CN112708567B - Fructosyltransferase and high-yield strain thereof - Google Patents

Abstract

The invention belongs to the field of microbial fermentation and enzyme engineering, and particularly relates to fructosyltransferase, construction of a high-yield strain of the fructosyltransferase and efficient fermentation preparation of the fructosyltransferase. The invention separates and obtains an aspergillus niger strain with better synthetic fructosyl transferase, and determines the fructosyl transferase and a coding gene thereof; further replacing the fructosyl hydrolase coding gene of the aspergillus niger by the fructosyl transferase coding gene of the aspergillus niger by a traceless gene exchange method to obtain a new strain of the fructosyl transferase aspergillus niger, establishing a fermentation production technology of the fructosyl transferase, and analyzing the application performance of the enzymatic preparation of the fructo-oligosaccharide.

Description

Fructosyltransferase and high-yield strain thereof

The technical field is as follows:

the invention belongs to the field of microbial fermentation and enzyme engineering, and particularly relates to fructosyltransferase, construction of a high-yield strain of the fructosyltransferase and efficient fermentation preparation of the fructosyltransferase.

Background art:

fructooligosaccharides (FOS) are important components of functional oligosaccharides, and have important application values in foods, feeds, daily chemicals, soil improvement, formula milk powder, dairy products, functional products and the like. The method for producing FOS on an industrial scale at present is a main method for producing FOS by taking sucrose as a raw material and catalyzing fructo-oligosaccharide by fructosyltransferase.

Fructosyltransferases belong to the glycoside hydrolase family GH32, and their enzyme molecular members comprise exo-fructosyl hydrolase, endo-fructosyl hydrolase, levanase, sucrase, fructosidase, etc., which are derived from bacteria, fungi, plants, etc. In the method, part of fructosidase from biological sources hydrolyzes sucrose molecules and transfers fructosyl to fructosyl of another sucrose molecule to form oligosaccharide based on sucrose molecules, which is also called fructosyl transferase, and FOS is produced by an enzyme method by taking sucrose as a raw material in industry. Therefore, obtaining the fructosyltransferase with high transglycosidic activity is an important link in the industrial production of FOS.

Most of fructosyltransferase with industrial application value is derived from aspergillus oryzae or aspergillus niger, and the main enzyme source for producing FOS by enterprises in China is obtained by fermenting aspergillus niger. Most of the fructosyl transferase produced by Aspergillus niger is expressed in cells, and contains a high level of sucrose hydrolase, and the fructosyl transferase needs to be released by cell disruption after fermentation is finished and needs to be further purified or immobilized so as to meet the industrial production of FOS (for example, Chinese patent: ZL 01128345.9); the preparation of fructo-oligosaccharide by using thalli or whole fermentation liquor as a catalyst and acting sucrose under the anaerobic fermentation condition (for example, Chinese patent: ZL200910018453.6) is also provided. The enzyme preparations obtained by the method all contain higher proportion of hydrolase activity, can generate hydrolysis action on substrate sucrose and product fructo-oligosaccharide, form glucose and fructose which are not beneficial to the production of fructo-oligosaccharide products, can reduce the conversion rate from sucrose to FOS, and can increase the complexity of the separation and purification of subsequent FOS products and the yield of products.

By analysis and functional characterization of the genome of Aspergillus niger CBS 513.88, Yuan et al (Yuan XL, et al, database mining and transport analysis of genes encoding in-modifying enzymes of Aspergillus niger, 2006,152:3061-73) found that this Aspergillus niger strain contains 5 enzyme molecules involved in the hydrolysis of fructosyl, wherein the enzyme molecules that were experimentally confirmed to be functional were sucrase (sucA), exoinulase (InuE) and endo-inulase (InuA/InuB), and the other two enzyme molecules (SucB and SucC) failed to determine their function, and also failed to resolve the fructosyltransferase involved in Aspergillus niger. The research result further indicates that the enzyme preparation prepared by adopting the wild type Aspergillus niger contains the enzyme activities of sucrase and inulase, and influences the conversion rate of the sucrose as the raw material for producing the fructo-oligosaccharide and the composition of the fructo-oligosaccharide.

The invention adopts techniques such as self-gene cloning, traceless gene replacement and the like, further integrates the aspergillus niger fructosyltransferase coding gene with optimal catalytic property through gene site-specific after defining the fructosyltransferase and the coding gene thereof in aspergillus niger, replaces fructosyl hydrolytic activity enzyme coding gene in the aspergillus niger genome, constructs and obtains the aspergillus niger new strain of high-yield high-transglycosidation activity fructosyltransferase, and establishes and optimizes through a fermentation process to obtain the high-efficiency preparation method of the fructosyltransferase. The above invention has not been reported.

The invention content is as follows:

the invention aims to obtain an Aspergillus niger production strain with higher single fructosyl transferase activity, which is used for the high-efficiency preparation of fructosyl transferase and the enzymatic preparation of fructo-oligosaccharide, thereby obviously improving the fermentation preparation level of the fructosyl transferase and the quality of an enzyme preparation, and obviously improving the conversion rate of the fructosyl transferase in the production of the fructo-oligosaccharide and the product quality of the fructo-oligosaccharide.

In order to realize the purpose, the invention separates and obtains an aspergillus niger strain with better synthetic fructosyl transferase, and determines the fructosyl transferase and a coding gene thereof; further replacing the fructosyl hydrolase coding gene of the aspergillus niger by the fructosyl transferase coding gene of the aspergillus niger by a traceless gene exchange method to obtain a new strain of the fructosyl transferase aspergillus niger, establishing a fermentation production technology of the fructosyl transferase, and analyzing the application performance of the enzymatic preparation of the fructo-oligosaccharide.

One of the technical schemes provided by the invention is An Aspergillus niger An-F308 strain capable of better synthesizing fructosyltransferase, which is obtained by natural separation culture and identification and has the capability of synthesizing the fructosyltransferase, and the strain is preserved in China general microbiological culture Collection center (CGMCC) at 11/26/2020, and the address is as follows: no.3 of Xilu No.1 Beijing, Chaoyang, Beijing, with the preservation number of CGMCC NO. 21028;

the second technical scheme provided by the invention is that the fructosyltransferase is synthesized by Aspergillus niger An-F308, and has An amino acid sequence shown as SEQID NO. 2;

further, the nucleotide sequence of the fructosyltransferase is shown as SEQ ID NO. 1;

the third technical scheme provided by the invention is An Aspergillus niger An-fru3III for high yield of fructosyltransferase, and the strain is obtained by replacing 2 fructosyl hydrolase encoding genes (inuA and inuE) in a genome with the fructosyltransferase shown in SEQ ID NO.1 on the basis of Aspergillus niger An-F308;

further, a traceless replacement technology is adopted for replacement;

further, the nucleotide sequence of the inuA is shown as SEQ ID NO. 3;

further, the nucleotide sequence of inuE is shown in SEQ ID NO. 4.

The fourth technical scheme provided by the invention is the application of Aspergillus niger An-fru3III in the production of fructosyltransferase, in particular to a method for producing fructosyltransferase by fermentation, which comprises the following steps:

the fermentation process comprises the following steps: according to the inoculation amount of 1-10%, the fermentation temperature is 25-36 ℃, the dissolved oxygen is maintained at 5-60% in the fermentation process, the pH is controlled to be 4-6 by using sulfuric acid or ammonia water in the fermentation process, and the fermentation time is 60-150 hours;

the fermentation medium comprises the following components: 3-20% of starch dextrin, 1-8% of bean cake powder, 0-5% of corn steep liquor, 0-3% of ammonium sulfate, 0.01-2% of calcium chloride and the balance of water;

under the condition, the enzyme production level of the fructosyl transferase of the An-fru3III strain reaches 4218-.

The fifth technical scheme provided by the invention is the application of fructosyltransferase shown in SEQ ID NO.2 in producing fructo-oligosaccharide;

when the concentration of sucrose is 400-1200 g/L, the pH value is 4.5-6.5, the reaction temperature is 55-75 ℃, the enzyme amount is 3-30U/g of sucrose, and the reaction time is 8-36 h, the conversion rate of kestose, nystose and nystose in the prepared fructo-oligosaccharide syrup can reach more than 65%.

Has the beneficial effects that:

1. according to the Aspergillus niger fructosyltransferase producing strain constructed by the invention, the enzyme production level under the shake flask condition is improved by 1.4 times compared with that of a producing strain, and only trace (3-10U/mL) fructosyl hydrolase activity is generated;

2. the fructosyltransferase provided by the invention has higher fructosyltransferase activity, only contains trace fructosyl hydrolase activity, and can greatly improve the conversion rate of fructooligosaccharide produced by taking sucrose as a raw material and the composition of fructooligosaccharide;

3. by applying the fructosyltransferase obtained by the invention, the conversion rate of kestose, nystose and nystose in the prepared fructo-oligosaccharide syrup can reach 65 percent, the later-stage decolorization, purification, refining and concentration processes of fructo-oligosaccharide can be greatly simplified, and the production cost of FOS is greatly saved.

Description of the drawings:

FIG. 1A sugar profile of the fructosyltransferase Fru3 catalyzing sucrose to form fructooligosaccharides;

FIG. 2 is a physical map of the recombinant plasmid pAUR-kan;

FIG. 3 physical map of recombinant plasmid pA: cfru 3;

FIG. 4 shows a physical map of the recombinant plasmid pE: cfru 3;

FIG. 5 enzyme production process for the fermentative production of fructosyltransferase by Aspergillus niger An-fru3 III.

The specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.

Unless otherwise specified, the main experimental methods involved in the present invention are as follows:

(1) screening and identification of fructosyltransferase producing strains

The method is carried out according to the conventional method in the laboratory (Zhuge Jianwang Zhengxiang, technical manual of industrial microorganism experiments, 1994). The natural sample is a soil sample collected from sugarcane plantation and orchard in Guangxi province. Properly diluting a soil sample with sterile normal saline, coating an YPD (1% yeast extract, 2% glucose and 2% peptone) plate (supplemented with 200mg/L penicillin), culturing at 28 ℃ for 2-3 days, culturing grown bacteria in an YPS culture medium (1% yeast extract, 2% sucrose and 2% peptone) for 1-2 days, taking fermentation supernatant, determining the composition and content of fructooligosaccharide in the fermentation supernatant by using an HPLC (the HPLC detection method is shown below), preserving the obtained strains with high fructooligosaccharide generation, and identifying by using a molecular identification method (sources, rapid classification and identification of industrial microbial resources based on molecular identification, university of doctor, Jiangnan, 2012).

(2) Separation and purification of Aspergillus niger total RNA

Aspergillus niger An-F308 is inoculated into YPD medium (2% yeast extract, 2% glucose, 2% peptone), cultured at 32 deg.C for 16h, collected thallus is frozen at-70 deg.C, ground in dry ice, and further processed by Thermofisher

Total RNA was recovered using a PlusRNA purification kit.

(3) Gene cloning of fructosyl transferase and related enzymes

The purified Aspergillus niger total RNA is subjected to reverse transcriptase kit

cDNA was synthesized using the Reverse Transcriptase AssayKit as a template and Fru1F/Fru 1R; fru2F/Fru 2R; fru3F/Fru 3R; primers Fru4F/Fru4R and Fru5F/Fru5R (Table 1), cDNA for fructosyl enzymeSpecific amplification is carried out to obtain corresponding products.

TABLE 1 nucleotide sequences of primers used in the present invention

(4) Fructosyltransferase and expression and function identification of related enzyme thereof

Cloning the obtained fructosyltransferase and coding genes of related enzymes thereof into pPIC9k to obtain corresponding recombinant plasmids according to the Pichia operation manual, linearizing the recombinant plasmids, then obtaining recombinant bacteria in Pichia pastoris GS115 in an electric shock transformation mode, respectively inoculating 50mL of BMGY at 30 ℃ and culturing at 180r/min according to the inoculation amount of 1 percent until OD is OD₆₀₀Centrifugation was carried out at 2-6 (about 16-18h), and the OD of the cells was resuspended in a volume of 1/5-1/10 primary medium of BMMY₆₀₀The enzyme was cultured in 0.5% methanol to induce the expression of the enzyme, and the enzyme activity of the prepared enzyme solution was measured and analyzed.

(5) Fructosyltransferase enzyme activity assay

The enzyme activity is defined as: the enzyme amount required to produce 1. mu. mol of kestose per minute under the above reaction conditions is defined as one unit of enzyme activity, based on the fact that the kestose produced in the system after the enzymatic reaction does not exceed 10% of the total sugar content.

The method is carried out according to the fructosyltransferase activity determination method described in the national standard GB/T23528-2009 fructo-oligosaccharide. The basic method comprises the following steps: adding enzyme solutions with different dilutions into 100g/L sucrose solution (dissolved in 0.2mol/L acetic acid buffer solution, pH 5.5), oscillating at 45 deg.C for 60min, and standing in boiling water bath for 10min to terminate the reaction; the supernatant was centrifuged and analyzed by HPLC for sugar profile analysis.

(6) Fructosyl hydrolase activity assay

One fructosyl hydrolase unit is defined as the amount of enzyme required to hydrolyze to 1. mu. mol glucose per minute under the conditions of the assay (pH 4.5, temperature 50 ℃) as one enzyme activity unit (U). The substrate is replaced by fructo-oligosaccharide and fructan (inulin), and the fructosyl hydrolysis activity of the enzyme to be detected is detected under the same conditions (namely the substrate is sucrose, and/or fructo-oligosaccharide, and/or fructan (inulin)).

Preheating 800 μ L of 6.75% (w/v) sucrose or fructo-oligosaccharide or fructan (inulin) solution at 50 deg.C for 5min, adding 200 μ L enzyme solution, reacting at 50 deg.C for 30min, heating at 85 deg.C for 10min to inactivate enzyme, and cooling to room temperature. Glucose content was determined using a biosensor.

(7) Construction of fructosyltransferase high-producing strain

Molecular cloning, DNA connection, transformation, nucleotide sequence determination, restriction mapping, Aspergillus niger genome preparation, PCR gene amplification and the like are carried out according to a conventional laboratory method. Genetic transformation of Aspergillus niger was carried out by electrotransformation according to the method established in the prophase of the subject group (Li Song, Wang Zheng Xiang. investigation of electrotransformation conditions of Aspergillus niger. J. Microbiol., 2010,30(1): 11-15).

The construction of the recombinant plasmid pAUR-kan was carried out by the following procedure. Plasmid pRS305K (Taxis C, KnopM. System of centromeric, episomal, and integral vectors based on drug resistance markers for Saccharomyces cerevisiae, 2006:40,73-78) was digested with EcoRI and PstI and filled-up with PfuDNA polymerase, and a sequence fragment of the Kan (G418) resistance-encoding gene of 1.25kb was gel-recovered, followed by ligation with plasmid pAUR135 (TaKaRa Co., Japan) which had been digested with PstI to remove the aurosomolin (Aba) resistance-encoding gene and filled-up, to obtain recombinant plasmid pAUR-Kan.

The construction of the gene-traceless replacement recombinant plasmid was carried out as follows. Aspergillus niger An-F308 genome DNA is taken as a template, PinuA-1/PinuA-2 is taken as a primer to amplify a promoter region (PinuA) of inuA by PCR, TinuA-1/TinuA-2 is taken as a primer to amplify a terminator and a downstream sequence (TiunA) thereof, then the obtained PinuA and TiunA fragments are fused with cfru3 (shown in SEQ ID NO. 1) obtained by amplification by taking BoxA-1/BoxA-2 as a primer by PCR amplification technology, the PinuA-cfru3-TiunA fragment is obtained by adopting the primer of PinuA-1/TinuA-2, and the fragment is cloned into a SmaI site of pAUR-kan to obtain a recombinant plasmid pApA:: cfru 3. The replacement recombinant plasmid pE of the inuE gene is obtained in the same way, and the plasmid is cfru 3.

Recombinant expression plasmids transformation of Aspergillus niger and selection of positive transformants were performed according to the instructions of pAUR135, TaKaRa. The constructed recombinant plasmids pA, cfru3 and pE, cfru3 are sequentially transformed into An Aspergillus niger An-F308 strain and screened, screening is firstly carried out on a plate containing 0.5g/L G418, and then on a plate containing 1% galactose, so as to obtain the recombinant strain of which cfru3 sequentially replaces inuA and inuE in the Aspergillus niger genome. The enzyme production level of the new strain was evaluated by fermentation in a shake flask and a 15L fermenter.

(8) Fructosyltransferase fermentation production assay

And (3) shaking flask fermentation: the fermentation is carried out in a 250mL triangular flask, the liquid loading amount is 25-50 mL, the culture medium is PDA (200g of potato, 20g of glucose and 1000mL of water), the initial pH value is 4.5-5.5, the fermentation temperature is 28-32 ℃, and the rotation speed is 180-250 r/min.

Fermentation in a fermentation tank: the fermentation medium comprises the following components: 3-20% of starch dextrin, 1-8% of bean cake powder, 0-5% of corn steep liquor, 0-3% of ammonium sulfate, 0.01-2% of calcium chloride and the balance of water; 1-10% of inoculation amount, fermentation temperature of 25-36 ℃, ventilation of 0.1-1.2 vvm, maintenance of dissolved oxygen of 5-60% in the fermentation process, control of pH to 4-6 by using sulfuric acid or ammonia water in the fermentation process, and fermentation time of 60-150 h. Sampling at regular time for fermentation, detecting and analyzing the enzyme activity level, and centrifuging/filtering to remove thalli after fermentation is finished to obtain the fructosyltransferase enzyme solution.

(9) Preparation and analysis of fructooligosaccharides

In a 30L reaction tank. The concentration of sucrose is 400-1200 g/L, the pH is 4.5-6.5, the reaction temperature is 55-75 ℃, the enzyme amount is 3-30U/g of sucrose, and the reaction time is 8-36 h; the sugar profile analysis of the reaction was carried out by HPLC using glucose, fructose, sucrose (product of Sigma Co.), kestose, nystose and nystose (Wako Co.) as standard references. The HPLC chromatographic conditions were: agilent 1200 high performance liquid chromatograph (Agilent Technologies, Palo Alto, Calif., USA) with a column for analysis of Prevail^TMCarbohydrate ES 5u sugar column (5.0 μm, 250X 4.6mm), column temperature 30 ℃; the detector is Alltech ELSD 2000s (G)race Davison Discovery Sciences, Deerfield, IL, USA), drift tube temperature 90 deg.C, gas flow 2.2L/min; the mobile phase is as follows: acetonitrile water 65:35(v/v) flow rate 1 mL/min. According to the requirements of national standard GB/T23528-2009, the percentage contents (%) of core indexes of kestose (GF2), nystose (GF3) and nystose (GF4) in the quality of fructo-oligosaccharide in the total sugar quality are calculated.

The present invention is further illustrated by the following examples.

Example 1: isolation and identification of fructosyltransferase-producing Aspergillus niger strains

A strain of filamentous fungus which has the capability of forming fructo-oligosaccharide by acting on sucrose is separated from soil samples collected from sugarcane plantations, orchards and the like in Guangxi province, is identified as Aspergillus niger, and is named as Aspergillus niger An-F308.

Further cloning genes cfru1, cfru2, cfru3, cfru4 and cfru5 related to fructosyl enzyme activity from cDNA of the Aspergillus niger An-F308 by adopting cDNA cloning technology and primers (Table 1); cloning the gene into pPIC9k, and genetically transforming into P.pastoris GS115 to obtain corresponding recombinant yeasts PP-fru1, PP-fru2, PP-fru3, PP-fru4 and PP-fru 5; further, by a shake flask fermentation method, enzyme solution preparation was carried out using the above 5 recombinant yeasts, and enzyme activity was measured using sucrose or levan (inulin) as a substrate, from which it was identified that the encoded product Fru3 (amino acid sequence SEQ ID NO:2) of the cfru3 gene (nucleotide sequence SEQ ID NO:1) exhibited mainly fructosyl transglycosylation activity, with almost NO sucrose and levan hydrolytic activity (approximately 0.1% of transglycosylation activity). The characteristic of the spectrum of sugars from Aspergillus niger fructosyltransferase Fru3 in the catalysis of sucrose to fructo-oligosaccharides is analyzed by HPLC, and the typical spectrum of sugars is shown in FIG. 1.

Example 2: construction of fructosyltransferase high-producing strain

Plasmid pRS305K was digested with EcoRI and PstI and filled in with Pfu DNA polymerase, and a 1.25kb sequence fragment of the Kan (G418) resistance-encoding gene was gel-recovered, followed by ligation with plasmid pAUR135 digested with PstI to remove the aureobasidin (Aba) resistance-encoding gene and filled in, to obtain recombinant plasmid pAUR-Kan (FIG. 2). Further, a promoter region (Pinus A) (adopting primers Pinus A-1 and Pinus A-2) and a terminator and a downstream sequence (Tiuna) (adopting primers TinuA-1 and TinuA-2) of inuA are amplified from the Aspergillus niger cDNA by adopting the primers listed in the table 1 and applying a PCR technology; adopting primers BoxA-1 and BoxA-2 to amplify cfru3 with a linker by PCR; the obtained PinuA, cfru3 and TiunA fragments are fused by a PCR amplification technology (primers PinuA-1 and TinuA-2 are adopted) to obtain an in-vitro recombinant mutation/expression cassette PinuA-cfru3-TiunA (nucleotide sequence SEQ ID NO:5), and the fragment is cloned into a SmaI site of pAUR-kan to obtain a recombinant plasmid pApA:: cfru3 (figure 3).

Similarly, a promoter region (Pinus-1 and Pinus-2) of inuE, a terminator and a downstream sequence (Tiune) (adopting primers TinuE-1 and TinuE-2) thereof are further amplified from the Aspergillus niger cDNA by a PCR technology; adopting a primer; boxE-1 and boxE-2, and cfru3 with a linker is amplified by PCR; the obtained Pinus E, cfru3 and Tiune fragments are fused by PCR amplification technology (primers Pinus E-1 and TinuE-2 are adopted) to obtain an in vitro recombinant mutation/expression cassette Pinus E-cfru3-Tiune (nucleotide sequence SEQ ID NO:6), and the fragment is cloned into the SmaI site of pAUR-kan to obtain a recombinant plasmid pE:: cfru3 (figure 4).

Electrically transforming the recombinant plasmid pA (cfru 3) into An Aspergillus niger An-F308 strain to obtain a recombinant, re-screening in a galactose-containing culture medium to remove a plasmid skeleton, and performing enzyme activity determination on the obtained recombinant by fructosyltransferase through a shake flask to obtain a recombinant bacterium Aspergillus niger An-fru3II, wherein the characteristic gene types of the recombinant bacterium are fru3, inuA1 and fru 3. The enzyme activity of the fructosyl transferase produced under the shake flask condition reaches 186U/mL, which is improved by 85 percent compared with the original strain. Further, the recombinant plasmid pE:: cfru3 is electrically transformed into Aspergillus niger An-fru3II, and by the same screening and shaking flask enzyme activity measurement, a new strain An-fru3III is obtained, which is characterized by the genotype of fru3, inuA:: fru3, inuE:: fru3, the synthesis level of the strain under the shaking flask condition reaches 243U/mL, which is improved by 1.4 times compared with the original strain, and only trace fructosyl hydrolase activity is produced (Table 2).

TABLE 2 Aspergillus niger An-fru3III fructosyltransferase Shake flask enzyme Activity

Example 3: fermentation production of fructosyltransferase by Aspergillus niger An-fru3III

The fermentation preparation was carried out in a 15L fully automatic fermenter. The inoculation amount is 5%, and the fermentation medium comprises the following components: 1% of corn steep liquor, 5% of starch dextrin, 3% of bean cake powder, 1% of ammonium sulfate, 0.5% of calcium chloride, the initial pH value is 5.0, the fermentation temperature is 28 ℃, 1vvm is ventilated, 5-60% of dissolved oxygen is maintained, the enzyme activity level of the fructosyltransferase is detected and analyzed by timing sampling, and after the fermentation is finished, the bacteria are removed by centrifugation/filtration, namely the fructosyltransferase liquid.

Aspergillus niger An-fru3III is fermented for 110h to reach the highest enzyme production level (figure 5), the enzyme activity of fructosyltransferase reaches 4588U/mL, and the enzyme activity of fructosyl hydrolase is 8.2U/mL.

Example 4: fructosyltransferase for producing fructo-oligosaccharide

The reaction was carried out in a 30L reaction tank, and the fructosyltransferase prepared in example 3 was added to sucrose at a concentration of 6U/g using a sucrose solution of 1050g/L, and the mixture was incubated at pH 6 and 65 ℃ for 24 hours. After the reaction, the sugar composition was analyzed by HPLC, and the ratio of fructo-oligosaccharide reached 65.36% at the highest (Table 3).

TABLE 3 content of fructooligosaccharide in fructooligosaccharide saccharide solution synthesized by enzymatic method

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.

SEQUENCE LISTING

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Gly Pro Thr Ala Phe Leu Tyr Ala Val Gln Trp His Glu Pro Phe Asp

195 200 205

Trp Gln Tyr Leu Gly Gln Leu Val Asp Phe Pro Leu Arg Phe Gln Pro

210 215 220

Ser Lys Lys Trp Cys Gly Asn Tyr Gly Ile Asn Trp Glu Cys Ala Asn

225 230 235 240

Phe Met Asn Leu Glu Ser Glu Ser Ala Ser Ala Thr Arg Thr Cys Leu

245 250 255

Ile Leu Gly Ala Glu Gly Asp Val Glu Arg Ala His Ile Glu Asn His

260 265 270

Gln Arg Pro Val Thr Val Pro Ala Arg Thr Val Arg Ser Leu Leu Trp

275 280 285

Met Phe Gly Asp Leu Ala Gly Ser Asn His Gln Thr Asp Asn Leu Lys

290 295 300

Cys Arg Phe Lys His Gly Gly Tyr Leu Asp His Gly Ser Leu Tyr Ala

305 310 315 320

Ala Ser Thr Phe Asn Asp Pro Ile Ser Gly Arg Arg Ile Leu Tyr Gly

325 330 335

Trp Ile Pro Glu Glu Asp Ile Thr Gly Gly His Ala Arg Glu Lys Gly

340 345 350

Trp Asn Gly Ser Leu Cys Leu Pro Arg Gln Leu Phe Leu Leu Ser Leu

355 360 365

Arg Asn Val Thr Arg Ala Val Arg Ser Lys Leu Gly Glu Ile Pro Ser

370 375 380

Ile Glu Met Val Ile Glu Ala Asp Gly Ser Thr Thr Leu Tyr Ser Leu

385 390 395 400

Gly Ile Arg Pro Val Ser Glu Ile Thr Gln Leu Arg Thr Lys Cys Ile

405 410 415

Gln Ala His Glu Ala Arg Pro Phe Ser Leu Pro Gln Ser Thr His Asn

420 425 430

Glu Ser Arg Ile Cys Arg Thr Ser Thr Ser Cys Trp Glu Leu Glu Ala

435 440 445

Thr Val Ser Leu Gly Val Ser Cys Glu Thr Val Gly Leu Arg Ile Arg

450 455 460

Cys Ala Gly Glu Glu Pro Val Gln Trp Val Ile Val Phe Ser Leu Val

465 470 475 480

Asp Glu Thr Ile Thr Ile Asp Arg Ser Arg Ser Ser Thr Arg Ser Asp

485 490 495

Val Asn Thr Cys Pro Glu Lys Gly Pro Phe Thr Leu Phe Tyr Val Thr

500 505 510

Asp Gly Thr Arg Thr Glu Lys Leu Glu Arg Phe His Leu Arg Val Ile

515 520 525

Val Asp Ala Asp Ile Val Glu Ile Tyr Ala Asn Asp Arg Phe Ala Leu

530 535 540

Ala Thr Met Met Tyr Pro Gly Ser His Lys Ala Glu Arg Glu Ile Val

545 550 555 560

Ala Phe Ala Thr Gly Asp Asn Glu Ser Ala Arg Phe Glu Gln Val Lys

565 570 575

Ile Trp Asp Gly Leu Asn Gly Met Glu Ala Leu Val Arg Asp Glu Gly

580 585 590

Thr Ala Lys Pro Asp Asn Gln Ser Leu

595 600

<210> 3

<211> 2640

<212> DNA

<213> Aspergillus niger An-F308

<400> 3

agctgcttga caactcccga tcccgtgaat atagcaatcc actctcggta cccggaatct 60

caccttgcat ctccaccatt ttaccaatcg cgttcgctcg ttcgactcct cacattcctt 120

gagtgagaaa ttagtgatct ctccgatgat tagtgatctc tccggatgaa tcggtcttgt 180

ttgtccacca ctaccgttcg atagaagtct cggcctcacg ctctccctga cacggatcgc 240

gttgtccttc cctttataaa aacctgcagg ggcgagcaaa catcacttcg gtataggcat 300

acctctatta actgtgaagg tcttatatga cggacaagga tgcgggagaa atttcgggga 360

aagtcaacgc tgggcatgcg actatcagtt tggggaaatg aaaggcgtct tggccatgat 420

atttatatag ctcgctttcc cgtgccagta gacggcttta cttttctgcc actcacaggt 480

ctctcagaga agtggacttc aagaatacca aattgttcgt tttgctttgt tactccaaga 540

tgttgaatcc gaaggttgcc tacatggtct ggatgacgtg cctgggttta acgttgccca 600

gccaggcgca gtctaatgat taccgtcctt cataccactt cacaccggac cagtactgga 660

tgaacgagcc aaacggcctg attaagatcg gatccacctg gcacctgttc tttcaacaca 720

atccgacggc caatgtatgg ggcaacatat gctgggggca cgctacgagc accgatctga 780

tgcactgggc acacaaaccc actgccattg cggatgagaa cggagtcgaa gcgtttaccg 840

gtacagccta ttatgatcca aacaatgcct ctggccttgg ggattcggca aacccaccct 900

acctggcctg gttcacaggt tataccgttt caagccaaac acaggaccag cgcctggctt 960

tcagtgtcga taacggggcg acgtggacca aatttcaagg caaccccatc atatcaacaa 1020

gccaggaagc accacatgat ataacgggcg gcctcgagag tcgggatcca aaggtattct 1080

tccatcgcca atcggggaac tggatcatgg ttctcgccca tggcgggcag gacaagctgt 1140

ctttctggac gtctgcagac accataaact ggacatggca gagtgacctg aagtccacct 1200

cgatcaacgg cctatcgtcc gatattacag ggtgggaagt ccccgacatg tttgaactcc 1260

cggttgaagg cactgaggag accacgtggg tggtgatgat gacgccggct gaaggatccc 1320

ctgccggtgg taacggggtc ttagctatca ccggttcttt tgacgggaaa agttttacgg 1380

cagatcccgt cgatgcttcg accatgtggc tggacaatgg gcgtgatttc gatggcgctc 1440

tgagctgggt gaacgtgcct gcgtccgatg gacggcggat tatcgccgcc gtcatgaata 1500

gctacggttc caacccgcct acagccacct ggaaagggat gctctccttt ccccggacac 1560

tgtcgctcaa gaaagttggg acgcagcagc actttgttca acagccgatc acagagttgg 1620

acataattag taccagtctg caaacactag aaaaccagac cattacccct ggccaaacat 1680

tgctatcatc gattcgggga actgctctcg atgttcgagt tgctttctac cctgatgctg 1740

gctcggttct gtccctcgcc gtccgaaagg gtgcttcgga gcaaacagtc attaagtaca 1800

cccagtcaga tgccacattg tcggttgatc gaacagagag tggagatacc tcgtatgacc 1860

cggccgcagg tggcgtccat accgccaagt tggaagagga cgacaccgga ctggtttcca 1920

tccgggtgtt ggtggatacg tgttctgtag aggtttttgg cggacaagga gaagccgtca 1980

tttccgacct catcttcccg agtgacagtt ctgatggcct ggccttggag gtaactggcg 2040

gaaatgcagt gctgcagtcg gtggacgtgc ggagtgtttc acttgaatga aggaggcgtg 2100

ggaggctgcc agacggggga aggggattaa gacagtgatt gaggggccta ggtaggaggg 2160

tgaggactgg atgttgaagt tgtactgtct gggctagaag tatactaata aaaatccatc 2220

tttcaagtgg aacgaataag ttggagatgc agagcaggca gggacccaat ccttgctcca 2280

acatggcatt cttttcgtac taagtagcta cctttccctg ctcaaaacct ttatgttgat 2340

aacccttcat aaatgctaac aggccacaca ttatatctcc cgcctaatcc ggacgcacgg 2400

gaatgatgag ggcgtagcac cggtctagca gtgaggctct ggatcatgaa gtgtctgcac 2460

tgtaggtaca cagtctccta ctaggatctt gttcaccggt taattgatgg aatgacagct 2520

tacagtgctt gacaagccta ggctggtggt ggctccgagt cacgctttcc agatcataat 2580

tccagggtaa tgcgagccaa gtgccattca tctcctggat gatcatgtaa cagacacgcc 2640

<210> 4

<211> 2737

<212> DNA

<213> Aspergillus niger An-F308

<400> 4

gagcagctat ccgctcaatc ccgaatgagg tccgtgttcg gtcgatgtca acaacacccc 60

acgcggggaa tgatacattg acgttggata accgcattcc ttcaaagacg caccccggat 120

ttgtaagaaa cacaaccggg gctgattgca caagtacaat tgagtcactc gatagcatat 180

atgtggacgg aaagggagag gaatttcaag ttcaagaaac aggaacttga agccagtgcg 240

tgtttacgga aaatatgggc cagatacgaa gatgaggcat taaacttcga tacggcgtca 300

aaacgtagaa gtttgtatat ctgggcgcat tattgcagac ccccaccccg ttgaacgaca 360

gacttggagt ccactggcgc acatataaac ccggtcatcc aatgtctcaa tcttcagaaa 420

tcatcttcag tcacagcacg atttctcaaa gggctcatta gcaatggctc gtcttttgaa 480

ggccgttact gtttgtgcgt tggcgggcat cgctcatgcc ttcaactatg accagcctta 540

ccgtggtcaa taccattttt caccccagaa gaactggatg aatgatccca atgggctttt 600

gtatcataat ggaacctacc atctattctt ccagtacaat cctggtggta tcgagtgggg 660

caacatatca tgggggcatg ctaccagtga ggaccttact cactgggagg agcagcccgt 720

tgcccttctg gcccgaggat acggcagcga tgtcaccgag atgtacttca gcggaagtgc 780

tgttgccgat gtcaataaca cgagtggctt cggaaaggac ggcaagacac ctctagttgc 840

catgtatact tcctatgtac ttgccccatc accgaaccat ctcctggagt gcatatcgct 900

aacagtaatt ccatagtacc ccgttgcaca gacattgccg agtggccaaa ccgtccaaga 960

ggaccagcaa tctcaatcca tcgcctacag tcttgatgac ggtctaacat ggacgacata 1020

cgatgccgcc aacccagtca tccccaaccc tccccagccc taccaagctc aataccagaa 1080

cttccgagac ccctttgtgt tctggcacga cgagtcccag aaatgggttg tcgttacaag 1140

tatagccgaa ctgcacaagc ttgcaattta tacatctgac aacctcaaag actggaagct 1200

agtgagcgaa ttcggtcctt acaatgcgca aggcggcgta tgggagtgcc ccggactttt 1260

caagctaccc cttgacgggg gaagctccac aaaatgggtt atcacgagcg gactgaaccc 1320

cggtggtcct ccaggcaccg tcggctctgg aacccagtac tttgtgggcg agttcgacgg 1380

aaccacattt acgcctgacg ccgatacggt atacccgggt aactcaaccg ccaactggat 1440

ggactggggc ccggacttct atgccgcggc tggctacaac ggcctctcaa ttaaagacca 1500

cgtccatatc ggctggatga acaactggca gtatggtgcg aacatcccca cctacccctg 1560

gcgcagcgcc atggccattc ctcgccacct ggctctgaag accatcaaca acaaaacaac 1620

gctagtccag cagccccagg aagcgtggtc ttccatctcg agcaagcatc cgctctattc 1680

gcgcacctac agtaccttct ctgaaggttc caccaacgca agcacgactg gagaaacgtt 1740

cagggtagat ctgagcttct ctgctacgtc gaaagcctca acatttgcaa tcgccctccg 1800

agcctccgcc aactttaccg agcagaccct tgctggctat gacttcgcca agcagcaaat 1860

cttcctcgac cggaccaagt caggggatgt gtcatttgat aacaccttcg cgagcgtcta 1920

tcatggaccc ttggtgccgg atagcactgg catggtgagg ttgagtatct tcgtcgacag 1980

gtccagcgtc gaggtattcg gaggccaagg tgagacgacc ttgacggctc agatctttcc 2040

tagcaatgat gcggttcacg cccgcctggt gtctactggt ggagctactg aggacgtccg 2100

cgttgatgtt cacaatatta cttcgacgtg gaattaacgt cttgttccta catgtggtag 2160

catcttgtgt ctgttggttt tactatttga ggttaagcgg tagatatact ctaaatcgaa 2220

ttgatatttc ttccacatgg tcattgaata aactagttac cttgcatcaa ggtaccggtg 2280

gccatgacgt cagccgcaat cccatatcgg gtaatgatca ggatcccgag gcatggctgg 2340

ttcacgtgcc ggctgcatga ggtacaagtc tatccgtgag ggggctgatt atagagttac 2400

cttttgctgt cagcatctgt tctttcatgt aaaaatactg aaatcgaaag gcattattaa 2460

atatttacac acgcaatatg tcggagaact acggcgacta tcagactgag atctatggcc 2520

gaggagcatt gacaggcgtc ctgcctaatg tcactaccga ccctcgctta cttgaaaaac 2580

aggcaacgaa ggccttgggc gctcgtagct tccactatgt ggctggcggt gctggagaga 2640

aggctacaat ggatagcaat cgactggctt ttcgacaatg gaagctgtga gttgagtcgt 2700

ttccgccgcg atattgcgat gtagagtcgg aagactg 2737

<210> 5

<211> 2895

<212> DNA

<213> Artificial sequence

<400> 5

agctgcttga caactcccga tcccgtgaat atagcaaacc actctcggta cccggaatct 60

caccttgcat ctccaccttt ttaccaatcg cgttcgctcg ttcgactcct cacattcctt 120

gagtgagaaa ttagtgatct ctccgatgat tagtgatctc tccggatgaa tcggtcttgt 180

ttgtccacca ctacccttcg atagaagtct cggcctcacg ctctccctga cacggatcgc 240

gttgtccttc cctttataaa gacctgcagg ggcgagcaaa catcacttcg gtataggcat 300

acctctatta actgtgaagg gcttatatga cggacaagga tccgggagaa atttcgggga 360

aagtcaacgc tgggcatgcg actatcagtt tggggaaatg aaaggcgtct tggccatgat 420

atttatatag ctcgcttttc cgtgccagta gacggcttta cttttctgcc actcacaggt 480

ctctcagaga agtggaattc aagaatacca aattgttcgt tttgctttgt tactccaaga 540

tgcccaaccg tggcagcaga cctggtcatc ctacggtcca catcaccgca ccgcattgga 600

ttaatgaccc ctgcgctcca ggctatgatc caaggactgg cctctatcat cttttctacc 660

aatgtaatcc atggggatgc gaatggggta acatgtcatg gggacatgca acgagtgaag 720

acctcctcca ctggaccgtt ccgtcaaacc agccagttct ggaaccagat catgactatg 780

acagggatgg tgtgtttacg ggatgctttc ttcctgtcat aagccatcca gacaatgagc 840

agctcacagt gttttactca tctgttcggc agctcccctt ccattggagc accccaccct 900

atccccggaa cgcagccggt ttatccatgg ccaacagcat cgacggagga aagacatgga 960

acaagtcaaa tagaaatccg atcgtccaag gtgagccaga gagcattcgg gtaaccggat 1020

tccgcgaccc gtacatcgcg ccctggccgg agatggataa acttcgaggg caacgatctc 1080

tctatggcat aatctctggc ggtattcaag acgcaggacc gactgccttc ctttatgccg 1140

tacagtggca cgagcctttc gactggcaat atctggggca gcttgtcgat ttcccactcc 1200

gatttcagcc gtcgaagaaa tggtgtggca actacgggat caattgggag tgcgccaact 1260

ttatgaatct agagtccgag agcgcatcgg ctacgcgtac atgtctgata cttggtgctg 1320

agggtgatgt tgaacgtgct catatcgaaa atcatcagcg accggtgacc gtgccggcac 1380

ggacagtaag gtccctgctg tggatgtttg gagacttggc tggaagcaat catcaaacgg 1440

ataacctgaa atgcagattc aaacatggag ggtaccttga ccatggttct ttatatgcgg 1500

ccagcacatt taatgatcca atatctggaa gacgaatttt gtatggatgg atccccgagg 1560

aagacattac aggaggccat gcacgtgaga aaggctggaa tggctctctg tgtctcccaa 1620

gacagctatt cctactctcg cttcgcaacg ttaccagggc ggtccgcagc aagttgggcg 1680

aaattcccag cattgagatg gtaatagaag ccgacggttc caccactctt tattcgttag 1740

gcatccgccc tgtatccgaa atcacccagt tgcggaccaa gtgtattcaa gcccatgaag 1800

cacggccctt ttctctccct cagtctactc acaacgaatc acgcatttgc cgcacttcaa 1860

cgtcgtgttg ggagcttgaa gctaccgtct cgcttggtgt tagctgcgag actgttgggt 1920

tacgcattcg atgcgcgggg gaagagcccg tccagtgggt catagtcttc tctcttgtgg 1980

atgagacgat tactattgat cggtcccgtt ccagtacccg ttctgacgtg aatacctgtc 2040

cagagaaagg tccatttacc ctattttatg tcacagatgg aacacgtaca gaaaaattag 2100

aaagatttca tctccgggtt atcgttgacg cagatattgt cgagatctac gccaacgacc 2160

gatttgccct cgcaacgatg atgtaccctg gaagtcacaa ggccgaaagg gagatagtcg 2220

cgttcgctac aggtgataat gagagcgcca gatttgagca ggtcaaaata tgggacggac 2280

taaacggaat ggaagctctg gtcagagatg aaggaactgc gaagccagat aatcaaagcc 2340

tatgaaggag gcgtgggagg ctgccagacg ggggaagggg attaagacag tgattgaggg 2400

gcctaggtag gagggtgagg actggatgtt gaagttgtac tgtctgggct agaagtatac 2460

taataaaaat ccatctttca agtggaacga ataagttgga gatgcagagc aggcagggac 2520

ccaatccttg ctccaacatg gcattctttt cgtactaagt agctaccttt ccctgctcaa 2580

aacctttatg ttgataaccc ctcataaatg ctaacaggcc acacattata tctcccgcct 2640

aatccggacg cacgggaatg atgagggcgt agcaccgggc tagcagtgag gctctggatc 2700

atgaagtgtc tgcactgtag ggacacagtc tcctactagg atcttgttca ccgggtaatt 2760

gatggaatga cagcttacag tgcttgacaa gcccaggctg gtggtggctc cgagtcacgc 2820

ttttcagatc ataattcccg ggtaatgcga gccaagtgcc attcatctcc tggatgatca 2880

tgtaacagac acgcc 2895

<210> 6

<211> 3019

<212> DNA

<213> Artificial sequence

<400> 6

gagcagctat ccgctcaatc ccgaatgagg tccgtgttcg gtcgatgtca acaacacccc 60

acgcggggaa tgatacattg acgttggata accgcatttc ttcaaagacg caccccggat 120

ttgtaagaaa cacaaccggg gctgattgca caagtacaat tgagtcactc gatagcatat 180

atgtggacgg aaagggagag gaaattcaag ttcaagaaac aggaacttga agccagtgcg 240

tgtttacgga aaatatgggc cagatacgaa gatgaggcat taaacttcga ttcggcgtca 300

aaacgtagaa gtttgtatat ctgggcgcat tattgcagac ccccaccccg ttgaacgaca 360

gacttggagt ccactggcgc acatataaac ccggtcatcc aatgtctcaa tcttcagaaa 420

tcatcttcag tcacagcacg atttctcaaa gggctcatta gcaatggctc gtcttttgaa 480

ggccgttact gtttgtgcgt tggcgggcat cgctcatgcc ttcaactatg accagcctta 540

ccgtggtcaa taccattttt caccccagaa gaactggatg aatgatccca atgcccaacc 600

gtggcagcag acctggtcat cctacggtcc acatcaccgc accgcattgg attaatgacc 660

cctgcgctcc aggctatgat ccaaggactg gcctctatca tcttttctac caatgtaatc 720

catggggatg cgaatggggt aacatgtcat ggggacatgc aacgagtgaa gacctcctcc 780

actggaccgt tccgtcaaac cagccagttc tggaaccaga tcatgactat gacagggatg 840

gtgtgtttac gggatgcttt cttcctgtca taagccatcc agacaatgag cagctcacag 900

tgttttactc atctgttcgg cagctcccct tccattggag caccccaccc tatccccgga 960

acgcagccgg tttatccatg gccaacagca tcgacggagg aaagacatgg aacaagtcaa 1020

atagaaatcc gatcgtccaa ggtgagccag agagcattcg ggtaaccgga ttccgcgacc 1080

cgtacatcgc gccctggccg gagatggata aacttcgagg gcaacgatct ctctatggca 1140

taatctctgg cggtattcaa gacgcaggac cgactgcctt cctttatgcc gtacagtggc 1200

acgagccttt cgactggcaa tatctggggc agcttgtcga tttcccactc cgatttcagc 1260

cgtcgaagaa atggtgtggc aactacggga tcaattggga gtgcgccaac tttatgaatc 1320

tagagtccga gagcgcatcg gctacgcgta catgtctgat acttggtgct gagggtgatg 1380

ttgaacgtgc tcatatcgaa aatcatcagc gaccggtgac cgtgccggca cggacagtaa 1440

ggtccctgct gtggatgttt ggagacttgg ctggaagcaa tcatcaaacg gataacctga 1500

aatgcagatt caaacatgga gggtaccttg accatggttc tttatatgcg gccagcacat 1560

ttaatgatcc aatatctgga agacgaattt tgtatggatg gatccccgag gaagacatta 1620

caggaggcca tgcacgtgag aaaggctgga atggctctct gtgtctccca agacagctat 1680

tcctactctc gcttcgcaac gttaccaggg cggtccgcag caagttgggc gaaattccca 1740

gcattgagat ggtaatagaa gccgacggtt ccaccactct ttattcgtta ggcatccgcc 1800

ctgtatccga aatcacccag ttgcggacca agtgtattca agcccatgaa gcacggccct 1860

tttctctccc tcagtctact cacaacgaat cacgcatttg ccgcacttca acgtcgtgtt 1920

gggagcttga agctaccgtc tcgcttggtg ttagctgcga gactgttggg ttacgcattc 1980

gatgcgcggg ggaagagccc gtccagtggg tcatagtctt ctctcttgtg gatgagacga 2040

ttactattga tcggtcccgt tccagtaccc gttctgacgt gaatacctgt ccagagaaag 2100

gtccatttac cctattttat gtcacagatg gaacacgtac agaaaaatta gaaagatttc 2160

atctccgggt tatcgttgac gcagatattg tcgagatcta cgccaacgac cgatttgccc 2220

tcgcaacgat gatgtaccct ggaagtcaca aggccgaaag ggagatagtc gcgttcgcta 2280

caggtgataa tgagagcgcc agatttgagc aggtcaaaat atgggacgga ctaaacggaa 2340

tggaagctct ggtcagagat gaaggaactg cgaagccaga taatcaaagc ctatgacgtc 2400

ttgttcctac atgtggtagc atcttgtgtc tgttggtttt actatttgag gttaagcggt 2460

agatatactc taaatcgaat tgatatttct tccacatggt cattgaataa actagttacc 2520

ttgcatcaag gtaccggtgg ccatgacgtc agccgcaatc ccatatcggg taatgatcag 2580

gatcccgagg catggctggt tcacgtgccg gctgcatgag gtacaagtct atccgtgagg 2640

gggctgatta tagagttacc ttttgctgtc agcatctgtt ctttcatgta aaaatcctga 2700

aatcgaaagg cattattaaa tattttcaca cgcaatatgt cggagaacta cggcgactat 2760

cagactgaga tctatggccg aggagcattg acaggcgtcc tgcctaatgt cactaccgac 2820

cctcgcttac ttgaaaaaca ggcaaagaag gccttgggcg ctcgtagctt caactatgtg 2880

gctggcggtg ctggagagaa ggctacaatg gatagcaatc gactggcttt tcgacaatgg 2940

aagctgtgag ttgagtcgtt tccgccgcga tattgcgatg tagagtcgga agactgacgc 3000

gtataacgct tgatgaaat 3019

Claims (3)

1. A strain of Aspergillus niger with high fructosyltransferase yield is characterized in that the Aspergillus niger specifically is Aspergillus niger An-fru3III, and is obtained by replacing 2 fructosyl hydrolase encoding genes-inuA and inuE in a genome with the fructosyltransferase shown in SEQ ID No.1 on the basis of Aspergillus niger An-F308;

the preservation number of the Aspergillus niger An-F308 is CGMCC NO. 21028;

the nucleotide sequence of the inuA is shown as SEQ ID NO. 3; the nucleotide sequence of the inuE is shown in SEQ ID NO. 4.

2. Use of An-fru3III according to claim 1 in the production of a fructosyltransferase.

3. Use according to claim 2, wherein the fructosyltransferase is produced by fermentation in a process which comprises:

the fermentation process comprises the following steps: according to the inoculation amount of 1-10%, the fermentation temperature is 25-36 ℃, the dissolved oxygen is maintained at 5-60% in the fermentation process, the pH is controlled at 4-6 in the fermentation process, and the fermentation time is 60-150 h.

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