Acidophilic bifunctional NSP enzyme and its application in improving the digestibility of grains and / or their by-products by monogastric animals

Abstract

The present invention belongs to the field of feed additive preparation, and specifically relates to an acidophilic bifunctional NSP enzyme and its application in improving the digestibility of grains and / or their by-products by monogastric animals, and the amino acid sequence of the acidophilic bifunctional NSP enzyme is shown in SEQ ID NO: 1. The NSP enzyme is an extremely acid-resistant bifunctional β-glucanase / xylanase, which can effectively hydrolyze barley, degrade β-glucan and xylan, thereby reducing the viscosity caused by NSP and improving the digestibility of barley, and the digestibility can be improved by about 8% compared with a control group (no enzyme added).

Description

Technical Field

[0001] The invention belongs to the field of feed additive preparation, and particularly relates to an acidophilic bifunctional NSP enzyme and application thereof in improving the digestibility of grains and / or their by-products by monogastric animals. Background Art

[0002] Cereals and their by-products contain non-starch polysaccharides (NSP) in varying proportions. In the digestive environment, NSP increases the viscosity of chyme, thereby forming a physical barrier that interferes with the interaction between digestive enzymes and their substrates, thereby affecting the absorption and digestion of nutrients.

[0003] Monogastric animals lack the enzymes to degrade NSP and are unable to digest these polysaccharides present in grain-based feeds. Barley has a high content of NSP, which, if directly used for monogastric animal feeding, may have a negative impact on animal production and lead to significant economic losses.

[0004] Adding NSP enzyme is one of the most effective strategies to reduce the adverse effects of NSP on the growth of monogastric animals. There are many types of NSP enzymes, including β-glucanase, xylanase, β-mannanase and pectinase. NSP enzyme can deconstruct the tight cell wall structure, release rich nutrients and digestive enzymes, and partially hydrolyze xylan and β-glucan into oligosaccharides, reduce the viscosity of the digestate, and improve the utilization of nutrients. In particular, the combined use of xylanase and β-glucanase is more effective than using either enzyme alone. Summary of the invention

[0005] Based on the above background, the purpose of this patent is to provide an acidophilic bifunctional β-glucanase / xylanase, which has the ability to effectively hydrolyze NSP in barley. The research and application of this enzyme will help improve the ability of monogastric animals to absorb and utilize grains such as barley, and promote the degradation of NSP in grains and their by-products.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides an acidophilic bifunctional NSP enzyme, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] Another aspect of the present invention provides a composition comprising the acidophilic bifunctional NSPase of the present invention.

[0009] Preferably, the above composition can be enzyme liquid or enzyme powder.

[0010] Another aspect of the present invention provides a feed additive comprising the acidophilic bifunctional NSPase of the present invention or the composition of the present invention.

[0011] In another aspect, the present invention provides a use of the acidophilic bifunctional NSPase of the present invention or the composition of the present invention in improving the digestibility of monogastric animals for grains and / or their by-products.

[0012] Preferably, the above application may include: application of the acidophilic bifunctional NSPase or the composition in degrading grains and / or their by-products.

[0013] Preferably, the above application may include: use of the acidophilic bifunctional NSP enzyme or the composition in degrading β-glucanase and / or xylanase in grains and / or their by-products.

[0014] Preferably, in the above application, the cereals include wheat, barley and wheat bran.

[0015] In another aspect, the present invention provides a method for degrading β-glucanase and / or xylanase in cereals and / or their by-products, comprising: mixing the acidophilic bifunctional NSP enzyme of the present invention or the composition of the present invention with the cereals and / or their by-products, and incubating for degradation.

[0016] Preferably, the incubation conditions in the above method include: a temperature of 37° C.-41° C. and a pH of 2.3-2.7.

[0017] The beneficial effects of the present invention include at least: the acidophilic bifunctional NSP enzyme provided by the present invention is an extremely acid-resistant bifunctional β-glucanase / xylanase, which can effectively hydrolyze barley, degrade β-glucan and xylan, thereby reducing the viscosity caused by NSP and improving the digestibility of barley, and the digestibility can be increased by about 8% compared with the control group (without enzyme addition). BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the pH dependence of RuCel143;

[0019] Figure 2 is the temperature dependence of RuCel143;

[0020] Figure 3 is the pH tolerance of RuCel143;

[0021] Figure 4 is the temperature tolerance of RuCel143;

[0022] Figure 5 To perform HPLC analysis of sodium carboxymethylcellulose hydrolysate using RuCel143;

[0023] Figure 6 To perform HPLC analysis of xylan hydrolysate using RuCel143;

[0024] Figure 7The HPLC chromatogram is a HPLC analysis of the hydrolyzate of sodium carboxymethyl cellulose by RuCel143; wherein G is glucose, G2 is cellobiose, G3 is cellotriose, G4 is cellotetraose, and G5 is cellopentose;

[0025] Figure 8 HPLC chromatogram of HPLC analysis of hydrolyzed products of xylan using RuCel143; wherein X1 is xylose, X2 is xylobiose, X3 is xylotriose, X4 is xylotetrose and X5 is xylopentose;

[0026] Fig. 9 The hydrolysis of wheat, barley and bran by RuCel143. DETAILED DESCRIPTION

[0027] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0028] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0029] The embodiment of the present invention provides an acidophilic bifunctional NSP enzyme, the amino acid sequence of which is shown in SEQ ID NO:1.

[0030] It should be noted that, after sequencing, the amino acid sequence of the acidophilic bifunctional NSP enzyme of the present invention is shown in SEQ ID NO: 1, which is as follows:

[0031] MIMKLKNILNTLSLAAVALVATGCQDTDAQTDVLEVDAPTLVSITPDDKGVLLFGEKTITVTFDKNIGFATKNASLITLNGKSVKYANVVGISKSLTIKADVNFDKTQKLHIPAGLIVGPQYKTYNQD IDVTWSIKPLADNAATQMTKKLGWGWNLGNHFDTSDMTYGYWDGVPTITSAPFDKLASAGAKTVRIPVTWTNHMDATNTIDAAYLNEVKAVVDLALNAGLNVIINTHHDSFETTLGECANDATKAEETA TLIQTLWTQVANKFQSYDEKLIFETFNEIHAGDNWSKGSDAENALLNQWNQLAIDAIRATGGNNATRWIAISHYAANVDQVIANLKIPENDDHIIVAAHCYDPYNFCLAPVASGTNSWGHNANAGSSV EGANEDYVIAQLYKLREAYIEKGIPCYLGEYGCVIQTTANANAFRKYYLEFYCRAAYLAGIPMFVWDNNGKVTGGDEENGYIDHATGDYIGDAEIVPMMVKACTDTDESYWFNNIWDKSPAYEAPAE*.

[0032] It should be noted that the sources of acidophilic bifunctional NSPase are wide and significantly affect their application effects. In the rumen of ruminants, a variety of microorganisms secrete enzymes that act on carbohydrates. This makes the rumen microbiota a rich gene pool for selecting NSP-degrading enzymes that specifically decompose NSP in grains and their by-products. It is worth noting that the rumen is maintained at a temperature of approximately 39°C-41°C, which is consistent with the body temperature of most animals; in addition, GH5 enzymes are part of the largest glycoside hydrolase (GH) family in the carbohydrate-active enzyme (CAZy) database, and they are highly specialized in function, especially enzymes in the GH5 family tend to have degradation effects on a variety of substrates. Therefore, multifunctional NSP-degrading enzymes from rumen microbiota may be identified in the GH5 family. This enzyme has good potential and application value as a feed additive in animal husbandry applications.

[0033] The present invention also provides a composition comprising the acidophilic bifunctional NSPase of the present invention.

[0034] It should be noted that the acidophilic bifunctional NSP enzyme of the present invention can be prepared into a composition for application, which can increase the scope of application and application efficiency.

[0035] In some specific examples, the composition may be enzyme liquid or enzyme powder.

[0036] It should be noted that the acidophilic bifunctional NSP enzyme can be prepared into an enzyme liquid or an enzyme powder for use, and the method for preparing the enzyme liquid or the enzyme powder is a method known in the art.

[0037] The embodiment of the present invention further provides a feed additive, which comprises the acidophilic bifunctional NSP enzyme of the present invention or the composition of the present invention.

[0038] It should be noted that the acidophilic bifunctional NSP enzyme of the present invention or the composition of the present invention may also be added with some feed additive auxiliary preparations to prepare feed additives, and feed additive auxiliary preparations are well known in the art.

[0039] The embodiments of the present invention also provide a use of the acidophilic bifunctional NSP enzyme of the present invention or the composition of the present invention in improving the digestibility of monogastric animals for grains and / or their by-products.

[0040] In some specific examples, the above application may include: use of an acidophilic bifunctional NSPase or composition in degrading grains and / or their by-products.

[0041] In some specific examples, the above application may include: use of an acidophilic bifunctional NSPase or composition in degrading β-glucanase and / or xylanase in grains and / or their by-products.

[0042] In some specific examples, in the above applications, the cereals include wheat, barley and wheat bran.

[0043] The embodiment of the present invention also provides a method for degrading β-glucanase and / or xylanase in cereals and / or their by-products, comprising: mixing the acidophilic bifunctional NSP enzyme of the present invention or the composition of the present invention with the cereals and / or their by-products, and incubating for degradation.

[0044] In some specific examples, the incubation conditions in the above method include: a temperature of 37°C-41°C, preferably 39°C, and a pH of 2.3-2.7, preferably 2.5.

[0045] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0046] 1. Production of RuCel143

[0047] (I) Cloning and expression of RuCel143

[0048] (1) The gene encoding RuCel143 was identified from rumen metagenomic data (NCBI accession number PRJNA806344);

[0049] (2) Then, using the liquid DNA as a template, a primer pair

[0050] 5'-CTTTAAGAAGGAGATATACGGATCCATGATTATGAAACTGAAGAAT-3' and 5'-AGTGGTGGTGGTGGTGGTGCTCGAGTTCAGCAGGTGCTTCATAGGC-3' were used to amplify the RuCel143 gene to obtain PCR products; the PCR amplification system is shown in Table 1 below, and the PCR amplification program is shown in Table 2 below.

[0051] Table 1 PCR amplification system (20 μL)

[0052] PCR amplification system volume ddH2O 7.8μL High-Fidelity DNA Polymerase 0.2μL F 0.5μL R 0.5μL DNA template 1μL PCR buffer 10μL

[0053] Table 2 PCR amplification program

[0054]

[0055] (3) The PCR product was cloned into the pET-28a vector using the pEASY-Basic seamless cloning kit (CU201-02, Beijing, China) and transformed into Escherichia coli Top10 cells by the heat shock method;

[0056] (4) pET-RuCel143 plasmid was extracted, amplified and verified by PCR, and sequenced. The amino acid sequence is shown in SEQ ID NO: 1, which is as follows:

[0057] MIMKLKNILNTLSLAAVALVATGCQDTDAQTDVLEVDAPTLVSITPDDKGVLLFGEKTITVTFDKNIGFATKNASLITLNGKSVKYANVVGISKSLTIKADVNFDKTQKLHIPAGLIVGPQYKTYNQD IDVTWSIKPLADNAATQMTKKLGWGWNLGNHFDTSDMTYGYWDGVPTITSAPFDKLASAGAKTVRIPVTWTNHMDATNTIDAAYLNEVKAVVDLALNAGLNVIINTHHDSFETTLGECANDATKAEETA TLIQTLWTQVANKFQSYDEKLIFETFNEIHAGDNWSKGSDAENALLNQWNQLAIDAIRATGGNNATRWIAISHYAANVDQVIANLKIPENDDHIIVAAHCYDPYNFCLAPVASGTNSWGHNANAGSSV EGANEDYVIAQLYKLREAYIEKGIPCYLGEYGCVIQTTANANAFRKYYLEFYCRAAYLAGIPMFVWDNNGKVTGGDEENGYIDHATGDYIGDAEIVPMMVKACTDTDESYWFNNIWDKSPAYEAPAE*.

[0058] (II) Preparation of RuCel143 enzyme solution

[0059] (1) The pET-RuCel143 plasmid was introduced into Escherichia coli BL21(DE3) by heat shock, and transformants were screened on LB agar plates containing kanamycin at 37°C overnight;

[0060] (2) Identify positive colonies using PCR and specific primers;

[0061] (3) Cultivate the bacterial suspension in LB liquid medium containing kanamycin at 37°C until OD 600 Reach 0.7;

[0062] (4) adding isopropyl-β-thiogalactoside (IPTG) at a final concentration of 0.2 mM to induce the expression of RuCel143;

[0063] (5) The culture was then transferred to 20°C and continued to be cultured for more than 18 hours;

[0064] (6) After the incubation, the mixture was centrifuged at 8000 rpm for 10 min at 4°C to collect the bacteria;

[0065] (7) Resuspend the bacterial pellet in PBS and disrupt it using ultrasound;

[0066] (8) using ultrasonic disruption to lyse the cells, then separating the supernatant from the precipitate by centrifugation, and taking the supernatant as the crude enzyme solution;

[0067] (9) RuCel143 crude enzyme solution was purified by affinity chromatography using a 5 mL bioscale micro Nuvia IMAC nickel charged column (Bio-Rad, Hercules, CA, USA) connected to a low pressure chromatography system (Bio-LP; Bio-Rad, Hercules, CA, USA); RuCel143 enzyme solution was purified.

[0068] 2. Analysis of Enzymatic Properties of RuCel143

[0069] (I) Substrate specificity

[0070] The substrate specificity of RuCel143 was investigated using a panel of five substrates, namely sodium carboxymethylcellulose, microcrystalline cellulose, xylan, chitosan, and pachyman, as follows:

[0071] The above-mentioned purified RuCel143 enzyme solution (3U) was added to 50mM citric acid-disodium hydrogen phosphate buffer containing 1% (w / v) substrate, and incubated in a shaking water bath at 40°C for 30 minutes; the reducing sugar concentration of the reaction solution was measured using alkaline 3,5-dinitrosalicylic acid (DNS), and the amount of reducing sugar was calculated using a standard curve; one unit (U) of enzyme activity was defined as the amount of enzyme required to release 1μmol of reducing sugar in 1min under the assay conditions; the test results are shown in Table 3 below.

[0072] Table 3 Substrate specificity of RuCel143

[0073] Substrate Primary Link Key Enzyme activity (U / mg) Sodium Carboxymethyl Cellulose 1,4-β-(Glucose) 0.459 Xylan 1,4-β-(Xylose) 0.163 Microcrystalline Cellulose 1,4-β-(Glucose) 0.029 Chitosan 1,4-β-(Glucose) 0.020 Poria polysaccharide 1,3-β-(Glucose) 0.049

[0074] As can be seen from Table 3 above, RuCel143 enzyme exhibited different degrees of activity on the five substrates, among which the activity of RuCel143 on sodium carboxymethyl cellulose and xylan was significantly higher than that on other substrates.

[0075] (II) pH dependence

[0076] According to the above test, sodium carboxymethyl cellulose and xylan were selected for characteristic analysis, as follows:

[0077] 1 mL of solution was used as the reaction system, including 1% of different substrates and citric acid-sodium hydrogen phosphate buffers of different pH values, and placed in a shaking water bath at 40° C. and incubated for 30 minutes. After the reaction, the released reducing sugar was measured.

[0078] The results are as follows Figure 1 As shown, for sodium carboxymethylcellulose, RuCel143 exhibited peak activity at pH 3.5 and maintained more than 80% of its activity in the pH range of 3.0-4.0; while for xylan, RuCel143 exhibited maximum xylanase activity at pH 4.0.

[0079] (III) Temperature dependence

[0080] Select the optimal pH buffer determined by the above experiment and keep other reaction components unchanged; set the temperature of the water bath to 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃, respectively, incubate for 30 minutes, and measure the released reducing sugar after the reaction.

[0081] The results are as follows Figure 2 As shown, the activity of RuCel143 towards sodium carboxymethyl cellulose was measured at pH 3.5, with the highest activity at 40°C, and within the temperature range of 30-50°C, the activity of the enzyme towards sodium carboxymethyl cellulose remained above 90%; however, above 50°C, the activity dropped sharply, resulting in complete inactivation at 70°C.

[0082] In contrast, the activity of RuCel143 on xylan evaluated at pH 4.0 reached a peak at 50 °C, the enzyme retained more than 75% of its xylanase activity in the range of 40 °C–60 °C, and exhibited residual activity at both 70 °C and 80 °C.

[0083] (IV) pH tolerance

[0084] In order to determine the pH tolerance of the enzyme, the purified RuCel143 enzyme solution (0.45 U of enzyme solution was used for sodium carboxymethyl cellulose as the degradation substrate; 0.09 U of enzyme solution was used for xylan as the degradation substrate) was preincubated in different pH buffers for 2 and 4 hours in the absence of substrate, and then the residual activity was measured under the optimal reaction conditions (β-glucanase pH 3.5, temperature 40°C; xylanase pH 4.0, temperature 50°C); the pH stability was determined by comparing with the residual activity of the untreated enzyme sample, which was set to 100%.

[0085] The results are as follows Figure 3 As shown, the pH stability of β-glucanase and xylanase activities of RuCel143 was evaluated in the pH range of 2.5-8.0; after incubation for 2 and 4 hours at different pH levels in this range, RuCel143 maintained more than 90% of its activity towards sodium carboxymethylcellulose; in contrast, within the pH range of 2.5-8.0, the activity of RuCel143 towards xylan increased with increasing pH.

[0086] (V) Temperature tolerance

[0087] In order to evaluate the thermal stability, the purified RuCel143 enzyme solution (0.45 U of enzyme solution was used for sodium carboxymethyl cellulose as the degradation substrate; 0.09 U of enzyme solution was used for xylan as the degradation substrate) was incubated at 50°C, 60°C, 70°C and 80°C for durations (5 min, 10 min, 20 min, 30 min, 1 h and 4 h), respectively, and the residual enzyme activity was measured under the optimal reaction conditions; the thermal stability of the enzyme was evaluated by comparing the residual activity of the control sample of the untreated enzyme, the latter being defined as 100%.

[0088] The thermal stability of RuCel143 to sodium carboxymethylcellulose and xylan Figure 4 As shown, the results showed that for sodium carboxymethyl cellulose, the activity of the enzyme was 102.9% after incubation at 50°C for 4 hours; at 60°C, 10 minutes of incubation resulted in 65.6% residual activity, while 20 minutes of incubation resulted in complete loss of activity; incubation at 70°C and 80°C for 5 minutes resulted in immediate inactivation.

[0089] For xylan, the activity was highest after incubation at 50°C for 10 min, and the residual activity gradually decreased to 84.0% after 4 h; after incubation at 60°C, 70°C and 80°C for 4 h, the activity remained at 46.5%, 22.3% and 28.1%, respectively.

[0090] 3. Analysis of enzymatic hydrolysis products of RuCel143

[0091] 1% (w / v) sodium carboxymethyl cellulose and wheat straw xylan were used as substrates for enzymatic hydrolysis, and the samples were incubated with the purified RuCel143 enzyme solution (0.45 U of enzyme solution was used for sodium carboxymethyl cellulose as the degradation substrate; 0.09 U of enzyme solution was used for xylan as the degradation substrate) under optimal conditions (β-glucanase pH 3.5, temperature 40°C; xylanase pH 4.0, temperature 50°C) with shaking for 30 minutes; the hydrolyzate was analyzed by high performance liquid chromatography (HPLC) for sodium carboxymethyl cellulose (including glucose, cellobiose, cellotriose, cellotetraose, and cellopentose) and wheat straw xylan (including xylose, xylobiose, xylotriose, and xylopentose); the HPLC conditions were as follows: equipped with an Agilent Eclipse XDB-C18 column (4.6×250 mm, 5 μm). Prior to chromatographic analysis, the product was derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP); the mobile phase flow rate was set at 1 mL / min, and the ultraviolet (UV) detector was set at a wavelength of 250 nm; the mobile phase consisted of a mixture of buffer A (25% 0.02 M ammonium acetate solution and 75% acetonitrile) and buffer B (83% 0.02 M ammonium acetate solution and 17% acetonitrile), and a gradient from 0% to 40% buffer B was used for separation; mannose was used as an internal standard.

[0092] Test results such as Figure 5 , Figure 6 , Figure 7 and Figure 8 As shown, the results show that for sodium carboxymethyl cellulose ( Figure 5 and Figure 6 ), the detection of glucose, cellobiose, cellotriose, cellotetraose and cellopentose showed that cellobiose was the most abundant, followed by cellotetraose, cellotriose and glucose, with concentrations of 193.5μg / ml, 142.3μg / ml, 80.9μg / ml and 53.8μg / ml, respectively; cellopentose was not detected.

[0093] For xylan ( Figure 7 and Figure 8 ), xylopentose was the main product, followed by xylotriose, xylotetraose, xylobiose and xylose; the concentrations of the five sugars produced were 621.6μg / ml for xyopentose, 33.8μg / ml for xylotriose, 32.7μg / ml for xylotetraose, 13.9μg / ml for xylobiose and 2.5μg / ml for xylose; this showed that RuCel143 could degrade CMC and xylan to produce oligosaccharides with prebiotic effects.

[0094] 4. Hydrolysis of wheat, barley and bran with RuCel143

[0095] The purified RuCel143 enzyme solution (3.6 U) was incubated with 1% (wheat, barley and wheat bran) substrate (i.e., 1% (w / v) substrate in 50 mM citric acid-sodium hydrogen phosphate buffer) at pH 2.5 and 39°C for 6 hours; the hydrolysis efficiency of RuCel143 on these substrates was determined by a control reaction without adding enzymatic reaction.

[0096] The results are as follows Fig. 9 As shown, at 39°C and pH 2.5, RuCel143 was effective in degrading wheat, barley and wheat bran; the enzyme had the highest degradation effect on barley, followed by wheat bran, and then wheat, with reducing sugar levels of 404.9 μg / ml, 287.3 μg / ml and 208.8 μg / ml, respectively.

[0097] 5. Adding RuCel143 to simulate wheat digestion in monogastric animals in vitro

[0098] In the following tests, relative viscosity = time for sample to pass through the viscometer / time for water to pass through the viscometer.

[0099] In the following tests, the in vitro digestion simulation steps are as follows:

[0100] (1) Add 3 g of barley and RuCel143 (356 U) into a 250 mL conical flask, add an equal amount of PBS as a control, and stir the mixture with a magnetic bar for 1 min;

[0101] (2) Then, 25 mL of phosphate buffer solution (0.1 M, pH 6.0) was added, and the pH was adjusted to 2.5 with 1 M hydrochloric acid or sodium hydroxide; to prevent bacterial contamination during the hydrolysis process, 0.5 mL of chloramphenicol (5 g / L ethanol) was added, followed by 1 mL of 20 g / L porcine pepsin solution (Shanghai Bioengineering, Shanghai);

[0102] (3) The flask was sealed with a silicon stopper and then slowly shaken at 39 °C for 2 h. For simulated intestinal digestion, after pepsin hydrolysis, 10 mL of phosphate buffer (0.2 M, pH 6.8) was added and the pH was adjusted to 6.8 with 1 M hydrochloric acid or sodium hydroxide.

[0103] (4) Subsequently, 1 mL of 50 g / L pancreatic enzyme solution (Shanghai Bioengineering, Shanghai) was added; the mixture was slowly shaken at 39 °C for 4 h;

[0104] (5) After digestion, the chyme was filtered with 200 mesh nylon cloth, and the filtrate was collected to determine the glucose and xylose contents and the viscosity. The residue was dried at 60°C for 24 h, weighed, and the digestibility was calculated.

[0105] The degradation of barley by RuCel143 during digestion in monogastric animals was evaluated by assessing several parameters including digestibility, viscosity, glucose and xylose concentrations, and the results are shown in Table 4.

[0106] Table 4 Parameters for simulating in vitro digestion of barley using RuCel143

[0107] project Control group RuCell43 P-value Digestibility 0.53±0.003 0.61±0.002 ＜0.001 Relative viscosity 1.41±0.010 1.38±0.010 0.021 Glucose concentration (mM) 21.72±1.388 24.49±1.117 0.055 Xylose concentration (mM) 7.73±0.316 12.26±1.755 0.012

[0108] As shown in Table 4, compared with the control group, the digestibility was significantly increased by about 8% after adding RuCel143; the viscosity was also significantly reduced, and the concentrations of glucose and xylose increased by about 2.77mM and 4.53mM, respectively. The results show that RuCel143 can effectively hydrolyze barley, degrade β-glucan and xylan, thereby reducing the viscosity caused by NSP and improving the digestibility of barley. In summary, RuCel143 is an extremely acid-resistant bifunctional β-glucanase / xylanase, which has important potential as a feed additive to improve the absorption and utilization of barley by monogastric animals.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. An acidophilic bifunctional NSP enzyme, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. A composition comprising the acidophilic bifunctional NSPase of claim 1.

3. The composition according to claim 2, characterized in that The composition is enzyme liquid or enzyme powder.

4. A feed additive, characterized in that Comprising the acidophilic bifunctional NSP enzyme of claim 1 or the composition of claim 2 or 3.

5. Use of the acidophilic bifunctional NSP enzyme according to claim 1 or the composition according to claim 2 or 3 for improving the digestibility of grains and / or their by-products by monogastric animals.

6. The use according to claim 5, characterized in that: Applications include: Use of an acidophilic bifunctional NSPase or composition for degrading grains and / or their by-products.

7. The use according to claim 6, characterized in that: Applications include: Use of an acidophilic bifunctional NSPase or composition for degrading β-glucanase and / or xylanase in cereals and / or their by-products.

8. The use according to any one of claims 5 to 7, characterized in that Cereals include wheat or barley; cereal by-products include bran.

9. A method for degrading β-glucanase and / or xylanase in cereals and / or their by-products, characterized in that: include: The acidophilic bifunctional NSP enzyme of claim 1 or the composition of claim 2 or 3 is mixed with grains and / or their by-products and incubated for degradation.

10. The method according to claim 9, characterized in that The incubation conditions include: temperature of 37°C-41°C and pH of 2.3-2.7.

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