enzyme double mutants G109D / K71R, G109D / K71S, and G109D / K71V are 9.04 U / g, 7.05 U / g, and 7.29 U / g, respectively.

[0101] In an optional embodiment, the amino acid sequence of the chain maltohexasaccharide generating enzyme mutant is shown in SEQ ID NO.3.

[0102] In an optional embodiment, the sugar solution contains more than 70% linear maltohexaose.

[0103] In an optional embodiment, the corn starch milk contains 5% to 30% corn starch by mass.

[0104] In an optional embodiment, the corn starch milk is prepared by adding corn starch to hot water at 60°C to 65°C and keeping it warm for 10 to 20 minutes.

[0105] In an optional embodiment, the pH of the corn starch milk is 4.5 to 7.5.

[0106] In an optional implementation, the liquefaction conditions are liquefaction at 80–90°C for 15–30 minutes.

[0107] In an optional embodiment, the amount of linear maltohexasaccharide synthase mutant added is 20 U / g to 45 U / g of dry substrate, and the enzymatic hydrolysis conditions are 60 to 70°C for 18 to 48 hours.

[0108] In an optional embodiment, the concentration of metal ions is 0.5 mM to 20 mM.

[0109] In an optional embodiment, the concentration of metal ions is 1 mM to 15 mM.

[0110] In an optional embodiment, the metal ion is a calcium ion with a concentration of 5 mM to 15 mM.

[0111] In an optional embodiment, the calcium ion concentration is 8–12 mM.

[0112] In an optional embodiment, a debranching enzyme can be added during enzymatic hydrolysis to synergistically hydrolyze the enzyme, with the amount of debranching enzyme added being 2 U / g to 10 U / g of dry substrate.

[0113] The debranching enzyme is any one or more of pullulanase, dextrin debranching enzyme, isoamylase, and oligosaccharide debranching enzyme.

[0114] In an optional embodiment, the debranching enzyme is pullulanase, and the amount added is 2-6 U / g dry substrate.

[0115] In an optional implementation, active dry yeast can be added after enzyme inactivation for further fermentation;

[0116] The active dry yeast is any one or more of baker's yeast, brewer's yeast, and beer yeast.

[0117] In an optional implementation, the amount of active dry yeast inoculated is 0.5% to 3.0% of the substrate mass, and the fermentation conditions are 30℃ to 37℃ for 2 to 12 hours.

[0118] In an optional implementation, the amount of active dry yeast inoculated is 1.0% to 2.0% of the substrate mass, and the fermentation conditions are 30℃ to 37℃ for 2 to 6 hours.

[0119] In optional embodiments, filtration is performed using a filter membrane; decolorization is performed using activated carbon; and desalination is performed using anion / cation exchange resins.

[0120] In some alternative embodiments, a method for improving the purity of maltohexasose in corn starch hydrolysate is provided, wherein a corn starch linear maltohexasose generating enzyme mutant and metal ions are added to corn starch milk, followed by liquefaction, enzymatic hydrolysis, and enzyme inactivation to obtain a reaction solution containing maltohexasose.

[0121] The amino acid sequences of the linear maltohexasaccharide synthase mutant are shown in SEQ ID NO.3–5.

[0122] The metal ion is any one or more of calcium ions, sodium ions, magnesium ions, and zinc ions.

[0123] In an optional embodiment, a debranching enzyme can be added during enzymatic hydrolysis to synergistically hydrolyze the enzyme, with the amount of debranching enzyme added being 2 U / g to 10 U / g of dry substrate.

[0124] The debranching enzyme is any one or more of pullulanase, dextrin debranching enzyme, isoamylase, and oligosaccharide debranching enzyme.

[0125] In an optional implementation, active dry yeast can be added after enzyme inactivation for further fermentation;

[0126] The active dry yeast is any one or more of baker's yeast, brewer's yeast, and beer yeast.

[0127] In an optional embodiment, the concentration of metal ions is 0.5 mM to 20 mM.

[0128] In an optional embodiment, the metal ion is a calcium ion with a concentration of 5 mM to 15 mM.

[0129] In an optional embodiment, the calcium ion concentration is 8–12 mM.

[0130] In an optional embodiment, the amount of linear maltohexasaccharide synthase mutant added is 20 U / g to 45 U / g of dry substrate, and the enzymatic hydrolysis conditions are 60 to 70°C for 18 to 48 hours.

[0131] Raw materials used in the examples:

[0132] Bacillus stearothermophilus STB04 and plasmid pST were published in the literature “Pan Sihui. Study on secretion expression, enzymatic properties and product synthesis of linear malt oligosaccharide generating enzyme in Bacillus subtilis [D]. Jiangnan University, 2018.”; plasmid pST / MFA-G109D was published in patent CN110229800A.

[0133] The macroporous weakly basic anion exchange resin was purchased from Shanghai Titan Technology Co., Ltd.

[0134] The macroporous strong acid styrene-type cation exchange resin was purchased from Shanghai Titan Technology Co., Ltd.

[0135] The active dry yeast was purchased from Angel Yeast Co., Ltd.

[0136] Pullulanase was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0137] The activated carbon was purchased from Shanghai Myriel Biochemical Technology Co., Ltd.

[0138] Test method:

[0139] 1. Method for determining the activity of linear maltodextrin generating enzyme:

[0140] Enzyme activity was determined using the 3,5-dinitrosalicylic acid (DNS) method. A 1% (w / v) soluble starch solution was prepared using C6H8O7-Na2HPO4 buffer (10 mM, pH 5.5) as the substrate. 100 μL of appropriately diluted enzyme solution was added to 900 μL of the substrate, and the reaction was carried out at 60 °C for 15 min. The reaction was terminated by adding 1.0 mL of DNS solution. After color development in a boiling water bath for 5 min, the mixture was immediately cooled in an ice bath. 2 mL of deionized water was added, and the mixture was shaken thoroughly. The absorbance was measured at 540 nm, and the reducing sugar content in the system was calculated based on the glucose standard curve. One enzyme activity unit (U) was defined as the amount of enzyme required to generate 1 μmol of reducing sugar (based on glucose) per minute.

[0141] 2. Methods for determining the content of components in the product:

[0142] The content of each component in the product was analyzed using high-performance anion exchange chromatography (HPAEC-PAD) under the following analytical conditions:

[0143] A CarboPac PA 200 column was used with 0.25M NaOH, 1M NaAc and ultrapure water as the mobile phase. The flow rate was set to 0.5 mL / min, the column temperature to 35℃, and the injection volume to 10 μL.

[0144] The calculation methods for the total substrate conversion rate and the percentage of each monosaccharide component in G1–G7 are as follows:

[0145] Monosaccharide percentage = (mass of monosaccharide components / total mass of G1 to G7) × 100%

[0146] Overall conversion rate = (total mass of G1 to G7 / dry basis mass of substrate) × 100%.

[0147] Example 1: Construction of a double mutant of linear maltodextrin-producing enzyme

[0148] 1. Constructing recombinant plasmids

[0149] The amino acid sequence of the linear maltohexaose-forming amylase (MFA) single mutant G109D is shown in SEQ ID NO.1 (nucleotide sequence is shown in SEQ ID NO.2). The nucleotide sequence of G109D was inserted between NcoⅠ and XhoⅠ in plasmid pST to construct the recombinant plasmid pST / MFA. G109D .

[0150] Using the recombinant vector pST / MFA G109D Using a template, the lysine at position 71 was mutated to arginine, serine, and valine, respectively, to construct the recombinant plasmid pST / MFA. G109D / K71R pST / MFA G109D / K71S pST / MFA G109D / K71V The primers are shown in Table 1.

[0151] Table 1 Primer sequences

[0152]

[0153] 2. Constructing recombinant strains

[0154] The recombinant plasmid obtained in step 1 was transformed into Escherichia coli JM109. The transformed Escherichiacoli JM109 was plated on LB agar medium containing 20 μg / mL kanamycin and cultured overnight at 37°C for 12 h. Single colonies were selected and inoculated into LB liquid medium containing 20 μg / mL kanamycin and cultured overnight at 37°C and 200 r / min. The plasmid was then extracted for identification and sequencing.

[0155] The recombinant plasmid with correct sequencing results was transformed into competent cells of the expression host Bacillus subtilis WB600 via chemical transformation. The resulting recombinant strains were named Bacillus subtilis WB600-pST / MFA. G109D / K71R , Bacillus subtilis WB600-pST / MFA G109D / K71S and Bacillus subtilis WB600-pST / MFA G109D / K71V .

[0156] 3. Mutant expression and purification

[0157] (1) Activation:

[0158] The recombinant strain obtained in step 2 was streaked on LB solid medium and cultured overnight in a 37°C incubator. Positive single colonies were picked and inoculated into 250 mL Erlenmeyer flasks containing 50 mL of LB liquid medium. Before inoculation, Erlenmeyer flasks with a final concentration of 20 μg / mL kanamycin were added and placed in a rotary shaker at 200 r / min and cultured at 37°C for 12 h to obtain the sparked seed culture.

[0159] (2) Fermentation:

[0160] The activated seed culture was transferred at an inoculation rate of 4% (v / v) to a 250 mL Erlenmeyer flask containing 50 mL of fermentation medium and cultured on a shaker for 48 h (200 r / min). Kanamycin was added to a final concentration of 20 μg / mL before inoculation. After fermentation, the fermentation broth was centrifuged at 4 °C and 10000 × g for 15 min, and the supernatant was collected to obtain the crude enzyme solution.

[0161] (3) Purification:

[0162] The fermentation supernatant was filtered through a 0.45 μm aqueous membrane and purified stepwise using a 5-mL HiTrap Phenyl HP column. The column was equilibrated with 25 mL of ultrapure water at a flow rate of 2 mL / min and the flow rate was kept constant. After loading 60 mL of the sample, the target protein on the column was eluted with 10 mM NaOH at a flow rate of 2 mL / min. The collected eluent was dialyzed in ultrapure water for 24 h, and then identified by enzyme activity determination and SDS-PAGE. Pure enzymes of the linear maltodextrin generating enzyme double mutants G109D / K71R (amino acid sequence as shown in SEQ ID NO.3), G109D / K71S (amino acid sequence as shown in SEQ ID NO.4), and G109D / K71V (amino acid sequence as shown in SEQ ID NO.5) were obtained and stored at -80℃.

[0163] The enzyme activities of the pure enzymes of the linear maltodextrin generating enzyme double mutants G109D / K71R, G109D / K71S and G109D / K71V were 9.04 U / g, 7.05 U / g and 7.29 U / g, respectively.

[0164] 4. Mutant thermal stability test

[0165] The thermostability of the mutant was tested at 60℃, 70℃, and 80℃ in a Gly-NaOH solution (pH 8.0) with an enzyme concentration of 1.32 mg / mL. The results are as follows: Figure 5 As shown in Table 2.

[0166] Table 2 Thermal stability test results

[0167]

[0168] The results showed that after treatment at 60–70 °C for 90 min, the residual enzyme activity still reached over 70%.

[0169] Example 2: Application of a linear maltodextrin-producing enzyme double mutant in the preparation of maltohexasaccharide

[0170] The linear maltodextrin generating enzyme double mutants G109D / K71R, G109D / K71S, and G109D / K71V prepared in Example 1 were analyzed for their enzymatic hydrolysis products. The steps are as follows:

[0171] Prepare a 20% (w / w) corn starch solution with pH 5.5. Add the linear maltodextrin synergist double mutants G109D / K71R, G109D / K71S and G109D / K71V at an enzyme dosage of 30 U / g, respectively, with the linear maltodextrin synergist single mutant G109D as the control. React at 70℃ for 24 h.

[0172] After the reaction, the enzyme was inactivated by boiling in a water bath for 30 min, centrifuged at 10000×g for 10 min, diluted and filtered through a 0.22μm syringe filter, and qualitative and quantitative analysis was performed using a mixture of G1 to G7 standards with a certain concentration gradient as a control.

[0173] The results are shown in Table 7, Table 3 and... Figure 1 As shown, the results indicate that G6 in G109D accounts for 43.85% of the product, and the substrate conversion rate is 63.65%. The double mutant of linear maltodextrin synthase significantly increased the proportion of maltohexasaccharide in the product, reaching 50.75% (G109D / K71R), 48.46% (G109D / K71S), and 46.08% (G109D / K71V), respectively. Among them, G109D / K71R showed the most significant improvement in the proportion and yield of linear maltohexasaccharide, increasing G109D by 15.74% and 22.97%, respectively.

[0174] Table 7 Product Detection Results

[0175] G1 2.53 0.51 0.98 1.89 G2 16.45 4.76 6.07 5.60 G3 14.57 9.18 9.97 9.86 G4 7.32 5.41 6.35 6.70 G5 10.79 16.31 22.33 24.90 G6 43.85 50.75 48.46 46.08 G7 4.50 13.08 5.84 4.96 Substrate conversion rate (%) 63.65 67.64 66.24 65.18

[0176] Note: G1 to G7 represent glucose, maltose, linear maltotriose, linear maltotetraose, linear maltopentose, linear maltohexaose, and linear maltoheptaose, respectively.

[0177] Table 3. Yield of linear maltohexaose

[0178] Straight-chain maltohexaose yield (%) 27.91 34.32 32.10 30.03 Increase in amount compared to G109D (%) - 22.97 15.01 7.60

[0179] Example 3: Preparation of linear maltohexasaccharide using calcium-assisted mutants

[0180] The linear maltodextrose-producing enzyme double mutant G109D / K71R prepared in Example 2 was used to prepare maltohexasaccharide by adding calcium ions. The steps are as follows:

[0181] (1) Prepare a 20% (w / w) corn starch milk at 60℃, adjust the pH to 5.5 and keep warm for 15 min;

[0182] (2) Add 30 U / g dry substrate (i.e., corn starch) G109D / K71R and 10 mM CaCl2 solution to the corn starch milk prepared in step (1), stir at 300 rpm, heat to 90℃ and liquefy for 15 min; then cool to 65℃ and continue the reaction for 24 h to obtain the enzymatic hydrolysate.

[0183] The reaction was terminated by heating the enzymatic hydrolysate in boiling water for 30 min and centrifuged at 10,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm aqueous membrane to remove impurities and then diluted. The distribution of small molecule sugars in the reaction solution was analyzed by ion chromatography.

[0184] The results are shown in Table 4 and Figure 2 As shown, the substrate conversion rate of the enzymatic hydrolysate was 81.11%, and the proportion of linear maltohexaose in the product was 60.53%.

[0185] Table 4. Detection results of calcium ion-assisted G109D / K71R reaction solution

[0186] Example 4: Preparation of linear maltohexasaccharide by pullulanase coordinated by calcium ion-assisted mutants

[0187] Based on Example 3, pullulanase was further added for synergistic hydrolysis, and the steps are as follows:

[0188] (1) Prepare a 20% (w / w) corn starch milk at 60℃, adjust the pH to 5.5 and keep warm for 15 min;

[0189] (2) Add 30 U / g dry substrate (i.e., corn starch) G109D / K71R and 10 mM calcium ions to the corn starch milk prepared in step (1), stir at 300 rpm, and heat to 90℃ for liquefaction for 15 min;

[0190] (3) Cool down to 65℃, add pullulanase of 4U / g dry substrate, and react synergistically at 300rpm and 65℃ for 24h to obtain synergistic enzymatic hydrolysate.

[0191] The reaction was terminated by heating the synergistic enzymatic hydrolysate in boiling water for 30 min, followed by centrifugation at 10,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm aqueous membrane to remove impurities and then diluted. The distribution of small molecule sugars in the reaction solution was analyzed by ion chromatography.

[0192] The results are shown in Table 5 and Figure 3 As shown, the substrate conversion rate of the synergistic enzymatic hydrolysate was 101.05%, the proportion of linear maltohexasose in the product was 53.77%, and the yield of linear maltohexasose was 54.33%.

[0193] Table 5. Detection results of calcium ion-assisted G109D / K71R synergistic pullulanase reaction solution.

[0194] Example 5: Preparation of linear maltohexasaccharide by coordinating pullulanase and active dry yeast with calcium ion-assisted mutants

[0195] Based on Example 4, active dry yeast was further added for synergistic hydrolysis, and the steps are as follows:

[0196] (1) Prepare a 20% (w / w) corn starch milk at 60℃, adjust the pH to 5.5 and keep warm for 15 min;

[0197] (2) Add 30 U / g dry substrate (i.e., corn starch) G109D / K71R and 10 mM calcium ions to the corn starch milk prepared in step (1), stir at 300 rpm, and heat to 90℃ for liquefaction for 15 min.

[0198] (3) Cool down to 65℃, add pullulanase of 4U / g dry substrate, and react synergistically at 300rpm and 65℃ for 24h to obtain synergistic enzymatic hydrolysate;

[0199] (4) After the enzyme inactivation of the synergistic enzymatic hydrolysate is in a boiling water bath for 30 min, the temperature is lowered to 35℃ and kept warm for 15-30 min. 1.5% of the dry substrate mass of active dry yeast is added to the reaction solution and fermented at 300 rpm for 4 h to obtain the fermentation broth.

[0200] The fermentation broth was passed through a 0.45 μm aqueous membrane to remove yeast, then through a 0.22 μm aqueous membrane to remove impurities and dilute. The distribution of small molecule sugars in the fermentation broth was analyzed by ion chromatography.

[0201] The results are shown in Table 6 and Figure 1 As shown, the final fermentation broth contained 70.27% G6.

[0202] Table 6. Detection results of calcium ion-assisted G109D / K71R synergistic pullulanase and active dry yeast fermentation broth.

[0203] Percentage of small molecule sugars (%) 0 0 0 6.3 16.47 70.27 6.96

[0204] Example 6: A method for preparing linear malthexasaccharide

[0205] Based on Example 5, the fermentation broth was further decolorized and desalted, as follows:

[0206] (1) Prepare a 20% (w / w) corn starch milk at 60℃, adjust the pH to 5.5 and keep warm for 15 min;

[0207] (2) Add 30 U / g dry substrate (i.e., corn starch) G109D / K71R and 10 mM calcium ions to the corn starch milk prepared in step (1), stir at 300 rpm, and heat to 90℃ for liquefaction for 15 min.

[0208] (3) Cool down to 65℃, add pullulanase of 4U / g dry substrate, and react synergistically at 300rpm and 65℃ for 24h to obtain synergistic enzymatic hydrolysate;

[0209] (4) After the enzyme inactivation of the synergistic enzymatic hydrolysate is in a boiling water bath for 30 min, the temperature is lowered to 35℃ and kept warm for 15-30 min. 1.5% of the dry substrate mass of active dry yeast is added to the reaction solution and fermented at 300 rpm for 4 h to obtain the fermentation broth.

[0210] (5) Add 3.0% activated carbon based on the weight of the fermentation broth to the fermentation broth, decolorize at pH 4.0 and 40℃ for 60 min, centrifuge and filter to remove yeast cells and activated carbon, and obtain a sugar solution with a light transmittance of more than 95%.

[0211] (6) The sugar solution is passed through D315 macroporous weak base anion exchange resin / macroporous strong acid styrene-type cation exchange resin to remove the anions / cations, so that the conductivity of the sugar solution is below 10 μs / cm.

[0212] The appearance of the sugar syrup is as follows Figure 4 As shown, the decolorized and desalted syrup is clear in color, and the proportion of linear maltohexasose is more than 70%. This method has advantages such as high substrate conversion rate, high yield of linear maltohexasose, high purity of linear maltohexasose, and low production cost.

[0213] Sequences used in the examples

[0214] amino acid sequence of SEQ ID NO.1G109D

[0215] AAPFNGTMMQYFEWYLPDDGTLWTKVANEANNLSSLGITALWLPPAYKGTSRSDVGYGVYDLYDLGEFNQKGTVRTKYGTKAQYLQAIQAAHAAGMQVYADVVFDHKGDADGTEWVDAVEVNPSDRNQEISGTYQIQAWTKFDFPGRGNTYSSFKWRWYHFDGVDWDESRKLSRIYKFRGIGKAWDWEVDTENGNYDYLMYADLDMDHPEVVTELKNWGKWYVNTTNIDGFRLDAVKHIKFSFFPDWLSYVRSQTGKPLFTVGEYWSYDINKLHNYITKTNGTMSLFDAPLHNKFYTASKSGGAFDMRTLMTNTLMKDQPTLAVTFVDNHDTEPGQALQSWVDPWFKPLAYAFILTRQEGYPGVFYGDYYGIPQYNIPSLKSKIDPLLIARRDYAYGTQHDYLDHSDIIGWTREGVTEKPGSGLAALITDGPGGSKWMYVGKQHAGKVFYDLTGNRSDTVTITSDGWGEFKVNGGSVSVWVPRKTTVSTITRPITTRPWTGEFVRWTEPRLVAWP

[0216] Nucleotide sequence of SEQ ID NO.2 G109D

[0217]

[0218] Amino acid sequence of SEQ ID NO.3 G109D / K71R

[0219] AAPFNGTMMQYFEWYLPDDGTLWTKVANEANNLSSLGITALWLPPAYKGTSRSDVGYGVYDLYDLGEFNQRGTVRTKYGTKAQYLQAIQAAHAAGMQVYADVVFDHKGDADGTEWVDAVEVNPSDRNQEISGTYQIQAWTKFDFPGRGNTYSSFKWRWYHFDGVDWDESRKLSRIYKFRGIGKAWDWEVDTENGNYDYLMYADLDMDHPEVVTELKNWGKWYVNTTNIDGFRLDAVKHIKFSFFPDWLSYVRSQTGKPLFTVGEYWSYDINKLHNYITKTNGTMSLFDAPLHNKFYTASKSGGAFDMRTLMTNTLMKDQPTLAVTFVDNHDTEPGQALQSWVDPWFKPLAYAFILTRQEGYPGVFYGDYYGIPQYNIPSLKSKIDPLLIARRDYAYGTQHDYLDHSDIIGWTREGVTEKPGSGLAALITDGPGGSKWMYVGKQHAGKVFYDLTGNRSDTVTITSDGWGEFKVNGGSVSVWVPRKTTVSTITRPITTRPWTGEFVRWTEPRLVAWP

[0220] Amino acid sequence of SEQ ID NO.4 G109D / K71S

[0221] AAPFNGTMMQYFEWYLPDDGTLWTKVANEANNLSSLGITALWLPPAYKGTSRSDVGYGVYDLYDLGEFNQSGTVRTKYGTKAQYLQAIQAAHAAGMQVYADVVFDHKGDADGTEWVDAVEVNPSDRNQEISGTYQIQAWTKFDFPGRGNTYSSFKWRWYHFDGVDWDESRKLSRIYKFRGIGKAWDWEVDTENGNYDYLMYADLDMDHPEVVTELKNWGKWYVNTTNIDGFRLDAVKHIKFSFFPDWLSYVRSQTGKPLFTVGEYWSYDINKLHNYITKTNGTMSLFDAPLHNKFYTASKSGGAFDMRTLMTNTLMKDQPTLAVTFVDNHDTEPGQALQSWVDPWFKPLAYAFILTRQEGYPGVFYGDYYGIPQYNIPSLKSKIDPLLIARRDYAYGTQHDYLDHSDIIGWTREGVTEKPGSGLAALITDGPGGSKWMYVGKQHAGKVFYDLTGNRSDTVTITSDGWGEFKVNGGSVSVWVPRKTTVSTITRPITTRPWTGEFVRWTEPRLVAWP

[0222] Amino acid sequence of SEQ ID NO.5 G109D / K71V

[0223] AAPFNGTMMQYFEWYLPDDGTLWTKVANEANNLSSLGITALWLPPAYKGTSRSDVGYGVYDLYDLGEFNQVGTVRTKYGTKAQYLQAIQAAHAAGMQVYADVVFDHKGDADGTEWVDAVEVNPSDRNQ EISGTYQIQAWTKFDFPGRGNTYSSFKWRWYHFDGVDWDESRKLSRIYKFRGIGKAWDWEVDTENGNYDYLMYADLDMDHPEVVTELKNWGKWYVNTTNIDGFRLDAVKHIKFSFFPDWLSYVRSQTGK PLFTVGEYWSYDINKLHNYITKTNGTMSLFDAPLHNKFYTASKSGGAFDMRTLMTNTLMKDQPTLAVTFVDNHDTEPGQALQSWVDPWFKPLAYAFILTRQEGYPGVFYGDYYGIPQYNIPSLKSKIDP LLIARRDYAYGTQHDYLDHSDIIGWTREGVTEKPGSGLAALITDGPGGSKWMYVGKQHAGKVFYDLTGNRSDTVTITSDGWGEFKVNGGSVSVWVPRKTTVSTITRPITTRPWTGEFVRWTEPRLVAWP

[0224] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing high-purity linear maltohexasaccharide solution, characterized in that, A linear maltohexasose generating enzyme mutant and metal ions were added to corn starch milk. The mixture was then liquefied, enzymatically hydrolyzed, and the enzyme was inactivated to obtain a reaction solution. The reaction solution was then filtered, decolorized, and desalted to obtain a linear maltohexasose sugar solution. The amino acid sequences of the linear maltohexasaccharide synthase mutant are shown in SEQ ID NO.3~5; The metal ion is calcium ion.

2. The method according to claim 1, characterized in that, The mass fraction of corn starch in corn starch milk is 5% to 30%.

3. The method according to claim 1, characterized in that, The liquefaction conditions are 80~90 ℃ for 15 min~30 min.

4. The method according to claim 1, characterized in that, The amount of linear maltohexasaccharide synthase mutant added was 20 U / g~45 U / g dry substrate, and the enzymatic hydrolysis conditions were 60~70℃ for 18 h~48 h.

5. The method according to claim 1, characterized in that, The concentration of metal ions ranges from 0.5 mM to 20 mM.

6. The method according to claim 5, characterized in that, The concentration of metal ions is 1 mM to 15 mM.

7. The method according to claim 6, wherein the metal ion is a calcium ion with a concentration of 5 mM to 15 mM.

8. The method according to claim 1, characterized in that, Debranching enzyme can be added during enzymatic hydrolysis to synergize with the hydrolysis. The amount of debranching enzyme added is 2 U / g to 10 U / g of dry substrate. The debranching enzyme is any one or more of pullulanase, dextrin debranching enzyme, isoamylase, and oligosaccharide debranching enzyme.

9. The method according to claim 8, characterized in that, The debranching enzyme is pullulanase, and the addition amount is 2~6 U / g dry substrate.

10. The method according to claim 1, characterized in that, After enzyme inactivation, active dry yeast can be added for further fermentation. The active dry yeast is any one or more of baker's yeast, brewer's yeast, and beer yeast.

11. The method according to claim 10, characterized in that, The amount of active dry yeast inoculated is 0.5% to 3.0% of the substrate mass, and the fermentation conditions are 30℃ to 37℃ for 2 to 12 hours.

12. The method according to claim 11, characterized in that, The amount of active dry yeast inoculated is 1.0%~2.0% of the substrate mass, and the fermentation conditions are 30℃~37℃ for 2 h~6 h.

13. The method according to any one of claims 1 to 12, characterized in that, Filtration is performed using a filter membrane; decolorization is performed using activated carbon; and desalination is performed using anion / cation exchange resins.

14. A method for improving the purity of maltohexaose in corn starch enzymatic hydrolysate, characterized in that, A linear maltohexasose synthase mutant of corn starch and metal ions were added to corn starch milk. The mixture was then liquefied, enzymatically hydrolyzed, and the enzyme was inactivated to obtain a reaction solution containing maltohexasose. The amino acid sequences of the linear maltohexasaccharide synthase mutant are shown in SEQ ID NO.3~5; The metal ion is calcium ion.

15. The method according to claim 14, characterized in that, During enzymatic hydrolysis, debranching enzyme can be added to synergistically hydrolyze the enzyme. The amount of debranching enzyme added is 2 U / g~10 U / g of dry substrate. The debranching enzyme is any one or more of pullulanase, dextrin debranching enzyme, isoamylase, and oligosaccharide debranching enzyme.

16. The method according to claim 15, characterized in that, After enzyme inactivation, active dry yeast can be added for further fermentation. The active dry yeast is any one or more of baker's yeast, brewer's yeast, and beer yeast.

17. The method according to any one of claims 14 to 16, characterized in that, The concentration of metal ions ranges from 0.5 mM to 20 mM.

18. The method according to any one of claims 14 to 16, characterized in that, The amount of linear maltohexasaccharide synthase mutant added was 20 U / g~45 U / g dry substrate, and the enzymatic hydrolysis conditions were 60~70℃ for 18 h~48 h.

19. The use of the method according to any one of claims 1 to 16 in the preparation of linear maltohexasaccharide.

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