Linear inulin-synthesizing bacterium and method for producing linear inulin

Abstract

The present invention addresses the problem of providing: a linear-type inulin-synthesizing bacterium of a related species of the genus Bacillus, which is characterized in that the content of the linear-type inulin-synthesizing bacterium is less than the content of the linear-type inulin-synthesizing bacterium, and the content of the linear-type inulin-synthesizing bacterium is less than the content of the linear-type inulin-synthesizing bacterium; and a method for producing the linear-type inulin using the linear-type inulin-synthesizing bacterium. A branched-chain inulinase gene or a predetermined branched-chain inulinase-like gene is deleted or inactivated, and an inulinase gene or a predetermined inulinase-like gene is present. As a solution, it is possible to produce only linear inulin by constructing a [delta] BI inulin-synthesizing strain in which a BI enzyme is deleted or inactivated by deleting or mutating all or part of the BI gene of an inulin-synthesizing strain, and allowing the culture supernatant of the constructed [delta] BI inulin-synthesizing strain to act on sucrose.

Description

Technical Field

[0001] This invention relates to linear inulin-producing bacteria and methods for manufacturing linear inulin. More specifically, it relates to a linear inulin-producing bacterium selected from one or more linear inulin-producing bacteria of the genera *Priscilla*, *Bacillus*, *Neobacillus*, *Fredinandcohnia*, *Parabacillus*, *Litchfieldia*, *Robertomarium*, *Mycobacterium*, *Haloxybacterium*, *Brucea*, *Bacillus-like*, and *Ammoniac*, as well as linear inulin-producing bacteria closely related to the genera *Bacillus* other than these linear inulin-producing bacteria. The bacterium is characterized by the absence or inactivation of a branched inulin synthase gene or a prescribed branched inulin synthase-like gene, and the presence of an inulin sucrase gene or a prescribed inulin sucrase-like gene. This invention also relates to methods for manufacturing linear inulin using at least one of these linear inulin-producing bacteria. Background Technology

[0002] Inulin is a polysaccharide (fructan) found in the roots of chicory or other plants in the Asteraceae family. It is characterized by a sucrose structure at its ends, with other fructoses polymerized at the fructose residue sites, and the inter-fructose bonds are primarily β-2,1 bonds. Broadly speaking, fructans containing only fructose and no glucose at their ends are also considered inulin. The degree of polymerization of inulin in plants generally ranges from 8 to 60. The synthesis pathway of inulin in plants involves the action of two enzymes: sucrose:sucrose-1-fructosyltransferase (1-SST), which transfers fructose residues between sucrose residues, and 2,1-fructan:2,1-fructan-1-fructosyltransferase (1-FFT), which transfers fructose residues between fructans with a degree of polymerization greater than 3. Linear inulin with β-2,1 bonds between fructose residues is poorly digestible and absorbable, but it can be utilized by Bifidobacteria in the digestive tract, improving the intestinal flora and intestinal environment. Therefore, it is added as a probiotic to health-conscious processed foods. In addition, inulin butter, which is made by dissolving inulin in water to form a creamy gel, has a texture close to that of fat and can therefore be used to improve the flavor of processed foods with reduced fat content.

[0003] Linear inulin is abundant in chicory roots and is used as a raw material in industrial manufacturing. The manufacturing process involves chicory cultivation, root harvesting, transportation to the factory, root cleaning and chopping, hot water extraction, filtration, purification, concentration, and spray drying. To manufacture linear inulin on an industrial scale, sufficient raw materials are essential, requiring ample arable land, a long cultivation period from spring to autumn, adequate fertilization, and disease prevention and appropriate pesticide application. Secondly, the inulin in the harvested roots easily decomposes and becomes low-molecular-weight during storage, and the roots themselves are prone to rotting during storage; therefore, the raw root processing must be completed in the shortest possible time. Consequently, linear inulin manufacturing requires large-scale machinery capable of processing large quantities of roots in a short period. For these reasons, linear inulin manufacturing requires significant labor and equipment, resulting in high production costs. The industry is seeking lower-priced linear inulin.

[0004] On the other hand, the synthesis of inulin from sucrose has been reported using an enzyme secreted by microorganisms called inulosucrase (hereinafter sometimes referred to as "IS"). Inulosucrase catalyzes the reaction that transfers the furanofructose β-2,1 group of sucrose to the terminal fructose residue of other sucroses or their glycosyl transfer products, thereby synthesizing inulin from sucrose. Most reports involve lactic acid bacteria as examples of inulin synthesis using inulosucrase produced through recombinant genetic technology or inulosucrase with improved purity through protein purification. For example, strains of Lactobacillus gasseri DSM20243, Lactobacillus gasseri DSM20604, Lactobacillus johnsonii NCC533, Lactobacillus reuteri 121, Lactobacillus reuteri TMW1.106, Leuconostoc citreum CW28, and Weissella confusa MBFCNC-2 (1) have been reported. In addition to lactic acid bacteria, inulin synthesis using inulin sucrase from Bacillus sp. 217C-11 and Salipaludibacillus agaradhaerens WDG185 has also been reported (Non-Patent Literature 1).

[0005] In industrial applications where microbial enzymes are used to manufacture inulin, the increased cost and difficulty in producing low-cost inulin arises from the need to purify the enzymes that catalyze the reaction. Currently reported inulin-sucrase-producing bacteria all secrete inulin-sucrase in the culture supernatant. In this invention, microorganisms that secrete inulin-sucrase in their culture medium and can produce inulin when the culture supernatant is mixed with sucrose and maintained at a certain temperature for a certain period are termed inulin-producing bacteria. If the culture supernatant of inulin-producing bacteria can be used directly as an enzyme solution without purification, it may be possible to manufacture inulin at a low cost. As an example of analyzing inulin generated from sucrose using the culture supernatant as an enzyme solution, Bacillus 217C-11 (Non-Patent Literature 2) is reported. The product described in this report, based on the inulin peak chromatogram and peak shape obtained by HPLC using the HPAEC-PAD method, is considered to be a composition containing inulin with a bonding mode different from that of linear inulin.

[0006] Existing technical documents

[0007] Non-patent literature

[0008] Non-patent literature 1: Biotechnology Advances, 37, 306-318 (2019)

[0009] Non-patent literature 2: J. Agric. Food Chem., 53, 1246-1253 (2005) Summary of the Invention

[0010] The problem that the invention aims to solve

[0011] In this invention, microbial culture supernatant (crude enzyme) is used as a substrate to generate inulin with a partially different structure through an enzymatic reaction, which is referred to as branched inulin. Branched inulin is a polymer in which the bonds between fructose molecules in inulin include bonds other than β-2,1. In nature, no microorganisms have been reported to be able to generate culture supernatants containing only linear inulin.

[0012] In other words, if the culture supernatant of inulin-producing bacteria is used as an enzyme solution to react with sucrose, branched inulin will inevitably be produced, making it impossible to produce only linear inulin. Using existing technology, to produce only linear inulin using bacterial enzymes, inulin sucrase protein obtained through partial or complete purification from the culture supernatant is required, thus significantly increasing industrial manufacturing costs. Furthermore, the reason why branched inulin is present in the inulin produced when the culture supernatant is used as an enzyme solution remains unknown, and no microorganisms have yet been discovered that can produce only linear inulin when the culture supernatant is used as an enzyme solution. On the other hand, by using recombinant genetic engineering to introduce the inulin sucrase gene into non-inulin-producing bacteria to produce inulin sucrase, it is technically easier to produce only linear inulin from sucrose. However, for the industrial application of recombinant microorganisms, strict management is required as an environmental safety measure, becoming a new cost factor. Moreover, the use of recombinant genetic engineering in the food sector requires careful safety verification, and consumer concerns about foods using recombinant genetic engineering remain deeply ingrained.

[0013] The present invention aims to provide one or more linear inulin-producing bacteria selected from *Priscilla*, *Bacillus*, *Neobacillus*, *Fredinandcohnia*, *Parabacillus*, *Litchfieldia*, *Robert Merah*, *Mycobacterium*, *Halophilic Bacillus*, *Brucea*, *Bacillus-like*, and *Ammoniac*, as well as linear inulin-producing bacteria closely related to the *Bacillus* genus other than these linear inulin-producing bacteria, and a method for manufacturing linear inulin using at least one of these linear inulin-producing bacteria. The linear inulin-producing bacteria are characterized by the absence or inactivation of a branched-chain inulin synthase gene or a specified branched-chain inulin synthase-like gene, and possess an inulin sucrase gene or a specified inulin sucrase-like gene.

[0014] Technical solutions for solving the problem

[0015] To solve the aforementioned problems, the inventors of this invention conducted repeated and in-depth research, and discovered that when it is desired to use the culture supernatant of inulin-synthesizing bacteria to generate only linear inulin from sucrose, the reason for generating branched inulin is that, in addition to inulin sucrase, which synthesizes linear inulin from sucrose, the culture supernatant also contains a branching inulin synthesis-contributed enzyme (hereinafter sometimes referred to as "BI"), which is an unknown enzyme that converts linear inulin into branched inulin. Branched inulin is synthesized by the combined action of these two enzymes. Based on this discovery, it was found that by deleting all or part of the BI gene of inulin-synthesizing bacteria, or by mutating it, linear inulin-synthesizing bacteria (hereinafter sometimes referred to as "ΔBI inulin-synthesizing strain") that lacks or is inactivated by the BI enzyme, and by reacting the culture supernatant of the constructed ΔBI inulin-synthesizing strain with sucrose, only linear inulin is generated, thus completing the inventions described below.

[0016] (1) A linear inulin-producing bacterium, selected from one or more of the following: *Priscilla*, *Bacillus*, *Neobacillus*, *Fredinandcohnia*, *Pseudomonas*, *Litchfieldia*, *Robert Merah*, *Mycobacterium*, *Halophilic Bacillus*, *Brucea*, *Bacillus* species, and *Ammoniac* species; and linear inulin-producing bacterium closely related to these *Bacillus* species, wherein the linear inulin-producing bacterium is characterized by:

[0017] The gene shown in at least one of (a) to (e) below is deleted or inactivated, and the user has at least one of the genes shown in (h) to (m) below.

[0018] (a) A gene encoding a protein consisting of a base sequence having more than 70% identity with the base sequence shown in sequence number 35.

[0019] (b) Genes that hybridize under strict conditions with the complementary strands of the gene consisting of the base sequence shown in sequence number 35.

[0020] (c) The gene encoding a protein consisting of the amino acid sequence shown in sequence number 36.

[0021] (d) Genes encoding proteins consisting of amino acid sequences with 1 to 10 amino acids deleted, substituted, added, or inserted in the amino acid sequence shown in sequence number 36.

[0022] (e) A gene encoding a protein consisting of an amino acid sequence having more than 67.9% identity with the amino acid sequence shown in sequence number 36;

[0023] (h) The gene with the base sequence shown in sequence number 33,

[0024] (i) A gene consisting of a base sequence having more than 70% identity with the base sequence shown in sequence number 33, and encoding a protein having activity of catalyzing the transfer of furanofructose β-2,1 of sucrose to the terminal fructose residue site of other sucrose or its glycosyl transfer products.

[0025] (j) The complementary strand of the gene consisting of the base sequence shown in sequence number 33 is hybridized under stringent conditions, and the gene encodes a protein having activity that catalyzes the transfer of the furanofructose β-2,1 of sucrose to the terminal fructose residue site of other sucrose or its glycosyl transfer products.

[0026] (k) Genes encoding proteins secreted outside the bacterial cell, consisting of the amino acid sequence shown in sequence number 34.

[0027] (l) A gene encoding an amino acid sequence consisting of 1 to 10 amino acids that are deleted, substituted, added, or inserted in the amino acid sequence shown in sequence number 34, and which is secreted outside the bacterial cell and has the activity of catalyzing the transfer of β-2,1 furanose of sucrose to the terminal fructose residue of other sucrose or its glycosyl transfer products.

[0028] (m) A gene that encodes an amino acid sequence having more than 67.3% identity with the amino acid sequence shown in sequence number 34 and that is secreted outside the bacterial cell by means of a reaction that catalyzes the transfer of the furanofructose β-2,1 of sucrose to the terminal fructose residue of other sucrose or its glycosyl transfer products.

[0029] (2) The linear inulin-producing bacteria as described in (1), characterized in that:

[0030] The linear inulin-producing bacteria described above have the characteristics shown in (f) or (g):

[0031] (f) Inoculate 1 platinum ring of bacterial cells into 3 mL of the culture medium with the following composition, and culture with shaking at 30°C for 24 hours and 130 rpm. Then add sucrose to the culture supernatant to make it Bx40, and react at 37°C for 24 hours to produce a linear inulin-producing bacterium that produces inulin with fructose bonds consisting only of β-2,1 bonds.

[0032] <Culture medium composition>

[0033] 10 g / L sucrose, 5 g / L soybean peptone, 1 g / L yeast extract, 0.5 g / L KH2PO4, adjusted to pH 7.2 with NaOH;

[0034] (g) After inoculating 1 platinum ring of bacteria into 3 mL of the culture medium with the composition described in (I) below and culturing with shaking at 30°C for 24 hours and 130 rpm, sucrose was added to the culture supernatant to make it Bx40, and the reaction was carried out at 37°C for 24 hours. When the resulting reaction product was analyzed by HPAEC-PAD under the conditions described in (II) below, it was characterized by the following: in the elution time of 20-30 minutes (degree of polymerization 9-16), a peak was observed at the same elution time as inulin composed only of β-2,1 bonds, and the area of ​​other peaks was less than 1% of the total peak area of ​​linear inulin-producing bacteria.

[0035] <(I) Culture medium composition>

[0036] 10 g / L sucrose, 5 g / L soybean peptone, 1 g / L yeast extract, and 0.5 g / L KH₂PO₄ were added, and the pH was adjusted to 7.2 with NaOH.

[0037] <(II) Conditions for HPAEC-PAD Analysis>

[0038] Column: CarboPac PA1 4×250mm; Guard column: CarboPac PA1 4×50mm; Injection volume: 5μL of 0.1% aqueous solution of sample; Flow rate: 1.0mL / min; Elution: NaOH 100mM constant, AcONa gradient (0~60min, 0→600mM); Detection: pulsed amperometric detection.

[0039] (3) The linear inulin-producing bacteria as described in (1), characterized in that:

[0040] Genes consisting of sequence number 35 or a sequence having at least 75% identity with that sequence number have the following characteristics:

[0041] Proteins consisting of the amino acid sequence encoded by the gene represented by sequence number 35 or having at least 75% identity with that sequence number, and proteins consisting of the amino acid sequence represented by sequence number 34, were added to a sucrose solution of Bx40 to make a concentration of 2 U / g sucrose. The mixture was reacted at 37°C for 24 hours. When the resulting reaction products were analyzed using HPAEC-PAD under the following conditions, the protein consisting of the amino acid sequence represented by sequence number 34 was obtained after elution time of 20 minutes (degree of polymerization ≥ 9). The reaction of the substance with sucrose solution of Bx40 yielded linear inulin with fructose bonds consisting solely of β-2,1 bonds. At the same elution time, more than 10 peaks of linear inulin, consisting of fructose residues in the linear inulin bound by bonds other than β-2,1 bonds, were detected. Furthermore, at elution times of 20–30 minutes (degree of polymerization 9–16), the proportion of branched inulin (total peak area of ​​branched inulin / total peak area × 100) was more than 10%.

[0042] <Conditions for HPAEC-PAD Analysis>

[0043] Column: CarboPac PA1 4×250mm; Guard column: CarboPac PA1 4×50mm; Injection volume: 5μL of 0.1% aqueous solution of sample; Flow rate: 1.0mL / min; Elution: NaOH 100mM constant, AcONa gradient (0~60min, 0→600mM); Detection: pulsed amperometric detection.

[0044] (4) The purified enzyme, crude enzyme or enzyme contents, culture medium, culture supernatant or cultured cells or treated products thereof obtained from any one of (1) to (3) linear inulin synthesizing bacteria.

[0045] (5) A method for manufacturing linear inulin, characterized in that:

[0046] It has a process for cultivating linear inulin synthesizing bacteria as described in any one of (1) to (3).

[0047] (6) A method for manufacturing linear inulin, characterized in that:

[0048] The process comprises reacting at least one of the purified enzyme, crude enzyme or enzyme contents, culture medium, culture supernatant or cultured cells or the treated products thereof obtained from linear inulin synthesizers as described in any one of (1) to (3) with sucrose to obtain a reaction product.

[0049] (7) The method for manufacturing linear inulin as described in (6), characterized in that:

[0050] When the above reaction products are analyzed by HPAEC-PAD under the following conditions, during an elution time of 20 to 30 minutes (degree of polymerization 9 to 16), the proportion of the above branched inulin with a structure in which any fructose residue site of the linear inulin with fructose bonds between fructose groups consisting of β-2,1 bonds has furan-fructose groups bonded by a bonding mode other than β-2,1 bonds (total peak area of ​​branched inulin / total peak area × 100) is less than 1%;

[0051] <Conditions for HPAEC-PAD Analysis>

[0052] Column: CarboPac PA1 4×250mm; Guard column: CarboPac PA1 4×50mm; Injection volume: 5μL of 0.1% aqueous solution of sample; Flow rate: 1.0mL / min; Elution: NaOH 100mM constant, AcONa gradient (0~60min, 0→600mM); Detection: pulsed amperometric detection.

[0053] (8) A method for manufacturing DFAIII, characterized in that:

[0054] Reaction of at least one of the purified enzyme, crude enzyme or enzyme contents, culture medium, culture supernatant or cultured cells or treated products thereof obtained from any one of the linear inulin synthesizing bacteria (1) to (3) with sucrose.

[0055] (9) The manufacturing method of DFAIII as described in (8), characterized in that:

[0056] Use inulin fructose transferase.

[0057] Invention Effects

[0058] Using the linear inulin-synthesizing bacteria of the present invention, linear inulin can be synthesized solely from sucrose using its culture supernatant. Furthermore, using the high-purity linear inulin obtained by the present invention, which is free of branched inulin, DFAIII, an oligosaccharide, can be manufactured in high yield and at low cost. Attached Figure Description

[0059] Figure 1 This graph represents the reaction products obtained by reacting the culture supernatant of inulin-producing bacteria selected from nature with Bx40 sucrose, as well as the peaks obtained by HPAEC-PAD analysis of inulin (Raftiline ST) from plants (chicory).

[0060] Figure 2 This is a diagram of a simplified molecular phylogenetic tree based on a partial 16S rDNA sequence of inulin-producing bacteria closely related to Bacillus derrenthes.

[0061] Figure 3This graph represents the reaction products obtained by reacting two sucrose metabolism-related enzymes purified from the culture supernatant of SD33 strain with Bx40 sucrose, as well as the peaks obtained by HPAEC-PAD analysis of inulin (Raftiline ST) from the plant.

[0062] Figure 4 This refers to the reaction products of fructan standard, inulin standard, peak A, and peak B. 13 A graph of C-nuclear magnetic resonance (NMR) signals.

[0063] Figure 5 This is a GC-MS peak diagram showing the reaction products of peak A, peak B, and peak 2 in peak B, representing their respective methylation analyses.

[0064] Figure 6 This is a graph showing the peaks obtained by HPAEC-PAD analysis of the reaction products obtained by reacting IS and BI of SD33 strain (obtained through recombinant Escherichia coli) with Bx40 sucrose.

[0065] Figure 7 It is a molecular phylogenetic tree constructed using standard strains of well-named microorganisms in the microbial community with high identity of IS and BI with SD33 strain, Ammoniac YIM78166 strain, Bacillus subtilis standard strain, and partial 16S rDNA base sequences of SD33 strain.

[0066] Figure 8 The peak diagrams are obtained by HPAEC-PAD analysis of the reaction products obtained from the reaction of the culture supernatants of SD33 strain and SD33_ΔBI strain with Bx40 sucrose.

[0067] Figure 9 The peak diagrams are obtained by analyzing the reaction products obtained by mixing the culture supernatant of SD33_ΔBI strain with various purified BI-like recombinant enzymes and reacting them with Bx40 sucrose, using HPAEC-PAD.

[0068] Figure 10 The peak diagrams are obtained by HPAEC-PAD analysis of the reaction products obtained by reacting the culture supernatant of SD33_ΔBI strain, OV191_IS (67.3% similarity to SD33 strain IS) and LL01_IS (68.8% similarity to SD33 strain IS) with Bx40 sucrose. Detailed Implementation

[0069] The following details the linear inulin-producing bacteria involved in this invention, the purified enzyme, crude enzyme or enzyme contents obtained from the linear inulin-producing bacteria involved in this invention, the culture medium, the culture supernatant or cultured cells or the treated products thereof, the method for manufacturing linear inulin, and the method for manufacturing DFAIII. The linear inulin synthesizing bacteria involved in this invention are selected from one or more linear inulin synthesizing bacteria of the genera *Priscilla*, *Bacillus*, *Neobacillus*, *Fredinandcohnia*, *Parabacillus*, *Litchfieldia*, *Robert Merah*, *Mycobacterium*, *Halophilic Bacillus*, *Bacillus brevis*, *Bacillus-like*, and *Ammoniac*, as well as linear inulin synthesizing bacteria of closely related species of the *Bacillus* genera other than these linear inulin synthesizing bacteria. The invention is characterized by the deletion or inactivation of a branched inulin synthase (BI) gene or a specified branched inulin synthase-like (BI-like) gene, and the presence of an inulin sucrase (IS) gene or a specified inulin sucrase-like (IS-like) gene.

[0070] The inventors of this invention isolated and collected a large number of inulin-producing bacteria from nature and explored in depth the bacteria that produce only linear inulin from sucrose using their culture supernatants. They studied in detail the inulin products produced using the culture supernatants of various inulin-producing bacteria as the object and sucrose as the substrate. However, all inulin-producing bacteria produced branched inulin, and no inulin-producing bacteria that produced only linear inulin were found.

[0071] Furthermore, in-depth research revealed the presence of an unknown protein in the culture supernatant of inulin-producing bacteria, contributing to the formation of branched inulin. Specifically, in addition to the enzyme IS (Inoculum Spectrostomum) that synthesizes linear inulin from sucrose, the culture supernatant of inulin-producing bacteria also contained an unknown enzyme that converts linear inulin into branched inulin. This unknown enzyme was identified as BI (Branch Breast Extract). It was further determined that branched inulin is synthesized through the combined action of these two enzymes, IS and BI.

[0072] To address this, a database of non-redundant protein sequences (NR) was used, and the Basic Local Alignment Search Tool (BLAST) was employed to search for bacterial species that carried base sequences encoding IS-like proteins but did not carry base sequences encoding the newly discovered BI-like proteins. However, no such bacterial species were found. Furthermore, it was discovered that any bacterium with an amino acid sequence encoding an amino acid sequence with more than 67.3% identity to the IS sequence shown in sequence number 34 also carried a base sequence encoding an amino acid sequence with more than 66.9% identity to the BI sequence shown in sequence number 36. Moreover, even when the protein with 67.9% identity to BI reacted with sucrose along with IS, branched inulin was generated. As bacterial species that carry base sequences encoding both IS-like and BI-like proteins and are capable of producing branched inulin, the genera *Priestia*, *Bacillus*, *Neobacillus*, *Fredinandcohnia*, *Metabacillus*, *Litchfieldia*, *Robertmurraya*, *Mycobacteroides*, *Alkalihalobacillus*, *Brevibacillus*, *Paenibacillus*, and *Ammoniphilus* were identified as closely related species to the genus *Bacillus*.

[0073] Next, the BI gene of the inulin-producing bacteria was deleted or mutated in whole or in part to construct a linear inulin-producing bacterium (hereinafter referred to as the ΔBI inulin-producing strain) with the BI enzyme missing or inactivated. When the culture supernatant of the constructed ΔBI inulin-producing strain was reacted with sucrose, only linear inulin was successfully produced, thus completing the present invention. As suitable strains for constructing the ΔBI inulin-producing strain, the previously mentioned Priestella, Bacillus, Neobacillus, Fredinandcohnia, Parabacillus, Litchfieldia, Robert Merah, Mycobacterium, Halophilic Bacillus, Brachybacterium, Bacillus-like bacteria, and Ammoniaphila can be used. In other words, it is possible to list Priestella, Bacillus, Neobacillus, Fredinandcohnia, Parabacillus, Litchfieldia, Robert Merah, Mycobacterium, Halophilic Bacillus, Brachybacterium, Bacillus-like Bacillus, and Ammoniaophila, which are closely related species of Bacillus. Furthermore, it can be said that without breeding improvement, it is impossible to use their culture supernatant to produce linear inulin.

[0074] In this invention, an "IS-like gene" refers to a gene corresponding to sequence number 33, which is a gene encoding an inulin sucrase activity that catalyzes the transfer of β-2,1 furanose saccharides of sucrose to the terminal fructose residues of other sucrose or their glycosyl transfer products. Specifically, examples include genes composed of base sequences having at least 70%, preferably 75%, more preferably 80%, further preferably 85%, particularly preferably 90%, and most preferably 95% or more identity with the base sequence of sequence number 33. Alternatively, it can also be a gene with the same function as the gene, composed of base sequences in which one or more base sequences are deleted, substituted, added, or inserted.

[0075] Furthermore, examples of genes that hybridize under stringent conditions to the complementary strand of a gene consisting of the base sequence shown in Serial No. 33, and that encode a protein with activity catalyzing the transfer of β-2,1 furanofructose of sucrose to the terminal fructose residue of another sucrose or its glycosyl transfer product. In this specification, "stringent conditions" refers to conditions where the complementary strand of a gene strand identical to the target sequence preferentially hybridizes with the target sequence, and the complementary strand of a gene strand not identical to the target sequence substantially does not hybridize. Specifically, stringent conditions can be exemplified by: overnight hybridization at 65°C, followed by washing with 2×SSC at room temperature for 5 minutes to remove non-specific reactions, and then repeating the washing process twice for 30 minutes each time with 0.2×SSC containing 0.1% SDS at 65°C. The 2×SSC consists of 0.3 mol / L NaCl and 30 mmol / L sodium citrate (pH 7.0).

[0076] Linear inulin-producing bacteria lacking or inactivating BI-like protein activity can be obtained by deleting or inactivating BI-like genes in the base sequence of the parent strain. Here, in this invention, "BI-like gene" refers to DNA including a transcriptional region containing an ORF and a transcriptional regulatory region such as a promoter of the gene. Specifically, it refers to the gene corresponding to sequence number 35, which is a gene encoding a protein of an enzyme that catalyzes the transfer of furanylfructose from sucrose to any fructose residue site in linear inulin via a bond other than a β-2,1 bond, to synthesize branched inulin. More specifically, examples include genes encoding proteins composed of amino acid sequences that have at least 70%, preferably 75%, more preferably 80%, further preferably 85%, particularly preferably 90%, and most preferably 95% or more identity with the base sequence of sequence number 35. Alternatively, it can also be a gene with the same function as the gene, formed by deleting, substituting, adding, or inserting one or more base sequences. "1 to several" can be, for example, 1 to 10, preferably 1 to 7, and more preferably 1 to 4. Furthermore, in this invention, "linear inulin" refers to a fructan with a structure formed by the polymerization of other fructose residues at the fructose residue site of sucrose via β-2,1 bonds, where the bonds between fructose residues consist solely of β-2,1 bonds. "Bonds other than β-2,1 bonds," as the text suggests, refers to "bonds other than β-2,1 bonds," meaning at least in addition to β-2,6 bonds, it also refers to bonds other than β-2,1 bonds such as β-2,3 and β-2,4 bonds.

[0077] In addition, in this invention, the gene that hybridizes with the gene consisting of the base sequence shown in sequence number 35 under strict conditions and encodes the protein of an enzyme that catalyzes the reaction of transferring the furanofructose group of sucrose to any fructose residue site of linear inulin via a bonding mode other than β-2,1 bond to synthesize branched inulin is also included in the BI-like gene.

[0078] In this invention, “deleting or inactivating BI-like genes or BI genes” can be exemplified by: introducing mutations into one or more bases in the base sequence of the gene, i.e., deleting part or all of the base sequence of the gene, replacing or inserting other base sequences into the base sequence, reducing the activity of transcribed or translated BI-like proteins or BI proteins, preventing the production of BI-like proteins or BI proteins, and introducing mutations into the base sequence encoding the signal peptide normally present at the 5' end of the BI-like gene or BI gene, thereby depriving the bacterial exocrine production capacity of BI-like proteins or BI proteins.

[0079] Next, the inulin sucrase of the present invention, which catalyzes the transfer of the furanofructose β-2,1 of sucrose to the terminal fructose residue of other sucroses or their glycosyl transfer products, is composed of the amino acid sequence shown in Serial No. 34. However, as long as it has this activity, a protein with an amino acid sequence having at least 67.3% identity with this amino acid sequence can be used. Specifically, proteins composed of amino acid sequences having at least 67.3%, preferably 70%, more preferably 75%, further preferably 80%, even more preferably 85%, particularly preferably 90%, and most preferably 95% identity can be listed. More specifically, proteins having at least 67.3%, preferably 68.0%, 68.6%, 68.8%, 70.3%, 70.9%, 71.7%, 73.4%, and 76.8% identity can be listed. Proteins composed of amino acid sequences with an affinity of 78.7%, 78.9%, 79.1%, 79.4%, 79.8%, 80.4%, 80.7%, 81.1%, 81.3%, 81.7%, 81.9%, 83.4%, 84.9%, 85.8%, 96.2%, 96.8%, 97.3%, 98.2%, 98.4%, or 98.6% or higher.

[0080] The enzyme in this invention that catalyzes the transfer of fructofuranose groups from sucrose to any fructose residue site in linear inulin via a bond other than β-2,1 bonds to synthesize branched inulin is composed of the amino acid sequence shown in Serial No. 36. However, as long as it possesses this activity, a protein with an amino acid sequence having at least 67.9% identity with this amino acid sequence can be used. Specifically, examples include proteins composed of amino acid sequences having at least 67.9%, preferably 70%, more preferably 75%, further preferably 80%, even more preferably 85%, particularly preferably 90%, and most preferably 95% or more identity. More specifically, examples include proteins with at least 67.9%, preferably 71.5%, 72.2%, 75.1%, 75.3%, 76.6%, 76.9%, 77.1%, and 77.3%. Proteins composed of amino acid sequences with an amino acid sequence that is 77.5% or higher, 78.3% or higher, 78.5% or higher, 78.7% or higher, 79.0% or higher, 80.8% or higher, 81.0% or higher, 84.8% or higher, 85.3% or higher, 85.4% or higher, 85.8% or higher, 86.0% or higher, 88.6% or higher, 89.7% or higher, 93.1% or higher, 93.7% or higher, 96.6% or higher, 98.7% or higher, 98.9% or higher, or 99.1% or higher.

[0081] Alternatively, it can be a protein with the same function as the protein, consisting of an amino acid sequence in which one or more amino acids are missing, substituted, added, or inserted in the amino acid sequences shown in Serial Numbers 34 and 36. Here, "one or more" can be, for example, 1 to 10, preferably 1 to 7, and more preferably 1 to 4.

[0082] Here, "loss or inactivation of BI-like protein or BI protein activity" can refer to knocking out the BI-like gene or the BI gene itself from the genome, or it can be achieved through base substitution, deletion, or insertion, introducing mutations that inactivate BI or reduce its activity. Alternatively, the base sequence encoding the signal peptide normally located at the 5' end of the BI-like gene or BI gene can be knocked out, resulting in the loss of BI's ability to be secreted in vitro.

[0083] In addition to methods that intentionally delete or inactivate BI-like genes or BI genes, methods for obtaining linear inulin-producing bacteria with lost or reduced BI activity after introducing random genomic mutations can also be listed.

[0084] Methods for intentionally deleting or inactivating BI-like genes or BI genes include, for example, homologous recombination. Specifically, a circular recombinant plasmid is obtained by inserting a DNA fragment containing the upstream and downstream sequences of the BI-like gene or BI gene into a suitable plasmid vector. This plasmid is then introduced into parental cells, and two cross-recombinations in the outer regions of the BI-like gene or BI gene result in the deletion of the BI-like gene in the parental genome. Alternatively, a straight-stranded DNA fragment containing an inactivating mutation (such as a base substitution or insertion) into the BI-like gene or BI gene can be constructed using methods such as PCR. This fragment is then introduced into parental cells, and two cross-recombinations occur in two regions outside the mutation site in the BI-like gene or BI gene of the parental genome, thereby replacing the BI-like gene or BI gene fragment with an inactivated BI gene. Furthermore, deletions, substitutions, and base insertions of the BI-like gene or BI gene can be performed using genome editing technologies.

[0085] On the other hand, methods for performing random gene deletion or inactivation include, for example, treating the parental strain with mutagens such as nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS), irradiation with UV, heavy ion beams, etc.

[0086] By culturing linear inulin-producing bacteria obtained by deleting or inactivating BI-like genes or BI genes using the above methods, a culture medium containing BI-like proteins or BI proteins that have been deleted or inactivated can be obtained.

[0087] When the culture supernatant of the culture medium is reacted with sucrose to obtain a reaction product, linear inulin constitutes the majority of the reaction product. When inulin is obtained from this reaction product and subjected to HPAEC-PAD analysis under the following conditions, more than 10 linear inulin peaks are detected at elution times after 20 minutes. It is speculated that branched inulin is generated similarly to the generation of inulin from sucrose using the culture supernatant of the parental strain described in paragraph 0043 below. The branched inulin peaks appear between the linear inulin peaks. At elution times of 20–30 minutes (degree of polymerization 9–16), the ratio of branched inulin (total peak area of ​​branched inulin / total peak area × 100) is less than 10%, preferably less than 7%, more preferably less than 5%, further preferably less than 3%, particularly preferably less than 1%, and most preferably less than 0.3%. In this invention, "reaction product" refers to a reaction product generated by transforming a substrate using the activity of IS or IS and BI. The reaction product generated by transforming a substrate using the activity of IS includes not only a reaction product containing linear inulin but also linear inulin itself. The reaction product generated by transforming a substrate using the activity of IS and BI includes not only a reaction product containing linear and branched inulin but also linear and branched inulin itself. Furthermore, the method for obtaining the linear or branched inulin contained in the reaction product is not particularly limited, and any method appropriately selected by those skilled in the art can be used.

[0088] <Conditions for HPAEC-PAD Analysis>

[0089] Column: CarboPac PA1 4×250mm; Guard column: CarboPac PA1 4×50mm; Injection volume: 5μL of 0.1% aqueous solution of sample; Flow rate: 1.0mL / min; Elution: NaOH 100mM constant, AcONa gradient (0–60 min, 0→600mM); Detection: Pulsed amperometric detection

[0090] As the parent strain used to obtain the linear inulin-producing bacteria of the present invention, any inulin-producing bacteria having a gene with a base sequence that is at least 60% identical to that of Serial No. 33 and Serial No. 35 is acceptable, without particular limitation. Specifically, bacteria of the genera *Priscilla*, *Bacillus*, *NeoBacillus*, *Fredinandcohnia*, *Parabacillus*, *Litchfieldia*, *Robert Merah*, *Mycobacterium*, *Halophyte*, *Brucea*, *Bacillus-like*, and *Ammoniac*, as well as closely related species of the *Bacillus* genus other than these, can be used.More specifically, examples include *Priestia megaterium*, *Bacillus sp.*, *Neobacillus bataviensis*, *Metabacillus bambusae*, *Robertmurraya korlensis*, *Fredinandcohnia onubensis*, *Litchfieldia salsa*, *Mycobacteroides abscessus subsp. abscessus*, *Brevibacillus sp.*, *Brevibacillus formosus*, *Brevibacillus brevis*, *Brevibacillus dissolubilis*, *Brevibacillus migulae*, and *Paenibacillus*. The following strains are listed: *Paenibacillus sp.*, *Paenibacillus elgii*, *Paenibacillus prosopidis*, *Paenibacillus tyrfis*, *Ammoniphilus sp.*, *Alkalihalobacillus krulwichiae*, *Salipaludibacillus agaradhaerens*, and more specifically, *Bacillus sp.* strain SD33 (accession number: NITE BP-03890). Based on molecular phylogenetic analysis using partial 16S rDNA sequences, except for the standard strains of the aforementioned microbial species of *Mycobacterium abscessus* subsp. *abscessus* and the ammoniaphile strain YIM78166, all occupy positions close to *Bacillus subtilis* (the most common genus of *Bacillus*). *Bacillus* and *Bacillus-like* genera are traditionally considered closely related to the genus *Bacillus*, and it is reasonable to consider the aforementioned microbial species, except for *Mycobacterium abscessus* subsp. *abscessus*, as closely related to the genus *Bacillus* at least.In addition, Bacillus sp. SD33 was deposited on May 22, 2023, at the National Institute for Technology Evaluation Patent Microbial Collection (2-5-8 Kazusa-Kamazutari, Kisarazu City, Chiba Prefecture, Japan), accession number: NITE BP-03890.

[0091] When the culture supernatant of the parent strain is reacted with sucrose to obtain a reaction product and inulin is obtained from the reaction product, the obtained inulin is analyzed by HPAEC-PAD under the following conditions: at an elution time of more than 10 peaks of branched inulin and more than 10 peaks of linear inulin are detected in each of the following conditions, and the peaks of branched inulin appear between the peaks of linear inulin. At an elution time of 20 to 30 minutes (degree of polymerization 9 to 16), the ratio of branched inulin (total peak area of ​​branched inulin / total peak area × 100) is more than 10%.

[0092] <Conditions for HPAEC-PAD Analysis>

[0093] Column: CarboPac PA1 4×250mm; Guard column: CarboPac PA1 4×50mm; Injection volume: 5μL of 0.1% aqueous solution of sample; Flow rate: 1.0mL / min; Elution: NaOH 100mM constant, AcONa gradient (0~60min, 0→600mM); Detection: pulsed amperometric detection.

[0094] The culture medium suitable for culturing the linear inulin synthetase of the present invention contains carbon sources, nitrogen sources, inorganic salts, etc., but is not limited to these. Depending on other needs, amino acids, vitamins, and other commonly used nutrient sources for culture can also be appropriately used. As the carbon source added to the culture medium, carbon sources known in the art, such as sucrose, glucose, fructose, maltose, and other sugars, can be used. However, when using Bacillus SD33 strain as the microorganism, a liquid culture medium with sucrose as the main carbon source is most preferred, thereby increasing the production of the inulin synthetase of the present invention. These carbon sources can be used alone or in combination at appropriate concentrations (e.g., 5–50 g / L). Furthermore, these carbon sources can be in isolated and purified forms, or in forms containing other substances. For example, when sucrose is used as a carbon source, sucrose concentrate or molasses can be used instead of purified sucrose. As nitrogen sources added to the culture medium, in addition to organic nitrogen sources such as peptone, meat extract, yeast extract, and corn steep liquor, inorganic nitrogen sources such as ammonium salts of sulfuric acid, nitric acid, and phosphate can be used alone or in combination, but are not limited to these. Furthermore, as inorganic salts, for example, sulfates, hydrochlorides, carbonates, nitrates, and phosphates of potassium, sodium, calcium, magnesium, manganese, and iron can be used alone or in combination, but are not limited to these.

[0095] The culture medium having the above composition has a pH of 6 to 9, preferably 7 to 8. Furthermore, the above-mentioned microorganisms can be cultured by shaking or aeration and stirring. The culture temperature is preferably 25 to 35°C. The culture time is only required to exceed or equal the time during which the microorganisms can proliferate, and is 5 to 96 hours, preferably 15 to 72 hours.

[0096] Next, the linear inulin-producing bacteria of the present invention secretes proteins outside the bacterial cell, i.e., secretes IS and / or IS-like substances outside the bacterial cell. These proteins can be proteins, i.e., enzymes, or enzyme-containing compounds. The proteins secreted outside the bacterial cell are: proteins composed of the amino acid sequence shown in Serial No. 34 and secreted outside the bacterial cell; proteins composed of amino acid sequences with 1 to 10 amino acids missing, substituted, added, or inserted in the amino acid sequence shown in Serial No. 34, and having catalytic activity in the reaction of transferring β-2,1 furanofructose of sucrose to the terminal fructose residue site of other sucrose or its glycosyl transfer products; and / or proteins composed of amino acid sequences having more than 67.3% identity with the amino acid sequence shown in Serial No. 34, and having catalytic activity in the reaction of transferring β-2,1 furanofructose of sucrose to the terminal fructose residue site of other sucrose or its glycosyl transfer products and secreted outside the bacterial cell. Examples of enzyme forms include purified enzymes and crude enzymes. When referring to enzyme contents, in addition to enzyme contents and processed forms of enzyme contents, they can also be culture media, culture supernatants, cultured cells, or processed forms of these. Furthermore, these purified enzymes, crude enzymes, enzyme contents, culture media, culture supernatants, cultured cells, or processed forms of these are all substances obtained from the linear inulin-producing bacteria involved in this invention, and the methods for obtaining them, including in vitro secretion by bacteria, can be appropriately selected by those skilled in the art.

[0097] In addition, the method for manufacturing inulin according to the present invention includes the following steps (i) and / or (ii).

[0098] (i) The process of culturing the linear inulin-producing bacteria involved in this invention;

[0099] (ii) A step of reacting at least one of the purified enzyme, crude enzyme or enzyme contents, culture medium, culture supernatant or cultured cells or treated products thereof obtained from the linear inulin synthesizing bacteria involved in the present invention with sucrose.

[0100] The above-mentioned step (i), namely "the step of culturing the linear inulin synthesizing bacteria involved in the present invention", is not particularly limited as long as it involves culturing the linear inulin synthesizing bacteria involved in the present invention. Regarding the culturing of the linear inulin synthesizing bacteria involved in the present invention, those skilled in the art can appropriately select possible culturing methods. For example, the following method can be listed: inoculating the linear inulin synthesizing bacteria involved in the present invention into a liquid culture medium with sucrose as the main carbon source, and culturing it for 15 to 72 hours under the conditions of pH 7 to 8 and 25 to 35°C.

[0101] The aforementioned step (ii), namely, "the step of reacting at least one of the purified enzyme, crude enzyme or enzyme content, culture medium, culture supernatant or cultured cells or treated products thereof obtained from the linear inulin synthesizing bacteria according to the present invention with sucrose," is a step of reacting the purified enzyme, crude enzyme or enzyme content, culture medium, culture supernatant or cultured cells or treated products thereof obtained by the above-described method with sucrose. The above-described "reaction" method is not particularly limited as long as it can produce linear inulin, and any reaction method that can be appropriately selected by those skilled in the art can be used. For example, such a "reaction" method can be described as follows: adding at least one of the purified enzyme, crude enzyme or enzyme content, culture medium, culture supernatant or cultured cells or treated products thereof obtained from the linear inulin synthesizing bacteria according to the present invention to a reaction solution containing sucrose, and allowing it to react.

[0102] In the above-described method for manufacturing linear inulin, which includes step (ii) namely, "the step of reacting at least one of the purified enzyme, crude enzyme or enzyme contents, culture medium, culture supernatant or cultured cells or the treated products thereof obtained by linear inulin synthesizing bacteria according to the present invention with sucrose to obtain a reaction product", the obtained reaction product can also be subjected to the HPAEC-PAD analysis described below in step (iii).

[0103] (iii) When the above reaction product (obtained through step (ii)) is subjected to HPAEC-PAD analysis under the following conditions, it is characterized in that: during the elution time of 20 to 30 minutes (degree of polymerization 9 to 16), the proportion of the above branched inulin with a structure in which any fructose residue site of linear inulin with fructose bonds consisting of β-2,1 bonds is bound to furan fructosyl groups by a bonding mode other than β-2,1 bonds is less than 1% (total peak area of ​​branched inulin / total peak area × 100);

[0104] <Conditions for HPAEC-PAD Analysis>

[0105] Column: CarboPac PA1 4×250mm; Guard column: CarboPac PA1 4×50mm; Injection volume: 5μL of 0.1% aqueous solution of sample; Flow rate: 1.0mL / min; Elution: NaOH 100mM constant, AcONa gradient (0~60min, 0→600mM); Detection: pulsed amperometric detection.

[0106] Secondly, by employing the linear inulin-synthesizing bacteria of the present invention, in the manufacture of substances using inulin and fructan as raw materials, such as difructosan type III, inulin oligosaccharides, and cyclic inulin oligosaccharides (cyclic inulin fructan), it is possible to directly manufacture them from sucrose by using them in combination with their culture supernatants. In particular, in the manufacture of DFAIII, in addition to inulin fructosyltransferase (hereinafter referred to as "IFT"), at least one of the following can be used (hereinafter referred to as "IFT et al."): purified enzyme, crude enzyme or enzyme content, culture medium, culture supernatant, or cultured cells or processed products thereof obtained from microorganisms that produce IFT. Furthermore, by combining at least one of the purified enzyme, crude enzyme or enzyme content, culture medium, culture supernatant, or cultured cells or processed products thereof obtained from the linear inulin-synthesizing bacteria according to the present invention with IFT et al., and reacting them with sucrose, DFAIII can be manufactured. By selecting such a method for manufacturing DFAIII, DFAIII can be directly manufactured from sucrose. Furthermore, in the aforementioned manufacturing of DFAIII, when IFT or the like is applied to linear inulin, DFAIII can be manufactured with higher efficiency compared to when IFT or the like is applied to inulin containing branched inulin.

[0107] In addition to microorganisms belonging to the genera *Arthrobacter*, *Kluyveromyces*, *Streptomyces*, *Enterobacter*, *Bacillus*, and *Microbacterium*, various bacteria, yeasts, filamentous fungi, and actinomycetes can also be appropriately used as microorganisms that generate IFT. Examples include *Arthrobacter ureafaciens*, *Arthrobacter ureafaciens* (IFO12140), *Arthrobacter ureafaciens* (ATCC21124), *Arthrobacter pastoris* (IFO12139), *Arthrobacter pastoris* T13-2, *Arthrobacter globiformis* (IFO12137), *Arthrobacter globiformis* C11-1, *Arthrobacter nictinovorans* GS-9, *Arthrobacter ilicis* OKU 17B, *Arthrobacter* sp., *Arthrobacter* H65-7, and *Arthrobacter* AHU1753 (FERM). BP-8296), Arthrobacter MCI-2493; Kluyveromyces marxianus (ATCC12424), Kluyveromyces marxianus CBS6556, Kluyveromyces marxianus var. marxianus, Kluyveromyces marxianus var. IFO1735; Streptomyces fumigatus, Streptomyces rochei, Streptomyces rochei E87, Streptomyces sp., Streptomyces MCI-2524; Pseudomonas fluorescens, Pseudomonas fluorescens No.949; Bacillus circulans, Bacillus circulans OKUMZ.31B, Bacillus circulans MCI-2554, Bacillus spp. Bacillus sp., Bacillus Snu-7; Aureobacterium sp., Aureobacterium MCI-2494; Microbacterium sp., Microbacterium AL-210; Enterobacter sp., Enterobacter S45; Aspergillus fumigatus; Penicillium purpurogenum.

[0108] Hereinafter, the linear inulin-producing bacteria involved in this invention, the purified enzyme obtained from the linear inulin-producing bacteria involved in this invention, the crude enzyme or enzyme content, the culture medium, the culture supernatant or cultured cells or the treated products thereof, the method for manufacturing linear inulin, and the method for manufacturing DFAIII will be described based on examples. Furthermore, the scope of the technology of this invention is not limited to the embodiments shown in these examples.

[0109] Example

[0110] Example 1: Searching for linear inulin-producing bacteria in nature

[0111] In this Example 1, strains that synthesize only linear inulin were searched from nature. The steps and results are described in detail below.

[0112] Soil samples collected from various parts of Japan were suspended in sterile water, spread onto basal agar medium, and incubated at 35°C for 24 hours. Colonies appearing on the agar medium were inoculated into 300 μL of basal medium and incubated statically at 35°C for 48 hours. 40 μL of the culture supernatant was added to 160 μL of 10 mM Na phosphate buffer (pH 7.0) containing 10% sucrose and allowed to react for 24 hours at 35°C. This was designated as Sample 1. 2 μL of exoinulinase from Aspergillus niger (prepared by Sigma-Aldrich) was added to 40 μL of Sample 1 and incubated at 35°C for 24 hours. This was designated as Sample 2. TLC analysis was performed on Samples 1 and 2. Strains whose polysaccharide plaques at the origin of Sample 1 disappeared in Sample 2 were identified as inulin-producing bacteria, yielding a total of 100 strains. The TLC analysis conditions were set as follows: "plate: TLC silica gel 60 (Merck); sample loading: 50 μg; mobile phase: 2-propanol:1-butanol:H2O = 2:2:1; development: 10 cm; color development: spray the reagent (p-anisaldehyde:acetic acid:concentrated sulfuric acid:ethanol = 13:5:18:478) and heat at 105 °C for 3 minutes."

[0113] Of the 100 strains obtained, the 20 strains with the highest enzyme secretion in the culture supernatant were screened. These 20 inulin-producing bacteria were then inoculated into 3 mL of basal medium and cultured with shaking at 30°C for 24 hours at 130 rpm. The culture supernatant was recovered by centrifugation at 12000 × g for 5 minutes. After determining the enzyme activity in the culture supernatant, Bx40 sucrose solution was mixed to make a concentration of 2 U / g sucrose, and the mixture was kept at 37°C for 24 hours. The reaction solution was then inactivated by incubating at 100°C for 5 minutes and provided for HPAEC-PAD analysis. Figure 1The peaks obtained from the reaction products and inulin (Raftiline ST) from the plant are represented by the peaks obtained from HPAEC-PAD analysis. The enzyme activity was calculated based on the amount of reducing sugar produced, as determined by the DNS method. Specifically, 100 μL of the reaction solution was prepared as an appropriately diluted enzyme solution (10 μL), 300 mM sucrose, and 40 mM MES-NaOH buffer (pH 6.5). After reacting at 37°C for 10 minutes, 100 μL of DNS reagent (16 g / L NaOH, 5 g / L 3,5-dinitrosalicylic acid, 300 g / L potassium sodium tartrate) was added, and the mixture was heated at 105°C for 20 minutes to allow color development. The absorbance at 535 nm was then measured. The amount of reducing sugar in the reaction solution was determined using a standard curve prepared using 0–5 mM D-glucose solution. The amount of enzyme producing 1 μmol of reducing sugar under these conditions was defined as 1 U. The HPAEC-PAD analysis in the following examples was performed using a Dionex HPAEC-PAD apparatus (ICS-3000) under the following conditions: column: CarboPac PA1 4×250 mm; guard column: CarboPac PA1 4×50 mm; injection volume: 5 μL of 0.1% aqueous solution of sample; flow rate: 1.0 mL / min; elution: constant NaOH 100 mM, AcONa gradient (0–60 min, 0→600 mM).

[0114] like Figure 1 As shown, the peaks of fructans with a degree of polymerization of 9 or higher (HPLC elution time range of 20-30 minutes) in the reaction solutions have different patterns. In all reaction solutions, peaks of polysaccharides that do not appear in inulin (Raftiline ST) from plants can be observed.

[0115] In addition, one strain of inulin-producing bacteria was identified as a Bacillus genus closely related to Bacillus drentensis through DNA base sequence analysis and molecular system analysis (16S rDNA full-length sequencing, commissioned to TechnoSuruga Lab Co., Ltd.). It should be noted that Bacillus drentensis was reclassified as Neobacillus drentensis in 2020 (Int. J. Syst. Evol. Microbiol., 70, 406–438 (2020)). Figure 2 The image shows a simplified molecular phylogenetic tree based on a partial 16S rDNA sequence of the identified *Bacillus* species closely related to *Bacillus derenthes*. Figure 2 As shown, this strain is designated as Bacillus SD33 (hereinafter referred to as SD33) (accession number: NITE BP-03890).

[0116] Example 2: Determination of the gene responsible for the generation of branched inulin

[0117] In this Example 2, the reason why the inulin-producing bacteria found in Example 1 can only produce linear inulin and branched inulin is clarified.

[0118] Strain SD33 was spread onto basal agar medium and incubated overnight at 35°C. Single colonies were inoculated into 6 mL of basal medium and pre-cultured at 30°C for 24 h at 130 rpm. The colonies were then inoculated in 2.5 mL portions into two 500 mL Erlenmeyer flasks containing the same medium and incubated at 30°C for 24 h at 130 rpm. The culture supernatant was then collected by centrifugation at 12000 × g for 20 min. Ammonium sulfate was added to the culture supernatant to 70% saturation and incubated overnight at 4°C with stirring. The ammonium sulfate precipitate in the culture supernatant was collected by centrifugation at 12000 × g for 20 min, dissolved in 10 mM sodium phosphate buffer (pH 7.0), and transferred to dialysis tubes. The solution was then dialyzed thoroughly with the same buffer to obtain the crude enzyme solution. The crude enzyme solution was loaded onto a Toyopearl DEAE 650M (2.5cm id×20cm, Tosoh Corporation) pre-equilibrated with 10mM sodium phosphate buffer (pH 7.0), and the non-adsorbed fraction was eluted with the same buffer. Activity of releasing reducing sugars from sucrose was detected in the non-adsorbed fraction; therefore, ammonium sulfate was added to the non-adsorbed fraction to a concentration of 80mM. This was then loaded onto a Toyopearl butyl 650M (2.5cm id×10cm, Tosoh Corporation) pre-equilibrated with 10mM sodium phosphate buffer (pH 7.0) containing 80mM ammonium sulfate. After eluting the non-adsorbed fraction with the same buffer, the adsorbed fraction was eluted using a linear gradient of ammonium sulfate concentration from 80mM to 0mM. Two activity peaks, A and B, indicating the activity of releasing reducing sugars from sucrose were obtained in this operation. For the fractional solutions of each obtained peak, the samples with added Bx40 sucrose to achieve a 2 U / g sucrose concentration were prepared as reaction solutions using the method described in Example 1. The crystal form of the products after enzyme inactivation was analyzed using HPAEC-PAD. Figure 3 The peaks represent the reaction products of peak A, the reaction products of peak B, and the peaks of inulin (Raftiline ST) from plants.

[0119] like Figure 3As shown, the peak shape of the reaction product at peak A is consistent with that of linear inulin, while the peak shape of the reaction product at peak B is consistent with that of inulin containing branched inulin. Next, the structures of each product were analyzed using nuclear magnetic resonance (NMR). First, 1 mL of 99.5% ethanol was added to each reaction solution to achieve a final concentration of 50%, precipitating the polysaccharides. The precipitate was then recovered by centrifugation at 12000×g for 2 minutes. 2 mL of 50% ethanol was added again to the recovered precipitate for resuspending, and the precipitate was recovered by centrifugation at 12000×g for 2 minutes. This operation was repeated 5 times to remove low-molecular-weight compounds. The resulting precipitate was freeze-dried, and inulin (from chicory, Raftiline HP) and fructan (produced by Zymomonas mobilis, Sigma Aldrich) were used as standards for further analysis. 13 C-NMR. In Figure 4 The terms "fructan standard", "inulin standard", "reaction product of peak A", and "reaction product of peak B" represent their respective properties. 13 C-NMR signal.

[0120] like Figure 4 As shown, all products exhibited essentially the same characteristics as the inulin standard. 13 C-NMR signals indicate that the reaction products of peaks A and B have the same basic structure as inulin, with some structural differences leading to the difference between linear and branched inulin. Since the protein in peak A is present in all peaks, it is inferred that the protein in peak A is IS, while the protein present only in peak B is identified as the causative enzyme BI, which causes the branched inulin formation.

[0121] Next, following the method described in *Nature Protocols, 7, 1590–1607 (2012)*, all free hydroxyl groups in 50 mg of the reaction products from peak A and peak B were methylated. After hydrolysis of the methylated polysaccharides, the remaining partially methylated sugars were reduced and acetylated using NaBD4 to obtain partially methylated aldose acetates (PMAAs). The obtained PMAAs were analyzed by GC-MS. GC-MS analysis was performed using a Shimadzu GC-MS apparatus (GCMS-QP2010Ultra). GC was performed under the following conditions: capillary column: BPX70 (25m × 0.22mm inner diameter, membrane = 0.25μm; SGE); injection volume: 1μL (split ratio 1 / 10); vaporization chamber temperature: 240℃; carrier gas: helium; flow rate: 1.0mL / min; column temperature: multi-stage temperature ramp (0–2 min: 170℃; 2–32 min: 170→260℃; 32–35 min: 260℃). MS was performed under the following conditions: ion source temperature: 230℃; interface temperature: 250℃; solvent elution time: 3 min; assay mode: scan; m / z: 100–350. Figure 5 The peaks in the GC-MS represent the reaction products of peak A, peak B, and peak 2 in peak B, representing various methylation analyses.

[0122] like Figure 5 As shown, the reaction product of peak B contains several GC-MS peaks that are not present in the reaction product of peak A. Among these, peak 2 shows major primary fragments at m / z 189 and 190, minor fragments at m / z 233 and 234, and a unique secondary fragment at m / z 129. Therefore, it is presumed to be 1,2,5,6-tetra-O-acetyl-(2-deuterated)-3,4-di-O-methylglucanol. That is, it is believed that branched inulin contains at least a β-2,6 branched structure.

[0123] Next, to obtain the gene sequence information of IS and BI, the N-terminal amino acid sequences of the proteins contained in peaks A and B were determined according to the following procedures. First, after SDS-polyacrylamide gel electrophoresis (10%) of each peak was performed according to standard methods, the samples were transferred to PVDF membranes and CBB stained. Then, the target band portions (all 44 kDa) of the membrane were excised and washed in the following order with MeOH, 50% MeOH, and ultrapure water. The N-terminal amino acid sequences were then analyzed using Procise 492 (Applied Biosystems). The results suggested that peak A was IS and peak B was a mixture of IS and BI. The N-terminal amino acid sequence of IS was determined to be Ala-Glu-Ile-Ser-Ser-Asp-Tyr-Thr-Ser-Ile-Trp, and the N-terminal amino acid sequence of BI was determined to be Glu-Asp-Val-Gly-Gln-Thr-Thr-Asn-Trp.

[0124] Next, the IS gene and its surrounding base sequence were determined using standard methods. Specifically, peak A was digested with lysyl peptidase, and the resulting peptide was separated by reverse-phase HPLC to analyze the internal sequence. Based on the internal sequence, several pairs of forward and reverse primers were prepared, and PCR was performed using genomic DNA from strain SD33 as a template. PCR for amplifying the DNA fragment was performed using PrimeSTAR max polymerase and an iCycle thermal cycler (BioRAD). The PCR reaction solution was prepared as follows: 1 μL of appropriately diluted template DNA, 20 pmol each of forward and reverse primers, and 25 μL of PrimeSTAR max Premix (2×) were added to bring the total reaction volume to 50 μL. The PCR reaction conditions were as follows: one cycle was performed at 98°C for 30 seconds, and 30 cycles were performed with three temperature changes: 10 seconds at 98°C, 5 seconds at 55°C, and 5 to 30 seconds at 72°C (adjusted according to the target amplification product, with a baseline of 5 seconds per 1kb).

[0125] Based on the sequence of the obtained amplified fragment, primers were prepared in the outward direction. The upstream and downstream base sequences were determined by adapter PCR using the LA in vitrocloning kit (Takara BIO). Here, "upstream" and "downstream" of the gene do not refer to the position relative to the origin of replication. Upstream refers to the region linked to the 5' side of the start codon of the target gene in each operation and step, while downstream refers to the region linked to the 3' side of the stop codon of the target gene in each operation and step, and so on.

[0126] The results are shown in sequence number 37. Open reading frames (ORFs) were searched in the surrounding sequences of the obtained IS gene, and the ORF presumed to be BI (hereinafter referred to as ORF1) was found to be located immediately downstream of IS. Furthermore, a signal peptide sequence was found to be present at the N-terminus of both ORFs.

[0127] Next, based on the base sequences of the mature proteins encoding the IS gene and ORF1 (excluding the signal peptide), an E. coli recombinase expression system was constructed. Specifically, using genomic DNA of SD33 strain as a template, PCR was performed using the primer pairs shown in Table 1 below for IS and ORF1 sequence extension to amplify the base sequences of each mature protein with a start codon added to the N-terminus and a His tag for affinity purification added to the C-terminus.

[0128] [Table 1]

[0129]

[0130] Furthermore, PCR for amplifying the DNA fragment was performed according to the description in Example 2. The obtained amplified fragment was ligated to the NdeI-His tag region of plasmid pET-22b (Merck Millipore) using TaKaRa InfusionHD (Takara BIO). The plasmid was produced using E. coli DH5α transformant and purified using alkaline SDS. After confirming the correctness of the plasmid sequence by sequence resolution using universal primers T7 promoter and T7 terminator, E. coli BL21 (λDE3) was transformed using the obtained plasmid and cultured on LB agar medium containing 200 μg / mL ampicillin at 37°C for 18 hours. The obtained colonies were inoculated into 3 mL of LB liquid medium containing 200 μg / mL ampicillin and incubated overnight at 37°C and 130 rpm. Then, the culture was inoculated at 1 / 100 volume into 50 mL of the same liquid medium and incubated at 37°C and 160 rpm until OD600 = 0.5. The culture was then incubated on ice for 10 minutes, and 50 μL of 0.1 M IPTG (final concentration 0.1 mM) was added. The culture was then incubated at 18°C ​​and 120 rpm for 18 hours. Next, the bacterial cells were recovered by centrifugation at 5000 × g for 10 minutes, resuspended in 10 mM sodium phosphate buffer (pH 7.5), and sonicated using a Sonifier 450 (Branson) (duty cycle 50, output 5, 2 min × 3). After further centrifugation at 12000×g for 10 minutes, the supernatant was loaded onto a Ni-chelate agarose gel chromatography column (φ1.0×4cm) pre-equilibrated with 10mM sodium phosphate buffer (pH 7.5) containing 50mM imidazole. After washing the non-adsorbed fraction with the same buffer, the imidazole concentration was increased to 250mM to elute the adsorbed proteins, which were then used as the IS and ORF1 purification enzymes.

[0131] When the IS purification enzyme is reacted with Bx40 sucrose to a concentration of 2 U / g sucrose, linear inulin is generated. Conversely, when the IS and ORF1 purification enzymes are mixed and reacted with Bx40 sucrose to a concentration of 2 U / g sucrose, branched inulin is generated, which is analyzed using the method described in Example 1. Figure 6 The peaks obtained by analyzing the reaction products obtained by reacting IS and BI of SD33 strain with Bx40 sucrose using HPAEC-PAD are shown in the figure.

[0132] like Figure 6As shown, it was confirmed that: after elution time of 20 minutes in this analysis, the number of branched inulin peaks detected between the linear inulin peaks was more than 10; and the ratio of branched inulin (total peak area of ​​branched inulin / total peak area × 100) in elution times of 20–30 minutes (degree of polymerization 9–16) was 23.9%, which is higher than 10%. Figure 6 Furthermore, when the ORF1 purification enzyme was reacted alone with Bx40 sucrose, glycosyltransfer products with a degree of polymerization of 3–5 were observed. Based on these findings, ORF1 was identified as the gene encoding the BI protein. The amino acid sequence similarity between IS (Sequence No. 34) and BI (Sequence No. 36) was 40.2%.

[0133] Example 3: Study of inulin-producing bacteria without carrying BI-like genes using BLAST search

[0134] In this Example 3, the inulin-producing bacteria that do not carry the gene encoding the causal protein (BI) identified in Example 2 were investigated by BLAST retrieval. It was found that "bacteria carrying genes encoding proteins that have a certain degree of similarity to IS all carry genes encoding proteins that have a certain degree of similarity to the causal protein (BI).

[0135] To explore inulin-producing bacteria that do not carry the BI-like gene, BLAST searches were performed on the amino acid sequences of IS and BI of SD33 strains shown in sequence numbers 34 and 36 using a non-redundant protein sequence database (nr). The results are shown in Table 2 below. In Table 2, microbial strains (recombinantly produced proteins) with base sequences encoding proteins with more than 67.3% identity to IS are indicated in bold.

[0136] [Table 2]

[0137]

[0138] First, strains encoding proteins with an amino acid sequence identical to that of IS (specifically, 67.3%, 68.0%, 68.6%, 68.8%, 70.3%, 70.9%, 71.7%, 73.4%, 76.8%, 78.7%, 78.9%, 79.1%, 79.4%, 79.8%, 80.4%, 80.7%, 81.1%, 81.3%, 81.7%, 81.9%, 83.4%, 84.9%, 85.8%, 96.2%, 96.8%, 97.3%, 98.2%, 98.4%, 98.6%) were identified (IS-like gene; sequence number 33). The results showed that all strains had a protein encoding a protein with more than 67.9% (specifically, 67.9%, 71.5%, 72.2%, 75.1%, 75.3%, 76.6%, 76.9%, 77.1%, 77.3%, 77.5%, 78.3%, 78.5%, 78.7%, 79.0%, 80.8%, 81.0%, 84.8%, 85.3%, 85.4%, 85.8%, 86.0%, 88.6%, 89.7%, 93.1%, 93.7%, 96.6%, 98.7%, 98.9%, and 99.1%) identical amino acid sequence to BI (BI-like gene; sequence number 35). Furthermore, the strains described in Table 2 above are consistent with strain SD33 in that they all have a BI-like gene located immediately downstream of the IS-like gene. In addition, regarding *Bacillus subtilis* and *Bacillus subtilis* OV191 strains, no stop codon was found in the IS-like gene, and no signal peptide-like sequence was found at the N-terminus of the BI-like gene; therefore, it is believed that they may be produced by linking BI-like and IS-like proteins. As described above, it has been determined that bacteria carrying IS-like genes with a certain degree of similarity also carry BI-like genes. Furthermore, the amino acid sequence similarity between IS and each BI-like protein, and between BI and each IS-like protein, is less than 50%.

[0139] Among the microorganisms shown in Table 2 above, the 16S rDNA partial base sequences of standard strains of microorganisms with clearly defined species names, ammoniaphile strains YIM78 and 166, Bacillus subtilis standard strain, and SD33 strain were subjected to multiple alignment using clustalW, and a molecular phylogenetic tree was constructed using FastTree v2.1.8 with default parameters. Figure 7 The diagram shows the molecular system that was created.

[0140] like Figure 7As shown, except for *Mycobacterium abscessus* subsp. *abscessus*, the standard strains of all microbial species and *Ammoniac* strains YIM78 and 166 are all positioned close to *Bacillus subtilis* (the most common *Bacillus* spp.). *Bacillus* spp. and *Bacillus-like* spp. are generally considered closely related to the *Bacillus* spp. Therefore, it is reasonable to consider at least the microbial species listed in Table 2 above, except for *Mycobacterium abscessus* subsp. *abscessus*, namely *Pristemonella*, *Bacillus* spp., *Neobacillus* spp., *Fredinandcohnia* spp., *Parabacterium* spp., *Litchfieldia* spp., *Robertomarella* spp., *Mycobacterium* spp., *Halophyte* spp., *Bacillus* spp., *Bacillus-like* spp., and *Ammoniac* spp., as closely related to the *Bacillus* spp.

[0141] Example 4: Construction of BI gene deletion plasmid, BI gene deletion strain and analysis of inulin production

[0142] In Example 4, a method for systematically breeding and improving the inulin-synthesizing bacterium SD33 isolated from nature into a strain suitable for the synthesis of linear inulin was described, including the steps for constructing the plasmid for deleting the BI gene of SD33 and the deleted strain. Furthermore, by deleting this gene, it was confirmed that linear inulin could be synthesized using the culture supernatant.

[0143] First, a summary of the BI gene deletion plasmid used in this method is described below. The BI gene deletion plasmid refers to a plasmid containing a subcloned BI gene deletion DNA fragment within a plasmid possessing any drug resistance gene. The BI gene deletion DNA fragment refers to a fragment consisting of approximately 0.5 kb of an upstream adjacent fragment (A) and approximately 0.5 kb of a downstream adjacent fragment (B) of the BI gene. First, the upstream fragment (A) and downstream fragment (B) of the BI-like gene are prepared by a first PCR, using primers designed to add a sequence of 10–30 bases upstream of the downstream fragment (B) to the downstream end of the upstream fragment (A). Table 3 below shows the primers used in Example 4.

[0144] [Table 3]

[0145]

[0146] Next, using the PCR fragments (A) and (B) prepared in the first step as templates, a second PCR was performed using the upstream primer of the upstream fragment and the downstream primer of the downstream fragment. This caused annealing between the downstream end of the upstream fragment and the upstream end of the downstream fragment, resulting in a DNA fragment formed by the ligation of (A) and (B). The DNA fragment obtained from the second PCR was then inserted into the plasmid using any method, thus obtaining a plasmid for the BI gene deletion. Specifically, genomic DNA from SD33 strain was used as a template, and PCR was performed using primers (1) and (2), (3) and (4) shown in Table 3 above. The two amplified fragments obtained were used as templates, and PCR was performed using primers (1) and (4) shown in Table 3 above. The PCR for DNA fragment amplification was performed according to the description in Example 2. The obtained amplified fragments were double-digested with restriction endonucleases SmaI and XbaI (Takara BIO) and subcloned into the SmaI-XbaI region of pHY300PLK (Takara BIO). The DNA Ligation Kit Mighty Mix (Takara BIO) was used for ligation. The plasmid was produced using a transformant of *E. coli* DH5α via alkaline SDS-PAGE. Using the obtained plasmid as a template, sequence analysis was performed using primers (5) shown in Table 3 above, confirming the insertion of the target base sequence. This plasmid was used as the plasmid for BI gene deletion.

[0147] Next, the SD33 strain was transformed using the obtained BI gene deletion plasmid. Through two rounds of homologous recombination, a strain lacking the BI gene was obtained. Specifically, 100 μL of the glycerol stock solution of the SD33 strain was inoculated into 5 mL of TM medium in a 300 mL Erlenmeyer flask and cultured at 30 °C and 130 rpm for 5 hours with shaking. The bacterial cells were recovered by centrifugation at 5000 × g for 2 minutes, and after washing with 5 mL of 50 mM Tris-HCl buffer (pH 7.5), the suspension was transferred to a 15 mL centrifuge tube and centrifuged at 5000 × g for 2 minutes. The bacterial cells were then resuspended in 5 mL of 50 mM Tris-HCl buffer (pH 8.5) and shaken at 37 °C and 85 rpm for 60 minutes. 500 μL of the suspension was collected by centrifugation at 5000 × g for 1 minute, and the supernatant was removed. 5 μL of a 300 ng / μL BI gene deletion plasmid solution was mixed with 50 μL of 70 mM phosphate buffer (pH 6.3) containing 0.5 M sodium sulfate. This mixture was added to a centrifuge tube containing bacterial cells, and the mixture was vortexed and allowed to stand for 5 minutes. 150 μL of 70 mM phosphate buffer (pH 6.3) containing 40% PEG was added to the solution in the centrifuge tube, and the mixture was vortexed. The cells were collected by centrifugation at 3000 × g for 5 minutes, resuspended in 1 mL of TM medium containing 20 mM MgCl2, transferred to 15 mL volumetric centrifuge tubes, and shaken at 37 °C and 85 rpm for 60 minutes. The bacterial cells were then collected by centrifugation at 5000 × g for 1 minute and inoculated into LB medium containing 10 μg / mL tetracycline. The cells were cultured at 37 °C for 3 days to obtain transformants.

[0148] The obtained colonies were inoculated into 3 mL of LB liquid medium containing 10 μg / mL tetracycline with one platinum loop and cultured at 30 °C and 130 rpm for 24 hours with shaking. 50 μL of the culture was spread onto the same agar medium and cultured at 35 °C for 24 hours. Using the obtained single colonies as templates, colony PCR was performed using the universal primers for pHY300PLK shown in Table 3 above, namely primer (5), and primers (6) designed based on the sequence near the transcription start position of the BI gene.

[0149] Colony PCR was performed using Emerald Amp Max Master Mix (Takara BIO). The colony PCR reaction solution was prepared by adding 1 μL of appropriately diluted bacterial suspension, 2 pmol each of forward and reverse primers, and 5 μL of Emerald Amp Max PCR Master Mix (2×Premix), bringing the total reaction volume to 10 μL. PCR reaction conditions were as follows: one cycle at 98°C for 30 seconds, followed by 30 cycles of three temperature variations: 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 1–5 minutes (adjusted according to the target amplification product, with a baseline of 1 minute per 1 kb). Sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific), and sequence analysis was performed using the Applied Biosystems 3730xl DNA analyzer (Thermo Fisher Scientific). The composition of the culture media used is described below. Unless otherwise specified, use commercially available premium grade reagents.

[0150] Using culture medium

[0151] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, (15 g / L agar powder)

[0152] Basic culture medium: 10 g / L sucrose, 5 g / L soybean peptone, 1 g / L yeast extract, 0.5 g / L KH₂PO₄, (15 g / L agar powder), adjusted to pH 7.2 with NaOH.

[0153] TM culture medium: 10 g / L glucose, 10 g / L soybean peptone, 5 g / L bonito extract, 2 g / L yeast extract, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·7H2O, 1 mg / mL ZnSO4·7H2O, adjusted to pH 7.0 with NaOH.

[0154] Colony PCR yielded approximately 500 bp amplified fragments, which were used as the first crossover recombinant strain. Colonies of the first crossover recombinant strain were inoculated into 3 mL of basal medium and cultured with shaking at 30°C and 130 rpm for 8–12 hours. Then, a 1 / 100 inoculation was performed into 3 mL of the same medium. This process was repeated four times. The culture was then spread onto the same agar medium and incubated overnight at 35°C. Colonies were then transferred by pressing an autoclaved agar plate onto a velvet cloth fixed to a substrate to allow adhesion, followed by pressing onto LB agar medium containing antibiotics and LB agar medium containing 10 μg / mL tetracycline. All colonies were incubated overnight at 35°C. In this experiment, colonies that grow on LB agar medium without antibiotics and do not grow on LB agar medium containing 10 μg / mL tetracycline were screened. These colonies were used as templates, and colony PCR was performed using primers (1) and (4). The strains that amplified fragments about 1000 bp shorter than the original SD33 strain were designated as BI gene deletion strains (hereinafter, SD33_ΔBI strain).

[0155] Next, SD33 and SD33_ΔBI strains were inoculated with one platinum ring in 3 mL of basal medium and cultured with shaking at 30°C and 130 rpm for 24 hours. The supernatant was recovered by centrifugation at 12000×g for 5 minutes. Sucrose was added to each culture supernatant to make Bx40, and the mixture was incubated at 37°C for 24 hours, followed by incubation at 100°C for 5 minutes to inactivate the enzyme. The sugar composition of the reaction solution was analyzed using HPAEC-PAD. Figure 8 The peaks obtained by analyzing the reaction products obtained from the reaction of the culture supernatants of SD33 strain and SD33_ΔBI strain with Bx40 sucrose were analyzed using HPAEC-PAD.

[0156] like Figure 8 As shown, in the reaction solution obtained using the culture supernatant of SD33 strain, branched inulin was also identified in addition to linear inulin. On the other hand, in the reaction solution using the culture supernatant of SD33_ΔBI strain, only linear inulin was identified, and during the elution time of 20-30 minutes, the area of ​​peaks other than linear inulin was less than 1% of the total peak area.

[0157] Example 5: Elucidation of sucrose reaction products of various identical BI and IS E. coli recombinases

[0158] In this Example 5, it was determined that even the protein with a relatively low similarity to the causative protein (BI) identified in Example 3, namely 67.9%, was also the cause of branched inulin production. As examples of inulin-producing bacteria carrying genes encoding the causative protein, Priestella, Bacillus, Neobacillus, Fredinandcohnia, Parabacillus, Litchfieldia, Robert Merah, Mycobacterium, Halophilic Bacillus, Brachybacterium, Bacillus-like bacteria, and Ammoniaphila were identified.

[0159] The base sequences encoding the amino acid sequences excluding the signal peptide and stop codon predicted by SignalP-5.0, as shown in bold in Table 2 above for each BI-like protein, were artificially synthesized (sequence numbers 38-47, commissioned to Integrated DNA Technologies). Furthermore, two unidentified bases in the base sequence information encoding the BI-like protein from Bacillus strain LL01 were inferred as guanidine bases based on the base sequences of related BI-like proteins, and the gene was artificially synthesized. Using the artificially synthesized base sequences as templates, PCR was performed using the corresponding primer pairs, following the same procedures as in Example 2, to construct an E. coli recombinase expression system, and the recombinase was purified. PCR for DNA fragment amplification was performed as described in Example 2. Additionally, the BI-like protein (ATN45517.1) from Agaricus WDG185 strain could not be normally expressed in E. coli, therefore no further experiments were performed. Table 4 below shows a list of primers used in Example 5.

[0160] [Table 4]

[0161]

[0162] Next, the purified BI-like recombinant enzymes and the culture supernatant (containing IS enzyme) of SD33_ΔBI strain were added to Bx40 sucrose solution to achieve a sucrose concentration of 2 U / g. After incubation at 37°C for 24 hours, the solutions were incubated at 100°C for 5 minutes to inactivate the enzymes. The sugar composition of the reaction solution was analyzed using HPAEC-PAD. The culture supernatant of SD33_ΔBI strain and the purified BI-like recombinant enzymes were mixed and reacted with Bx40 sucrose. Figure 9 The peaks obtained from the reaction products are represented by the peaks obtained from HPAEC-PAD analysis. Furthermore, enzyme activity was calculated according to the description in Example 1.

[0163] like Figure 9As shown, the inulin generated in the culture supernatant of the SD33_ΔBI strain alone is linear. Furthermore, Table 5 below shows the ratio of branched inulin (total peak area of ​​branched inulin / total peak area × 100) during elution time of 20–30 minutes (degree of polymerization 9–16) when the culture supernatant of each purified BI-like recombinase and the SD33_ΔBI strain are mixed and reacted with Bx40 sucrose.

[0164] [Table 5]

[0165]

[0166] As shown in Table 5 above, the proportion of branched inulin in elution times of 20–30 minutes (degree of polymerization 9–16) was 0.3%. On the other hand, as Figure 9 As shown, by adding any BI-like recombinase with at least 67.9% similarity to sequence number 36, branched inulin peaks were detected among the linear inulin peaks after elution time of 20 minutes, with a number of more than 10 peaks. Furthermore, as shown in Table 5 above, the proportion of the aforementioned branched inulin in elution times of 20–30 minutes (degree of polymerization 9–16) exceeded 10% when using any BI. In addition, when each BI-like recombinase was reacted alone with Bx40 sucrose, it produced glycosyltransfer products with a degree of polymerization of 3–5, similar to the BI of strain SD33.

[0167] Example 6: A study on the correlation between IS gene identity and the synthetic activity of linear inulin.

[0168] In this Example 6, it was determined that even proteins with a very low similarity to the IS protein identified in Example 2, namely 67.3%, possess inulin sucrase activity for producing linear inulin.

[0169] Based on the base sequences encoding the IS-like protein of Bacillus subtilis strain OV191 (67.3% identity with the amino acid sequence of SD33 strain IS, SEQ ID NO: 34) and the IS-like protein of Bacillus subtilis strain LL01 (68.8% identity with the amino acid sequence of SD33 strain IS, SEQ ID NO: 34) (hereinafter referred to as "OV191_IS" and "LL01_IS", respectively), as in Example 5, base sequences encoding the amino acid sequence excluding the signal peptide and stop codon predicted by SignalP-5.0 were synthesized (SEQ ID NO: 48, 49), and an E. coli recombinase expression system was constructed. The recombinase was then purified. Figure 10 Peaks obtained by HPAEC-PAD analysis of the reaction products obtained by reacting the culture supernatant of SD33_ΔBI strain, OV191_IS, and LL01_IS with Bx sucrose.

[0170] like Figure 10As shown, the inulin produced when the two recombinant enzymes were reacted with Bx40 sucrose solution exhibited fewer peaks on the high degree of polymerization side, but similarly showed the peak shape of linear inulin as when using the SD33_ΔBI strain. Based on the above, it can be concluded that even proteins with as low as 67.3% and 68.8% identity with the SD33 strain's IS possess inulin sucrase activity for producing linear inulin.

[0171] Example 7: Determination of DFAIII yield in one-pot synthesis

[0172] In this Example 7, it was determined that even a protein with a very low similarity to the causal protein (BI) identified in Example 3, namely 67.9%, was the cause of the generation of branched inulin, and was the cause of the reduced yield of DFAIII from sucrose in the culture supernatant of the BI gene-deleted strain SD33 from Example 4 and inulin fructosyltransferase.

[0173] When inulin is acted upon by inulin fructosyltransferase (IFT), DFAIII is generated. However, the yield of DFAIII is lower when inulin containing branched inulin is used compared to when linear inulin is used. In a one-pot reaction of DFAIII with IS and IFT added to sucrose solution, if the culture supernatant of wild-type strains such as SD33 is used as IS, this culture supernatant also contains BI, thus the yield of DFAIII is lower compared to when purified IS is used. First, the one-pot reaction was compared using the culture supernatant of SD33 and the culture supernatant of SD33_ΔBI strain.

[0174] In a Bx40 sucrose solution of IFT (produced by Japan Beet Sugar Manufacturing Co., Ltd.) containing Arthrobacter H65-7 strain at a sucrose ratio of 4 U / g sucrose, culture supernatant of (1) SD33 strain (2 U / g sucrose) or (2) SD33_ΔBI strain (1 U / g sucrose) was added. After incubation at 50°C for 90 hours, the reaction was stopped by incubation at 100°C for 10 minutes. Next, the reaction solution was diluted 10-fold with ultrapure water for analysis of its sugar composition. To acid hydrolyze the unreacted sucrose in the reaction solution, 100 μL of 1N HCl was added to 1 mL of the 10-fold dilution. After incubation at 60°C for 2 hours, 100 μL of 1N NaOH was added to restore the pH before HPLC analysis. HPLC conditions were set as follows: column: Shodex SUGAR KS-801 (guard column: SUGAR KS-G); column temperature: 60℃; solvent: 1 / 20000N NaOH; flow rate: 1.0 mL / min; detection: RI; injection volume: 5 μL of 1 w / v% aqueous solution. Based on the obtained peak areas, the sugar composition % of DFAIII (equivalent to the DFAIII synthesis yield relative to sucrose) was calculated using the following formula. The DFAIII synthesis yields when each purified BI-like recombinase was reacted with sucrose are shown in Table 6 below.

[0175] [Calculation formula]

[0176] Sugar composition % of DFAIII = [Peak area of ​​disaccharides after decomposition] / [Total peak area of ​​sugars before decomposition] × 100

[0177] [Table 6]

[0178]

[0179] The DFAIII synthesis yield using the culture supernatant of SD33 strain shown in Table 6 above was 27.0%, which was significantly improved to 38.6% when using the culture supernatant of SD33_ΔBI strain.

[0180] Next, to clarify the identity of the BI that reduced the reaction yield of the one-pot DFAIII reaction, the following experiment was conducted using the BI-like recombinase prepared in Example 5. In a Bx40 sucrose solution containing IFT (Japan Beet Sugar Manufacturing Co., Ltd.) to make it 4 U / g sucrose, a mixture of culture supernatant of SD33_ΔBI strain with 4 U / g sucrose and purified BI-like recombinase (1 U / g sucrose each) was added, and the enzyme reaction was carried out under the same reaction conditions as described above. The DFAIII synthesis yield was then measured. The DFAIII synthesis yield relative to sucrose is shown in Table 6 above. When any BI-like recombinase was mixed, the DFAIII yield was reduced compared to when only the culture supernatant of SD33_ΔBI strain was used. This confirmed that BI-like proteins with 67.9% or more identity with the BI of SD33 strain possess the same enzymatic activity as the BI of SD33 strain, thus reducing the DFAIII yield in the one-pot DFAIII synthesis reaction.

[0181] Finally, the reaction yield of the one-pot DFAIII reaction was investigated whether combining BI and IS from other strains reduced the reaction yield. IS (1 U / g sucrose) from Bacillus subtilis strain OV191, or a combination of IS and BI (1 U / g sucrose each), was added to a Bx40 sucrose solution containing IFT (4 U / g sucrose). The reaction was maintained at 50°C for 90 hours, followed by a 10-minute incubation at 100°C to stop the enzyme reaction. The DFAIII synthesis yield relative to sucrose was then measured. Similarly, the enzyme reaction was performed using a combination of IS and BI from Bacillus subtilis strain LL01, and the DFAIII synthesis yield was measured. Table 7 below shows the DFAIII synthesis yield when the recombinases of IS and BI from Bacillus subtilis strain OV191 and Bacillus subtilis strain LL01 were reacted with sucrose.

[0182] [Table 7]

[0183]

[0184] The DFAIII synthesis yields are shown in Table 7. Based on the above, it was confirmed that, similar to the SD33 strain, the DFAIII yield decreased when using IS and BI from Bacillus subtilis strain OV191 and from Bacillus subtilis strain LL01.

[0185] . PCT / RO / 134 form

Claims

1. A linear inulin-producing bacterium, selected from one or more of the following: *Priscilla*, *Bacillus*, *Neobacillus*, *Fredinandcohnia*, *Pseudomonas*, *Litchfieldia*, *Robert Merah*, *Mycobacterium*, *Halophilic Bacillus*, *Brucea*, *Bacillus-like*, and *Ammoniac* species; and linear inulin-producing bacteria closely related to the *Bacillus* genus other than these linear inulin-producing bacteria. The linear inulin-producing bacterium is characterized by: The gene shown in at least one of (a) to (e) below is deleted or inactivated, and the user has at least one of the genes shown in (h) to (m) below. (a) A gene encoding a protein consisting of a base sequence having more than 70% identity with the base sequence shown in sequence number 35. (b) Genes that hybridize under strict conditions with the complementary strands of the gene consisting of the base sequence shown in sequence number 35. (c) The gene encoding a protein consisting of the amino acid sequence shown in sequence number 36. (d) Genes encoding proteins consisting of amino acid sequences with 1 to 10 amino acids deleted, substituted, added, or inserted in the amino acid sequence shown in sequence number 36. (e) A gene encoding a protein consisting of an amino acid sequence having more than 67.9% identity with the amino acid sequence shown in sequence number 36; (h) The gene with the base sequence shown in sequence number 33, (i) A gene consisting of a base sequence having more than 70% identity with the base sequence shown in sequence number 33, and encoding a protein having activity of catalyzing the transfer of furanofructose β-2,1 of sucrose to the terminal fructose residue site of other sucrose or its glycosyl transfer products. (j) Hybridization under stringent conditions of the complementary strand of a gene consisting of the base sequence shown in sequence number 33, and the gene encoding a protein having activity that catalyzes the transfer of β-2,1 furanofructose of sucrose to the terminal fructose residue site of other sucrose or its glycosyl transfer products. (k) Genes encoding proteins secreted outside the bacterial cell, consisting of the amino acid sequence shown in sequence number 34. (l) A gene encoding an amino acid sequence consisting of 1 to 10 amino acids that are deleted, substituted, added, or inserted in the amino acid sequence shown in sequence number 34, and which is secreted outside the bacterial cell and has the activity of catalyzing the transfer of β-2,1 furanose of sucrose to the terminal fructose residue of other sucrose or its glycosyl transfer products. (m) A gene that encodes an amino acid sequence having more than 67.3% identity with the amino acid sequence shown in sequence number 34 and that is secreted outside the bacterial cell by means of a reaction that catalyzes the transfer of the furanofructose β-2,1 of sucrose to the terminal fructose residue of other sucrose or its glycosyl transfer products.

2. A linear inulin-producing bacterium, characterized in that, The linear inulin-producing bacteria described above have the characteristics shown in (f) or (g): (f) Inoculate 1 platinum ring of bacterial cells into 3 mL of the culture medium with the following composition, and culture with shaking at 30°C for 24 hours and 130 rpm. Then add sucrose to the culture supernatant to make it Bx40, and react at 37°C for 24 hours to produce a linear inulin-producing bacterium that produces inulin with fructose bonds consisting only of β-2,1 bonds. Culture medium composition: 10 g / L sucrose, 5 g / L soybean peptone, 1 g / L yeast extract, 0.5 g / L KH2PO4, adjusted to pH 7.2 with NaOH; (g) Inoculate 1 platinum ring of bacterial cells into 3 mL of the culture medium with the composition described in (I) below, and culture with shaking at 30°C for 24 hours at 130 rpm. Add sucrose to the culture supernatant to make it Bx40, and react at 37°C for 24 hours. When the resulting reaction product is analyzed by HPAEC-PAD under the conditions described in (II) below, it is characterized by: observing a peak at the same elution time as inulin composed only of β-2,1 bonds during an elution time of 20–30 minutes (degree of polymerization 9–16), and the area of ​​other peaks being less than 1% of the total peak area of ​​linear inulin-producing bacteria. (I) Culture medium composition: 10 g / L sucrose, 5 g / L soybean peptone, 1 g / L yeast extract, and 0.5 g / L KH₂PO₄ were added, and the pH was adjusted to 7.2 with NaOH. (II) Conditions for HPAEC-PAD analysis: Column: CarboPac PA1 4×250mm; Guard column: CarboPac PA1 4×50mm; Injection volume: 5μL of 0.1% aqueous solution of sample; Flow rate: 1.0mL / min; Elution: NaOH 100mM constant, AcONa gradient (0~60min, 0→600mM); Detection: pulsed amperometric detection.

3. The linear inulin-producing bacteria as described in claim 1, characterized in that, A gene consisting of sequence number 35 or a base sequence having more than 75% identity with that sequence number has the following characteristics: Proteins consisting of the amino acid sequence encoded by the gene represented by sequence number 35 or having at least 75% identity with that sequence number, and proteins consisting of the amino acid sequence represented by sequence number 34, were added to a sucrose solution of Bx40 to make a concentration of 2 U / g sucrose. The mixture was reacted at 37°C for 24 hours. When the resulting reaction products were analyzed using HPAEC-PAD under the following conditions, the protein consisting of the amino acid sequence represented by sequence number 34 was obtained after elution time of 20 minutes (degree of polymerization ≥ 9). The reaction of the substance with sucrose solution of Bx40 yielded linear inulin with fructose bonds consisting solely of β-2,1 bonds, exhibiting more than 10 peaks at the same elution time. Furthermore, more than 10 branched inulin peaks were detected between the linear inulin peaks, exhibiting a structure where any fructose residue site of the linear inulin is bound to a furanylfructose group via a bonding method other than β-2,1 bonds. During an elution time of 20–30 minutes (degree of polymerization 9–16), the proportion of branched inulin (total peak area of ​​branched inulin / total peak area × 100) was more than 10%. Conditions for HPAEC-PAD analysis: Post: CarboPac PA1 4×250mm; Protective post: CarboPac PA1 4×50mm; Injection volume: 5 μL of 0.1% aqueous solution of sample; Flow rate: 1.0 mL / min; Elution: NaOH 100mM constant, AcONa gradient (0-60 min, 0→600mM); Detection: pulsed amperometric detection.

4. A purified enzyme, crude enzyme or enzyme content, culture medium, culture supernatant or cultured cells or treated products thereof obtained from the linear inulin synthetogenic bacteria according to any one of claims 1 to 3.

5. A method for manufacturing linear inulin, characterized in that, The procedure includes the cultivation of linear inulin-producing bacteria as described in any one of claims 1 to 3.

6. A method for manufacturing linear inulin, characterized in that, The method comprises a step of reacting at least one of the purified enzyme, crude enzyme or enzyme contents, culture medium, culture supernatant or cultured cells or the treated products thereof obtained from the linear inulin synthesizing bacteria according to any one of claims 1 to 3 with sucrose to obtain a reaction product.

7. The method for manufacturing linear inulin as described in claim 6, characterized in that, When the reaction products were analyzed by HPAEC-PAD under the following conditions, during an elution time of 20–30 minutes (degree of polymerization 9–16), the proportion of branched inulin (where the fructose residues of linear inulin, where the fructose bonds between fructose molecule are β-2,1 bonds, and any fructose residue site is bound with a furan-fructose group by a bonding mode other than β-2,1 bonds) was less than 1% (total peak area of ​​branched inulin / total peak area × 100). Conditions for HPAEC-PAD analysis: Post: CarboPac PA1 4×250mm; Protective post: CarboPac PA1 4×50mm; Injection volume: 5 μL of 0.1% aqueous solution of sample; Flow rate: 1.0 mL / min; Elution: NaOH 100mM constant, AcONa gradient (0-60 min, 0→600mM); Detection: pulsed amperometric detection.

8. A method for manufacturing DFAIII, characterized in that, At least one of the purified enzyme, crude enzyme or enzyme contents, culture medium, culture supernatant or cultured cells or treated products thereof obtained from the linear inulin synthetus of any one of claims 1 to 3 is reacted with sucrose.

9. The method for manufacturing DFAIII as described in claim 8, characterized in that, Use inulin fructose transferase.

Images