A recombinant fusion xylanase and a method for preparing arabinoxylan with different molecular weights and structural characteristics using the same

Abstract

The present invention relates to the field of bioengineering technology, and in particular to a recombinant fusion xylanase and a method for preparing arabinoxylans of different molecular weights and structural characteristics using the same. The present invention is designed from two directions: the selection and transformation of hydrolases and the transformation of enzyme substrates, to obtain arabinoxylans of specific molecular weights and structural characteristics, and to clarify the corresponding relationship between the molecular weight of xylanase and the branching degree of substrate arabinoxylan and the enzyme-catalyzed hydrolysis product. The present invention provides a basis for the industrial preparation of functional arabinoxylans of specific molecular weight using corn husks as raw materials, is conducive to the resource utilization of food processing by-products, extends the food processing industry chain, drives the development of the food economy, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a recombinant fusion xylanase and a method for preparing arabinoxylan with different molecular weights and structural characteristics by using the recombinant fusion xylanase. Background Art

[0002] Corn is one of the three major staple foods in my country. Corn husk is a byproduct of corn deep processing, accounting for 10% to 15% of the total weight of corn, and its hemicellulose content is about 70%. The main chain branching degree of xylan in corn husk fiber is very high, and more than 70% of the main chain xylose residues have one or more branching substitution groups.

[0003] Corn husk fiber can be degraded into arabinoxylan (AX), which is a natural polysaccharide. According to its different physical and chemical properties and biological activities, it is widely used in food thickening, stabilization, emulsification, etc. For example, it can be used in cold food, beverages, dairy products, condiments, cakes, candies, brewing, food preservation and refrigeration and other food industries. As a dietary fiber, arabinoxylan is an important part of a healthy diet, which can promote intestinal peristalsis and help digestion. The biological activity of arabinoxylan, its molecular weight and molecular structure determine its physical and chemical properties and biological functions, which in turn affect the application value and application field of AX.

[0004] The preparation methods of arabinoxylan mainly include chemical method, physical method and enzymatic method. The enzymatic method has the advantages of mild conditions, strong enzymatic hydrolysis specificity, controllable products, and maximum recovery of effective ingredients. At present, the research mainly focuses on the modification and optimization of single enzyme molecules, and few studies focus on the effects of enzyme molecular weight and substrate side chain steric hindrance on enzyme hydrolysis products. Summary of the invention

[0005] The invention is designed from two directions: the selection and modification of hydrolases and the modification of enzyme substrates, and obtains arabinoxylan with specific molecular weight and structural characteristics, and clarifies the corresponding relationship between the molecular weight of xylanase and the branching degree of substrate arabinoxylan and the enzyme-catalyzed hydrolysis product.

[0006] Based on this, the following technical solution is proposed.

[0007] First, the present invention provides a recombinant fusion xylanase, which includes a single-domain xylanase and a protein domain without substrate catalysis / binding function, wherein the protein domain without substrate catalysis / binding function includes but is not limited to lipase, protease, amylase, phytase, glucanase, mannanase and other enzymes.

[0008] In a specific implementation process, the protein domain without substrate catalysis / binding function is fused and expressed at the N-terminus or C-terminus of the single-domain xylanase.

[0009] Preferably, the protein without substrate catalysis / binding function is lipase.

[0010] Preferably, the recombinant fusion xylanase comprises a single-domain xylanase, a linker and a lipase connected in sequence.

[0011] Preferably, the single domain xylanase is Scxyn22, Paxyn28 or Scxyn39.

[0012] Preferably, the amino acid sequence of Scxyn22 is shown as SEQ ID No.1; the amino acid sequence of Paxyn28 is shown as SEQ ID No.2; the amino acid sequence of Scxyn39 is shown as SEQ ID No.3; the amino acid sequence of Linker is shown as SEQ ID No.4; and the amino acid sequence of the protein without substrate catalysis / binding function is shown as SEQ ID No.5.

[0013] Furthermore, the present invention provides a method for preparing arabinoxylan with different molecular weights and structural characteristics, comprising: hydrolyzing corn fiber gum with single-domain xylanase Scxyn22, Paxyn28, Scxyn39 or the recombinant fusion xylanase to obtain arabinoxylan with different molecular weights and structural characteristics.

[0014] Preferably, for the same single-domain xylanase, the larger the molecular weight of the recombinant fusion xylanase used in hydrolyzing corn fiber gum, the smaller the weight average molecular weight of the obtained arabinoxylan and the narrower the molecular weight distribution.

[0015] Preferably, the method further comprises: firstly using arabinofuranosidase Scabf32 to enzymatically hydrolyze corn fiber gum, then inactivating arabinofuranosidase Scabf32 to obtain an enzymatic hydrolysis product; and then using the single-domain xylanase or the recombinant fusion xylanase to hydrolyze the enzymatic hydrolysis product.

[0016] Through synergistic effects, the corn fiber gum substrate is first hydrolyzed using the arabinofuranosidase Scabf32 that can hydrolyze the arabinose branches of corn fiber gum, reducing the substrate branch ratio and density and increasing the action space of xylanase. The modified enzymatic hydrolysis product is then catalyzed and hydrolyzed using xylanases of different molecular weights to prepare arabinoxylan with specific molecular weight and structural characteristics.

[0017] Preferably, the weight average molecular weight of the obtained arabinoxylan is below 90 KDa; the number average molecular weight is below 50 KDa; and the peak molecular weight is below 80 g / mol.

[0018] Preferably, the reaction pH of Scxyn22 is 3.8-4.2; the reaction pH of Paxyn28 is 3.8-4.2; the reaction pH of Scxyn39 is 5.8-6.2; the reaction pH of the recombinant fusion xylanase containing Scxyn22 is 3.8-4.2; the reaction pH of the recombinant fusion xylanase containing Scxyn39 is 5.8-6.2; the reaction pH of Scabf32 is 5.0-6.0.

[0019] Preferably, the reaction temperature of Scxyn22 is 48-52°C; the reaction temperature of Paxyn28 is 58-62°C; the reaction temperature of Scxyn39 is 68-72°C; the reaction temperature of the recombinant fusion xylanase containing Scxyn22 is 58-62°C; the reaction temperature of the recombinant fusion xylanase containing Scxyn39 is 48-52°C; the reaction temperature of Scabf32 is 58-62°C.

[0020] Preferably, the method for preparing the corn fiber gum comprises: (1) Weigh corn husk powder, add n-hexane, stir, filter, wash, dry, treat with α-amylase, then add saccharifying enzyme, add neutral protease to the above treatment solution, inactivate after enzymatic hydrolysis, and filter.

[0021] (2) Weigh the sample obtained in step (1), add NaOH, Ca(OH)2 and water, heat, and centrifuge to separate the supernatant and precipitate after the reaction; (3) Add H2O2 to the supernatant obtained in step (2) to adjust the pH of the solution to alkaline, stir at room temperature, then adjust the pH to acidic, centrifuge and separate the supernatant, add anhydrous ethanol to the supernatant to obtain a white flocculent precipitate CFG1.

[0022] (4) Add distilled water to the precipitate obtained in step (2), then add H2O2, adjust the pH to alkaline, heat, cool and centrifuge to separate the supernatant, adjust the pH of the supernatant to acidic, centrifuge and separate the supernatant, then add anhydrous ethanol to obtain a white flocculent precipitate CFG2.

[0023] (5) The white flocculent precipitate CFG1 and the white flocculent precipitate CFG2 obtained in the above steps (3) and (4) are mixed, and freeze-dried to obtain the corn fiber gum.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The present invention is designed from two directions: the selection and modification of hydrolases and the modification of enzyme substrates, and obtains arabinoxylan with specific molecular weight and structural characteristics, and clarifies the corresponding relationship between the molecular weight of xylanase and the branching degree of substrate arabinoxylan and the enzyme-catalyzed hydrolysis product. The present invention provides a basis for the industrial preparation of functional arabinoxylan with specific molecular weight using corn husk as raw material, which is helpful for the resource utilization of food processing by-products, extends the food processing industry chain, drives the development of food economy, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the construction of Scxyn22-NFP and Scxyn39-NFP fusion proteins.

[0026] Figure 2 It is an SDS-PAGE electrophoresis diagram; wherein, lane 1: Scxyn22, 2: Paxyn28, 3: Scxyn39, 4: Scxyn22-NFP, 5: Scxyn39-NFP, 6: Scabf32.

[0027] Figure 3 This is the result of determining the optimum pH value of the recombinant enzyme.

[0028] Figure 4 This is the result of the optimum temperature determination of the recombinase.

[0029] Figure 5 This is the apparent viscosity diagram of CFG enzymatic hydrolysis products.

[0030] Figure 6 This is the apparent viscosity diagram of the CFG-abf32 enzymatic hydrolysate. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are 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 are within the scope of protection of the present invention.

[0032] If no specific techniques or conditions are specified in the examples, all are carried out according to conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.

[0033] The materials and reagents in the following examples are as follows: 1. Strains and vectors: expression host Pichia pastorisGS115, expression plasmid vector pPIC9K; 2. Enzymes and biochemical reagents: Endonucleases were purchased from New England Biolabs, recombinant enzymes were purchased from Quanshijin, birchwood xylan was purchased from Sigma, wheat arabinoxylan was purchased from Megazyme, and corn husks were purchased from Hebei Guangyu Starch Company. Other reagents were domestic analytical reagents (all available from ordinary biochemical reagent companies). 3. Culture medium: (1) BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (v / v), 1.34% YNB, 0.00004% Biotin; (2) BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, and 0.5% methanol (v / v).

[0034] Example 1 Construction of Scxyn22-NFP and Scxyn39-NFP fusion proteins

[0035] Scxyn22 (SEQ ID No.6), Scxyn39 (SEQ ID No.8) and NFP genes (non-functional protein, lipase in this embodiment, the gene sequence is shown in SEQ ID No.10, the lipase is derived from lipase B (CALB) of Candida antarctica), and the xylanase gene is fused with the NFP gene by fusion PCR to obtain two recombinant fusion genes Scxyn22-linker-NFP and Scxyn39-linker-NFP (linker nucleic acid sequence is shown in SEQ ID No.9); the schematic diagram of the construction of Scxyn22-NFP and Scxyn39-NFP fusion proteins is shown in FIG. Figure 1 shown.

[0036] Example 2 Construction of Pichia pastoris expression recombinant vector

[0037] The full length of 7 genes, including Scxyn22, Paxyn28 (SEQ ID No.7), Scxyn39, Scabf32 (amino acid sequence is shown in SEQ ID No.11, nucleic acid sequence is shown in SEQ ID No.12), Scxyn22-linker-NFP and Scxyn39-linker-NFP, were amplified by PCR. The recombinant vector for Pichia pastoris expression was a yeast expression vector pPIC9K (Invitrogen, USA, catalog number V17520) in which the above 7 gene sequences were replaced. Eco R Ⅰ and Not Ⅰ DNA molecules between the double restriction sites. The recombinant vector was transformed into TransI-T1 competent cells, and the transformants were selected for sequencing verification. The transformants verified by sequencing were the recombinant yeast expression plasmids pPIC9K-Scxyn22, pPIC9K-Paxyn28, pPIC9K-Scxyn39, pPIC9K-Scabf32, pPIC9K-Scxyn22-NFP and pPIC9K-Scxyn39-NFP. The amplification of the recombinase gene and the construction of the recombinant vector were referred to Liu Yuchun et al. Transcriptome analysis of Schizophyllum schizophyllum fermented with corn husk fiber and heterologous expression of recombinant α-L-arabinofuranosidase [J]. Food and Fermentation Industries, 2019, 45(23): 21-28.

[0038] Example 3 Construction, induced expression and purification of Pichia pastoris engineered bacteria

[0039] The recombinant yeast expression plasmids pPIC9K-Scxyn22, pPIC9K-Paxyn28, pPIC9K-Scxyn39, pPIC9K-Scabf32, pPIC9K-Scxyn22-NFP and pPIC9K-Scxyn39-NFP were cleaved with endonuclease SacI was linearized and transformed into Pichia pastoris GS115 to obtain recombinant yeast strains GS115 / pPIC9K-Scxyn22, GS115 / pPIC9K-Paxyn28, GS115 / pPIC9K-Scxyn39, GS115 / pPIC9K-Scabf32, GS115 / pPIC9K-Scxyn22-NFP and GS115 / pPIC9K-Scxyn39-NFP. The positive transformants were picked and transferred to 50 mL Erlenmeyer flasks containing 5 mL BMGY medium and cultured at 30 ℃ and 200 rpm for 36 h; the fermentation broth was centrifuged at 3000g for 5 min, the supernatant was discarded, and the precipitated bacteria were resuspended in 5 mL BMMY medium and then induced at 30 ℃ and 200 rpm for 180 h. After induction culture, the fermentation broth supernatant was collected after centrifugation at 10000×g for 10 min. The fermentation supernatant (crude enzyme solution) was dialyzed for 16 h with 20 mM pH 5.0 citric acid-sodium citrate buffer (buffer A, citric acid monohydrate and trisodium citrate dihydrate), and then purified using an anion exchange chromatography column HiTrap QFF (Cytiva, Marlborough, MA, USA) at a flow rate of 0.5 mL / min to obtain pure enzyme. After loading, the sample was eluted with buffer A (flow rate of 1.0 mL / min), and then linearly eluted with buffer A containing 0-500 mM NaCl for 10 column volumes, and the enzyme solution was collected to obtain the purified recombinant lipase (pure enzyme solution). The purified recombinant enzyme was detected by SDS-PAGE electrophoresis ( Figure 2 ). The electrophoresis results showed that the molecular weight of Scxyn22 was 22 KDa, the molecular weight of Paxyn28 was 28 KDa, the molecular weight of Scxyn39 was 39 KDa, the molecular weight of Scxyn22-NFP was 53.5 KDa, and the molecular weight of Scxyn39-NFP was 71.5 KDa, a total of 5 xylanolytic enzymes with different molecular weights, and the molecular weight of arabinofuranosidase Scabf32 was 32 KDa.

[0040] Example 4 Enzyme Activity Analysis

[0041] The enzymatic activity of xylanase was detected as follows: 200 μL of the enzymatic reaction system contained 190 μL of substrate (0.5%, m / v, wheat arabinoxylan) and 10 μL of appropriately diluted recombinant xylanase solution, and the reaction was kept at the optimal temperature and optimal pH for 5 min, followed by the addition of 300 μL of DNS reagent and boiling in boiling water for 5 min. After the sample was cooled, the absorbance at 540 nm was measured. One unit of enzyme activity (U) is defined as the amount of enzyme required to generate 1 μmol of reducing end per minute under given conditions.

[0042] The enzyme activity detection method of arabinofuranosidase is as follows: 25 μL 4 mM p-nitrophenyl α-L-arabinofuranoside (pNPAF) solution, 15 μL 0.1 M pH 6.0 citric acid buffer solution, and 10 μL of appropriately diluted enzyme solution are added. Reaction conditions: The mixture is placed in a 40°C water bath for 10 min, and 150 μL 1 M Na2CO3 is quickly added to terminate the reaction, and then the absorbance is measured at 405 nm. The enzyme solution inactivated in a boiling water bath for 10 min is used as a blank control. A standard curve is prepared with p-nitrophenol to calculate the p-nitrophenol content produced in the enzymatic reaction system. Definition of enzyme activity: The amount of enzyme required to produce 1 μM p-nitrophenol per minute is one unit of enzyme activity.

[0043] 1. Determination of the optimal pH of recombinant enzyme

[0044] The recombinant enzyme purified in Example 3 was subjected to enzymatic reaction at different pH values ​​to determine its optimum pH. The substrate was dissolved in buffers of different pH values, which were: 0.1 mol / L Gly-HCl buffer solution, 1.0-3.0; 0.1 mol / L citric acid buffer solution, 3.0-7.0; 0.1 mol / L NaH2PO4-Na2HPO4 buffer solution, 6.0-8.0; 0.1 mol / L Tris-HCl buffer solution, 7.5-8.5; 0.1 mol / L Gly-NaOH buffer solution, 8.5-10.0. The activity of the recombinant enzyme was determined at 40°C.

[0045] The results showed that the optimal reaction pH of the recombinant xylanase Scxyn22 was about 4.0; the optimal reaction pH of the recombinant xylanase Paxyn28 was about 4.0; the optimal reaction pH of the recombinant xylanase Scxyn39 was about 6.0; the optimal reaction pH of the recombinant enzyme Scxyn22-NFP was about 4.0; the optimal reaction pH of the recombinant enzyme Scxyn39-NFP was about 6.0; the optimal reaction pH of the recombinant enzyme Scabf32 was about 5.0-6.0 ( Figure 3 ).

[0046] 2. Determination of the optimal temperature of recombinase

[0047] The optimum temperature of the recombinant enzymes was determined by performing enzymatic reactions in 0.1 mol / L citric acid buffer at the optimum pH buffer system and at different temperatures. The results showed that the optimum temperature of the recombinant xylanase Scxyn22 was around 50°C; the optimum temperature of the recombinant xylanase Paxyn28 was around 60°C; the optimum temperature of the recombinant xylanase Scxyn39 was around 70°C; the optimum temperature of the recombinant enzyme Scxyn22-NFP was around 60°C; the optimum temperature of the recombinant enzyme Scxyn39-NFP was around 50°C; the optimum temperature of the recombinant enzyme Scabf32 was around 60°C ( Figure 4 The conditions for catalytic hydrolysis of corn fiber gum (CFG) were determined according to the optimal pH and temperature of the recombinant enzyme.

[0048] Example 5 Extraction of corn fiber gum

[0049] 1) Dry and crush corn husks and pass through a 60-mesh sieve. Weigh 50 g corn husk powder, add 500 mL n-hexane, stir for 2 h, and filter with a Buchner funnel. Wash the sample with an appropriate amount of anhydrous ethanol, filter and dry, add distilled water at a solid-liquid ratio of 1:10 (w / v), adjust the pH to 6.0, add thermostable α-amylase to the system, treat in a 95°C water bath for 30 min, reduce the water bath temperature to 55°C, add saccharifying enzyme, react for 30 min, and use I2-KI solution to check the starch hydrolysis. Add neutral protease to the above-mentioned treatment solution, react for 1 h at a water bath temperature of 55°C and a pH of 7, heat to 100°C to inactivate the enzyme for 5 min, filter with gauze, wash the filter residue twice with distilled water, and dry at 60°C for use.

[0050] 2) Weigh 50 g of the sample obtained in 1), add 2 g of NaOH and 1.9 g of Ca(OH)2, add 0.5 L of distilled water, mix and boil in a boiling water bath for 1 h. Cool the reaction solution to room temperature, centrifuge at 6000×g for 20 min, and separate the supernatant A1 and the precipitate B1.

[0051] 3) The pH value of the supernatant A1 was adjusted to 4.0, and the supernatant was separated by centrifugation at 10,000 × g for 30 min; 2 volumes of anhydrous ethanol were added to the supernatant to obtain a white flocculent precipitate CFG1.

[0052] 4) Add an appropriate amount of distilled water to the precipitate B1, adjust the pH to 4.0-4.5, and then centrifuge at 10,000 × g for 30 min. After separating the supernatant, add 2 times the volume of anhydrous ethanol to obtain a white flocculent precipitate CFG2.

[0053] 5) Dissolve CFG1 and CFG2 in distilled water, mix them, and freeze-dry them to obtain corn fiber gum CFG.

[0054] Example 6 Preparation of arabinoxylan by hydrolyzing CFG with xylanases of different molecular weights

[0055] According to the optimum pH determined in Example 4, buffers with different pH values ​​were prepared and CFG substrate was dissolved at a concentration of 0.25% (w / v). Recombinant enzymes Scxyn22, Paxyn28, Scxyn39, Scxyn22-NFP, and Scxyn39-NFP with the same unit enzyme activity were added, respectively, and the enzymes were inactivated by water bathing at the optimum temperature determined in Example 4 for 24 h, and then in a boiling water bath. The hydrolyzate was used for the analysis of monosaccharide component composition and molecular weight characteristics.

[0056] Example 7 Synergistic hydrolysis of CFG by arabinofuranosidase and xylanases of different molecular weights

[0057] 200 mg CFG was dissolved in 30 mL citric acid buffer (pH 4.0), and the recombinant enzyme Scabf32 was added. The mixture was incubated in a 40°C water bath for 24 h and then inactivated in a boiling water bath. Two volumes of anhydrous ethanol were then added, and the flocculent precipitate was separated by centrifugation. CFG-abf32 was obtained after freeze-drying.

[0058] According to the optimum pH determined in Example 4, buffers with different pH values ​​were prepared and CFG-abf32 was dissolved at a concentration of 0.25% (w / v). Recombinant enzymes Scxyn22, Paxyn28, Scxyn39, Scxyn22-NFP, and Scxyn39-NFP with the same unit enzyme activity were added, respectively, and the enzymes were inactivated by water bathing at the optimum temperature determined in Example 4 for 24 h, and then in a boiling water bath. The hydrolyzate was used for the analysis of monosaccharide component composition and molecular weight characteristics.

[0059] Example 8 Analysis of Monosaccharide Composition

[0060] The monosaccharide components in the hydrolyzate of the above example were determined using an Agilent 1200 with an ultraviolet detector, and the results are shown in Table 1. The chromatographic conditions were as follows: the chromatographic column was Agilent C18 (4.6 mm×250 mm×5 μm), the mobile phase A was 0.1 mol / L KH2PO4 (pH 6.8), the mobile phase B was acetonitrile, the mobile phase gradient was mobile phase A: mobile phase B = 82:18, isocratic elution, the flow rate was 1.0 mL / min, and the column temperature was 25°C.

[0061] Table 1 Monosaccharide composition characteristics

[0062] The results showed that the molar ratios of xylose, arabinose, galactose, glucuronic acid, glucose, galacturonic acid, rhamnose, ribose, and fucose in CFG were 1279:1324:337:311:49:18:10:4:1. The molar ratios of xylose, arabinose, galactose, glucuronic acid, glucose, galacturonic acid, rhamnose, ribose, and fucose in CFG-abf32 were 336.6:259.4:81.4:67.8:12.5:3.6:2.6:1.0:4.4 (Table 1).

[0063] The molar ratio of arabinose / xylose of the reaction substrate CFG is 1.03, that is, the side chain substitution degree (Ara / Xyl) is 1.03; the molar ratio of arabinose / galactose is 3.92. After treatment with arabinofuranosidase Scabf32, the molar ratio of arabinose / xylose of CFG-abf32 is significantly reduced to 0.77, and the molar ratio of arabinose / galactose is 3.19. Therefore, after hydrolysis by arabinofuranosidase, the branching degree of CFG-abf32 is reduced, providing space for xylanase to bind to the substrate.

[0064] Example 9 Molecular Weight Characteristic Analysis

[0065] Gel permeation chromatography was used to determine the molecular weight characteristics of CFG and various hydrolysis products, and the results are shown in Table 2. The chromatographic conditions were: high performance liquid chromatograph Waters 1525, differential detector Waters 2414, chromatographic column PL aquqgel-OHMIXED-H 8μm, mobile phase 0.2 M NaNO3, flow rate 1 mL / min, column temperature 30℃, sample volume 20 μL.

[0066] Table 2 Molecular weight characteristic parameters

[0067] The results showed that the molecular weight of CFG was 372.1 KDa. Recombinant enzymes Scxyn22, Paxyn28, Scxyn39, Scxyn22-NFP, and Scxyn39-NFP hydrolyzed CFG to produce five kinds of arabinoxylans, which were 361.3 KDa, 307.6 KDa, 192.8 KDa, 310.6 KDa, and 165.0 KDa, respectively. Recombinant enzymes Scxyn22, Paxyn28, Scxyn39, Scxyn22-NFP, and Scxyn39-NFP hydrolyzed CFG-abf32 to produce five kinds of arabinoxylans with lower weights, which were 85.5 KDa, 57.1 KDa, 34.5 KDa, 62.7 KDa, and 32.1 KDa, respectively. The molecular weight order of the arabinoxylans in the hydrolysis products was CFG>C1>C4>C2>C3>C5>A1>A4>A2>A3>A5. This result showed that a series of arabinoxylans with different molecular weights were obtained by controlling the molecular weight of xylanase and the degree of branching of xylan substrate.

[0068] Example 10 Rheological Characteristics Analysis

[0069] The rheological properties of arabinoxylan determine its application areas. For example, low molecular weight arabinoxylan affects the water holding capacity of dough, and high molecular weight arabinoxylan significantly affects the extensibility of dough. Therefore, this example analyzes the rheological characteristics of the enzymatic hydrolysis product.

[0070] According to the optimal pH determined in Example 4, buffer solutions of different pH values ​​were prepared and the CFG substrate and the arabinofuranosidase hydrolysis product (CFG-abf32) were dissolved at a concentration of 2% (w / v). Excess amounts of recombinant enzymes Scxyn22, Paxyn28, Scxyn39, Scxyn22-NFP, and Scxyn39-NFP were added respectively, and the mixture was incubated in a water bath for 48 h at the optimal temperature determined in Example 4 to allow the substrate to be fully hydrolyzed.

[0071] The apparent viscosity was measured using a rotational rheometer (AR2000ex). A parallel plate measuring system with a diameter of 40 mm was selected, the sample stage temperature was set to 25 °C, the plate spacing was 1.0 mm, and the shear rate was varied from 0.1 s -1 Increase to 100 s -1 The results showed that the hydrolysates with different molecular weights had different apparent viscosity characteristics. The apparent viscosity of CFG hydrolysates was in the order of CFG>C4>C1>C2>C3, and the apparent viscosity of C3 and C5 was similar ( Figure 5 The apparent viscosity of the hydrolyzed products of CFG-abf32 is in the order of CFG-abf32>A4>A1>A2>A5>A3 ( Figure 6). The apparent viscosity order of the CFG hydrolysate was similar to that of the CFG-abf32 hydrolysate, and the results showed that the apparent viscosity of substrates with different arabinose / xylose ratios changed consistently after being hydrolyzed by xylans with different molecular weights. The apparent viscosity order of the hydrolysate was not completely consistent with the molecular weight order obtained in Example 9 (CFG>C1>C4>C2>C3>C5>A1>A4>A2>A3>A5), which may be because the apparent viscosity of polysaccharides is related to the number and complexity of polysaccharide branches in addition to the molecular weight.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant fusion xylanase, characterized in that: It includes single domain xylanases and protein domains without substrate catalytic / binding functions.

2. The recombinant fusion xylanase according to claim 1, characterized in that: The protein without substrate catalysis / binding function is lipase.

3. The recombinant fusion xylanase according to claim 2, characterized in that: The recombinant fusion xylanase comprises a single-domain xylanase, a linker and a lipase which are connected in sequence.

4. The recombinant fusion xylanase according to claim 1, characterized in that: The single domain xylanase is Scxyn22, Paxyn28 or Scxyn39.

5. The recombinant fusion xylanase according to any one of claims 1 to 4, characterized in that: The amino acid sequence of Scxyn22 is shown in SEQ ID No.1; the amino acid sequence of Paxyn28 is shown in SEQ ID No.2; the amino acid sequence of Scxyn39 is shown in SEQ ID No.3; the amino acid sequence of Linker is shown in SEQ ID No.4; the amino acid sequence of the protein without substrate catalysis / binding function is shown in SEQ ID No.

5.

6. A method for preparing arabinoxylans with different molecular weights and structural characteristics, characterized in that: include: The single-domain xylanase Scxyn22, Paxyn28, Scxyn39 or the recombinant fusion xylanase described in any one of claims 1 to 5 is used to hydrolyze corn fiber gum to obtain arabinoxylan with different molecular weights and structural characteristics.

7. The method according to claim 6, characterized in that For the same single-domain xylanase, the larger the molecular weight of the recombinant fusion xylanase used in hydrolyzing corn fiber gum, the smaller the weight average molecular weight of the obtained arabinoxylan and the narrower the molecular weight distribution.

8. The method according to claim 6, characterized in that Also includes: The corn fiber gum is firstly enzymatically hydrolyzed by using arabinofuranosidase Scabf32, and then the arabinofuranosidase Scabf32 is inactivated to obtain an enzymatic hydrolysis product; and then the enzymatic hydrolysis product is hydrolyzed by using the single-domain xylanase or the recombinant fusion xylanase.

9. The method according to claim 8, characterized in that The weight average molecular weight of the obtained arabinoxylan is below 90 KDa, the number average molecular weight is below 50 KDa, and the peak molecular weight is below 80 g / mol.

10. The method according to any one of claims 6 to 9, characterized in that: The reaction pH of Scxyn22 is 3.8~4.2; the reaction pH of Paxyn28 is 3.8~4.2; the reaction pH of Scxyn39 is 5.8~6.2; the reaction pH of the recombinant fusion xylanase containing Scxyn22 is 3.8~4.2; the reaction pH of the recombinant fusion xylanase containing Scxyn39 is 5.8~6.2; the reaction pH of Scabf32 is 5.0~6.0; And / or, the reaction temperature of Scxyn22 is 48~52°C; the reaction temperature of Paxyn28 is 58~62°C; the reaction temperature of Scxyn39 is 68~72°C; the reaction temperature of the recombinant fusion xylanase containing Scxyn22 is 58~62°C; the reaction temperature of the recombinant fusion xylanase containing Scxyn39 is 48~52°C; the reaction temperature of Scabf32 is 58~62°C.

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