Strain for producing inulin candy transferase and application thereof

Abstract

The invention belongs to the technical field of food biology, and discloses a strain for producing inulin glycosyl transferase and application of the strain. The bacterial strain is classified and named as Paenarthrobacter aurescens QL1.001, and is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M 2024550, and the preservation date is March 25, 2024. By optimizing an enzyme production fermentation process, the strain can generate high-activity inulin fructotransferase, the enzyme activity can reach 150-160 U / mL, the prepared inulin fructotransferase has excellent thermal stability, difructose anhydride III can be obtained by reacting the inulin fructotransferase with high-concentration inulin, the conversion time is shorter, conversion can be completed in 30 min, and the conversion rate can reach 95%.

Description

Technical Field

[0001] The invention belongs to the field of food biotechnology, and particularly relates to a strain producing inulin fructotransferase and application thereof. Background Art

[0002] Difructose anhydride Ⅲ (DFA Ⅲ) is a new type of non-reducing disaccharide with a molecular weight of 324 and a melting point of 163.7 ℃. It exists in small amounts in plants such as chicory and Jerusalem artichoke in nature, and in small amounts in the processing of honey and coffee. DFA Ⅲ is a low-calorie sweetener with a sweetness of 52% of sucrose and a calorie content of only 1 / 15 of sucrose. DFA Ⅲ is heat-resistant and acid-resistant. After being kept at 100 ℃ and pH 2.0 for 30 min, there is almost no decomposition. When DFA Ⅲ is stored at 37 ℃ and pH 3.0 for 3 months, only 2% of DFA Ⅲ decomposes. DFA Ⅲ also has the functions of promoting mineral absorption, promoting bone growth, promoting the growth and reproduction of intestinal flora, improving constipation, and resisting dental caries, which makes it have broad application prospects in the food industry.

[0003] Since DFA Ⅲ is present in low concentrations in nature, the cost of extracting and separating it is high; chemical preparation of DFA Ⅲ also has problems such as more by-products, difficulty in separation and purification, and environmental pollution. Therefore, the product is currently mainly prepared by biological methods, using inulin fructotransferase to catalyze the decomposition of inulin to generate DFA Ⅲ. This method has a high conversion rate, is safe and reliable, and has a wide source of inulin, making it suitable for large-scale industrial production.

[0004] Existing wild fungi produce indica saccharotransferase but the enzyme activity is low, the conversion time is long and other problems. Cho et al. screened a high-yield indica saccharotransferase strain from the soil of Flavobacterium spp. Flavobacterium sp.LC-413, with an enzyme activity of up to 78 U / mL, was screened by Yokota et al. Arthrobacter The inulin saccharotransferase produced by sp.H65-7 has an activity of up to 90 U / mL. Patent document CN200810196709.8 discloses a strain producing inulin saccharotransferase, with an enzyme activity of 2-100 U / mL; Patent document CN 201410327029.0 discloses a strain of Arthrobacter chlorophenol that produces inulin saccharotransferase, with an enzyme activity of 2-100 U / mL, a conversion time of 3-24 hours, and a conversion rate of more than 75%. If the inulin saccharotransferase activity is low and the conversion time is long, it cannot fully meet the needs of industrial applications.

[0005] The present invention obtains a new strain capable of high-yield inulin fructotransferase through screening. By optimizing the enzyme production fermentation process, the strain can produce highly active inulin fructotransferase, with an enzyme activity of up to 150-160 U / mL. The inulin fructotransferase has excellent thermal stability. At the same time, the enzyme reacts with high-concentration inulin to obtain difructose anhydride III, with a shorter conversion time, the conversion can be completed in 30 minutes, and the conversion rate can reach 95%. The present invention is proposed based on the above findings. Summary of the invention

[0006] In order to make up for the deficiencies of the prior art, the present invention provides a strain producing inulin saccharotransferase and application thereof.

[0007] The present invention is achieved through the following technical solutions: In the first aspect, the present invention provides a strain producing inulin sucrose transferase, which is classified and named as aurobacterium aureum ( Paenarthrobacter aurescens )QL1.001, has been deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 2024550, and the deposit date: March 25, 2024.

[0008] Furthermore, the strain produces inulin fructotransferase with an enzyme activity of 150-160 U / mL. The inulin fructotransferase converts inulin to produce difructose anhydride III, and the conversion is completed in 30 minutes with a conversion rate of 95%.

[0009] Furthermore, the inulin fructotransferase target gene sequence determination result is SEQ ID NO. 1, and the encoded amino acid sequence is SEQ ID NO. 2.

[0010] In a second aspect, the present invention provides use of a strain producing inulin fructotransferase in producing inulin fructotransferase.

[0011] The use of the above-mentioned inulin fructotransferase-producing strain in producing inulin fructotransferase comprises the following steps: (1) Seed culture: Arthrobacter aureus Paenarthrobacter aurescens QL1.001 was inoculated into seed culture medium (peptone 3-12 g / L, NaCl 3-8 g / L, beef extract 1-5 g / L, pH 6.5-7.5) and cultured in a shaking flask at 25-30°C and 180-220 rpm for 12-24 h; (2) Fermentation culture: Fermentation culture includes shake flask fermentation culture or fed-batch fermentation culture; Shake flask fermentation: inoculate the seed culture solution into the fermentation medium (inulin 5-35 g / L, NaNO3 1-5 g / L, yeast extract powder 0.1-1 g / L, MgSO4 0.1-0.5 g / L, KH2PO4 0.2-2 g / L, FeSO4·7H2O 0.005-0.02 g / L, pH 6.5-7.5), and culture in a shake flask at 25-30 ℃, 180-220 rpm for 72-120 h; Fed-batch fermentation: inoculate the seed culture solution into a fermentation tank containing fermentation medium at an inoculation amount of 1%-10%, control the initial pH at 6.0-8.0, stir at 300-600 rpm during fermentation, and aerate at 1-3 vvm. When the fermentation time is 6-48 h, add 5-25 g / L of carbon source inulin, and culture at 25-30 °C for 72-120 h. (3) Post-fermentation treatment: The bacteria in the fermentation broth are removed by centrifugation to obtain a crude enzyme solution of inulin fructotransferase, which is then concentrated to 10-20 times using an ultrafiltration membrane.

[0012] Furthermore, in step (1), the seed culture medium is 10 g / L peptone, 5 g / L NaCl, 3 g / L beef extract, and pH 7.0.

[0013] Furthermore, in step (2), the fermentation medium is 25 g / L inulin, 3 g / L NaNO3, 0.3 g / L yeast extract, 0.2 g / L MgSO4, 0.8 g / L KH2PO4, 0.005 g / L FeSO4·7H2O, and pH 7.0.

[0014] In a third aspect, the present invention provides an application of inulin fructotransferase in the production of difructose anhydride III, and the application of inulin fructotransferase in the production of difructose anhydride III comprises the following steps: adding inulin fructotransferase to an inulin solution with a concentration of 10-200 g / L for catalytic conversion, the amount of enzyme to substrate is 0.5-20 U / g, pH 4.0-8.0, the conversion reaction temperature is 30-70 ° C, the conversion reaction time is 0.5-24 h, and an enzyme reaction solution containing difructose anhydride III is obtained. The obtained enzyme reaction solution is concentrated to a solid content of 30% by a rotary evaporator, purified by DTF-01 ion exchange resin, and vacuum dried at 40-60 ° C to obtain difructose anhydride III powder.

[0015] Compared with the prior art, the present invention has the following advantages: The present invention provides a new strain capable of high-yield inulin fructosyltransferase. By optimizing the enzyme production fermentation process, the strain can generate highly active inulin fructosyltransferase, with an enzyme activity of up to 150-160 U / mL. At the same time, the enzyme reacts with high-concentration inulin to obtain difructose anhydride III, with a shorter conversion time, which can be completed in 30 minutes, and a conversion rate of up to 95%. In addition, the inulin fructosyltransferase has excellent thermal stability, and the enzyme can convert inulin into difructose anhydride III more quickly, shortening the conversion time, increasing the production rate, and reducing the production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described below in conjunction with the accompanying drawings.

[0017] Figure 1 Arthrobacter aureus Paenarthrobacter aurescens QL1.001 Colony morphology; Figure 2 HPLC chromatogram, where (A) is the standard sample of difructose anhydride III; (B) is the QL 1.001 enzyme reaction solution; Figure 3 This is a diagram of the fed-batch fermentation test. DETAILED DESCRIPTION

[0018] Arthrobacter aureus Paenarthrobacter aurescens QL1.001 is used to ferment and produce inulin fructotransferase and enzymatically convert it into difructose anhydride III, but the present invention is not limited to the examples listed. In addition, the genetic resource of this application, Arthrobacter aureus ( Paenarthrobacter aurescens )The data collector of QL1.001 is Zhao Meng, the collection location is Jinan City, Shandong Province, and the acquisition time is March 2024.

[0019] Example 1 Screening and acquisition of strains (1) Primary screening: 1 g of soil sample from Jerusalem artichoke garden was placed in 50 mL of enrichment medium (inulin 10 g / L, (NH4)2SO4 1.4 g / L, KH2PO4 2 g / L, CaCl2 0.3 g / L, MgSO4·7H2O 0.3 g / L, FeSO4·7H2O 0.005 g / L, MnSO4·H2O 0.002 g / L, ZnSO4·7H2O 0.002 g / L, CoCl2 0.002 g / L, urea 0.3 g / L, peptone 1 g / L, Tween 80 2 g / L, pH 7.0) and cultured in a shaking flask at 30 ℃ for 24 h. The enriched soil solution was centrifuged at 10,000 g for 20 min, and the supernatant was diluted in gradients and inoculated onto screening medium plates (inulin 10 g / L, NaNO3 2 g / L, yeast extract 0.2 g / L, MgSO4 0.5 g / L, KH2PO4 0.5 g / L, FeSO4·7H2O 0.01 g / L, agar 20 g / L, pH 7.0), and cultured at 30°C for 48-72 h. A single colony with a transparent circle around it was picked from the plate and streaked three times to obtain the QL1.001 strain. The colony morphology is shown in the figure below. Figure 1 shown.

[0020] (2) Rescreening: The above strains were transferred to seed culture medium, activated at 30°C and 200 rpm for 24 h, and then inoculated into a flask containing fermentation medium at a 4% inoculation rate, and cultured at 30°C and 200 rpm for 72 h. The fermentation broth was centrifuged at 8000 g, 30 min, 4°C, and the supernatant was taken. The inulin fructotransferase activity was determined by HPLC to identify the strain as one that produces inulin fructotransferase. The results showed that the fermentation broth produced by strain QL1.001 had the ability to hydrolyze inulin and produce difructose anhydride III. The HPLC determination results were as follows: Figure 2 shown.

[0021] (3) The selected strains were identified by 16S rRNA gene sequence analysis. Paenarthrobacter aurescens , and its 16S rRNA sequence is shown in SEQ ID NO. 3, as follows:

[0022] The 16S rRNA gene sequence of QL1.001 was compared with that of strain QL1.001 by BLAST on NCBI. Paenarthrobacter aurescens The closest relationship is the ANI index of 99.97%.

[0023] Example 2 Method for producing inulin saccharotransferase by strains The following steps are involved: (1) Seed culture: Arthrobacter aureus ( Paenarthrobacter aurescens ) QL1.001 was inoculated into seed culture medium (peptone 3-12 g / L, NaCl 3-8 g / L, beef extract 1-5 g / L, pH 6.5-7.5), and cultured in a shaking flask at 25-30°C and 180-220 rpm for 12-24 h; (2) Fermentation culture: Fermentation culture includes shake flask fermentation culture or fed-batch fermentation culture; Shake flask fermentation: inoculate the seed culture solution into the fermentation medium (inulin 5-35 g / L, NaNO3 1-5 g / L, yeast extract powder 0.1-1 g / L, MgSO4 0.1-0.5 g / L, KH2PO4 0.2-2 g / L, FeSO4·7H2O 0.005-0.02 g / L, pH 6.5-7.5), and culture in a shake flask at 25-30 ℃, 180-220 rpm for 72-120 h; Fed-batch fermentation: inoculate the seed culture solution into a fermentation tank containing fermentation medium at an inoculation amount of 1%-10%, control the initial pH at 6.0-8.0, stir at 300-600 rpm during fermentation, and aerate at 1-3 vvm. When the fermentation time is 6-48 h, add 5-25 g / L of carbon source inulin, and culture at 25-30 °C for 72-120 h. (3) Post-fermentation treatment: The bacteria in the fermentation broth are removed by centrifugation to obtain a crude enzyme solution of inulin fructotransferase, which is then concentrated to 10-20 times using an ultrafiltration membrane.

[0024] In specific implementation, this embodiment adopts: (1) Seed culture: Arthrobacter aureus ( Paenarthrobacter aurescens ) QL1.001 was inoculated into seed culture medium (10 g / L peptone, 5 g / L NaCl, 3 g / L beef extract, pH 7.0) and cultured in a shaking flask at 28 °C and 200 rpm for 24 h; (2) Fermentation culture: Fermentation culture includes shake flask fermentation culture or fed-batch fermentation culture; Shake flask fermentation: The seed culture solution was inoculated into the fermentation medium (inulin 25 g / L, NaNO3 3 g / L, yeast extract 0.3 g / L, MgSO4 0.2 g / L, KH2PO4 0.8 g / L, FeSO4·7H2O 0.005 g / L, pH 7.0) and cultured in a shake flask at 28 °C and 200 rpm for 72 h. Fed-batch culture: see Example 4 for details; (3) Post-fermentation treatment: The bacteria in the fermentation broth are removed by centrifugation to obtain a crude inulin fructotransferase solution, which is then concentrated to 10-20 times using an ultrafiltration membrane.

[0025] Example 3 Identification of difructose anhydride III, a high-purity product of inulin fructotransferase The crude inulin fructotransferase was purified by DEAE-Sepharose Fast Flow ion exchange chromatography and Superdex200 10 / 300 GL gel filtration (for specific steps, refer to Bioresource Technology 102 (2011) 1757–1764doi:10.1016 / j.biortech.2010.08.093) to obtain high-purity inulin fructotransferase. A high-purity difructose anhydride III sample was prepared according to the method described in the invention content, and the freeze-dried sample was dissolved in heavy water (D2O), and 3-trimethylsilyl-1-propanesulfonic acid sodium salt (DSS) was used as a standard, and a nuclear magnetic resonance system was used for analysis. 13 C NMR analysis. The results are shown in Table 1. The number of sample peaks of the main reaction product is 12, corresponding to the chemical shift values ​​of 12 carbon atoms in difructose anhydride III. The values ​​are consistent with those reported in the literature (Journal of Fermentation and Bioengineering, 1991, 72: 258-261). Therefore, it can be determined that the prepared product is difructose anhydride III.

[0026] Table 1 High purity difructose anhydride III 13 C NMR chemical shift values .

[0027] Note: a The value of DFA III is the chemical shift value of difructose anhydride III in the literature.

[0028] Example 4 Fed-batch fermentation A method for synthesizing difructose anhydride III using a strain having high inulin fructotransferase production, comprising the following steps: (1) Activation culture of bacterial strains: Arthrobacter aureus ( Paenarthrobacter aurescensQL1.001) were inoculated into the culture medium and activated for 24 h to obtain activated seeds; (2) Seed culture: The activated seeds were inoculated into a shake flask containing seed culture medium and cultured at 28°C and 200 rpm for 24 h to obtain the seed culture solution; (3) Fed-batch fermentation: The seed culture was inoculated into a fermentation tank containing fermentation medium at an inoculation rate of 4%. The initial pH was controlled at 7.0. During the fermentation process, the stirring speed was 500 rpm and the aeration rate was 2 vvm. When the fermentation time was 24 h, 15 g / L of carbon source inulin was added and the culture was carried out at 28 °C for 120 h. Samples were taken during the fermentation process to detect enzyme activity and OD 600 , online monitoring of pH value and dissolved oxygen DO.

[0029] The results are as follows Figure 3 As shown, enzyme activity and OD 600 As the fermentation time increases, the enzyme activity gradually increases, and at 72 h, the enzyme activity reaches the highest; as the biomass increases, a large amount of oxygen is consumed, and the dissolved oxygen DO will decrease; the pH of the fermentation liquid gradually increases as the fermentation time increases.

[0030] Example 5 Effect of enzyme-substrate ratio on enzymatic conversion to produce difructose anhydride III Prepare different concentrations (50-200 g / L) of inulin solution and add Arthrobacter aureus ( Paenarthrobacter aurescens ) The purified inulin saccharotransferase from fermentation culture of QL1.001 was subjected to catalytic conversion for 24 h, with an enzyme-substrate ratio of 0.05-10 U / g, a conversion reaction temperature of 60 ℃, and a pH of 5.5, and the conversion rate was determined. The results are shown in Table 2. As the enzyme-substrate ratio increases, the conversion rate gradually increases, and when the enzyme-substrate ratio is 5 U / g, the conversion rate has stabilized.

[0031] Table 2 Effect of enzyme-substrate ratio on enzymatic conversion to produce difructose anhydride III .

[0032] Example 6 Effect of conversion time on enzymatic conversion to produce difructose anhydride III Prepare different concentrations (10-200 g / L) of inulin solution and add Arthrobacter aureus ( Paenarthrobacter aurescens) The purified inulin glycosyltransferase from fermentation culture of QL1.001 was catalyzed for 24 h, the dosage of the enzyme to the substrate was 5 U / g, the reaction temperature was 60 ℃, and the pH was 5.5, and the conversion rate was determined. The results are shown in Table 3. When the conversion time was 30 min, the conversion rate was basically stable and the conversion time was short. And with the increase of inulin concentration, the conversion rate gradually decreased. When the inulin concentration was 10 g / L, the conversion rate was as high as 95%, and when the inulin concentration was 200 g / L, the conversion rate was still above 80%.

[0033] Table 3 Effect of conversion time on enzymatic conversion to produce difructose anhydride III .

[0034] Example 7 Thermostability of Inulin Fructose Transferase In the pH 5.5 citrate buffer solution, inulin fructotransferase was reacted at 50, 60, 70, 75, and 80 ℃ with inulin as substrate for 4 h. Samples were taken at different time processes to determine the enzyme activity and calculate the residual enzyme activity. The results are shown in Table 4. The residual enzyme activity of inulin fructotransferase can still be maintained at more than 90% when maintained at 50, 60, and 70 ℃ for 3 h, and the residual enzyme activity is still 58.03% when maintained at 75 ℃ for 4 h. Therefore, this enzyme has excellent thermal stability.

[0035] Table 4 Thermostability of inulin sucrose transferase .

[0036] Example 8 Paenarthrobacter aurescens Nucleotide sequence and gene cloning of the gene encoding inulin fructotransferase (1) Primer synthesis and design: Upstream primer P1: 5'- CGC GGATCCA TGGGAATTGATAAGACGCTTGA -3′; Downstream primer P2: 5'- CCC AAGCTT TCAGGGCGTGGGCCGAATGG -3′; The primers carry restriction sites for BamH I and Hind III and three protective bases. The restriction site of the upstream primer P1 is "GGATCC", and the restriction site of the downstream primer P2 is "AAGCTT". (2) Amplification of target gene: extraction Paenarthrobacter aurescensQL1.001 bacterial genomic DNA; using bacterial genomic DNA as a template, primers P1 and P2 were used to amplify the target gene; PCR reaction conditions: 95 ℃ pre-denaturation for 33 min; 95 ℃ denaturation for 15 s, 60 ℃ annealing for 15 s, 72 ℃ extension for 60 s, 32 cycles; 72 ℃ extension for 5 min; (3) Construction of recombinant plasmid: The PCR recovered product and the expression vector pET-28a(+) were double-digested with two restriction endonucleases, BamH I and Hind III, and the digested products were purified and recovered; the target gene after digestion and the purified product of the expression vector were ligated under the action of T4 ligase to obtain the recombinant plasmid pET-28a(+)-IFT; (4) Construction of recombinant genetic engineering bacteria: The recombinant plasmid pET-28a(+)-IFT was transformed into competent Escherichia coli DH5α, spread on LB solid medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C to obtain preliminary positive clones. The preliminary positive clones were picked and placed in LB liquid medium (containing kanamycin) and shaken at 37°C for 12-16 h. The plasmid was extracted and double-digested with restriction endonucleases BamH Ⅰ and Hind Ⅲ. The plasmid was judged to be a positive recombinant plasmid based on the electrophoresis results. The plasmid was sent to Shanghai Biotechnology Co., Ltd. for sequencing. The target gene sequence was SEQ ID NO. 1, and the amino acid sequence encoded by it was SEQ ID NO. 2.

[0037] SEQ ID NO. 1 1353 DNA Paenarthrobacter aurescens Inulin glycosyltransferase

[0038] SEQ ID NO. 2 450 PRT Paenarthrobacter aurescens Inulin fructotransferase MGIDTTLDMNNTSRRKLVGAGAVGTLAAALSLGMSPKAEAAEGAKASPLNSPNVYDVTAWRIKGQPKVTAESDIGAVINDIIADIKKRQSTPETRPGAVVIIPPGDYDLHTQVVVDVDYLTIAGFGHGFFSRSIKDNVDTTGWLNLQPGGSHIRVLTPPTSPQAFLVRRDGSPRLSGIVFKDFCLDGVSFVPDGNSYKNGKTGIDVASDNDSIHITGMGFVYLEHALIVRGADALRVHDNMVAECGNCVELTGAGQATIVSDNLMGAGPEGVTLLAENHEGLLVTGNNFFPRGRSLLEFTGCNRCSVTSNRFQGFYPGMMRLLNGCKENLITSNHFRRGTEGFPPFIDRTNGLDDLYGVIHAMGDNNLISNNLFAYDVPPGKIAPAGAQPTIMLIAGGDGNVIATNHVISNVDTQHVVLDGSATRSKVLDSGAASTITSYSPDTAIRPTP; Met Gly Ile Asp Thr Thr Leu Asp Met Asn Asn Thr Ser Arg Arg Lys LeuVal Gly Ala GlyAla Val Gly Thr Leu Ala Ala Ala Leu Ser Leu Gly Met Ser ProLys Ala Glu Ala Ala Glu Gly Ala Lys Ala Ser Pro Leu Asn Ser Pro Asn Val TyrAsp Val Thr Ala Trp ArgIle Lys Gly Gln Pro Lys Val Thr Ala Glu Ser Asp IleGly Ala Val Ile Asn Asp Ile Ile Ala Asp Ile Lys Lys Arg Gln Ser Thr Pro GluThr Arg Pro Gly Ala Val Val IleIle Pro Pro Gly Asp Tyr Asp Leu His Thr GlnVal Val Val Asp Val Asp Tyr Leu Thr Ile Ala Gly Phe Gly His Gly Phe Phe SerArg Ser Ile Lys Asp Asn Val Asp Thr ThrGly Trp Leu Asn Leu Gln Pro Gly GlySer His Ile Arg Val Leu Thr Pro Pro Thr Ser Pro Gln Ala Phe Leu Val Arg ArgAsp Gly Ser Pro Arg Leu Ser Gly Ile Val Phe LysAsp Phe Cys Leu Asp Gly ValSer Phe Val Pro Asp Gly Asn Ser Tyr Lys Asn Gly Lys Thr Gly Ile Asp Val AlaSer Asp Asn Asp Ser Ile His Ile Thr Gly Met Gly Phe ValTyr Leu Glu His AlaLeu Ile Val Arg Gly Ala Asp Ala Leu Arg Val His Asp Asn Met Val Ala Glu CysGly Asn Cys Val Glu Leu Thr Gly Ala GlyGln Ala Thr Ile Val SerAsp Asn LeuMet Gly Ala Gly Pro Glu Gly Val Thr Leu Leu Ala Glu Asn His Glu Gly Leu LeuVal Thr Gly Asn Asn Phe Phe Pro Arg Gly Arg Ser Leu Leu Glu Phe Thr GlyCysAsn Arg Cys Ser Val Thr Ser Asn Arg Phe Gln Gly Phe Tyr Pro Gly Met Met ArgLeu Leu Asn Gly Cys Lys Glu Asn Leu Ile Thr Ser Asn His Phe Arg Arg Gly ThrGluGly Phe Pro Pro Phe Ile Asp Arg Thr Asn Gly Leu Asp Asp Leu Tyr Gly ValIle HisAla Met Gly Asp Asn Asn Leu Ile Ser Asn Asn Leu Phe Ala Tyr Asp ValPro Pro Gly Lys Ile Ala Pro Ala Gly Ala Gln Pro Thr Ile Met Leu Ile Ala GlyGly Asp Gly AsnVal Ile Ala Thr Asn His Val Ile Ser Asn Val Asp Thr Gln HisVal Val Leu Asp Gly Ser Ala Thr Arg Ser Lys Val Leu Asp Ser Gly Ala Ala SerThr Ile Thr Ser Tyr SerPro Asp Thr Ala Ile Arg Pro Thr Pro。

Claims

1. A strain producing inulin saccharotransferase, characterized in that: Its taxonomic name is Arthrobacter aureus ( Paenarthrobacter aurescens )QL1.001, has been deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO:M2024550, and the deposit date: March 25, 2024.

2. The strain producing chrysanthemum saccharosyltransferase according to claim 1, characterized in that: The strain produces inulin fructosyltransferase with an enzyme activity of 150-160 U / mL. The inulin fructosyltransferase converts inulin to produce difructose anhydride III. The conversion is completed in 30 minutes with a conversion rate of 95%.

3. The strain producing chrysanthemum saccharosyltransferase according to claim 2, characterized in that: The inulin fructotransferase target gene sequence determination result is SEQ ID NO. 1, and the encoded amino acid sequence is SEQ ID NO.

2.

4. Use of a strain producing inulin saccharotransferase in producing inulin saccharotransferase, characterized in that: The inulin fructotransferase-producing strain is the inulin fructotransferase-producing strain according to claim 1.

5. Use of the strain producing inulin saccharotransferase according to claim 4 in producing inulin saccharotransferase, characterized in that: The use of the strain producing inulin saccharotransferase in producing inulin saccharotransferase comprises the following steps: (1) Seed culture: Arthrobacter aureus Paenarthrobacter aurescens QL1.001 was inoculated into the seed culture medium and cultured in a shaking flask at 25-30 °C and 180-220 rpm for 12-24 h; the seed culture medium was peptone 3-12 g / L, NaCl 3-8 g / L, beef extract 1-5 g / L, pH 6.5-7.5; (2) Fermentation culture: Fermentation culture includes shake flask fermentation culture or fed-batch fermentation culture; Among them, shake flask fermentation culture: the seed culture solution is inoculated into the fermentation medium, and the shake flask is cultured at 25-30 ℃ and 180-220 rpm for 72-120 hours; Fed-batch fermentation: inoculate the seed culture solution into a fermentation tank containing fermentation medium at an inoculation amount of 1%-10%, control the initial pH at 6.0-8.0, stir at 300-600 rpm during fermentation, and aerate at 1-3 vvm. When the fermentation time is 6-48 h, add 5-25 g / L of carbon source inulin, and culture at 25-30 °C for 72-120 h. Post-fermentation treatment: remove the bacteria in the fermentation liquid by centrifugation to obtain the crude enzyme solution of inulin fructotransferase, and concentrate the crude enzyme solution to 10-20 times through an ultrafiltration membrane.

6. Use of the strain producing inulin saccharotransferase according to claim 5 in producing inulin saccharotransferase, characterized in that: The seed culture medium in step (1) is 10 g / L peptone, 5 g / L NaCl, 3 g / L beef extract, and pH 7.

0.

7. Use of the strain producing inulin saccharotransferase according to claim 5 in producing inulin saccharotransferase, characterized in that: In step (2), the fermentation medium comprises 5-35 g / L inulin, 1-5 g / L NaNO3, 0.1-1 g / L yeast extract, 0.1-0.5 g / L MgSO4, 0.2-2 g / L KH2PO4, 0.005-0.02 g / L FeSO4·7H2O, and pH 6.5-7.

5.

8. Use of the strain producing inulin saccharotransferase according to claim 7 in producing inulin saccharotransferase, characterized in that: In step (2), the fermentation medium is 25 g / L inulin, 3 g / L NaNO3, 0.3 g / L yeast extract, 0.2 g / L MgSO4, 0.8 g / L KH2PO4, 0.005 g / L FeSO4·7H2O, and pH 7.

0.

9. An application of inulin fructotransferase in the production of difructose anhydride III, characterized in that: The inulin fructotransferase is the inulin fructotransferase produced in claim 5.

10. The use of inulin fructotransferase in the production of difructose anhydride III according to claim 9, characterized in that: Application of the inulin fructose transferase in the production of difructose anhydride III The method comprises the following steps: adding inulin glycosyltransferase to an inulin solution with a concentration of 10-200 g / L for catalytic conversion, wherein the amount of the enzyme to the substrate is 0.5-20 U / g, the pH value is 4.0-8.0, the conversion reaction temperature is 30-70°C, and the conversion reaction time is 0.5-24 h, to obtain an enzyme reaction solution containing difructose anhydride III, the enzyme reaction solution is concentrated by a rotary evaporator to a solid content of 30%, purified by a DTF-01 ion exchange resin, and vacuum dried at 40-60°C to obtain difructose anhydride III powder.

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