Fructooligosaccharide synthesis engineering strain capable of efficiently expressing high-activity glucose oxidase as well as construction method and application of fructooligosaccharide synthesis engineering strain

Abstract

The invention relates to the technical field of gene engineering, in particular to a fructo-oligosaccharide synthesis engineering strain capable of efficiently expressing high-activity glucose oxidase as well as a construction method and application of the fructo-oligosaccharide synthesis engineering strain. The invention relates to a fructo-oligosaccharide synthesis engineering strain capable of efficiently expressing high-activity glucose oxidase, which takes aspergillus niger ATCC 20611 as a starting strain and can express glucose oxidase and beta-fructofuranosidase. A genome of the fructo-oligosaccharide synthesis engineering strain contains a high-activity glucose oxidase expression cassette FG5C. According to the engineering strain, the activity of glucose oxidase is remarkably improved, the fructo-oligosaccharide preparation efficiency is improved, operation is easy and convenient, cost is low, and high application value is achieved; the method disclosed by the invention has guiding significance for preparing high-purity fructo-oligosaccharide by an enzyme method, and can effectively promote market promotion and industrial development of fructo-oligosaccharide.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to an oligofructose synthesis engineering strain capable of efficiently expressing highly active glucose oxidase, and a construction method and application thereof. Background Art

[0002] Fructooligosaccharides (FOS) are also known as cane oligosaccharides, with a structural formula of GF n (G and F represent glucose and fructose, respectively, and n is the number of fructose groups), generally composed of a mixture of 1 to 3 fructose groups bound to sucrose molecules, such as GF2, GF3, and GF4. Fructo-oligosaccharides maintain the balance of intestinal flora by regulating beneficial bacteria such as bifidobacteria, and have the effects of moistening the intestines, lowering blood lipids, and improving immunity. They have been widely used in food, beverages, and medicine. In industry, microbial enzymes are mainly used to mass-produce fructo-oligosaccharides. High-concentration sucrose is used as raw material, and the synthesis is catalyzed by β-fructofuranosidase (EC3.2.1.26, also known as fructosyltransferase Fructosyltransferase, EC 2.4.1.9) produced by microorganisms with fructosyl transfer activity. However, glucose, a byproduct, accumulates during the synthesis process. Glucose competitively inhibits the transfructosylation activity of β-fructofuranosidase, thereby limiting the complete conversion of sucrose. Therefore, the final product still contains 25%-30% glucose and 13%-25% unconverted sucrose and a small amount of fructose, which is not suitable for diabetics and patients with dental caries. Therefore, eliminating or converting glucose in the product to produce high-purity oligofructose is of great significance in increasing the application range of the product.

[0003] The expression of glucose oxidase in oligofructose synthesis strains can realize the one-step enzymatic preparation of oligofructose with high purity by mycelium, which has good industrial application value. Chinese invention patent CN 107418903 A uses glucose oxidase and the C-terminal domain of β-fructofuranosidase (FopA) to express and construct the engineering strain GOC-4. The glucose oxidase activity of the engineering strain GOC-4 reaches 90.0 U / g, and the β-fructofuranosidase activity reaches 380 U / g. The mycelium of the engineering strain GOC-4 is directly used to prepare oligofructose, and the oligofructose content in the product can reach 71.2%. Compared with the purity of the product obtained by the reaction of the current most important oligofructose production strain Aspergillus niger ATCC 20611, although there is a certain improvement, the activity of glucose oxidase and β-fructofuranosidase is not high, so the purity of the prepared oligofructose still has room for further improvement. Summary of the invention

[0004] Aiming at the technical problems that the activity of glucose oxidase and β-fructofuranosidase of the engineered strain GOC-4 is not high and the purity of the prepared oligofructose is not ideal, the present invention provides an engineered strain for synthesizing oligofructose that efficiently expresses high-activity glucose oxidase and its construction method and application. The engineered strain of the present invention significantly improves the activity of glucose oxidase, improves the efficiency of preparing oligofructose, is easy to operate and low in cost, and has strong application value; the present invention has guiding significance for the preparation of high-purity oligofructose by enzymatic method, and can effectively promote the market promotion and industrial development of oligofructose.

[0005] The technical solution of the present invention is as follows: In a first aspect, the present invention provides an engineered strain for oligofructose synthesis that efficiently expresses high-activity glucose oxidase, using Aspergillus niger ATCC 20611 as a starting strain, and capable of expressing glucose oxidase and β-fructofuranosidase; The genome of the oligofructose synthesis engineering strain contains a high-activity glucose oxidase expression cassette FG5C, which contains mind (α-glucosidase gene) upstream homology arm, Aspergillus niger β-fructofuranosidase promoter P fopA Its signal peptide SP fopA , high activity glucose oxidase gene gox-5M , C-terminal domain gene of β-fructofuranosidase FopA fopA- C, Aspergillus nidulans tryptophan terminator T trpC as well as mind downstream homology arm; in, gox-5M The amino acid sequence is shown in SEQ ID No. 1, mind Upstream and downstream homology arms, promoter P fopA Its signal peptide SP fopA and the C-terminal domain encoding gene of β-fructofuranosidase FopA fopA -C from Aspergillus niger ATCC20611 genome, terminator T trpC The gene was derived from plasmid pAN7-1.

[0006] The β-fructofuranosidase promoter P used in the present invention fopA It is a sucrose-inducible promoter of Aspergillus niger, which is induced by sucrose concentration but not inhibited by the metabolite glucose, and can drive high expression of glucose oxidase; the gene encoding high-activity glucose oxidase GOX gox-5M It is a rationally designed sequence in this study. Compared with ordinary glucose oxidase, it reduces the volume of the active site of glucose oxidase, which has a positive impact on the catalytic efficiency. Therefore, the designed glucose oxidase has higher activity.

[0007] In a second aspect, the present invention provides a method for constructing an engineered strain for oligofructose synthesis that efficiently expresses highly active glucose oxidase, comprising the following steps: Glucose oxidase GOX gox-5M The gene encoding the C-terminal domain of β-fructofuranosidase FopA fopA -C fusion to obtain fusion gene gox-5M-fopA -C, then mind (α-glucosidase gene) upstream homology arm, promoter P fopA Its signal peptide SP fopA , terminator T trpC and mind The downstream homology arms are connected to form a high-activity glucose oxidase expression cassette FG5C, and the high-activity glucose oxidase expression cassette FG5C is transformed into the genome of Aspergillus niger ATCC 20611 to obtain; in, gox-5M The amino acid sequence is shown in SEQ ID No. 1, mind Upstream and downstream homology arms, promoter P fopA Its signal peptide SP fopA and the C-terminal domain encoding gene of β-fructofuranosidase FopA fopA -C from Aspergillus niger ATCC20611 genome, terminator T trpC The gene was derived from plasmid pAN7-1.

[0008] In a third aspect, the present invention further provides a use of the oligofructose synthesis engineered strain as described in the first aspect or the oligofructose synthesis engineered strain obtained by the construction method as described in the second aspect in the preparation of oligofructose.

[0009] Furthermore, the application method comprises the following steps: (1) Inoculating the oligofructose synthesis engineered strain into a fermentation medium to obtain mycelium; (2) Mycelium was added to the reaction system, and the activity of β-fructofuranosidase and substrate sucrose in the reaction system met 5-9 U / g to prepare oligofructose.

[0010] Furthermore, in step (1), 1×10 5 ~1×10 7 The oligofructose synthesis engineering strain was inoculated into the fermentation medium at an inoculation amount of spores / mL, the culture temperature was controlled at 30-40°C, the culture speed was 180-220 rpm, and the mycelium was collected after 36-60 hours of culture.

[0011] Furthermore, in step (1), 1×10 6The oligofructose synthesis engineering strain was inoculated into the fermentation medium with an inoculation amount of spores / mL, and the culture temperature was controlled at 30°C, the culture speed was 200 rpm, and the culture time was 48 h.

[0012] Furthermore, the fermentation medium comprises 40 g / L sucrose, 15 g / L yeast extract, 0.5 g / L KCl, 2 g / L NaNO3, 5 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, and a pH value of 5.0.

[0013] Furthermore, the reaction system of step (2) includes 350-550 g / L sucrose and 10-15 g / L CaCO3, the reaction temperature is controlled at 40±5°C, the reaction speed is 180-220 rpm, and the reaction time is 5-20 h.

[0014] Furthermore, the reaction system of step (2) includes 450 g / L sucrose, 10 g / L CaCO3, a mycelium mass of 9 U β-fructofuranosidase activity / g sucrose, a reaction temperature of 35°C, a reaction speed of 200 rpm, and a reaction time of 20 h.

[0015] The beneficial effects of the present invention are: Compared with the prior art engineering strain GOC-4, the oligofructose synthesis engineering strain provided by the present invention achieves high expression of glucose oxidase, and its activity is higher, the effect is better, and the inhibitory effect of byproduct glucose is further reduced when catalyzing sucrose to synthesize oligofructose, promoting the forward conversion of oligofructose preparation, further improving the purity of oligofructose in the product, and greatly reducing the production cost of oligofructose. Experiments have confirmed that: using the method of the present invention, the glucose oxidase activity of the oligofructose synthesis engineering strain that efficiently expresses high-activity glucose oxidase reaches 127.3 U / g, and the β-furanosidase activity reaches 450 U / g. The mycelium is directly used to prepare oligofructose, and its content reaches 86.5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 The high-activity glucose oxidase gene in Example 1 gox-5M and amplification electrophoresis of expression cassette FG5C; A in the figure is gox-5M A is the electrophoresis diagram of amplification, B is the electrophoresis diagram of amplification of expression cassette FG5C.

[0018] Figure 2 Schematic diagram of the high-activity glucose oxidase expression cassette FG5C in Example 2.

[0019] Figure 3 This is a plate test diagram of the glucose oxidase activity of the oligofructose synthesis engineering strain in Example 2.

[0020] Figure 4 It is the result of glucose oxidase activity determination of the oligofructose synthesis engineering strain in Example 3; Figure A is the result of glucose oxidase activity determination of mycelium of engineering strains GOC-1 and GOC-2 when they were fermented for 24 h, 48 h and 72 h, respectively, and Figure B is the result of glucose oxidase activity determination of mycelium of engineering strain GOC-1 fermented for 48 h at 30°C, 35°C, 40°C, 45°C and 50°C, respectively.

[0021] Figure 5 It is the result of the β-fructofuranosidase activity determination of the oligofructose synthesis engineering strain in Example 4; in the figure, A is the glucose standard curve of the β-fructofuranosidase activity determination by the 3,5-dinitrosalicylic acid method, and B is the β-fructofuranosidase activity result of the mycelium of the engineering strain GOC-1 and the starting strain ATCC 20611 fermented and cultured for 48 hours.

[0022] Figure 6 It is the result of determining the sugar content in the oligofructose product prepared by the engineering strain GOC-1 in Example 4; in the figure, A is the result of determining the glucose content in the product prepared by the reaction of the engineering strain GOC-1 and the starting strain ATCC 20611 for 5 h, 10 h and 20 h, and B is the result of determining the oligofructose content in the product prepared by the reaction of the engineering strain GOC-1 and the starting strain ATCC 20611 for 20 h. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0024] The glucose oxidase gene used in the following examples gox-5MAfter being designed by ourselves, it was synthesized by Beijing Qingke Biotechnology Co., Ltd. and its amino acid sequence is (SEQ ID No.1).

[0025] Aspergillus niger ATCC 20611 used in the following examples was purchased from the American Type Culture Collection, and plasmid pAN7-1 was purchased from VTT Technical Research Centre of Finland. ptrA The plasmid pME2892 expressing the expression cassette was prepared according to the method described in the academic paper Deletion and Allelic Exchange of the Aspergillus fumigatus veA Locus via a Novel Recyclable Marker Module (Krappmann S, Bayram O, Braus GH. Eukaryotic Cell, 2005, 4(7):1298. DOI:10.1128 / EC.4.7.1298-1307.2005.).

[0026] The primer information used in the following examples is shown in Table 1.

[0027] Table 1 Primer sequences

[0028] The MM liquid culture medium used in the following examples includes 20 g / L glucose, 5 g / L (NH4)2SO4, 15 g / L KH2PO4, 0.6 g / L MgSO4·7H2O, 0.6 g / L anhydrous CaCl2, 2 g / L peptone, 0.005 g / L FeSO4·7H2O, 0.0016 g / L MnSO4·H2O, 0.0014 g / L ZnSO4·7H2O, and 0.002 g / L CoCl2, and was sterilized at 115°C for 30 min before use; The transformation upper medium composition included glucose 10 g / L, MgSO4·7H2O 1 g / L, KH2PO4 10 g / L, (NH4)2SO4 6 g / L, C6H5Na3O 73 g / L, FeSO4·7H2O 0.005 g / L, MnSO4·H2O 0.0016 g / L, ZnSO4·7H2O 0.0014 g / L, CoCl2 0.002 g / L, sorbitol 182.18 g / L, and agarose 6 g / L; The transformation lower layer medium composition includes glucose 10 g / L, MgSO4·7H2O 1 g / L, KH2PO4 10 g / L, (NH4)2SO4 6 g / L, C6H5Na3O 73 g / L, FeSO4·7H2O 0.005 g / L, MnSO4·H2O 0.0016 g / L, ZnSO4·7H2O 0.0014 g / L, CoCl2 0.002 g / L, agar powder 20 g / L, sterilized at 115℃ for 30 min before use; The preparation method of PDA medium is as follows: 200 g of peeled potatoes were chopped, boiled with water for 30 min, filtered through 8 layers of gauze, added with 20 g of glucose, fixed to 1 L with distilled water, natural pH, added with 2% agar powder, and sterilized at 115°C for 30 min before use; The components of Czapek medium include NaNO3 3 g / L, K2HPO4 1 g / L, MgSO4·7H2O 0.5 g / L, KCl 0.5 g / L, FeSO4 0.01 g / L, sucrose 30 g / L, solids need to add 2% agar powder, and sterilize at 115℃ for 30 min before use; The fermentation medium contained 40 g / L sucrose, 15 g / L yeast extract, 0.5 g / L KCl, 2 g / L NaNO3, 5 g / L K2HPO4, and 0.5 g / L MgSO4·7H2O. The pH value was 5.0 and the medium was sterilized at 115°C for 30 min before use.

[0029] Example 1 Construction of high-activity glucose oxidase expression cassette FG5C Firstly, the chromosome DNA of Aspergillus niger ATCC 20611 was used as template and aglA-U-1F / aglA-U-2214R were used as primers to amplify the promoter P fopA 2.2 kb upstream homology arm of the adapter mind -U; PfopA-2116UF / fopA-57R were used as primers to amplify gox-5M 2.2 kb promoter P fopA Its signal peptide SP fopA ; fopA-1438F / fopA-1965R were used as primers to amplify the T trpC 0.5kb of adapter fopA -C gene fragment; a 2.1 kb downstream homology arm was amplified using aglA-D-1F / aglA-D-2079R as primers mind -D.

[0030] Synthetic high-activity glucose oxidase gene gox-5M As template, gox-5M-1F / gox-5M-1767R were used as primers to amplify fopA-C 1.8 kb highly active glucose oxidase gene gox-5M (like Figure 1 As shown in A, M represents 1 kb DNA marker).

[0031] Plasmid pAN7-1 was used as template and TtrpC-1044DF / TtrpC-1805DR were used as primers to amplify the downstream homology arm. mind -0.8 kb terminator T of D linker trpC .

[0032] Then, the six fragments were fused using Double joint PCR technology to obtain a 9.5 kb high-activity glucose oxidase expression cassette FG5C (such as Figure 1 As shown in B, M represents 1 kb DNA marker).

[0033] Example 2 Construction of an engineered strain for oligofructose synthesis expressing highly active glucose oxidase Genetic transformation of Aspergillus niger ATCC 20611 using the PEG / CaCl2-mediated protoplast transformation method and the use of the pyrithiamine resistance gene ptrA For screening markers, such as Figure 2 As shown, the transformation purpose is to transform the expression cassette FG5C into the Aspergillus niger ATCC 20611 genome mind The specific method is as follows: (1) Preparation of protoplasts of Aspergillus niger ATCC 20611: Aspergillus niger ATCC 20611 spores were inoculated into 50 mL MM liquid medium (the final spore concentration was 1 × 10 6 / mL), 30℃, 200 rpm for 30 h; collect the mycelium by centrifugation and rinse with sterile water, take 1 g (wet weight) of mycelium into 20 mL of phosphate buffer (pH = 5.6) containing 1% lysozyme (w / v), shake gently to evenly disperse the mycelium, enzymolyze at 30℃, 200 rpm for 2 h, and collect the protoplasts by centrifugation at 2500 rpm.

[0034] (2) Transformation of Aspergillus niger ATCC 20611 protoplasts with the high-activity glucose oxidase expression cassette FG5C: The expression cassette FG5C prepared in Example 1 was combined with a ptrA 10 μg of each plasmid pME2892 of the expression cassette was mixed with 100 μL of Aspergillus niger ATCC 20611 protoplasts, and 50 μL of polyethylene glycol 6000 solution (25%) was added, and the mixture was placed on ice for 20 min. Then, 2 mL of polyethylene glycol 6000 solution (25%) was added, and the mixture was placed at room temperature for 5 min. Finally, 4 mL of phosphate buffer (pH 6.5) containing 1 M sorbitol was added, and the mixture was gently mixed. Then, the above solution was added to 10 mL of transformation upper medium containing 2 μg / mL pyrithiamine hydrobromide (purchased from Sigma), and the mixture was poured into a pre-solidified transformation lower medium plate. After cooling and solidification, the plate was cultured at 30°C for 3 days. The well-growing transformants were selected and inoculated into PDA medium for sporulation.

[0035] (3) Screening of engineered strains expressing highly active glucose oxidase: The transformants were screened using the glucose oxidase activity plate test method. The specific method was as follows: inoculate the transformant spores onto a Czapek solid medium plate and culture at 30°C for 24 h; prepare a reaction mixture: 10 mg / mL glucose, 0.6U / mL peroxidase, 0.08 mg / mL o-dianisidine, and 10 mg / mL agar powder; pour the prepared reaction mixture onto a plate with mycelium and cover the entire plate, and culture at 30°C for 4 h; strains with obvious brown halos around the colonies indicate successful expression of highly active glucose oxidase. Figure 3 As shown, compared with Aspergillus niger ATCC 20611 as the negative control, obvious brown halos were produced around the colonies of transformants GOC-1 and GOC-2, indicating that the FG5C expression cassette was successfully transformed into their genomes and glucose oxidase was expressed. Therefore, transformants GOC-1 and GOC-2 can be used as oligofructose synthesis engineering strains expressing highly active glucose oxidase for subsequent analysis applications.

[0036] Example 3 Determination of glucose oxidase activity of oligofructose synthesis engineering strain The engineered strains GOC-1 and GOC-2 obtained in Example 2 were mixed with fresh spores (1×10 6 / mL) were inoculated into 100 mL of fermentation medium and cultured in shake flasks at 30°C and 200 rpm. Samples were taken every 24 h and the mycelium was collected by filtration for the determination of glucose oxidase activity.

[0037] Glucose oxidase activity determination method: 2.6 mL of phosphate buffer (pH 6.0) containing 0.1 mg / mL o-dianisidine, 0.3 mL of 180 mg / mL glucose solution, 0.1 mL of 0.5 mg / mL horseradish peroxidase solution, mixed evenly, added 0.02 g mycelium, reacted at 30°C for 30 s, measured the absorbance at a wavelength of 460 nm and recorded it as A0, read the absorbance at 1.5 min of reaction and recorded it as A1, and obtained ∆A 460 =A1-A0, enzyme activity (X, U / g) is calculated as: X=∆A 460 ×f×B / (11.3×A×t), where f is the dilution factor of the enzyme solution, B is the total reaction volume (3 mL), 11.3 is the extinction coefficient, A is the sample mass (g), and t is the reaction time (min).

[0038] Glucose oxidase activity assay results Figure 4As shown in A, the glucose oxidase activity of the engineered strains GOC-1 and GOC-2 was the highest at 48 h of culture, and the glucose oxidase activity of the engineered strain GOC-1 reached 103.9 U / g; while the glucose oxidase activity of the starting strain ATCC 20611 was almost undetectable, indicating that the glucose oxidase activity of the engineered strains GOC-1 and GOC-2 came from the glucose oxidase expression cassette FG5C gox-5M Successful expression of the gene. Because the glucose oxidase activity of the engineered strain GOC-1 was the highest, GOC-1 was selected as the engineered strain for subsequent applications.

[0039] The glucose oxidase activity of the engineered strain GOC-1 was further measured at 30°C, 35°C, 40°C, 45°C and 50°C. Figure 4 As shown in B, the glucose oxidase activity of GOC-1 was the highest at 35°C, reaching 127.3 U / g, indicating that the optimal catalytic temperature of the enzyme was 35°C.

[0040] Example 4 Application of the oligofructose synthesis engineered strain GOC-1 in the preparation of oligofructose Fresh spores of the engineered strain GOC-1 and the starting strain ATCC 20611 were cultured at a concentration of 1 × 10 6 The culture medium was inoculated with 100 μg / mL and cultured at 30 °C and 200 rpm for 48 h, and the mycelium was collected.

[0041] The method for determining the activity of β-fructofuranosidase is as follows: weigh 0.02 g of mycelium and mix with 2 mL of 25% (w / v) sucrose solution (prepared with 0.1 mol / L citric acid phosphate buffer solution with a pH value of 5.0) and 2 mL of phosphate buffer with a pH value of 5.0, react at 50°C for 2 h, terminate the reaction in a boiling water bath for 5 min, dilute the reaction supernatant appropriately, take 1 mL and add it to an ELISA tube, then add 2 mL of 10 mg / mL 3,5-dinitrosalicylic acid (DNS) solution, boil for 2 min, cool with cold water, add distilled water to 25 mL, shake well, and measure the OD value at 540 nm. The blank control is to directly take 1 mL of distilled water and mix it with 2 mL of DNS and perform the same experimental conditions.

[0042] The amount of enzyme required to catalyze the reaction of substrate sucrose to produce 1 μmol of reducing sugar per minute is defined as one unit of enzyme activity. The preparation of the glucose standard curve uses the same reducing sugar determination system. The glucose concentration of the standard curve is between 0 mg / mL and 1.5 mg / mL. The glucose standard curve is as follows Figure 5 As shown in A.

[0043] The results of β-fructofuranosidase activity assay were as follows Figure 5As shown in B, the enzyme activity of the engineered strain GOC-1 reached 450 U / g, which was not significantly different from that of the starting strain.

[0044] The mycelium of the engineered strain GOC-1 cultured for 48 h was directly used to prepare oligofructose. The concentration of substrate sucrose was 450 g / L. According to the enzyme activity of the engineered strain GOC-1 reaching 450 U / g, the mycelium amount of the engineered strain GOC-1 was 9 U β-fructofuranosidase activity / g sucrose. The reaction temperature was adjusted to 35°C, the reaction speed was 200 rpm, the amount of CaCO3 added was 10 g / L, and the reactions were carried out for 5 h, 10 h and 20 h, respectively.

[0045] The reaction products were analyzed by HPLC under the following conditions: the liquid phase instrument was LC-6A (Shimadzu, Japan), the chromatographic column was Agilent Zorbax NH2column (5 μm, 4.6 mm×250 mm), the differential detector was Waters 2414, the mobile phase was 70% acetonitrile aqueous solution, the injection volume was 10 μL, and the flow rate was 1.0 mL / min. Finally, the content of each sugar component in the product was determined based on the retention time and peak area. The negative control was performed under the same experimental conditions with the mycelium of the starting strain ATCC20611 cultured for 48 hours.

[0046] The results of sugar content analysis in oligofructose preparation products are as follows Figure 6 As shown in Figure 2, the glucose content in the reaction product of the engineered strain GOC-1 was 132.1 mg / mL at 20 h, which was 54.1% lower than that of the starting strain ATCC 20611 (287.6 mg / mL). Figure 6 In A), the oligofructose content synthesized by the engineered strain GOC-1 can reach 86.5% ( Figure 6 (B), which was 66% higher than the starting strain (52.1%).

[0047] Example 5 Application of engineered strain GOC-1 in the preparation of oligofructose Fresh spores of the engineered strain GOC-1 and the starting strain ATCC 20611 were cultured at a concentration of 1 × 10 5 / mL was inoculated into the fermentation medium, cultured at 30°C and 180 rpm for 36 h, and the mycelium was collected; the method for determining the β-fructofuranosidase activity of the mycelium and the preparation of the glucose standard curve were the same as those described in Example 4. Under this condition, the β-fructofuranosidase activity of the engineered strain GOC-1 reached 345 U / g.

[0048] The mycelium of the engineered strain GOC-1 cultured for 48 h was directly used to prepare oligofructose. The substrate sucrose concentration was 350 g / L. According to the enzyme activity of the engineered strain GOC-1 reaching 345 U / g, the mycelium amount of the engineered strain GOC-1 was 5 U β-fructofuranosidase activity / g sucrose. The reaction temperature was adjusted to 40°C, the reaction speed was 180 rpm, the CaCO3 addition amount was 12 g / L, and the reaction was carried out for 20 h.

[0049] The reaction products were analyzed by HPLC under the same conditions as described in Example 4. Under these conditions, the glucose content in the reaction product of the engineered strain GOC-1 was 182.8 mg / mL at 20 h, and the content of synthesized oligofructose was 65.7%.

[0050] Example 6 Application of the oligofructose synthesis engineered strain GOC-1 in the preparation of oligofructose Fresh spores of the engineered strain GOC-1 and the starting strain ATCC 20611 were cultured at a concentration of 1 × 10 7 / mL was inoculated into the fermentation medium, cultured at 30°C and 220 rpm for 60 h, and the mycelium was collected; the method for determining the β-fructofuranosidase activity of the mycelium and the preparation of the glucose standard curve were the same as those described in Example 4. Under this condition, the β-fructofuranosidase activity of the engineered strain GOC-1 reached 393 U / g.

[0051] The mycelium of the engineered strain GOC-1 cultured for 48 h was directly used to prepare oligofructose. The concentration of substrate sucrose was 550 g / L. According to the enzyme activity of the engineered strain GOC-1 reaching 393 U / g, the mycelium amount of the engineered strain GOC-1 was 7 U β-fructofuranosidase activity / g sucrose. The reaction temperature was adjusted to 45°C, the reaction speed was 220 rpm, the amount of CaCO3 added was 15 g / L, and the reaction was carried out for 20 h.

[0052] The reaction products were analyzed by HPLC under the same conditions as described in Example 4. Under these conditions, the glucose content in the reaction product of the engineered strain GOC-1 was 157.8 mg / mL at 20 h, and the content of synthesized oligofructose was 70.1%.

[0053] In summary, the engineered strain of the present invention can efficiently express highly active glucose oxidase, and plays a role in removing the inhibitory effect of byproduct glucose in the process of oligofructose preparation, which is beneficial to the forward progress of the oligofructose preparation reaction, thereby obtaining oligofructose products with higher purity.

[0054] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. An engineered strain for oligofructose synthesis that efficiently expresses highly active glucose oxidase, characterized in that: Aspergillus niger ATCC 20611 is used as the starting strain, and is capable of expressing glucose oxidase and β-fructofuranosidase; The genome of the oligofructose synthesis engineering strain contains a high-activity glucose oxidase expression cassette FG5C, which contains aglA Upstream homology arm, Aspergillus niger β-fructofuranosidase promoter P fop Its signal peptide SP fop , high activity glucose oxidase gene gox-5M , C-terminal domain gene of β-fructofuranosidase FopA fopA- C, Aspergillus nidulans tryptophan terminator T trpC as well as aglA downstream homology arm; in, gox-5M The amino acid sequence is shown in SEQ ID No. 1, aglA Upstream and downstream homology arms, promoter P fop Its signal peptide SP fop and the C-terminal domain encoding gene of β-fructofuranosidase FopA fop -C from Aspergillus niger ATCC 20611 genome, terminator T trpC The gene was derived from plasmid pAN7-1.

2. A method for constructing an engineered strain for oligofructose synthesis that efficiently expresses highly active glucose oxidase, characterized in that: The steps include: Glucose oxidase GOX gox-5M The gene encoding the C-terminal domain of β-fructofuranosidase FopA fop -C fusion to obtain fusion gene gox-5M-fopA -C, then aglA Upstream homology arm, promoter P fop Its signal peptide SP fop , terminator T trpC and aglA The downstream homology arms are connected to form a high-activity glucose oxidase expression cassette FG5C, and the high-activity glucose oxidase expression cassette FG5C is transformed into the genome of Aspergillus niger ATCC 20611 to obtain; in, gox-5M The amino acid sequence is shown in SEQ ID No. 1, aglA Upstream and downstream homology arms, promoter P fop Its signal peptide SP fop and the C-terminal domain encoding gene of β-fructofuranosidase FopA fop -C from Aspergillus niger ATCC 20611 genome, terminator T trpC The gene was derived from plasmid pAN7-1.

3. Use of the oligofructose synthesis engineered strain as claimed in claim 1 or the oligofructose synthesis engineered strain obtained by the construction method as claimed in claim 2 in the preparation of oligofructose.

4. The use according to claim 3, characterized in that The application method includes the following steps: (1) Inoculating the oligofructose synthesis engineered strain into a fermentation medium to obtain mycelium; (2) Mycelium is added to the reaction system, and the activity of β-fructofuranosidase and substrate sucrose in the reaction system meet 5-9 U / g to prepare oligofructose.

5. The use according to claim 4, characterized in that In step (1), 1×10 5 ~1×10 7 The oligofructose synthesis engineering strain was inoculated into the fermentation medium at an inoculation amount of spores / mL, the culture temperature was controlled at 30-40°C, the culture speed was 180-220 rpm, and the mycelium was collected after 36-60 h of culture.

6. The use according to claim 4, characterized in that In step (1), 1×10 6 The oligofructose synthesis engineering strain was inoculated into the fermentation medium with an inoculation amount of spores / mL, and the culture temperature was controlled at 30°C, the culture speed was 200 rpm, and the culture time was 48 h.

7. The use according to claim 4, characterized in that The composition of the fermentation medium included sucrose 40 g / L, yeast extract 15 g / L, KCl 0.5 g / L, NaNO3 2 g / L, K2HPO4 5 g / L, MgSO4·7H2O 0.5 g / L, and the pH value was 5.

0.

8. The use according to claim 4, characterized in that The reaction system of step (2) includes 350-550 g / L sucrose and 10-15 g / L CaCO3, the reaction temperature is controlled at 40±5°C, the reaction speed is 180-220 rpm, and the reaction time is 5-20 h.

9. The use according to claim 4, characterized in that The reaction system of step (2) includes 450 g / L sucrose, 10 g / L CaCO3, a mycelium mass of 9 U β-fructofuranosidase activity / g sucrose, a reaction temperature of 35°C, a reaction speed of 200 rpm, and a reaction time of 20 h.

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