Preparation and Application of an Inulinase and Its Encoding Gene

Abstract

The present invention discloses an endoinulinase gene derived from Flavobacterium sp. and a preparation method and application of the enzyme. That is, by using the technical methods of genome mining and genetic engineering, the gene of the endoinulinase is cloned into an Escherichia coli expression vector to obtain an Escherichia coli recombinant strain capable of heterologously expressing the enzyme. The endoinulinase prepared by heterologous expression of this strain can effectively degrade inulin. The endoinulinase provided by the present invention can be widely applied in the fields of agriculture, feed additives, medicine, and the preparation of fructooligosaccharides, etc.

Description

Technical Field

[0001] The present invention relates to a gene sequence of endoinulinase, a preparation method thereof and an application thereof. The present invention provides a recombinant plasmid and a recombinant genetically engineered strain of the endoinulinase and an application thereof in inulin degradation. The endoinulinase provided by the present invention can be widely applied to fields such as agriculture, feed additives, medicine and the preparation of sucrose fructooligosaccharides. Background Art

[0002] Inulin, also known as inulin, is a rich source of renewable raw materials. Inulin mainly comes from plants, including 11 families such as Compositae, Campanulaceae, Gentianaceae in dicotyledonous plants, and Liliaceae, Gramineae in monocotyledonous plants. Inulin is abundantly present in the roots and tubers of plants such as Jerusalem artichoke, chicory, and dahlia. Among them, Jerusalem artichoke is the most typical source plant of inulin, and the tuber contains more than 70% of inulin. Inulin is a linear polysaccharide formed by the connection of fructose through β-2,1 glycosidic bonds, with a glucose residue connected by an α-2,1 glycosidic bond at its reducing end, a degree of polymerization of 2-60, and a molecular weight between 3500 and 5500. At present, China's inulin industry mostly stays at the primary processing level, and there is an urgent need for high-value processing technologies. Converting inulin into sucrose fructooligosaccharides is an important prerequisite for the deep processing industry of inulin.

[0003] Inulinase is a class of hydrolases that can hydrolyze β-2,1-D-fructooligosaccharide glycosidic bonds. According to the different ways of acting on substrates, it is divided into exoinulinase and endoinulinase. Exoinulinase can catalytically hydrolyze β-D-fructofuranose residues from the non-reducing end of inulin molecules, and its substrates can be inulin, sucrose, and fructan; endoinulinase can be used to produce fructooligosaccharides. Fructooligosaccharides with a degree of polymerization of 2-7 are a kind of functional polysaccharide with various physiological functions, such as improving the microflora in the intestine and increasing the number of beneficial bacteria such as Bifidobacterium in the intestinal flora.

[0004] Inulinase has a very wide range of sources, and it can be produced in many plants, the digestive tracts of animals, and microorganisms. However, the content of inulinase produced by plants and animal digestive tracts is relatively low and cannot meet the requirements of industrial production. In contrast, inulinase from microbial sources has become a research hotspot at home and abroad due to its advantages such as low production cost, short enzyme production cycle, and high enzyme production. According to reports, there are about 10 genera and more than 20 species of yeasts, about 17 genera and more than 40 species of filamentous fungi, and about 12 genera and more than 10 species of bacteria that can produce inulinase. Among them, yeasts can produce more and higher-activity inulinase compared with filamentous fungi and bacteria. However, all inulinases from yeast sources are exo-inulinases, and no endo-inulinase has been found. Aspergillus sp. and Penicillium sp. in filamentous fungi are also good sources for producing inulinase, which can produce both endo-enzymes and exo-enzymes. Among them, Aspergillus sp. is the most studied fungal microorganism. Although the yield and activity of inulinase from bacterial sources are far lower than those of yeasts and filamentous fungi and it is not the preferred microorganism for producing inulinase, some strains can grow in relatively harsh environments, and the inulinase produced has a wider activity range. For example, the growth temperature of Bacillus stearothermophilus strain G. stearothermophilus is between 41 - 69 °C, and the temperature range of the inulinase produced is between 30 - 75 °C, with an optimum temperature of 60 °C. Therefore, inulinase from bacterial sources has certain application value in industrial production. However, most inulinases from bacterial sources are exo-type, and only a few are endo-type. Compared with the series of fructooligosaccharides with different degrees of polymerization produced by the degradation of inulin by endo-inulinase, the degradation products of inulin by exo-inulinase are relatively single, which is inconvenient for the high-value utilization of inulin. Therefore, endo-inulinase from bacterial sources has certain research value. Summary of the Invention

[0005] The first object of the present invention is to provide a novel endo-inulinase FsInuA derived from Flavobacterium sp. and its encoding gene.

[0006] The second object of the present invention is to provide a method for preparing the novel endo-inulinase FsInuA.

[0007] The third object of the present invention is to provide a recombinant expression plasmid containing the endo-inulinase FsInuA gene and a recombinant genetic engineering strain.

[0008] The fourth object of the present invention is to provide an application of the novel endo-inulinase FsInuA in inulin degradation.

[0009] The endoinulinase FsInuA provided by the present invention is derived from Flavobacterium sp. S20, with the deposit address: Institute of Microbiology, Chinese Academy of Sciences, deposit number: CGMCC NO. 5026, deposit date: July 5, 2011. The endoinulinase FsInuA-encoding gene (named FsInuA) amplified therefrom has one or more of the following nucleotide sequence characteristics:

[0010] 1) The deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing;

[0011] 2) The deoxyribonucleic acid (DNA) sequence encoding the amino acid sequence of SEQ ID NO.2 in the sequence listing;

[0012] 3) A deoxyribonucleic acid (DNA) sequence with a homology of 80% or more to the deoxyribonucleic acid (DNA) sequence defined by SEQ ID NO.1 and capable of encoding a protein that degrades inulin;

[0013] 4) A nucleotide sequence obtained by substituting, deleting or adding one or several nucleotides to the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing and encoding an endoinulinase with activity.

[0014] The present invention also provides the amino acid sequence of endoinulinase FsInuA, having one or more of the following characteristics:

[0015] 1) The amino acid residue sequence from positions 1 to 829 starting from the amino terminus in SEQ ID NO.2 in the sequence listing;

[0016] 2) An amino acid sequence formed by substituting, deleting or adding one or two or more amino acids to the amino acid residues from positions 1 to 829 starting from the amino terminus in SEQ ID NO.2 in the sequence listing and having the same endoinulinase activity.

[0017] The amino acid sequence of endoinulinase FsInuA of the present invention and its nucleotide coding sequence can also be artificially synthesized according to the predicted amino acid sequence of endoinulinase FsInuA and its nucleotide coding sequence.

[0018] The method for preparing recombinant enzyme FsInuA is to clone the endoinulinase gene into a recombinant expression vector, introduce it into a host cell, and obtain the recombinantly expressed endoinulinase.

[0019] The above-mentioned endoinulinase gene has one or more of the following nucleotide sequence characteristics:

[0020] 1) A deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing;

[0021] 2) A deoxyribonucleic acid (DNA) sequence encoding the amino acid sequence of SEQ ID NO.2;

[0022] 3) A nucleotide sequence encoding an endoinulinase with endoinulinase activity obtained by substituting, deleting or adding one or more nucleotides to the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing;

[0023] The expression vector for recombinant expression of endoinulinase FsInuA may be an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a Lactobacillus expression vector, a Streptomyces expression vector, a phage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, a mammalian cell expression vector, etc.

[0024] The recombinant bacterium or transgenic cell line for recombinant expression of endoinulinase FsInuA may be an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a Lactobacillus host cell, a filamentous fungus host cell, an insect cell, a mammalian cell.

[0025] The gene sequence of endoinulinase FsInuA of the present invention was cloned from Flavobacterium sp.S20 by PCR technology. The coding region of this gene is 2490bp long and contains multiple domains.

[0026] The endoinulinase provided by the present invention can be applied in the degradation of inulin, including one or two of the following applications:

[0027] 1) Application in cleaving the glycosidic bond of inulin to obtain monosaccharides, fructooligosaccharides or sucrose fructooligosaccharides;

[0028] 2) Application in cleaving the glycosidic bond of fructan to obtain monosaccharides or fructooligosaccharides;

[0029] 3) Application in the co-cleavage of fructan glycosidic bonds after mixing with other inulinases or fructanases.

[0030] The endoinulinase FsInuA obtained by recombinant expression from Escherichia coli of the present invention can efficiently degrade inulin, and has good activity under the conditions of 37°C and pH7.4 with an inulin substrate, and the specific activity is 60U / mg.

[0031] The endoinulinase FsInuA of the present invention can be widely applied in the fields of agriculture, feed additives, medicine and the preparation of sucrose fructooligosaccharides, etc.

[0032] The present invention discloses an endoinulinase gene derived from Flavobacterium sp., a preparation method and application of the enzyme. That is, by using the technical methods of genome mining and genetic engineering, the gene of the endoinulinase is cloned into an Escherichia coli expression vector to obtain an Escherichia coli recombinant strain capable of heterologously expressing the enzyme. The endoinulinase prepared by heterologous expression of this strain can effectively degrade inulin. Brief Description of the Drawings

[0033] Figure 1 : Agarose gel electrophoresis detection of the endoinulinase gene FsInuA. Lane 1: nucleic acid standard; Lane 2 - amplification product of FsInuA.

[0034] Figure 2 : Detection of the expression and purification of the endoinulinase FsInuA. The samples added to each lane are: Lane 1 - soluble supernatant of FsInuA, Lane 2 - the flow-through solution after passing the column three times of FsInuA, Lane 3 - eluting the column with Binding buffer, Lane 4 - flow-through solution eluted with 20 mM imidazole, Lane 5 - flow-through solution eluted with 60 mM imidazole, Lane 6 - protein molecular weight standard.

[0035] 1 - crude enzyme solution, 2 - flow-through solution after three times, 3 - elution with Binding buffer, 4 - elution with 20 mM imidazole, 5 - elution with 60 mM imidazole, 6 - Marker

[0036] Figure 3 : Product analysis of the degradation of inulin by the endoinulinase FsInuA. Detailed Embodiments

[0037] Sequence Listing

[0038] Information of SEQ ID No.1

[0039] (a) Sequence Characteristics

[0040] Length: 2490 bp nucleotides

[0041] Type: nucleotide

[0042] Strand type: single strand

[0043] (b) Molecular type: DNA

[0044] Sequence description: SEQ ID NO.1

[0045] ATGAAATCTCAAAATGACCCAAGTCCAACTCCCAATCCAACTCCCAAT

[0046] CCGTCGGTGGCTACAGCCCTTTTATCGTTTAATTTTAACGAGACATCTG

[0047] GACAATCGCTTTCAGAAACTAAGACGAATGCGAGTTTTAGCATTAACG

[0048] GACCTGCTGGTTCTGCTGAACGAATAAGTGGTGTTGAAGGCAATGCG

[0049] CTCCGGGTGAATGGTTTTTATGGAATGCGGGTTTACGACAATATGATTG

[0050] CCGAAGCGGACAAGCAGGGGCTGCGCCTGATCCTACCTTTTATCGATC

[0051] ACTGGTGGTGGTGGGGCGGCCGCGAGCAGCTGGCGGCGTTTTATCAT

[0052] GAAAAGCCGGCAGACTTTTATCGCACCGACAGTCAAACCTACCGGGC

[0053] CTATCTCGACGTGATCCGTCAGGTGATCACCCGGACCAACAGCAAAG

[0054] TTGGGACAAATGTGATTCAAATTTTATCGGATCAAGAAGTAGCCTTGA

[0055] AAAAGTGGAGTTTTATTGCATTAAACATAAATGCTTCAAACGGTTCTG

[0056] CTTCGTTATATCTGAATGGGGTTCAAATCAAAAGTACCACGTTTGATGC

[0057] CGGGACTCTTTTATGGGATAATAATGCCACAATTTATATTGGAAAAGAA

[0058] TCTAAATCCAAAACAATTGCCGGGTTTGACACTAATGGATTAACAGGA

[0059] GCCATTGATCAGGTAGCTCTTTGGAATAAAGAATTGACAGCTGCCGAA

[0060] ATACTTACGCAGTACACTAAATACGCCCCTGCGGATCCAGATCTAAAA

[0061] ATCCCCAGTGCTCGATTTGCCAATGACATTCATCGTCCAAAATACCACT

[0062] TGCTTCCATCAGCTGGGTGGACCAATGAATCGCACGGTTTGCTCTATAT

[0063] AGACAATAAATACCATATATTTAGTCAAAGAAATTTCAATGGACCTTAT

[0064] TTGGAACATATCAATTGGGGTCATTATGTTAATAGTGATTTAATAAACT

[0065] GGGAGGAAAAAACACAAGTTTTGTGGCCACATCTGCCGGGCTTCCCT

[0066] GTGGAGGGAAAATCCAACCAGGAGATGGAGGTGGTGAACCTGGTGC

[0067] GTGCCGCCTCGGCGCAGATGGCCGGGCAGGAGAGAGTGCCGCCGCT

[0068] GCCGAAGCCGGACGCGCCGAAGCTGCGGGAGACGGACTCGCCGTTT

[0069] GCCATCAACTGGAATGCTCCAACAACATCGGCCAATGCTGATTTTAGA

[0070] GATCCGTTTGTGTTTCAACACAATTCAGAATGGTATATGATGATAGGAA

[0071] CAGGCTTGAGAAGTGGTACATCAAGAGGAGGATTGTATTTGTATAAAT

[0072] CAACTTCATCCGATTTTAAAAATTGGTCGCCACAAGGCACAATGTTGG

[0073] AAGGAAATCCTGCTGTGGATGGAACTGGTGATTTTTGGGAAATGCCAA

[0074] TTTATTATAATTTTGGTGCCAAATCCATTGTACTAATCAATAAGCTTCCT

[0075] AATGCAAACGCTTTGTATTGGACAGGGACTTTTAATGGTTCACAATTC

[0076] GTTAGAGACAATCCCGTTCCGGAAAGATTGGATGTGATAAATCAATTG

[0077] CTGTCTCCTTCCATTCATCCTGATGTCAATGGTAATTTGACAGCCATTG

[0078] GAATTATTCCTGATGGTGTCAGTTCGGCAAAACACAAAGAGCAAGGT

[0079] TGGGCACACACCTTTAGTTTGCCTAGAGTTTGGACACTGGTTAATGGA

[0080] AAAATAAAACAAGTTCCCCATCCCAACGTATTGAATCTTAGAGGTGCT

[0081] TCGAAAAATTTCACCAATGTAGTTTTTGATCAAAACAGTTCGAATGTT

[0082] TTAAATAATTCAACTGGATCTCAAGAGCTGAAAGCCGGCGACAACCG

[0083] CATCGACTTTGGCAAATTCGCCGGCGACTGGGGATATATGTTTATCAAA

[0084] TCGATCGAAGTGATTGCAGAGTACTATGATTTTGCAACAAGTAGTTTT

[0085] GTTGTCGATAGAAGTAAATCTTCTATTCTTACAGGTGTTCCGCTGTCGA

[0086] ATCAATCTACAAATTATGTGCTGCCTGCTGGAAATATTAATTGGAGAAT

[0087] TTTTGTAGATGCCTCAGTAATTGAAGTTTTTGTAAATGAAGAATTGGCT

[0088] TTTGCAACACGCTCTTTCCCATCTACAGGAAATAATTTAATCGATTTAT

[0089] ACCGCCGACGACGGCCTGCTTAAAAGCCGTAAAACCGTCAGGGTAAC

[0090] GGTAGCGAAAGCCGCGGGCAAAGCGCCTGCCGACTACTGCCGTTTTA

[0091] CGGGCGAAATCTTTGACGTCAGCCGGGGCAAGATTGCGCTTCAAGTG

[0092] TCAATTTCAACATTAAATTTGGCCACATCACCGAAGCAACTCCCGTTT

[0093] ACGGCTATATCTTTGATATAACAGGGTTTCCAGTGAAAAAGATTTTAAA

[0094] AATGCTGGATGAAAATAATTCAATCGTTCATTGGGATGGAATAATGGAC

[0095] AATGGAAATAAAGCCGTCAGAGGTGTTTATATCATCAAAGGTTTATTG

[0096] AAAAACGAATTGTTTGATGCTAAAATAATTGTTGAATGA

[0097] Information of SEQ ID No.2

[0098] (a) Sequence characteristics

[0099] Length: 829 amino acids

[0100] Type: Amino acid

[0101] Chain type: single chain

[0102] (b) Molecular type: protein

[0103] Sequence description: SEQ ID NO.2

[0104] MKSQNDPSPTPNPTPNPSVATALLSFNFNETSGQSLSETKTNASFSINGPA

[0105] GSAERISGVEGNALRVNGFYGMRVYDNMIAEADKQGLRLILPFIDHWW

[0106] WWGGREQLAAFYHEKPADFYRTDSQTYRAYLDVIRQVITRTNSKVGTN

[0107] VIQILSDQEVALKKWSFIALNINASNGSASLYLNGVQIKSTTFDAGTLLW

[0108] DNNATIYIGKESKSKTIAGFDTNGLTGAIDQVALWNKELTAAEILTQYTKY

[0109] APADPDLKIPSARFANDIHRPKYHLLPSAGWTNESHGLLYIDNKYHIFSQ

[0110] RNFNGPYLEHINWGHYVNSDLINWEEKTQVLWPHLPGFPVEGKSNQEM

[0111] EVVNLVRAASAQMAGQERVPPLPKPDAPKLRETDSPFAINWNAPTTSAN

[0112] ADFRDPFVFQHNSEWYMMIGTGLRSGTSRGGLYLYKSTSSDFKNWSPQ

[0113] GTMLEGNPAVDGTGDFWEMPIYYNFGAKSIVLINKLPNANALYWTGTF

[0114] NGSQFVRDNPVPERLDVINQLLSPSIHPDVNGNLTAIGIIPDGVSSAKHKE

[0115] QGWAHTFSLPRVWTLVNGKIKQVPHPNELKAGDNRIDFGKFAGDWGY

[0116] MFIKSIEVIAEYEIVATVNPGTATKVGFSLNKNTATGEHTLIYYDFATSSFV

[0117] VDRSKSSILTGVPLSNQSTNYVLPAGNINWRIFVDASVIEVFVNEELAFAT

[0118] RSFPSTGNNLIDLTADDGLLKSRKTVRVTVAKAAGKAPADYCRFTGEIFD

[0119] VSRGKIAPSSVNFNIKFGHITEATPVYGYIFDITGFPVKKILKMLDENNSIV

[0120] HWDGIMDNGNKAVRGVYIIKGLLKNELFDAKIIVE

[0121] Example 1 Cloning of the full-length gene of endo-inulinase and construction of recombinant plasmid

[0122] After performing multiple sequence alignment and analysis on the endo-inulinase gene sequences in the The National Center for Biotechnology Information (NCBI) database by gene mining, the endo-inulinase gene was selected from the genus Flavobacterium. Primers FsInuA-F: 5’-taagaaggagatatacatatgATGAAATCTCAAAATGACCCAAGTC-3’; FsInuA-R:

[0123] 5’-gtggtggtggtggtgctcgagTTCAACAATTATTTTAGCATCAAACAA-3’ were designed. Using the genomic DNA extracted from Flavobacterium as a template, the gene sequence encoding the mature protein of endo-inulinase was amplified. The PCR reaction conditions were: 94°C for 2 min, 1 cycle; 94°C for 30 s, 68°C for 30 s (decreasing 0.5°C for each cycle), 72°C for 2 min 30 s, 30 cycles; 72°C for 5 min, 1 cycle. After agarose gel electrophoresis analysis of the PCR products (see Figure 1) The target gene was recovered by gel extraction using a Gel Extraction Kit (Thermo, AP-GX-250). To facilitate gene recombination and ligation, NdeI and XhoI restriction enzyme sites were introduced into the designed forward and reverse primers, respectively. The PCR-cleaned products FsInuA and the expression vector pET21a (Novagen, 69740-3) were digested with NdeI (Thermo, FD0585) and XhoI (Thermo, FD0694) respectively. After the digestion products were recovered using a PCR Clean-up Kit (Axygen, AP-PCR-250), they were ligated with T4 DNA Ligase (Thermo, EL0011) (ligation system: 5 μL, including 0.5 μL of T4 DNA Ligase, 0.5 μL of 10× T4 DNA Ligase Buffer, 1 μL of pET21a, and 3 μL of PCR product). The ligation condition was overnight ligation at room temperature. 5 μL of the ligation product was used to transform E. coli TOP10 competent cells, which were then spread on solid Luria-Bertani medium containing 100 μg / mL ampicillin and cultured at 37°C for 16 h. Single colonies were picked and verified by colony PCR using identification primers (F: 5'-TAATACGACTCACTATAGGG-3', R: 5'-GCTAGTTATTGCTCAGCGG-3'). The correctly amplified single colonies were inoculated into liquid Luria-Bertani medium containing 100 μg / mL ampicillin and cultured, and the plasmids were extracted and sent to BGI for sequencing. The sequencing results showed that the FsInuA gene shown in SEQ ID NO 1 was inserted between the NdeI and XhoI restriction enzyme sites of pET21a, and the insertion direction was correct, proving that the recombinant plasmid was successfully constructed. This recombinant plasmid was named pET21a-FsInuA.

[0124] Example 2 Sequence Analysis of Endoinulinase Gene

[0125] The sequencing results were analyzed using the Basic Local Alignment Search Tool (BLAST) in the GenBank database, multiple sequence alignment was performed using DNAMAN software, and sequence information was analyzed using Vector NTI.

[0126] The obtained endo-inulinase gene (named FsInuA) has a coding region of 2490 bp, and its nucleotide sequence is shown in SEQ ID NO 1. FsInuA encodes 829 amino acids and a stop codon, and its amino acid sequence is shown in SEQ ID NO 2. The theoretical molecular weight of the protein is 90.11 kDa, and the predicted isoelectric point is 6.69. The amino acids encoded by FsInuA contain multiple domains: a concanavalin a-like lectin / glucanase superfamily domain, and a Glycoside hydrolase (GH) 32 superfamily domain (which contains a CBM (Carbohydrate Binding Module) 6-CBM35_Like superfamily domain in the middle). These domains are often related to the GH catalytic module and work together to complete the degradation of inulin.

[0127] Example 3 Recombinant expression and purification of FsInuA gene in Escherichia coli

[0128] Transform pET21a-FsInuA into E. coli BL21(DE3) competent cells, spread them on solid Luria-Bertani medium containing 100 μg / mL ampicillin, and culture at 37 °C for 16 h. Pick a single colony into 10 mL of liquid Luria-Bertani containing 100 μg / mL ampicillin and culture overnight at 37 °C. Inoculate according to an inoculation amount of 1% into 100 mL of liquid Luria-Bertani containing 100 μg / mL ampicillin. When OD 600nm is about 0.8, add IPTG with a final concentration of 0.1 mM and culture at 16 °C for 20 h. After collecting the bacteria, purify the expressed protein through Ni-NTA. Detect the expression and purification of endo-inulinase FsInuA by polyacrylamide gel electrophoresis. The results are as Figure 2 shown. The position of the purified endo-inulinase FsInuA coincides with the predicted molecular weight.

[0129] Example 4 Activity determination of endo-inulinase FsInuA

[0130] Using 450 μL of 0.5% (w / v, g / ml) inulin as the substrate, add 50 μL of the recombinant enzyme FsInuA and react for 30 min. Use the 3,5-dinitrosalicylic acid (DNS) method to determine its activity. The enzyme activity unit is defined as: the amount of enzyme required to release 1 μmol of reducing sugar (calculated as fructose) per minute is one enzyme activity unit (U). The protein concentration is measured using the BCA Protein Concentration Assay Kit from Beyotime.

[0131] Under the conditions of 30 °C and pH 7.4, the specific activity of FsInuA was measured to be 60 U / mg.

[0132] Product Analysis of Inulin Degraded by Recombinant Enzyme FsInuA in Example 5

[0133] After mixing 2% (w / v, g / ml) inulin with recombinant enzyme FsInuA at a ratio of 50:1 (volume ratio), the reaction was carried out at 30 °C, and different sampling time points were set. After removing the protein in the degradation products by the Sevage method, high-performance liquid analysis was performed. As Figure 3 shown, the degradation of inulin by FsInuA can generate a series of oligosaccharides with different degrees of polymerization, and the final degradation product is fructooligosaccharide with DP = 2 - 5. Therefore, FsInuA can be used for the preparation of inulin oligosaccharides and research related to inulin degradation, including fields such as agriculture, feed addition, medicine, and the preparation of inulin oligosaccharides. Sequence Listing <110> Dalian Institute of Chemical Physics, Chinese Academy of Sciences <120> Preparation and Application of an Inulinase and Its Encoding Gene <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 2490 <212> DNA <213> Artificial Sequence <400> 1 atgaaatctc aaaatgaccc aagtccaact cccaatccaa ctcccaatcc gtcggtggct 60 acagcccttt tatcgtttaa ttttaacgag acatctggac aatcgctttc agaaactaag 120 acgaatgcga gttttagcat taacggacct gctggttctg ctgaacgaat aagtggtgtt 180 gaaggcaatg cgctccgggt gaatggtttt tatggaatgc gggtttacga caatatgatt 240 gccgaagcgg acaagcaggg gctgcgcctg atcctacctt ttatcgatca ctggtggtgg 300 tggggcggcc gcgagcagct ggcggcgttt tatcatgaaa agccggcaga cttttatcgc 360 accgacagtc aaacctaccg ggcctatctc gacgtgatcc gtcaggtgat cacccggacc 420 aacagcaaag ttgggacaaa tgtgattcaa attttatcgg atcaagaagt agccttgaaa 480 aagtggagtt ttattgcatt aaacataaat gcttcaaacg gttctgcttc gttatatctg 540 aatggggttc aaatcaaaag taccacgttt gatgccggga ctcttttatg ggataataat 600 gccacaattt atattggaaa agaatctaaa tccaaaacaa ttgccgggtt tgacactaat 660 ggattaacag gagccattga tcaggtagct ctttggaata aagaattgac agctgccgaa 720 atacttacgc agtacactaa atacgcccct gcggatccag atctaaaaat ccccagtgct 780 cgatttgcca atgacattca tcgtccaaaa taccacttgc ttccatcagc tgggtggacc 840 aatgaatcgc acggtttgct ctatatagac aataaatacc atatatttag tcaaagaaat 900 ttcaatggac cttatttgga acatatcaat tggggtcatt atgttaatag tgatttaata 960 aactgggagg aaaaaacaca agttttgtgg ccacatctgc cgggcttccc tgtggaggga 1020 aaatccaacc aggagatgga ggtggtgaac ctggtgcgtg ccgcctcggc gcagatggcc 1080 gggcaggaga gagtgccgcc gctgccgaag ccggacgcgc cgaagctgcg ggagacggac 1140 tcgccgtttg ccatcaactg gaatgctcca acaacatcgg ccaatgctga ttttagagat 1200 ccgtttgtgt ttcaacacaa ttcagaatgg tatatgatga taggaacagg cttgagaagt 1260 ggtacatcaa gaggaggatt gtatttgtat aaatcaactt catccgattt taaaaattgg 1320 tcgccacaag gcacaatgtt ggaaggaaat cctgctgtgg atggaactgg tgatttttgg 1380 gaaatgccaa tttattataa ttttggtgcc aaatccattg tactaatcaa taagcttcct 1440 aatgcaaacg ctttgtattg gacagggact tttaatggtt cacaattcgt tagagacaat 1500 cccgttccgg aaagattgga tgtgataaat caattgctgt ctccttccat tcatcctgat 1560 gtcaatggta atttgacagc cattggaatt attcctgatg gtgtcagttc ggcaaaacac 1620 aaagagcaag gttgggcaca cacctttagt ttgcctagag tttggacact ggttaatgga 1680 aaaataaaac aagttcccca tcccaacgta ttgaatctta gaggtgcttc gaaaaatttc 1740 accaatgtag tttttgatca aaacagttcg aatgttttaa ataattcaac tggatctcaa 1800 gagctgaaag ccggcgacaa ccgcatcgac tttggcaaat tcgccggcga ctggggatat 1860 atgtttatca aatcgatcga agtgattgca gagtactatg attttgcaac aagtagtttt 1920 gttgtcgata gaagtaaatc ttctattctt acaggtgttc cgctgtcgaa tcaatctaca 1980 aattatgtgc tgcctgctgg aaatattaat tggagaattt ttgtagatgc ctcagtaatt 2040 gaagtttttg taaatgaaga attggctttt gcaacacgct ctttcccatc tacaggaaat 2100 aatttaatcg atttataccg ccgacgacgg cctgcttaaa agccgtaaaa ccgtcagggt 2160 aacggtagcg aaagccgcgg gcaaagcgcc tgccgactac tgccgtttta cgggcgaaat 2220 ctttgacgtc agccggggca agattgcgct tcaagtgtca atttcaacat taaatttggc 2280 cacatcaccg aagcaactcc cgtttacggc tatatctttg atataacagg gtttccagtg 2340 aaaaagattt taaaaatgct ggatgaaaat aattcaatcg ttcattggga tggaataatg 2400 gacaatggaa ataaagccgt cagaggtgtt tatatcatca aaggtttatt gaaaaacgaa 2460 ttgtttgatg ctaaaataat tgttgaatga 2490 <210> 2 <211> 829 <212> PRT <213> Artificial Sequence <400> 2 Met Lys Ser Gln Asn Asp Pro Ser Pro Thr Pro Asn Pro Thr Pro Asn 1 5 10 15 Pro Ser Val Ala Thr Ala Leu Leu Ser Phe Asn Phe Asn Glu Thr Ser 20 25 30 Gly Gln Ser Leu Ser Glu Thr Lys Thr Asn Ala Ser Phe Ser Ile Asn 35 40 45 Gly Pro Ala Gly Ser Ala Glu Arg Ile Ser Gly Val Glu Gly Asn Ala 50 55 60 Leu Arg Val Asn Gly Phe Tyr Gly Met Arg Val Tyr Asp Asn Met Ile 65 70 75 80 Ala Glu Ala Asp Lys Gln Gly Leu Arg Leu Ile Leu Pro Phe Ile Asp 85 90 95 His Trp Trp Trp Trp Gly Gly Arg Glu Gln Leu Ala Ala Phe Tyr His 100 105 110 Glu Lys Pro Ala Asp Phe Tyr Arg Thr Asp Ser Gln Thr Tyr Arg Ala 115 120 125 Tyr Leu Asp Val Ile Arg Gln Val Ile Thr Arg Thr Asn Ser Lys Val 130 135 140 Gly Thr Asn Val Ile Gln Ile Leu Ser Asp Gln Glu Val Ala Leu Lys 145 150 155 160 Lys Trp Ser Phe Ile Ala Leu Asn Ile Asn Ala Ser Asn Gly Ser Ala 165 170 175 Ser Leu Tyr Leu Asn Gly Val Gln Ile Lys Ser Thr Thr Phe Asp Ala 180 185 190 Gly Thr Leu Leu Trp Asp Asn Asn Ala Thr Ile Tyr Ile Gly Lys Glu 195 200 205 Ser Lys Ser Lys Thr Ile Ala Gly Phe Asp Thr Asn Gly Leu Thr Gly 210 215 220 Ala Ile Asp Gln Val Ala Leu Trp Asn Lys Glu Leu Thr Ala Ala Glu 225 230 235 240 Ile Leu Thr Gln Tyr Thr Lys Tyr Ala Pro Ala Asp Pro Asp Leu Lys 245 250 255 Ile Pro Ser Ala Arg Phe Ala Asn Asp Ile His Arg Pro Lys Tyr His 260 265 270 Leu Leu Pro Ser Ala Gly Trp Thr Asn Glu Ser His Gly Leu Leu Tyr 275 280 285 Ile Asp Asn Lys Tyr His Ile Phe Ser Gln Arg Asn Phe Asn Gly Pro 290 295 300 Tyr Leu Glu His Ile Asn Trp Gly His Tyr Val Asn Ser Asp Leu Ile 305 310 315 320 Asn Trp Glu Glu Lys Thr Gln Val Leu Trp Pro His Leu Pro Gly Phe 325 330 335 Pro Val Glu Gly Lys Ser Asn Gln Glu Met Glu Val Val Asn Leu Val 340 345 350 Arg Ala Ala Ser Ala Gln Met Ala Gly Gln Glu Arg Val Pro Pro Leu 355 360 365 Pro Lys Pro Asp Ala Pro Lys Leu Arg Glu Thr Asp Ser Pro Phe Ala 370 375 380 Ile Asn Trp Asn Ala Pro Thr Thr Ser Ala Asn Ala Asp Phe Arg Asp 385 390 395 400 Pro Phe Val Phe Gln His Asn Ser Glu Trp Tyr Met Met Ile Gly Thr 405 410 415 Gly Leu Arg Ser Gly Thr Ser Arg Gly Gly Leu Tyr Leu Tyr Lys Ser 420 425 430 Thr Ser Ser Asp Phe Lys Asn Trp Ser Pro Gln Gly Thr Met Leu Glu 435 440 445 Gly Asn Pro Ala Val Asp Gly Thr Gly Asp Phe Trp Glu Met Pro Ile 450 455 460 Tyr Tyr Asn Phe Gly Ala Lys Ser Ile Val Leu Ile Asn Lys Leu Pro 465 470 475 480 Asn Ala Asn Ala Leu Tyr Trp Thr Gly Thr Phe Asn Gly Ser Gln Phe 485 490 495 Val Arg Asp Asn Pro Val Pro Glu Arg Leu Asp Val Ile Asn Gln Leu 500 505 510 Leu Ser Pro Ser Ile His Pro Asp Val Asn Gly Asn Leu Thr Ala Ile 515 520 525 Gly Ile Ile Pro Asp Gly Val Ser Ser Ala Lys His Lys Glu Gln Gly 530 535 540 Trp Ala His Thr Phe Ser Leu Pro Arg Val Trp Thr Leu Val Asn Gly 545 550 555 560 Lys Ile Lys Gln Val Pro His Pro Asn Glu Leu Lys Ala Gly Asp Asn 565 570 575 Arg Ile Asp Phe Gly Lys Phe Ala Gly Asp Trp Gly Tyr Met Phe Ile 580 585 590 Lys Ser Ile Glu Val Ile Ala Glu Tyr Glu Ile Val Ala Thr Val Asn 595 600 605 Pro Gly Thr Ala Thr Lys Val Gly Phe Ser Leu Asn Lys Asn Thr Ala 610 615 620 Thr Gly Glu His Thr Leu Ile Tyr Tyr Asp Phe Ala Thr Ser Ser Phe 625 630 635 640 Val Val Asp Arg Ser Lys Ser Ser Ile Leu Thr Gly Val Pro Leu Ser 645 650 655 Asn Gln Ser Thr Asn Tyr Val Leu Pro Ala Gly Asn Ile Asn Trp Arg 660 665 670 Ile Phe Val Asp Ala Ser Val Ile Glu Val Phe Val Asn Glu Glu Leu 675 680 685 Ala Phe Ala Thr Arg Ser Phe Pro Ser Thr Gly Asn Asn Leu Ile Asp 690 695 700 Leu Thr Ala Asp Asp Gly Leu Leu Lys Ser Arg Lys Thr Val Arg Val 705 710 715 720 Thr Val Ala Lys Ala Ala Gly Lys Ala Pro Ala Asp Tyr Cys Arg Phe 725 730 735 Thr Gly Glu Ile Phe Asp Val Ser Arg Gly Lys Ile Ala Pro Ser Ser 740 745 750 Val Asn Phe Asn Ile Lys Phe Gly His Ile Thr Glu Ala Thr Pro Val 755 760 765 Tyr Gly Tyr Ile Phe Asp Ile Thr Gly Phe Pro Val Lys Lys Ile Leu 770 775 780 Lys Met Leu Asp Glu Asn Asn Ser Ile Val His Trp Asp Gly Ile Met 785 790 795 800 Asp Asn Gly Asn Lys Ala Val Arg Gly Val Tyr Ile Ile Lys Gly Leu 805 810 815 Leu Lys Asn Glu Leu Phe Asp Ala Lys Ile Ile Val Glu 820 825

Claims

1. An endoinulinase gene, the nucleotide sequence of which is: A deoxyribonucleic acid (DNA) sequence encoding the amino acid sequence of SEQ ID NO.

2.

2. An endoinulinase encoded by the endoinulinase gene according to claim 1, characterized in that: The amino acid sequence of which is: The amino acid residue sequence from the amino terminus of positions 1-829 of SEQ ID NO.2 in the sequence listing.

3. The preparation method of the endoinulinase according to claim 2, characterized in that: The endoinulinase gene is cloned into a recombinant expression vector and introduced into a host cell to obtain recombinantly expressed endoinulinase; The above endoinulinase gene, the nucleotide sequence of which is: A deoxyribonucleic acid (DNA) sequence encoding the amino acid sequence of SEQ ID NO.

2.

4. The method for preparing endoinulinase according to claim 3, wherein: 1) The expression vector for recombinantly expressing endoinulinase refers to one or more of an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacterium expression vector, a Streptomyces expression vector, a phage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, a mammalian cell expression vector; 2) The host cell, that is, the recombinant bacterium or transgenic cell line used for recombinantly expressing endoinulinase, refers to an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a lactic acid bacterium host cell, an actinomycete host cell, a filamentous fungus host cell, an insect cell, a mammalian cell.

5. Use of the endoinulinase according to claim 2 in degrading inulin.

6. Use of the endoinulinase according to claim 2 in degrading inulin, characterized in that: Use in obtaining one or more of fructooligosaccharides with a degree of polymerization of 2-5 by cleaving the glycosidic bond of inulin.

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