JP2021177712A - Method for producing inulin using inulosucrase from bacillus krulwichiae - Google Patents

Abstract

To provide a method for efficiently producing inulin from sucrose, leading to waste reduction and cost reduction.SOLUTION: There is provided an inulosucrase isolated and recombinantly expressed from Bacillus krulwichiae, comprising: sequences consisting of specific amino acid sequences and variants thereof created by recombinant techniques.SELECTED DRAWING: Figure 1

Description

The present invention relates to a novel inulosucrase gene and a method for producing inulin using a novel inulosucrase obtained by recombinantly expressing the gene.

Inulin is a type of polysaccharide and is mainly contained in the tubers of some plants (chicory, dahlia, Jerusalem artichoke, burdock, garlic, etc.). Its structure is different from starch, which is a polymer of glucose, and D-fructose is polymerized on the fructose side of sucrose by β- (2 → 1) binding. Its molecular weight varies depending on the chain length of fructose, and inulin is a general term for two or more fructose polymerized. Usually, the chain length is often expressed by the number of polymerizations of fructose (expressed as GF10, etc.), but those with a short chain length are 1-kestose (GF2), 1-nistose (GF3), 1-fructose furanosyl. Named Nistose (GF4). Plant-derived inulin generally has a chain length of about GF30 to GF50. Inulin is mainly produced by extracting and purifying from tubers of plants such as chicory and Jerusalem artichoke, but only a few methods have been studied to produce inulin from sucrose using microbial enzymes.

Patent No. 4667672 Special table 2017-536131

Inulin is known to have a remarkable effect of improving the intestinal flora, and is in high demand as a functional food material. Inulin is abundantly contained in tubers of plants (chicory, Jerusalem artichoke, etc.), but at present, the main method for producing inulin is extraction and purification from plants, which has problems such as high cost and a large amount of waste. As another method, there is a method of producing inulin from sucrose using a microbial enzyme, but only a few studies have been conducted and it cannot be said that it is an efficient production method.

The entire genome sequence of the Bacillus cruluwiche JCM11691 strain has been published, and this genome contains a gene thought to encode the novel inulosucrase (InuBK) shown in SEQ ID NO: 3.

When this gene encoding InuBK was isolated and inserted into a plasmid for secretory expression and expressed using Brevibatillas chocinensis as a host, the accumulation of a protein of about 50 kDa estimated to be the protein was confirmed.

The gene encoding InuBK derived from the Bacillus cruluwiche genome of the present invention, a polypeptide recombinantly expressed from a synthetic nucleic acid, was identified as inulosucrase as a result of examining its enzymatic activity and properties.

The activity (specific activity) of the enzyme of novel inulosucrase (InuBK) is described in the prior patent (Patent Document 2 and Kralj S, et al., (2018) "Synthesis of fructooligosacchararides (FosA) and inulin (InuO) by GH68 fructosyltransferases". from Bacillus agaradhaerens strain WDG185. "Carbohydr. Polym., 179, 350-359) is at least 1.4 times higher. By utilizing this novel inulosucrase, inulin can be produced from sucrose more efficiently than before. A composition comprising inulosucrase by a host cell comprising this gene and / or its expression product, an active fragment thereof, synthetic nucleic acids and plasmids encoding all or part of InuBK, variants thereof and those genes or synthetic nucleic acids. Is provided.

According to the present invention, the inulosucrase InuBK produced by the Bacillus cruluwiche ATCC11691 strain is allowed to act in a neutral to slightly alkaline (pH 6.0-9.0) buffer containing sucrose at 50 to 55 ° C. As a result, inulin, which is in high demand as a functional food material, can be easily and efficiently produced with less steps than the production method using plant extraction, and with less waste. This inulosucrase has a higher specific activity than the prior art, and is advantageous because it can produce inulin with a smaller amount of enzyme.

Inulin formation by recombinant InuBK. Arrows indicate the positions of GF3 (nistose) and GF15, respectively. The X-axis is the retention time (minutes), and the Y-axis is the detector response value (nc). Time course of inulin synthesis from sucrose by InuBK (0 hours, 1 hour, 3 hours, 8 hours). The arrows indicate the positions of sucrose (Suc), 1-kestose (GF2), and nistose (GF3), respectively. The units of the X-axis and Y-axis are the same as in Figure 1. The figure which shows the progress of the inulin formation reaction by InuBK. Mix 40 ml of 20% sucrose, 50 mM general purpose buffer, 50 μg / ml of purified InuBK, sucrose (〇), glucose (△) at 0, 1, 2, 3, 4, 6, 8 hours, The concentration of fructose (▼) is shown. It is a figure which shows that the polysaccharide produced by InuBK is inulin. The polysaccharide produced by InuBK from sucrose was subjected to ethanol precipitation (twice), centrifuged, and then redissolved in water and lyophilized to obtain a purified polysaccharide powder (purified inulin). Dissolve 0.5 mg of this powder in 10 μl of water and mix with 10 μl of makilbane buffer (pH 4.5) to exoinulinase (2000 U / ml) and endoinulinase (300 U / ml) (both manufactured by Megazyme). Was added in an amount of 1 μl each. Similarly, 0.5 mg of powder was dissolved in 10 μl of water, mixed with 10 μl of 0.1 M MES buffer (pH 6.0), and 1 μl of endolevanase (450 U / ml, manufactured by Megazyme) was added. All enzyme reaction solutions were incubated at 40 ° C. for 2 hours and subjected to thin layer chromatography. Lane 1; no treatment, lane 2; endoinulinase treatment, lane 3; exoinulinase treatment, lane 4; endrevanase treatment. It is a figure which showed by NMR analysis that the polysaccharide produced by InuBK from a sucrose solution is inulin. Top; Inulin produced by InuBK, Medium; Commercial inulin (manufactured by DKSH Japan), Bottom; Commercial levan (manufactured by Megazyme).

The inulosucrase gene identified in the present invention can be hosted by Brevibacillus chocinensis or Vatilas satiris in its expression.

The appropriate medium to be used is MT medium for Brevibacillus chocinensis and L medium for Vaticus sachilis, but it is necessary to add an appropriate antibiotic to the medium depending on the plasmid to be used to retain the expression plasmid.

The culture conditions are a temperature of 30-33 ° C., and shaking culture (for example, rotary shaking at a rotation speed of 250 rpm, reciprocating shaking at about 180 times / minute. The appropriate culture time is about 24 to 36 hours.

Sufficient inulosucrase activity is accumulated in the culture broth after culturing, but since medium components and bacterial cell metabolites are mixed, ammonium sulfate precipitation, ion exchange chromatography, and hydrophobicity are required as necessary. It is preferable to purify and use it by a method such as sex chromatography.

This inulosucrase gene is a plasmid or artificial nucleic acid constructed so that a histidine tag (a peptide in which 6 consecutive histidine residues are fused and expressed by an artificial gene) is fusedly expressed at the C-terminal of the enzyme after expression. Even if it is expressed by, the enzyme activity is maintained in a sufficient amount. The culture medium containing this histidine tag fusion inulosucrase may be purified by affinity chromatography (for example, using a nickel-binding column).

Cloning of the InuBK gene A genome was extracted from the Bacillus cruluwiche JCM11691 strain, and the InuBK gene was PCR amplified (1395 bp, the enzyme used was KOD plus polymerase) using the prima shown in Sequence Listings 1 and 2 as a template. The obtained fragment was inserted into the BamHI-XbaI (XbaI smoothed with Klenow fragment) site of PNI DNA (manufactured by Takara Bio). The obtained plasmid was designated as pNIInuBKChis.

Transformation of Brevibatillas chocinensis by InuBK The Brevibatillas chocinensis strain (purchased from Takara Bio) was transformed by the New Tris peg method using pNIInuBKChis. The transformed cells were coated on MT agar medium containing neomycin (30 μg / ml), and the colonies grown by culturing at 37 ° C. were selected as transformants. This transformed strain was designated as Brevibacillus chocinensis InuBKhis strain.

InuBK-expressing Brevibatillas chocinensis strain InuBKhis was cultured in TM medium (containing 10 μg / ml neomycin) by shaking at 37 ° C. (250 rpm, rotary). After culturing for 24 hours, the cells were removed by centrifugation (9500 rpm, 10 minutes).

InuBK Purification This precipitate was dissolved in a column test solution (50 mM Tris, 20 mM imidazole (pH 7.2)), and the insoluble material was filtered off (sulfate precipitate solution). This ammonium sulfate precipitation solution was applied to a histrap column (manufactured by GE Healthcare), and further washed with a column test solution having a volume 10 times the volume (50 mM) of the column. Then, the adsorbed proteins were sequentially fractionated and eluted with a concentration gradient from column test solution-eluent (50 mM tris / 600 mM imidazole) = 100% -0% to 0% -100%. The active fraction was detected by SDS-PAGE and activity measurement, and dialyzed against a general-purpose buffer (50 mM, pH 7.0) to prepare a purified enzyme solution. The protein concentration of the enzyme was quantified by the Bradford method using bovine serum albumin (Fraction V, SIGMA) as a standard.

High performance anion exchange chromatography
The InuBK reaction solution was appropriately diluted with pure water and then subjected to an analysis using an HPAEC device (ICS-5000 manufactured by Dionex) equipped with a pulsed current measurement detector. Moving layer A: 0.15 M NaOH-5 mM NaOAc, B: 0.15 M NaOH-600 mM NaOAc, sample injection volume: 50 μl, column temperature 30 ° C, column: Carbopac PA-1 (Thermo Fisher, 4 × 250 mm). The concentration gradient was as follows. Moving layer A: 100% (0 minutes), moving layer A: 50%, moving layer B: 50% (70 minutes). The applied pulse of the detector is standard quad potential: +0.1 volt (0-0.4 s); -2.0 volt (0.41-0.42 s); 0.6 volt (0.43 s); -0.1 volt (0.44-0.5 s). The data was analyzed by Chromeleon software (manufactured by Thermo Fisher).

Inulin production Purified InuBK was incubated with 20% (W / V) sucrose at a concentration of 50 μg / ml at 50 ° C. and reacted in 50 mM general-purpose buffer to produce inulin. Sucrose decreased over time, and glucose increased in correlation with it. Fructose did not increase much (Fig. 3).

When this reaction solution (after 8 hours of reaction) was subjected to high-performance anion exchange chromatography (HPAEC) and analyzed, the produced inulin chain length was at least in the range of GF2 to GF25 (Fig. 1).

When the reaction solution was sampled over time for up to 8 hours and HPAEC analysis was performed, the extension of the inulin chain was completed from 3 hours to 8 hours at the latest under these reaction conditions (Fig. 2).

In a volume of 40 ml, mix sucrose in a universal buffer (50 mM, pH 7.0) to 30% (W / V) and purified InuBK 50 μg / ml (both final concentrations), and mix at 50 ° C. overnight (about). 14 hours) Keeped warm. After heat insulation, twice the amount of ethanol was added, and the mixture was kept at ice temperature overnight. The cloudy solution was centrifuged (900 × g, 1 hour), the supernatant was removed, and the precipitate was air-dried (ethanol precipitation). The dried product was redissolved in an appropriate amount (about 30 ml) of water, and then ethanol precipitation was repeated. The obtained dried product was dissolved in an appropriate amount of water and then freeze-dried. The weight of the obtained dried product (purified inulin) was measured and found to be 1.6 g.

Characteristic analysis Thin layer chromatography Purified inulin was dissolved in water at a concentration of 1 mg / 20 μl, spotted on a silica gel 60 plate (manufactured by Merck), and a moving layer (1-butanol: ethanol: water = 5: 5: 3) was applied. Deployed using. Coloring liquid (
Anisaldehyde 1.3 ml, acetic acid 0.5 ml, concentrated sulfuric acid 1.8 ml, ethanol 47.8 ml) was sprayed and color-developed at 110 ° C. for about 15 minutes.

Freeze-dried inulinase treated with exoinulinase, endoinulinase, endrevanase 0.5 mg (1 mg / 20 μl) was mixed with 10 μl macylvain buffer (pH 4.5) to exoinulinase (2000 U / ml). , Endoinulinase (300 U / ml) (both manufactured by Megazyme) were added in an amount of 1 μl each. Similarly, 0.5 mg of FOS was mixed with 10 μl of 0.1 M MES buffer (pH 6.0), and 1 μl of endolevanase (450 U / ml, manufactured by Megazyme) was added. All enzyme reaction solutions were incubated at 40 ° C. for 2 hours and subjected to thin layer chromatography.

As a result, no change was observed in untreated inulin, while inulin digested with endoinulinase was degraded, and degraded polysaccharide was detected at a molecular weight estimated to be between monosaccharide and inulin. When digested with exoinulinase, spots were detected only at the positions of monosaccharides. In endolevanase, the polysaccharide was hardly degraded. This result showed that inulin was not levan but reliable inulin (Fig. 4).

The method for measuring the total glucose transfer activity when sucrose was used as a substrate was to measure the amount of glucose released during the reaction for a unit time. In addition, the amount of fructose released during the reaction was measured and used as the hydrolysis activity. One unit was the amount that released 1 μmol of glucose or fructose per minute. The value obtained by subtracting the hydrolysis activity from the total activity was defined as the transfer activity.

As a result of measuring the activity of each enzyme per 1 mg of enzyme with an enzyme concentration of 10 μg / ml, a substrate concentration of 30%, and a universal buffer solution (50 mM, pH 7.0), the total activity was 87 units, the transfer activity was 78 units, and hydrolysis. The activity was 9 units.

Each magnetic resonance method
Purified inulin samples were dissolved in deuterium water (manufactured by Sigma-aldrich) to 1% (weight / volume). Commercially available chicory inulin (Olaf Tea GR, DKSH Japan) and commercially available levan (manufactured by Megazyme) were used as controls. As a result, the NMR pattern of purified inulin closely resembled that of commercial chicory inulin and was clearly different from that of commercial levan (Fig. 5).

INDUSTRIAL APPLICABILITY The present invention provides a method for producing high-purity and high-quality inulin in a large amount more efficiently than before.

HPAEC High Performance Anion Exchange Chromatography
NMR nuclear magnetic resonance method
Inulosucrase gene of inuBK Bacillus kluluwiche JCM11691 strain
InuBK Inulosucrase transcribed and translated from inuBK

Sequence listing
SEQUENCE LISTING

<110> Nihon Origo Co. Ltd.
Toyama Pref.
Yokoi, kenji

<120> New Inulin synthesis prodedure using the inulosucrase of Bacillus
krulwichiae

<130> Demo No.

<160> 4

<170> PatentIn version 3.5

<210> 1
<211> 35
<212> DNA
<213> Artificial Sequence

<220>
<223> Artificial Sequence

<400> 1
acaggatcca aaatcaaaac tcgtaaaaag gtagg 35


<210> 2
<211> 46
<212> DNA
<213> Artificial Sequence

<220>
<223> Artificial sequence

<400> 2
ttagtggtga tggtgatgat gctttaaaga tctaccgtaa ggaccg 46


<210> 3
<211> 1389
<212> DNA
<213> Bacillus krulwichiae


<220>
<221> misc_feature
<222> (1) .. (1389)
<223> Levansucrase

<300>
<308> J113868
<309> 2020-02-20
<313> (1) .. (1389)

<300>
<308> NZ_CP020814
<309> 2019-12-17
<313> (1) .. (1389)

<400> 3
atgaaaatca aaactcgtaa aaaggtagga aaattagttt tatgtgctgc gattttagcg 60

agcagtctga caagcattag cgttgctgcg agctctaact ggagtattga ggatgattac 120

acggcatcat ggtccagaca gcaagcagag aaagtggctc taacggaaga gacgacagcc 180

ccaatcatcg atttggactt tgaagatgta gctccggatg tgtgggtttg ggatacttgg 240

ccgcttcaaa acagagacgg ttcccttgcg aacgtgaaag gttacagaat cgcattcgca 300

ttggttgcac cacgtactta tacttggcat gaccgtcata ctgaagcaag aatcggcatg 360

ttctactcta aaaacggcaa ggattggacg tatgctggaa ttccatacga ctatgacaac 420

gcgttagggc acatgcaatg ggctggatct gctatgttgg acgagaaagg aaaagtgcat 480

ttcttctata cagcaactag tgatatgaat gccaatggcg gtaaagaatt taatcaagac 540

ggatgggtgc aaagagcgga acaacgccta gctaagacga catttgatat cagtgcagac 600

aaaaatggcg tgcatctgac aaatgaaggg gatcaccaaa ttcttcttga agcagatggc 660

catcattatg aaacgatcga acagttccaa gagcacggaa atatcatcac tggattccgc 720

gatccgtttt tcttcaaaga tccgaataca ggtgaagaat acattatttg ggaaggtcaa 780

gcaggcacta acagaaatga tatcaaaccg gaaaatatcg gggataaaga ataccgcgaa 840

tcacacaacg ttcctgatca tgcgaaattt tataacggaa atatcggaat tgcgaaagta 900

cttgacaacg acgtaactaa acttgaaata ttaccgccgc ttcttgagtc ggttggggtt 960

aaccatcagt tagaacgtcc gcatgttgtg gtaaaagacg acacttacta cctgcttaca 1020

atcagtcatg aatttacgtt tgcaccaggt ttaacaggtc cggatggatt gtacggtttt 1080

gtcggcgagg gaagcttgcg cacagattat aagccagtaa atggcacagg tcttgttgtt 1140

gccaatccgg cggaaaaacc gtttcaagct tattcatggt gggcggctcc agatggccag 1200

gttatcagct tcattaatga acctgtagat gagaatggac aagttaaatt tggcggaaca 1260

tttgcaccga cgctgaaggt atcctttgac ggtgacaaaa caaagatcgt gaaagaaatg 1320

caagctggag aaatcaaacc attcggtcct tacggtagat ctttaaagca tcatcaccat 1380

caccactaa 1389


<210> 4
<211> 456
<212> PRT
<213> Bacillus krulwichiae


<220>
<221> Levansucrase
<222> (1) .. (456)

<400> 4

Met Lys Ile Lys Thr Arg Lys Lys Val Gly Lys Leu Val Leu Cys Ala
1 5 10 15


Ala Ile Leu Ala Ser Ser Leu Thr Ser Ile Ser Val Ala Ala Ser Ser
20 25 30


Asn Trp Ser Ile Glu Asp Asp Tyr Thr Ala Ser Trp Ser Arg Gln Gln
35 40 45


Ala Glu Lys Val Ala Leu Thr Glu Glu Thr Thr Ala Pro Ile Ile Asp
50 55 60


Leu Asp Phe Glu Asp Val Ala Pro Asp Val Trp Val Trp Asp Thr Trp
65 70 75 80


Pro Leu Gln Asn Arg Asp Gly Ser Leu Ala Asn Val Lys Gly Tyr Arg
85 90 95


Ile Ala Phe Ala Leu Val Ala Pro Arg Thr Tyr Thr Trp His Asp Arg
100 105 110


His Thr Glu Ala Arg Ile Gly Met Phe Tyr Ser Lys Asn Gly Lys Asp
115 120 125


Trp Thr Tyr Ala Gly Ile Pro Tyr Asp Tyr Asp Asn Ala Leu Gly His
130 135 140


Met Gln Trp Ala Gly Ser Ala Met Leu Asp Glu Lys Gly Lys Val His
145 150 155 160


Phe Phe Tyr Thr Ala Thr Ser Asp Met Asn Ala Asn Gly Gly Lys Glu
165 170 175


Phe Asn Gln Asp Gly Trp Val Gln Arg Ala Glu Gln Arg Leu Ala Lys
180 185 190


Thr Thr Phe Asp Ile Ser Ala Asp Lys Asn Gly Val His Leu Thr Asn
195 200 205


Glu Gly Asp His Gln Ile Leu Leu Glu Ala Asp Gly His His Tyr Glu
210 215 220


Thr Ile Glu Gln Phe Gln Glu His Gly Asn Ile Ile Thr Gly Phe Arg
225 230 235 240


Asp Pro Phe Phe Phe Lys Asp Pro Asn Thr Gly Glu Glu Tyr Ile Ile
245 250 255


Trp Glu Gly Gln Ala Gly Thr Asn Arg Asn Asp Ile Lys Pro Glu Asn
260 265 270


Ile Gly Asp Lys Glu Tyr Arg Glu Ser His Asn Val Pro Asp His Ala
275 280 285


Lys Phe Tyr Asn Gly Asn Ile Gly Ile Ala Lys Val Leu Asp Asn Asp
290 295 300


Val Thr Lys Leu Glu Ile Leu Pro Pro Leu Leu Glu Ser Val Gly Val
305 310 315 320


Asn His Gln Leu Glu Arg Pro His Val Val Val Lys Asp Asp Thr Tyr
325 330 335


Tyr Leu Leu Thr Ile Ser His Glu Phe Thr Phe Ala Pro Gly Leu Thr
340 345 350


Gly Pro Asp Gly Leu Tyr Gly Phe Val Gly Glu Gly Ser Leu Arg Thr
355 360 365


Asp Tyr Lys Pro Val Asn Gly Thr Gly Leu Val Val Ala Asn Pro Ala
370 375 380


Glu Lys Pro Phe Gln Ala Tyr Ser Trp Trp Ala Ala Pro Asp Gly Gln
385 390 395 400


Val Ile Ser Phe Ile Asn Glu Pro Val Asp Glu Asn Gly Gln Val Lys
405 410 415


Phe Gly Gly Thr Phe Ala Pro Thr Leu Lys Val Ser Phe Asp Gly Asp
420 425 430


Lys Thr Lys Ile Val Lys Glu Met Gln Ala Gly Glu Ile Lys Pro Phe
435 440 445


Gly Pro Tyr Gly Arg Ser Leu Lys
450 455

SEQ ID NO: 1-Primer sequence
5'-ACAGGATCCAAAATCAAAACTCGTAAAAAGGTAGG-3'
SEQ ID NO: 2-Primer sequence
5'-TTAGTGGTGATGGTGATGATGCTTTAAAGATCTACCGTAAGGACCG-3'
Nucleotide sequence of Bacillus kluluwiche JCM11691 gene encoding SEQ ID NO: 3-InuBK:

SEQ ID NO: 4-Amino acid sequence of Bacillus kluluwiche JCM11691 strain InuBK
MKIKTRKKVGKLVLCAAILASSLTSISVAASSNWSIEDDYTASWSRQQAEKVALTEETTAPIIDLDFEDVAPDVWVWDTWPLQNRDGSLANVKGYRIAFALVAPRTYTWHDRHTEARIGMFYSKNGKDWTYAGIPYDYDNALGHMQWAGSAMLDEKGKVHFFYTATSDMNANGGKEFNQDGWVQRAEQRLAKTTFDISADKNGVHLTNEGDHQILLEADGHHYETIEQFQEHGNIITGFRDPFFFKDPNTGEEYIIWEGQAGTNRNDIKPENIGDKEYRESHNVPDHAKFYNGNIGIAKVLDNDVTKLEILPPLLESVGVNHQLERPHVVVKDDTYYLLTISHEFTFAPGLTGPDGLYGFVGEGSLRTDYKPVNGTGLVVANPAEKPFQAYSWWAAPDGQVISFINEPVDENGQVKFGGTFAPTLKVSFDGDKTKIVKEMQAGEIKPFGPYGRSLK

Claims (6)

An isolated, recombinantly expressed inulosucrase from Bacillus krulwichiae containing amino acids 31-456 of SEQ ID NO: 4, those variants created by recombinant techniques. Nucleic acid derived from Bacillus krulwichiae JCM11691, which encodes the entire or part of the amino acid sequence of inulosucrase according to claim 1, shown in SEQ ID NO: 3. The composition containing the inulosucrase according to claim 1. The synthetic nucleic acid encoding the inulosucrase according to claim 1. The vector containing the synthetic nucleic acid according to claim 4. A host cell comprising the synthetic nucleic acid according to claim 4 or the vector according to claim 5.

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