Application of man-Ta protein and coding gene thereof in degradation of beta-mannan

Abstract

The invention discloses application of man-Ta protein and a coding gene thereof in degradation of beta-mannan, and relates to the technical field of degradation of beta-mannan. According to the invention, the beta-mannase coding gene man-Ta is screened, and the protein coded by the gene can be used for degrading beta-mannan. Further enzymatic characteristic analysis shows that the beta-mannase man-Ta has the characteristics of high specific activity, high temperature resistance, wide pH and temperature application range and the like, and lays a foundation for developing a novel feed beta-mannase preparation, improving the feed utilization rate and assisting in green and efficient breeding of livestock and poultry.

Description

Technical Field

[0001] The present invention relates to the technical field of β-mannan degradation, and more specifically to the application of man-Ta protein and its encoding gene in the degradation of β-mannan. Background Art

[0002] β-mannan is a linear polysaccharide of hemicellulose with the highest content in plant feed ingredients after xylan, which is composed of β-1,4-mannoside bonds. β-mannan has high hydrophilicity, and when dissolved in water, it will swell to form a gel-like substance and has strong viscosity. After monogastric animals eat plant feed, because their bodies do not have enzymes to decompose β-mannan, the viscous mannan reduces the mixing efficiency of gastrointestinal motility on chyme, affecting the contact between digestive enzymes and substrates and the penetration and diffusion of digestion products into the small intestinal epithelial villi. Therefore, β-mannan is an antinutritional factor that hinders animal feed digestion and nutrient absorption.

[0003] β-mannanase is an endoglycoside hydrolase that can hydrolyze the β-1,4-glycosidic bond within the mannan molecule. It has a wide range of sources and has been found in some animals, plants and microorganisms. β-mannanase has good application prospects in the animal feed industry. Adding β-mannanase to feed can remove the antinutritional effect of mannan in animals and improve the absorption and utilization efficiency of animals for feed. In addition, as a new type of prebiotic, its degradation product mannan oligosaccharide (also called oligomannose) can improve intestinal function and enhance immunity by promoting the proliferation of intestinal probiotic flora and reducing the generation of toxic metabolites.

[0004] The digestive physiological conditions of livestock and poultry require that feed enzymes should have the characteristics of adapting to both the strong acidic environment of gastric juice (pH value in the range of 2.2 - 3.5) and the neutral environment of intestinal juice in the small intestine (pH value in the range of 5.0 - 7.0) to play a better effect. In addition, the feed pelleting process requires treatment at 80°C for 3 - 5 minutes, which requires feed enzymes as feed additives to have high specific activity characteristics under gastrointestinal temperature (about 40°C) conditions, and also requires good high-temperature tolerance. Therefore, exploring β-mannanase with high temperature resistance and a wide pH application range is of great significance for the development of efficient feed β-mannanase preparations.

[0005] Currently, there are indeed technologies for producing β-mannanase by genetic engineering and molecular biology means, by searching for relevant genes or proteins and fermenting to produce β-mannanase; however, when improving the thermal stability of the enzyme through genetic modification, it may lead to a decrease in its catalytic efficiency (such as specific activity) or a reduction in substrate affinity. This is because the rigid structure that stabilizes the enzyme protein may limit the conformational changes of its active center, affecting the binding or catalytic process with the substrate. Moreover, due to the limitations of heterologous system expression, when expressed in common hosts such as Escherichia coli and yeast, insufficient yields may occur due to codon bias, low protein folding efficiency or toxicity problems. Thermophilic enzymes are prone to form insoluble inclusion bodies in the host, requiring additional steps for renaturation, increasing costs. Therefore, the core contradiction in the production of thermostable β-mannanase by genetic engineering lies in how to improve the thermal stability without sacrificing catalytic performance, while solving the bottlenecks of heterologous expression and industrial production.

[0006] Therefore, providing a gene that can encode a thermostable and highly thermally stable β-mannanase is a technical problem that those skilled in the art urgently need to solve. Summary of the Invention

[0007] In view of this, the present invention provides the application of man-Ta protein and its encoding gene in the degradation of β-mannan.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] On the one hand, an embodiment of the present invention provides the use of man-Ta protein, the encoding gene of man-Ta protein, or a substance containing the encoding gene of man-Ta protein, or a substance that upregulates the expression level of the encoding gene of man-Ta protein, and the use includes any one of the following:

[0010] A1) Application in animals for catalytically producing mannan oligosaccharides with β-mannan as a substrate;

[0011] A2) Application in the preparation of related products on the basis of A1);

[0012] Among them, the man-Ta protein is one of the following:

[0013] B1) A protein with an amino acid sequence as shown in SEQ ID NO.2;

[0014] B2) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to B1) and having 80% identity with 1) and the same function;

[0015] B3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of B1) or B2).

[0016] Preferably, the coding gene of the man-Ta protein is the coding gene expressing the nucleic acid molecule shown in SEQ ID NO.1.

[0017] Preferably, the substance containing the coding gene of the man-Ta protein includes any one of the following:

[0018] D1) An expression cassette containing the nucleotide sequence shown in SEQ ID NO.1;

[0019] D2) A recombinant vector containing the nucleotide sequence shown in SEQ ID NO.1 or a recombinant vector containing the expression cassette shown in D1);

[0020] D3) A recombinant microorganism containing the nucleotide sequence shown in SEQ ID NO.1, or containing the expression cassette shown in D1), or containing the recombinant vector described in D2);

[0021] D4) A transgenic plant containing the nucleotide sequence shown in SEQ ID NO.1, or containing the expression cassette shown in D1), or containing the recombinant vector described in D2).

[0022] Preferably, in D2), the recombinant vector is pET-28a-man-Ta; the recombinant microorganism includes E.coli BL21 / pET-28a-man-Ta.

[0023] Preferably, the substance for upregulating the expression level of the coding gene of the man-Ta protein is the substance that performs at least one of the following 6 regulations:

[0024] E1) Regulation carried out at the gene transcription level;

[0025] E2) Regulation carried out after gene transcription, that is, regulation of splicing or processing of the primary transcript of the gene;

[0026] E3) Regulation of RNA transport of the gene, that is, regulation of the transport of mRNA of the gene from the nucleus to the cytoplasm;

[0027] E4) Regulation of translation of the gene;

[0028] E5) Regulation of mRNA degradation of the gene;

[0029] E6) Post-translational regulation of the gene, that is, regulation of the activity of the protein translated from the gene.

[0030] Preferably, in A2), the related products include animal feeds, health products or drugs for animal intestinal regulation; wherein, the intestinal regulation is to degrade β-mannan in plant feed ingredients into mannan oligosaccharides, thereby promoting the proliferation of intestinal probiotics.

[0031] Preferably, the man-Ta protein is derived from the artificially synthesized Thermofilum adornatus.

[0032] The second aspect of the embodiments of the present invention provides a product, which is any one of the following:

[0033] 1) An animal feed containing the man-Ta protein described above; the animal is livestock and poultry;

[0034] 2) A health product or drug containing the man-Ta protein described above; the health product or drug is used for animal intestinal regulation, to degrade β-mannan in plant feed ingredients into mannan oligosaccharides, thereby promoting the proliferation of intestinal probiotics.

[0035] Preferably, the health product or drug further includes a pharmaceutically acceptable excipient.

[0036] The third aspect of the embodiments of the present invention provides a method for producing mannan oligosaccharides. The process includes: cloning the coding gene of the man-Ta protein onto the expression vector pET-28a, transforming Escherichia coli E.coil BL21 to obtain the genetically engineered strain E.coliBL21 / pET-28a-man-Ta; then inducing the expression of the engineered bacterium E.coli BL21 / pET-28a-man-Ta with IPTG and purifying it by His-tag affinity to obtain the recombinant man-Ta protein; then catalyzing with β-mannose as the substrate to obtain mannan oligosaccharides.

[0037] Compared with the prior art, the technical effects achieved by the present invention are as follows: By searching the GenBank database, the present invention discovered a predicted β-mannanase-encoding gene from the genome of the thermophilic microorganism Thermofilum adornatus, named man-Ta, and commissioned Sangon Biotech (Shanghai) Co., Ltd. to synthesize the man-Ta gene. The man-Ta gene was cloned into the expression vector pET-28a and transformed into Escherichia coli BL21(DE3), obtaining the genetically engineered strain E. coli BL21(DE3) / pET-28a-man-Ta. After induction and expression of the engineered strain E. coli BL21(DE3) / pET-28a-man-Ta with IPTG and purification by His-tag affinity chromatography, the recombinant protein man-Ta was obtained. The DNS method for measuring the enzyme activity of the recombinant protein showed that it could degrade the substrate β-mannan, demonstrating its β-mannanase activity. Further enzymatic property analysis showed that the β-mannanase man-Ta had characteristics such as high specific activity, high temperature resistance, and wide pH and temperature application ranges, laying a foundation for the identification and development of a new type of feed β-mannanase preparation to improve feed utilization rate and facilitate green and efficient breeding of livestock and poultry.

[0038] Furthermore, the optimal reaction temperature and pH of the recombinant β-mannanase man-Ta were 60 °C and 4.0, and it showed good stability in the range of 40 °C to 90 °C and pH 2.5 to 6.5. Mg 2+ and Fe 2+ significantly enhanced the enzyme activity, while Na + 、Zn + 、K + and Cu 2+ and other metal ions had no obvious effect on the enzyme activity; EDTA inhibited the enzyme activity by 33%, and SDS almost completely abolished the enzyme activity; when locust bean gum was used as the substrate, the Km value of the enzyme was 5.86 mg / mL and the Vmax was 91.74 μmol / min.mg, indicating high catalytic efficiency and substrate affinity. The above enzymatic properties indicate that the newly discovered β-mannanase man-Ta can withstand the high-temperature environment during feed pelleting and adapt to the gastrointestinal environment of livestock and poultry, and is suitable as an enzyme for feed additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings according to the provided drawings without creative efforts.

[0040] Figure 1 The attached figure shows the PCR amplification result of the man-Ta gene; M: DNA marker; 1: PCR product of the man-Ta gene.

[0041] Figure 2 The attached figure shows the verification results of single and double enzyme digestion of the pET-28a-man-Ta plasmid; M: DNA marker; 1: Single enzyme digestion of the recombinant vector; 2: Double enzyme digestion of the recombinant vector.

[0042] Figure 3 The attached figure shows the map of the recombinant expression vector pET-28a-man-Ta of Escherichia coli.

[0043] Figure 4 The attached figure shows the SDS-PAGE detection results of the induced expression and purification of the recombinant protein; M: Protein molecular weight marker; 1 and 2: Supernatants of cell lysates of E. coli BL21(DE3) / pET-28a-man-Ta without IPTG induction and E. coli BL21(DE3) / pET-28a-man-Ta with IPTG induction, respectively; 3: Purified β-mannanase man-Ta.

[0044] Figure 5 The attached figure shows the effects of temperature and pH on the recombinant β-mannanase man-Ta; (A) Optimum temperature of the recombinant β-mannanase man-Ta; (B) Temperature stability of the recombinant β-mannanase man-Ta; (C) Optimum pH of the recombinant β-mannanase man-Ta; (D) pH stability of the recombinant β-mannanase man-Ta.

[0045] Figure 6 The attached figure shows the effects of metal ions and chemical reagents on the recombinant β-mannanase man-Ta.

[0046] Figure 7 The attached figure shows the enzymatic reaction kinetic curve of β-mannanase man-Ta. Detailed implementation manners

[0047] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0048] Materials used in the embodiments of the present invention

[0049] Plasmids, strains:

[0050] The pET-28a plasmid was used as the expression vector for the β-mannanase gene.

[0051] Escherichia coli BL21(DE3) was used as the host strain for the expression of the β-mannanase gene. Escherichia coli was grown in LB liquid medium at 220 r / min and 37 °C, or on LB agar plates at 37 °C.

[0052] LB medium: 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of NaCl, made up to 1 L with distilled water. 1.5% agar was added to the solid medium based on the existing formula.

[0053] Example 1 Prediction and artificial synthesis of the man-Ta gene

[0054] The gene predicted to encode β-mannanase derived from Thermofilum adornatus was named man-Ta, and its open reading frame (ORF) sequence consisted of 2,106 nucleotides as follows:

[0055]

[0056] Its ORF encodes a protein consisting of 701 amino acids, and its amino acid sequence from the N-terminus to the C-terminus of the protein is as follows:

[0057] MSRIVSLALIAVIIIAMAIAYGYPSVSPQNKPKKLLIYYGYPSLFNGSTTI

[0058] QGAARLFDKYDIVVLGAGLESPEHPDHLNALKIIQRSRATFYGYVQSTLPL

[0059] DEAKKRIDLWTDMGVAGIFLDEFGFDYLVGRVASNKQQARLHQKELIDY

[0060] VKSKNMRVAINFWNPDDVFGPVGGVSLNLSGVEIVIESAVYGYGEKKWE

[0061] YIDHYYKMASAAKAVNASTWCIVSTKASTSPLNKMIGYDALTYIYGGCDA

[0062] VAIQEDYGEDSNVFYPFSEPSNASMYFLGVNVPLWGDFPKGWLENLLDQL

[0063] KDIGFNSVQYTVYYEMDSPVFSVIHTSSVTVPDDQLGYAIDAAKQRGFKVF

[0064] LRIGLNVKNSWSGEVNPLNRDFWFKSYEAILLKYAEIAQEKSVDAYIIGTE

[0065] LSMLQADPSWRPLILKVKAKYSGPISYSANWGHEATIFTDMLDFIGVDAY

[0066] YPILNITSWDIVHETRIEPLVFLYGKPVVFLEIGYRSVENPGLQPWDWQRRG

[0067] KPDQEAQASLWEIFFLKESRRINGFFYWDEGSWKEDETGYNVLGKLAEKV

[0068] FRKYVPLLSIQQQLESKKCINETELQAYKTFLQSCIENATKYILDAETYKSL

[0069] YESCLQNCTYLQTRLSQVINDYMDTMETLLKLQAQYKELAVKLSRTETAL

[0070] GYSIAINILLVVLLLFVSLLYIRSKRQNKNEREKTSQEKAEQQLS, as shown in SEQ I

[0071] D NO.2.

[0072] Construction of Recombinant Expression Vector pET-28a-man-Ta in Example 2 and Transformation of Escherichia coli

[0073] Shanghai Sangon Biotech Co., Ltd. was commissioned to synthesize the man-Ta gene. Using the artificially synthesized Thermofilum adornatus gene man-Ta as a template, the man-Ta gene was amplified using the forward primer man-Ta-F and the reverse primer man-Ta-R, with a 6X His-tag for purification carried downstream, and NdeI and XhoI restriction enzyme cleavage site sequences carried at both the upstream and downstream ends. The primer sequences are as follows:

[0074] man-Ta-F: 5'-attagatatcatgtctaggattgtatcgcttgc-3', as shown in SEQ ID NO.3; man-Ta-R: 5'-attactcgaggctcaattgctgttcagcctt-3', as shown in SEQ ID NO.4.

[0075] The results of agarose gel electrophoresis are as Figure 1 shown. The band size of the PCR amplification product is consistent with the man-Ta gene, indicating successful amplification of the β-mannanase man-Ta gene.

[0076] The PCR amplification product and the pET-28a expression vector were double-digested with restriction endonucleases NdeI and XhoI, and the DNA products were purified. The purified products were ligated using the DNA Ligation Kit Ver.2.1, and the ligation products were transformed into E.coli BL21(DE3) competent cells by heat shock transformation. The cells were spread on LB solid medium containing kanamycin and incubated upside down in a 37°C constant temperature incubator for 12 h. Transformants were picked and plasmids were extracted for single and double digestion verification. The correct transformants were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The verification results are as Figure 2 shown. The band size is consistent with the expectation, indicating successful construction of the recombinant expression vector pET-28a-man-Ta. The map of pET-28a-man-Ta is shown in Figure 3 .

[0077] Example 3: Induced Expression and Purification of Recombinant Protein man-Ta

[0078] Inoculate the engineered strain E. coli BL21(DE3) / pET-28a-man-Ta into a 25 mL Erlenmeyer flask containing 5 mL of LB liquid medium and culture it overnight for 12 h in a constant temperature shaker to prepare a seed solution; transfer the seed solution to a 250 mL Erlenmeyer flask containing 50 mL of LB medium according to a 1:10 ratio and shake it for culture. When the OD 600 reaches 0.6, add IPTG with a final concentration of 1 mM to the medium. After inducing expression for 6 h, centrifuge the bacterial solution at 12,000 r / min and 4 °C for 10 min to collect the cell pellet.

[0079] The protein purification process is as follows:

[0080] Step 1: Add binding buffer (0.1 M Tris-HCl, pH 8.0) with a volume 1 / 10 of the cell pellet volume and lysozyme with a final concentration of 100 μg / mL to the cell pellet, fully suspend the cells to obtain a cell suspension. After ice-bathing the cell suspension for 30 min, ultrasonically disrupt it for about 10 min (ultrasonic for 5 s / interval 10 s), and centrifuge at 12,000 r / m and 4 °C for 15 min to obtain the supernatant of the cell lysate.

[0081] Step 2: Equilibrate Ni-Charged MagBeads with 10 mM Tris-HCl (pH 8.0) containing 0.5 M NaCl, and add the equilibrated Ni-Charged MagBeads to the supernatant of the collected cell lysate, and mix quietly at 4 °C for 1 h;

[0082] Step 3: Discard the supernatant, and wash the Ni-Charged MagBeads with 10 mM Tris-HCl (pH 8.0) containing 20 mM imidazole and 0.5 mM NaCl;

[0083] Step 4: Discard the supernatant, add 10 mM Tris-HCl (pH 8.0) containing 300 mM imidazole and 0.5 mM NaCl, mix quietly at 4 °C for 1 h, and collect the supernatant.

[0084] Prepare a denaturing polyacrylamide gel for SDS-PAGE according to the conventional method. Add the above-mentioned E. coli cell pellet lysate and protein purification supernatant samples to 5X loading buffer in proportion, boil for 10 min, and load the samples at a loading volume of 15 μL / well for electrophoresis. Stain the SDS-PAGE gel with Coomassie Brilliant Blue R250 and then decolorize it. The results are as Figure 4As shown, a clear specific target protein band could be observed at 75 kDa in the IPTG-induced sample, which was consistent with the predicted theoretical protein molecular weight. The recombinant β-mannanase man-Ta has 6×His-tag at both ends of the peptide chain, and Ni-Charged MagBeads were used for the purification of recombinant β-mannanase. The SDS-PAGE detection results of the purified product indicated the successful purification of β-mannanase.

[0085] Example 4 Determination of β-mannanase activity of recombinant protein man-Ta

[0086] The DNS method specified in the national standard GB / T 36861-2018 "Determination of β-mannanase activity in feed additives - Spectrophotometry" of the People's Republic of China was used to determine the enzyme activity of its recombinant protein man-Ta. The specific steps are as follows:

[0087] Dilute the enzyme solution to be measured with 0.1 mol / L acetic acid-sodium acetate buffer solution at pH 5.5. Pipette 10 mL of the enzyme solution and add 2 g of mannan solution. React in a water bath at 37 °C for 30 min. After the reaction, add 5 mL of DNS reagent and immediately boil for 5 min to terminate the reaction. Quickly cool the reaction solution to room temperature with cold water and make up the volume to 25 mL with water. Measure the absorbance value at a wavelength of 540 nm. Calculate the activity of β-mannanase according to the linear regression equation. The amount of enzyme required to catalyze the release of 1 μmol of reducing sugar from the substrate per minute is defined as one enzyme activity unit (U).

[0088] The recombinant enzyme man-Ta was respectively under the conditions of 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C and 90 °C, and the enzyme activity at the corresponding temperature was determined according to the DNS method. The highest enzyme activity was set as 100%, and the relative enzyme activity at other temperatures was calculated to determine the optimal temperature of β-mannanase; the enzyme solution to be measured was incubated at four temperature conditions of 50 °C, 60 °C, 70 °C and 80 °C respectively. Take the enzyme solution incubated at each temperature every 0.5 h to measure the enzyme activity. Calculate the relative enzyme activity at each temperature condition with the highest enzyme activity as the control to analyze the temperature stability of the enzyme.

[0089] The results are as Figure 5 shown in A below. It was found that its enzyme activity was the highest at 60 °C, indicating that its optimal action temperature was 60 °C. In the range of 40 °C - 90 °C, its relative enzyme activity could remain above 45%, and it could tolerate the high temperature conditions of feed pelleting. Through the analysis of its temperature stability test, the results Figure 5As shown in B in [reference], after being incubated at 60 °C, 70 °C, and 80 °C for 5 h, more than 50% of the enzyme activity could still be detected, while only about 20% of the enzyme activity could be detected in the enzyme solution incubated at 50 °C for 5 h. It can be seen that β-mannanase has the property of high temperature resistance.

[0090] Example 6 Determination of the Optimal pH and pH Stability of Recombinant β-Mannanase man-Ta

[0091] Prepare the enzyme activity assay reaction system with buffer solutions of pH 2, 3, 4, 5, 6, 7, 8, and 9, and measure the enzyme activity at the corresponding pH under the condition of 60 °C. Set the highest enzyme activity as 100%, calculate the relative enzyme activity at other pH values, and determine the optimal pH of this enzyme; prepare the sample enzyme solutions with buffer solutions of pH 3, 4, 5, and 6 respectively, take the enzyme solutions stored under each pH condition for enzyme activity measurement every 0.5 h, calculate the relative enzyme activity under each pH condition with the highest enzyme activity as the control, and analyze the pH stability of the recombinant enzyme. The results are as Figure 5 shown in C in [reference], Figure 5 and D in [reference]. Under the condition of the optimal reaction temperature, the enzyme activity is the highest at pH 4.0. When pH > 6.5, the enzyme activity drops to about 50% or less of the highest activity. In this experiment, the sample enzyme solutions and reaction systems were prepared with buffer solutions of pH 3, 4, 5, and 6 respectively. The enzyme solutions stored under each pH condition were taken for enzyme activity measurement every 30 min. It was found that the enzyme activity stability at pH 4.0 was the strongest. The recombinant enzymes at pH 3, 4, and 5 could still maintain more than 60% of the enzyme activity after incubation for 5 h. However, after incubation for 5 h, the pH stability of the recombinant enzyme at pH 6 decreased, but the lowest remaining enzyme activity was also maintained above 30%. It can be seen that this enzyme has strong pH stability.

[0092] Example 7 Effects of Metal Ions and Chemical Reagents on β-Mannanase

[0093] Under the conditions of the optimal temperature and pH, measure the enzyme activities after adding different metal ion solutions or other chemical reagents with a final concentration of 1 mM respectively. Set the enzyme activity measured under the condition of not adding metal ion solutions or other chemical reagents as 100%, calculate the relative enzyme activities under each condition, and study their effects on the activity of β-mannanase. The results are as Figure 6 shown in [reference], Mg 2+ and Fe 2+ have a significant enhancing effect on the enzyme activity, while other metal ions such as Na + , Zn + , K + and Cu 2+ have almost no effect on the enzyme activity. In addition, EDTA inhibits 33% of the enzyme activity, while SDS almost completely abolishes the enzyme activity.

[0094] Determination of Kinetic Constants of Recombinant β-Mannanase man-Ta in Example 8

[0095] Mannan solutions with concentrations of 1, 2, 3, 4, 5, 6, and 7 mg / mL were prepared using the optimal pH buffer, and were separately reacted under the optimal temperature conditions. The Michaelis constant Km and the maximum reaction rate Vmax of β-mannanase man-Ta were calculated using the double-reciprocal plotting method. According to the double-reciprocal plotting method, the kinetic curve of β-mannanase man-Ta with locust bean gum as the substrate was plotted. From Figure 6 the calculated Km value of β-mannanase man-Ta was 5.86 mg / mL and the Vmax value was 91.74 μmol / min﹒mg. See Figure 7 .

[0096] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the various embodiments, reference can be made to each other.

[0097] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of the man-Ta protein, the coding gene of the man-Ta protein, a substance containing the coding gene of the man-Ta protein, or a substance that upregulates the expression level of the coding gene of the man-Ta protein, characterized in that, The uses include any one of the following: A1) Application in animals for catalytically producing mannan oligosaccharides using β-mannan as a substrate; A2) Application in preparing related products on the premise of A1); Among them, the man-Ta protein is one of the following: B1) A protein with an amino acid sequence shown in SEQ ID NO.2; B2) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to B1), having 80% identity with 1) and having the same function; B3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of B1) or B2).

2. The use according to claim 1, wherein The coding gene of the man-Ta protein is the coding gene expressing the nucleic acid molecule shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that, Substances containing the coding gene of the man-Ta protein include any one of the following: D1) An expression cassette containing the nucleotide sequence shown in SEQ ID NO.1; D2) A recombinant vector containing the nucleotide sequence shown in SEQ ID NO.1 or a recombinant vector containing the expression cassette shown in D1); D3) A recombinant microorganism containing the nucleotide sequence shown in SEQ ID NO.1, or containing the expression cassette shown in D1), or containing the recombinant vector described in D2); D4) A transgenic plant containing the nucleotide sequence shown in SEQ ID NO.1, or containing the expression cassette shown in D1), or containing the recombinant vector described in D2).

4. The use according to claim 3, characterized in that, In D2), the recombinant vector is pET-28a-man-Ta; the recombinant microorganism is E.coli BL21 / pET-28a-man-Ta.

5. The use according to claim 1, wherein Substances for upregulating the expression level of the coding gene of the man-Ta protein are substances for performing at least one of the following 6 regulations: E1) Regulation carried out at the gene transcription level; E2) Regulation carried out after gene transcription, that is, regulation of the splicing or processing of the primary transcript of the gene; E3) Regulation of the RNA transport of the gene, that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm; E4) Regulation of the translation of the gene; E5) Regulation of the degradation of the mRNA of the gene; E6) Regulation after gene translation, that is, regulation of the activity of the protein translated from the gene.

6. The use according to claim 1, characterized in that, In A2), the related products include animal feed, health products for animal intestinal regulation, or drugs; among them, the intestinal regulation is to degrade β-mannan in plant-based feed raw materials into mannan oligosaccharides, thereby promoting the proliferation of intestinal probiotics.

7. The use according to claim 1, characterized in that, The man-Ta protein is derived from the artificially synthesized Thermofilum adornatus.

8. A product, characterized in that, The product is any one of the following: 1) Animal feed containing the man-Ta protein described in claim 1; the animal is livestock and poultry; 2) Health products or drugs containing the man-Ta protein described in claim 1; the health products or drugs are used for animal intestinal regulation, degrade β-mannan in plant-based feed ingredients into mannan oligosaccharides, and further promote the proliferation of intestinal probiotics.

9. The product according to claim 8, wherein The health products or drugs also include pharmaceutically acceptable excipients.

10. A method for producing mannan oligosaccharide, characterized in that, The process includes: cloning the coding gene of the man-Ta protein described in claim 1 into the expression vector pET-28a, transforming Escherichia coli E.coil BL21 to obtain the genetically engineered strain E.coliBL21 / pET-28a-man-Ta; then inducing the expression of the engineered strain E.coli BL21 / pET-28a-man-Ta with IPTG and purifying it by His-tag affinity to obtain the recombinant man-Ta protein; then catalyzing with β-mannose as the substrate to obtain mannan oligosaccharides.

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