FvAbnA protein with arabinase activity and application of FvAbnA protein

Abstract

The invention discloses FvAbnA protein with arabinase activity and application of the FvAbnA protein, and belongs to the technical field of biological enzymes. The invention finds that the FvAbnA protein from fusarium verticillium has arabinase activity, is high in enzyme activity and good in stability, and can efficiently degrade araban for the first time; when being combined with cellulase, the bacillus subtilis can synergistically improve the straw degradation efficiency, and has a wide application prospect in the aspects of efficiently developing and utilizing crop straw biomass as livestock feed and industrial ethanol raw materials.

Description

Technical Field

[0001] The invention relates to the technical field of biological enzymes, and in particular to a FvAbnA protein with arabinanase activity and an application thereof. Background Art

[0002] Crop straw is one of the important sources of animal husbandry feed due to its low price, wide availability and rich nutrition. In particular, the demand for straw feed is huge in the breeding process of ruminants such as cattle and sheep. However, the high crude fiber content and strong polymer structure of crop straw make it difficult for animals to quickly digest and fully absorb, which greatly limits the application of straw as feed and needs to be solved urgently.

[0003] Arabinan is a type of cell wall structural polysaccharide widely found in gramineous plants such as wheat, rice, corn and sorghum. It is composed of L-arabinose residues linked by α-1,5-glycosidic bonds and is an important component of the plant cell wall's anti-degradation barrier. Arabinan can also form complex poly-derivatives with other sugars, such as galactoarabinan, arabinoxylan, and arabinogalacturonic acid. The complex structure and tight binding of these natural organic polymer compounds are one of the important reasons why straw is difficult to effectively degrade and utilize.

[0004] Arabinase (EC3.2.1.99) is an endonuclease that specifically degrades arabinan, also known as "1,5-α-L-arabinanase". It hydrolyzes α-1,5-glycosidic bonds to decompose high-molecular-weight arabinan into arabino-oligosaccharides with a degree of polymerization of 2 to 5. These oligosaccharides can be absorbed and utilized by animals as prebiotics, and can also be used for the fermentation and production of bioethanol. They have important application value in the feed industry and the bioethanol industry, and have become a new hot spot in the field of bioenzyme research.

[0005] At present, arabinanase is mainly derived from bacteria, such as thermophilic denitrifying Bacillus, Bacillus licheniformis, Bacillus polymyxa, Bacillus crispatus, etc. However, there are still few fungal resources that produce arabinanase, and further exploration and development are urgently needed. Studies have shown that Fusarium verticillioides can secrete a variety of biomass degrading enzymes including cellulase, xylanase and ligninase, making it play an important role in the development of crop straw. However, arabinanase in Fusarium verticillioides has not yet been studied and utilized. Therefore, it is of great practical significance to explore the arabinanase with high activity and good stability in Fusarium verticillioides and apply it to the feed utilization of crop straw and industrial ethanol production, which can reduce the waste of biomass resources, improve feed utilization, and alleviate energy shortage. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide a FvAbnA protein with arabinanase activity and its application. The present invention has found that the FvAbnA protein from Fusarium verticillium has arabinanase activity, has high enzyme activity and good stability, and can efficiently degrade arabinan; when used in combination with cellulase, it can synergistically improve the degradation efficiency of straw.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides the use of FvAbnA protein in any one of the following (1)-(3):

[0009] (1) Degradation of arabinan;

[0010] (2) Synergistically degrade straw with cellulase;

[0011] (3) preparing additives for straw feed;

[0012] The amino acid sequence of the FvAbnA protein is shown in SEQ ID No.1.

[0013] In the above application, the FvAbnA protein degrades arabinan by hydrolyzing α-1,5-glycosidic bonds.

[0014] In the above application, the straw includes but is not limited to: rice straw, wheat straw, corn straw and sorghum straw.

[0015] The second aspect of the present invention provides the use of the FvAbnA gene in producing a biological enzyme having arabinanase activity;

[0016] The FvAbnA gene is a nucleic acid molecule as shown in the following i) or ii):

[0017] i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID No. 2;

[0018] ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 1 other than i).

[0019] The third aspect of the present invention provides the use of a recombinant expression vector or an engineered bacterium containing the FvAbnA gene in producing a biological enzyme having arabinanase activity.

[0020] Preferably, the recombinant expression vector is a yeast eukaryotic expression vector pPIC9K containing the FvAbnA gene.

[0021] The fourth aspect of the present invention provides a combined enzyme for degrading straw, wherein the combined enzyme is composed of FvAbnA protein and cellulase in a mass ratio of 1:(3-20).

[0022] Preferably, the combined enzyme consists of FvAbnA protein and cellulase in a mass ratio of 1:(5-10).

[0023] The FvAbnA protein and cellulase are compounded and used in a certain ratio, which has a significant synergistic degradation effect on straw and can greatly increase the amount of reducing sugars produced by degrading straw.

[0024] The fifth aspect of the present invention provides the use of the above enzyme combination in the preparation of straw feed additives.

[0025] A sixth aspect of the present invention provides a method for collaboratively degrading straw, comprising the following steps:

[0026] The above enzyme combination was added to the straw substrate and incubated at 50-60°C for 4-8h.

[0027] In the above method, the straw substrate is rice straw or wheat straw.

[0028] Beneficial effects of the present invention:

[0029] (1) The present invention first discovered that the FvAbnA protein from Fusarium oxysporum has arabinanase activity and can degrade arabinan by hydrolyzing α-1,5-glycosidic bonds. In addition, the FvAbnA protein has high enzyme activity and strong thermal stability and acid-base stability.

[0030] (2) The FvAbnA protein of the present invention can synergistically degrade crop straw with cellulase, and has broad application prospects in the efficient development and utilization of crop straw biomass as livestock feed and industrial ethanol raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : SDS-PAGE electrophoresis of FvAbnA protein.

[0032] Figure 2 : Investigation of the optimal hydrolysis reaction temperature (a) and the optimal hydrolysis reaction pH (b) of FvAbnA protein.

[0033] Figure 3 : Temperature stability (a) and pH stability (b) of FvAbnA protein.

[0034] Figure 4 :FvAbnA protein and cellulase synergistically degrade wheat straw.

[0035] Figure 5:FvAbnA protein and cellulase synergistically degrade rice straw. DETAILED DESCRIPTION

[0036] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0037] Terminology Note:

[0038] Straw feed: refers to the fiber feed made from crushed straw of rice, wheat, corn, sweet sorghum, cotton, etc., which is the main feed for ruminants.

[0039] As mentioned above, arabinan is one of the important reasons why straw is difficult to effectively degrade and utilize. Arabinanase can decompose high-molecular-weight arabinan into arabino-oligosaccharides with a degree of polymerization of 2 to 5 by hydrolyzing α-1,5-glycosidic bonds, and thus has become a hot topic in the field of bioenzyme research. Fungi as enzyme-producing strains have the advantages of stable properties and low degradation, and are an important choice for enzyme-producing strains, but there are currently few fungal resources that produce arabinanase.

[0040] In view of this, the present invention has conducted an in-depth study on the pseudo-verticillioides fungus resources. The hypothetical protein FVEG_10735[Fusarium verticillioides 7600](XP_018758065.1) is a protein with unknown function in pseudo-verticillioides 7600. The present invention analyzes the structure of the FVEG_10735 protein and then performs a truncation process. The truncated protein is named FvAbnA protein, and its amino acid sequence is shown in SEQ ID No. 1, which is as follows:

[0041] KPAQKSKTFSNVEIFDPPSNYNDPQVLYARPLELSDGTLATWENYSPEPPHVWFPIVRSKDGGKTWKPFSKVQDTQNNWGLRYQPQLYELPRRFGKYPKGTVLISGSSIPTDLSETLIEVYASLDKGETWEFVSHVALGGEAKPEHGLTPVWEPFLMTYKEKLILYYSDQRDNATHSQKLVH QTTTTDLKKWSKVVEDTKYDDYYARPGMPTVAKLPNNEYIYTYEYGGGPNPPAGSTYWFPVFYRLSKDPQKFLNKPGQQIISTDGARPAGSPYVVWTPYGGKNGTIVVSCGTMSEIFTNQALGDASAWKKWSVPQPTAYTRALMTFKKDPDLLMIMGAGILPPAGGNNTVSASVVRLSEIMKA.

[0042] The nucleotide sequence of the FvAbnA gene encoding the protein is shown in SEQ ID No. 2, and is as follows:

[0043]

[0044] The present invention firstly utilizes a yeast eukaryotic expression system in combination with an AKTA protein purification system to obtain a high-purity recombinant FvAbnA protein, and then investigates the enzyme activity and stability of the FvAbnA protein. It is found that the FvAbnA protein can specifically hydrolyze arabinan and has good stability, and has high stability under high temperature and neutral and acidic conditions.

[0045] Further research found that the FvAbnA protein can synergistically degrade straw with cellulase, greatly increasing the amount of reducing sugars produced by degrading straw. Therefore, the FvAbnA protein is of great significance for the utilization of crop straw as feed, and thus the present invention is proposed.

[0046] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments. The test materials used in the embodiments of the present invention are all conventional test materials in the field and can be purchased through commercial channels. Among them:

[0047] The F. verticillioides used in the examples of the present invention is the standard strain Fv7600 of F. verticillioides.

[0048] The culture medium and its components used in the embodiments of the present invention are as follows:

[0049] BMGY medium: yeast powder: 1.0 g, peptone: 2.0 g, YNB: 1.34 g, 0.1 mol / L pH 7.4 phosphate buffer, glycerol: 1.0 mL, add distilled water to 100 mL.

[0050] BMMY medium: yeast powder: 1.0 g, peptone: 2.0 g, YNB: 1.34 g, 0.1 mol / L pH 7.4 phosphate buffer, add 1 mL of methanol to every 100 mL of medium after high temperature sterilization.

[0051] Arabinan was produced by Macklin Company, product number A994581-1G.

[0052] Cellulase was produced by Sigma-Aldrich Company, product number C8546-500G.

[0053] Example 1: Expression and purification of FvAbnA protein

[0054] 1. Test method:

[0055] Extract the mycelial RNA of the standard strain Fv7600 of Fusarium verticillium, design the specific primer of FvAbnA gene, the sequence of the specific primer is as follows:

[0056] FvAbnA-F: CCGGAATTCAAGCCTGCTCAGAAGAGCAAGA; (SEQ ID No. 3)

[0057] FvAbnA-R:TTGCGGCCGCTCAGTGGTGGTGGTGGTGGTGAGCCTTCATGATCTC AGA. (SEQ IDNo.4)

[0058] The amplified product with EcoRI and NotI restriction sites at both ends was obtained by RT-PCR amplification, and the amplified product was ligated to the yeast expression vector pPIC9K that had been double-digested with EcoRI and NotI to obtain the yeast expression vector pPIC9K / FvAbnA carrying the target gene.

[0059] The yeast expression vector pPIC9K / FvAbnA was linearized with restriction endonuclease SalI, and then transformed into yeast competent cells by electroporation to obtain yeast engineering bacteria.

[0060] The activated yeast engineering bacteria were inoculated into BMGY medium and cultured at 28°C with shaking until the bacterial solution OD 600 When the pH was 1.6, the culture solution was transferred to BMMY medium after centrifugation to start inducing expression, and 1 mL of methanol was added every 12 h for 7 consecutive days. The culture solution was centrifuged and the supernatant crude enzyme solution was collected; the supernatant crude enzyme solution was precipitated, dialyzed and separated and purified by nickel column (HisTrap HP column (GE Healthcare, Buckinghamshire, United Kingdom)).

[0061] The molecular weight of the separated and purified protein was detected by SDS-PAGE denaturing electrophoresis. The operation steps and staining and decolorization process refer to the instructions of the gel preparation kit (produced by Kangwei Century Biotechnology Co., Ltd., item number CW0022M). After the electrophoresis is completed, the useless concentrated gel is removed, and the separation gel part is stained with Coomassie brilliant blue and decolorized. After the decolorization is completed, the gel is placed on the film observation light for observation and analysis of whether the required protein is expressed and the separation and purification is successful.

[0062] 2. Test results:

[0063] The results of SDS-PAGE electrophoresis of the purified protein are as follows Figure 1 shown.

[0064] The purified protein was sequenced and its amino acid sequence was shown in SEQ ID No. 1. The purified protein was named FvAbnA protein.

[0065] Example 2: Performance evaluation of FvAbnA protein

[0066] 1. Determination of FvAbnA protein concentration:

[0067] The corresponding parameters (extinction coefficient and relative molecular weight) were set on the spectrophotometer, 1.5 μL of pure water was first drawn to adjust the zero, and 1.5 μL of the enzyme solution purified in Example 1 was drawn to measure the protein concentration.

[0068] The results showed that the concentration of FvAbnA protein was 5.25 mg / mL, indicating that the Pichia pastoris expression system successfully expressed the FvAbnA protein.

[0069] The concentration of FvAbnA protein was adjusted to 0.2 mg / mL and used as enzyme solution for subsequent investigations on enzyme activity, optimal reaction temperature, optimal reaction pH, stability, etc.

[0070] 2. Investigation of the optimal hydrolysis conditions of enzymes:

[0071] Preliminary experiments revealed that the FvAbnA protein had specific hydrolysis activity on arabinan, and the optimal hydrolysis conditions (optimal reaction temperature, optimal reaction pH) of the FvAbnA protease solution were further investigated.

[0072] (1) Investigation of the optimum reaction temperature:

[0073] 150 μL of the reaction system (50 μL of arabinan + 50 μL of buffer + 50 μL of enzyme solution) was reacted at different temperatures (30°C-90°C) at pH = 7.0 for 30 min, an equal volume of DNS was added and boiled for 8 min, and after cooling to room temperature, the absorbance of the sample at 540 nm was measured using a spectrophotometer; the enzyme inactivated by boiling at 100°C was used as the OD 540 The enzyme activity was calculated by comparing with the arabinose standard curve. The relative enzyme activity under different temperature conditions was determined and analyzed.

[0074] The relative enzyme activity is: the enzyme activity value of the sample with the highest enzyme activity in the treatment is calibrated as 100%, and the value of enzyme activity of each treatment sample / enzyme activity of the highest sample is the relative enzyme activity.

[0075] The results showed that the optimal hydrolysis temperature of FvAbnA was 55°C ( Figure 2 a).

[0076] (2) Investigation of the optimal reaction pH:

[0077] 150 μL of reaction system (50 μL of arabinan + 50 μL of buffer + 50 μL of enzyme solution) was reacted at 55°C for 30 min under different pH conditions (pH 3-11), and an equal volume of DNS was added and boiled for 8 min. After cooling to room temperature, the absorbance of the sample at 540 nm was measured using a spectrophotometer. The enzyme inactivated by boiling at 100°C was used as the OD 540 The enzyme activity was calculated by comparing with the arabinose standard curve. The relative enzyme activity under different pH conditions was determined and analyzed.

[0078] The results showed that the optimal hydrolysis pH of FvAbnA was 5 ( Figure 2 b).

[0079] 3. Determination of enzyme activity under optimal hydrolysis conditions:

[0080] The unit of enzyme activity is defined based on the amount of substrate reduced or product generated per unit time under optimal conditions. In 1961, the International Commission on Enzymology unified the definition: Under standard conditions, the amount of enzyme that catalyzes the conversion of 1 μmol substrate in one minute is one unit of enzyme activity, i.e., the international unit (IU).

[0081] 150 μL of reaction system (50 μL of arabinan + 50 μL of buffer + 50 μL of enzyme solution) was reacted at 55°C, pH = 5.0 for 30 min. 150 μL of DNS was added and boiled in water for 8 min, cooled to room temperature, and OD was measured by spectrophotometry. 540 The enzyme was inactivated by boiling at 100℃ as the OD value. 540 The standard curve of arabinose was prepared with different concentrations of L-arabinose. The enzyme activity was calculated by comparing with the standard curve of arabinose. The data analyzed in this study were obtained by at least three independent repeated experiments.

[0082] The results showed that FvAbnA specifically hydrolyzed arabinan with an enzyme activity of 428.59 IU / mg.

[0083] 4. Determination of enzyme thermal stability:

[0084] After the enzyme solution was pretreated at different temperature conditions (50°C, 60°C, 70°C, 80°C) for 0-180 min, the treated enzyme solution was prepared into a 150 μL reaction system (50 μL arabinan + 50 μL Buffer + 50 μL enzyme solution), reacted for 30 min at pH = 5, an equal volume of DNS was added and boiled for 8 min, and after cooling to room temperature, the absorbance of the sample at 540 nm was detected by a spectrophotometer to determine and analyze the relative enzyme activity.

[0085] The results showed that after treatment at 50°C for 180 minutes, the enzyme activity of FvAbnA did not change significantly; under high temperature conditions of 60°C-80°C, the enzyme activity of FvAbnA gradually decreased with the extension of treatment time. After treatment at 70°C and 80°C for 60 minutes, the residual enzyme activity of FvAbnA remained above 30% and 60% respectively; but after treatment at 80°C for 150 minutes, the enzyme activity of FvAbnA was almost undetectable ( Figure 3 a).

[0086] In summary, it was shown that FvAbnA had good thermal stability and could maintain high enzyme activity for a long time under high temperature conditions.

[0087] 5. Determination of pH stability of enzyme:

[0088] After the enzyme solution was pretreated at different pH conditions (3, 5, 7, 9, 11) for 0-96h, the treated enzyme solution was prepared into a 150μL reaction system (50μL arabinan + 50μL Buffer + 50μL enzyme solution), reacted at 55°C for 30min, an equal volume of DNS was added and boiled for 8min, and after cooling to room temperature, the absorbance of the sample at 540nm was detected by a spectrophotometer to determine and analyze the relative enzyme activity.

[0089] The results showed that FvAbnA could maintain full enzyme activity for a long time (96 hours) under neutral conditions (pH 7). In addition, FvAbnA's acid resistance was higher than its alkali resistance. After being treated at pH 5 and pH 3 for 96 hours, the enzyme activity of FvAbnA remained above 70% and 50%, respectively. After being treated at pH 11 for 96 hours, the remaining enzyme activity of FvAbnA exceeded 30% ( Figure 3 b).

[0090] In summary, it was shown that FvAbnA had high stability under neutral and acidic conditions.

[0091] Example 3: Investigation of the synergistic effect of FvAbnA protein and cellulase

[0092] 1. Test method:

[0093] (1) Substrate pretreatment

[0094] The rice and wheat straw samples were treated with 2% (m / v, g / 100 mL) sodium hydroxide solution at 120°C for 40 min, with 100 mL of sodium hydroxide solution added for every 10 g of sample. After cooling to room temperature, the samples were rinsed with ddH2O until the pH was neutral, and then dried at 60°C to constant weight for further use.

[0095] (2) FvAbnA cooperates with cellulase to promote the degradation of wheat and rice straw

[0096] Preparation of enzyme premix:

[0097] Cellulase was produced by Sigma-Aldrich Company, with the product number C8546-500G, and was prepared into a cellulase solution with a concentration of 0.3 mg / mL.

[0098] The FvAbnA protein was prepared as in Example 1.

[0099] The total volume of each reaction system was 300 μL, including: 200 μL of 0.3 mg / mL cellulase solution and the corresponding mass ratio of α-L-arabinanase FvAbnA, 10 mg straw substrate (pretreated wheat straw or rice straw), and 0.05 mol / L CH3COOH-CH3COONa buffer (pH 5.0) was used to make up to a total volume of 300 μL.

[0100] The reaction system was incubated at 55 °C for 6 h, 300 μL of DNS was added and boiled in water for 8 min, cooled to room temperature, and OD was measured by spectrophotometry. 540 The value was compared with the arabinose standard curve to calculate the amount of reducing sugar produced.

[0101] The control experiments were conducted without adding enzyme, adding commercial cellulase enzyme solution alone, and adding α-L-arabinanase FvAbnA alone. The mass ratios of α-L-arabinanase FvAbnA and cellulase were 1:3, 1:5, 1:10, 1:15, 1:20, and 1:25, respectively.

[0102] Degree of synergy = amount of reducing sugar produced by FvAbnA and cellulase in degrading straw / (amount of reducing sugar produced by FvAbnA in degrading straw + amount of reducing sugar produced by cellulase in degrading straw)

[0103] 2. Test results:

[0104] The results showed that under the above experimental conditions, the amount of reducing sugar produced by cellulase alone when degrading wheat straw was 3.86mM, while the amount of reducing sugar produced by 0-20μg of FvAbnA alone when degrading wheat straw did not exceed 0.87mM. When cellulase and FvAbnA degraded wheat straw together at different mass ratios, the two had a significant synergistic degradation effect, which could greatly increase the amount of reducing sugar produced by degrading wheat straw. Among them, when the mass ratio of FvAbnA to cellulase was 1:5, the amount of reducing sugar produced reached the maximum (21.45mM), and the synergy also reached the maximum of 4.87 ( Figure 4 ).

[0105] In addition, the amount of reducing sugar produced by cellulase alone when degrading rice straw was 7.56mM, while the amount of reducing sugar produced by 0-20μg of FvAbnA alone when degrading rice straw did not exceed 0.59mM. When cellulase and FvAbnA were used to degrade rice straw in different mass ratios, the two had a significant synergistic degradation effect, which could greatly increase the amount of reducing sugar produced by degrading rice straw. Among them, when the mass ratio of FvAbnA to cellulase was 1:10, the amount of reducing sugar produced reached the maximum (26.54mM), and the synergy also reached the maximum of 3.37 ( Figure 5 ).

[0106] In summary, FvAbnA can significantly enhance the ability of cellulose to degrade wheat and rice straw. When the two are compounded in a certain proportion, they can effectively synergistically hydrolyze the straw and produce reducing sugars.

[0107] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of the FvAbnA protein in any of the following (1)-(3): (1) Degradation of arabinan; (2) Synergistically degrade straw with cellulase; (3) preparing an additive for straw feed; The amino acid sequence of the FvAbnA protein is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that: The FvAbnA protein degrades arabinan by hydrolyzing α-1,5-glycosidic bonds.

3. The use according to claim 1, characterized in that: The straw is selected from rice straw, wheat straw, corn straw and / or sorghum straw.

4. Application of the FvAbnA gene in producing a biological enzyme having arabinanase activity; the FvAbnA gene is a nucleic acid molecule as shown in i) or ii) below: i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID No. 2; ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 1 other than i).

5. Use of a recombinant expression vector or an engineered bacterium containing the FvAbnA gene according to claim 4 in producing a biological enzyme having arabinanase activity.

6. The use according to claim 5, characterized in that: The recombinant expression vector is a yeast eukaryotic expression vector pPIC9K containing the FvAbnA gene.

7. A combined enzyme for degrading straw, characterized in that: The combined enzyme is composed of the FvAbnA protein according to claim 1 and cellulase in a mass ratio of 1:(3-20).

8. The combined enzyme according to claim 7, characterized in that: The combined enzyme is composed of FvAbnA protein and cellulase in a mass ratio of 1:(5-10).

9. Use of the combined enzyme according to claim 7 or 8 in the preparation of straw feed additives.

10. A method for collaboratively degrading straw, characterized in that: The following steps are involved: Add the combined enzyme of claim 7 or 8 to the straw substrate and incubate at 50-60° C. for 4-8 hours.

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