Acidophilic and halophilic GH12 xyloglucanase gene as well as expression protein and application thereof

Abstract

The invention discloses an acidophilic and halophilic GH12 xyloglucanase gene as well as an expression protein and application thereof. The DNA (Deoxyribose Nucleic Acid) sequence of the acidophilic and halophilic GH12 xyloglucanase gene is as shown in SEQ ID NO.1, and the amino acid sequence of the acidophilic and halophilic xyloglucanase expressed by the acidophilic and halophilic GH12 xyloglucanase gene is as shown in SEQ ID NO.2. The xyloglucanase disclosed by the invention shows the maximum enzyme activity at the pH value of 4.0 and the temperature of 40 DEG C and has remarkable pH stability, and the activity is still kept at 80% or above in the presence of 1.71 M NaCl and 10% (v / v) ethanol. The acidophilic and halophilic GH12 xyloglucanase disclosed by the invention can be used for effectively degrading xyloglucan and producing xyloglucan oligosaccharide, and is expected to become a catalyst in food and biological industries.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to an acidophilic and halophilic GH12 xyloglucanase gene, an expression protein thereof and an application thereof. Background Art

[0002] Hemicellulose is the second most abundant renewable polysaccharide in plants and plays an important role in promoting the development of sustainable biotechnology. Xyloglucan, the main component of hemicellulose, is composed of a β-1,4-linked D-glucose backbone and α-1,6-xylose side chains. Xyloglucan oligosaccharides are recognized as prebiotics, with biological activities such as lowering blood sugar and blood lipid levels. Due to the complex structure of xyloglucan, methods are required to degrade xyloglucan into xyloglucan oligosaccharides. Common methods for producing xyloglucan oligosaccharides include physical, chemical, and enzymatic hydrolysis. Enzymatic hydrolysis is widely used in the degradation of xyloglucan due to its advantages such as low environmental pollution, mild reaction conditions, and high efficiency.

[0003] Xyloglucanases (EC 3.2.1.151) are key enzymes that hydrolyze xyloglucans by cleaving β-1,4-glycosidic bonds to produce xyloglucan oligosaccharides. According to the Carbohydrate-Active Enzymes (CAZymes) database (http: / / www.cazy.org / ), these enzymes are classified into glycoside hydrolase (GH) families 74, 45, 44, 16, 12, 9, and 5. GH12 family xyloglucanases, in particular, stand out for their remarkable functional properties and have been widely used in industries such as feed, food, biofuels, and papermaking. For example, in the brewing process of alcoholic beverages, xyloglucans produced by brewing raw materials can cause clogging of filter membranes, thereby affecting the quality of alcoholic beverages. Therefore, the use of acid- and ethanol-resistant xyloglucanases can effectively address this problem. However, according to currently available literature, most xyloglucanases have low activity under acidic conditions and are unable to maintain high activity in high ethanol and salt concentrations, limiting their application in industries such as food processing and feed fermentation.

[0004] Therefore, according to the current situation, it is necessary to construct a highly active xyloglucanase that can operate under acidic, high ethanol concentration and high salt concentration conditions, expand the industrial application range of xyloglucanase, enable it to effectively degrade xyloglucan, and reduce the cost of biomass conversion. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies of the existing technology and provide an acidophilic and halophilic GH12 xyloglucanase gene and its expression protein and application.

[0006] The object of the present invention is achieved through the following technical solutions:

[0007] The present invention provides an acidophilic and halophilic GH12 xyloglucanase gene. The DNA sequence of the acidophilic and halophilic GH12 xyloglucanase gene is shown as SEQ ID NO.1.

[0008] The present invention also provides an acidophilic and halophilic GH12 xyloglucanase, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] The present invention also provides a recombinant vector comprising the DNA sequence shown in SEQ ID NO.1.

[0010] The present invention also provides a recombinant strain comprising the above-mentioned recombinant vector.

[0011] The present invention also provides a primer for amplifying the acidophilic and halophilic GH12 xyloglucanase gene. The primer pair used is shown in SEQ ID NO. 3-4:

[0012] Tth-1: 5'-GAATTCTTAACAATTCTCGACAAGCGG-3'

[0013] Tth-2: 5'-TCTAGATTATTGAACAGAAATAGT-3'.

[0014] The present invention also provides the use of the acidophilic and halophilic GH12 xyloglucanase in the degradation of xyloglucan, biotransformation, food or feed industries.

[0015] The beneficial effects of the present invention are as follows: the acidophilic and halophilic GH12 xyloglucanase expressed by the acidophilic and halophilic GH12 xyloglucanase gene provided by the present invention has the characteristics of acidophilicity and halophilicity, and shows maximum activity at pH values ​​of 4.0 and 40°C; the xyloglucanase maintains a peak activity of more than 40% at a pH value of 3.0-6.0, and maintains a peak activity of more than 60% between 20-55°C; and under acidic conditions, the xyloglucanase shows extremely high stability, maintaining an activity of more than 70%, 90% and 80% at pH values ​​of 2, 3 and 4, respectively; and remains stable for 1 hour within the temperature range of 20-50°C, with the activity maintained at more than 80%; these characteristics make the xyloglucanase suitable for use in environments such as the feed industry, where the operating temperature is about 40°C and the pH value is 4.8; at the same time, Pb 2+ 、Mn 2+ 、Cu 2+ and SDS could significantly inhibit the activity of the xyloglucanase, while 5mMFe 2+The presence of can improve its activity; in addition, in the presence of 1.71M NaCl and 10% ethanol, the activity of the xyloglucanase is still maintained at more than 80%; the xyloglucanase has a high affinity for the substrate, among which the K m Value and V max The values ​​were 1.29 mg / mL and 1032.1 μmol / min / mg, respectively, while the K m Value and V max The values ​​were 2.35 mg / mL and 68.1 μmol / min / mg, respectively. The above-mentioned characteristics make the acidophilic and halophilic GH12 xyloglucanase provided by the present invention have greater advantages than existing xyloglucanases. For example, the halophilicity of the xyloglucanase makes it very suitable for the production of seafood and salty foods. For example, the tolerance of the xyloglucanase to ethanol makes it have broad application prospects in the production of ethanol, beer fermentation, and feed production and fermentation. In short, the above-mentioned characteristics make the xyloglucanase have great industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the PCR verification image of the recombinant strain TaXEG12-X33;

[0017] Figure 2 Schematic diagram of the enzymatic activity of xyloglucanase TaXEG12 using tamarind xyloglucan as substrate;

[0018] Figure 3 This is the SDS-PAGE protein electrophoresis diagram of xyloglucanase TaXEG12;

[0019] Figure 4 This is the optimum pH result diagram of xyloglucanase TaXEG12;

[0020] Figure 5 This is the pH stability result of xyloglucanase TaXEG12;

[0021] Figure 6 This is the optimum temperature result diagram of xyloglucanase TaXEG12;

[0022] Figure 7 This is the thermal stability result diagram of xyloglucanase TaXEG12;

[0023] Figure 8 This is the result diagram of the effect of ethanol concentration on the activity of xyloglucanase TaXEG12;

[0024] Figure 9 This is the result graph showing the effect of NaCl concentration on the activity of xyloglucanase TaXEG12;

[0025] Figure 10 The kinetic parameters of xyloglucanase TaXEG12 using tamarind xyloglucan as substrate;

[0026] Figure 11 This is the kinetic parameter diagram of xyloglucanase TaXEG12 using barley β-glucan as substrate. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to illustrate the present invention, rather than to represent all embodiments. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0028] For experimental methods where specific experimental conditions are not specified, conventional experimental conditions or those recommended by the manufacturer are generally followed. Materials and reagents used were commercially available unless otherwise specified.

[0029] 1. Strain: Trichoderma asperellum ND-1 (GenBank No.: MH496612) was isolated from a soil sample collected in Chifeng (Inner Mongolia, China).

[0030] 2. Vector: Pichia pastoris X-33 and pPICZαA vector used for protein production were purchased from Invitrogen.

[0031] 3. Matrix: Tamarind xyloglucan, beechwood xylan, and barley β-glucan were purchased from Megazyme (Wicklow, Ireland). Sodium carboxymethylcellulose (CMC-Na) and sodium alginate were from Sigma-Aldrich (USA). The present invention is further described below with reference to specific examples, but the scope of the present invention is not limited thereto.

[0032] Example 1: Preparation of the acidophilic and halophilic GH12 xyloglucanase gene XEG12

[0033] The acidophilic and halophilic GH12 xyloglucanase gene, XEG12, was discovered in the genome of T. asperellum ND-1 (GenBank ID: MH496612), isolated from a soil sample collected in Chifeng (Inner Mongolia Autonomous Region, China). The protein encoded by the acidophilic and halophilic GH12 xyloglucanase gene, XEG12, was analyzed using the SignalP server (http: / / www.cbs.dtu.dk / services / signalP), revealing an 18-amino acid signal peptide (MKFIHLVSAFFTANTAAA). Primers for amplifying the acidophilic and halophilic GH12 xyloglucanase gene (excluding the signal peptide) are shown in SEQ ID NOs. 3-4:

[0034] Tth-1: 5'-GAATTCTTAACAATTCTCGACAAGCGG-3'

[0035] Tth-2: 5'-TCTAGATTATTGAACAGAAATAGT-3'.

[0036] The native gene sequence of the acidophilic and halophilic GH12 xyloglucanase gene XEG12 is shown in SEQ ID NO: 1, specifically:

[0037] atgaagtttatacatctggtgtcagccttttttactgccaatacggcagctgctttaacaattctcgacaagcgggctacaacctggtgcgatgcctttggtagtctccaagccggcccatatactgtctttcataataactggggtgcaaataccgcctcatctggatctcagtgtactaccttttcaactttgaatggcaattctgtgcaatggtctacagattggagttgggcagggggcgccgggcacgtaaagtcttattccaacgtggccctggaaaaggttaacaagaagctatctgacattcaacgtattcctactacatggacctggagttatactggatcaaatatggtagcagatgtatcttttgatttgtggctcgccccaaccgcatcgtccaataacgagtacgaaattatgatctgggttggttcatacggtggagcaggcccaatttcgtccactggaagtactccaattgctacacccactattctcggcacaaagtggaaactcttcaaaggcccaaatggagatacaacagtcttttcgtttgttgcgccaagtaacattaagaactggaacggcgatctgaaggcattttttacttatttgacttcaaaggaaggcgttagtgttgggatggttgtcacaagccttcaagccggaactgagcctttctctggcaccaacgctcactttaaaacaactgcatatactatttctgttcaataa。

[0038] Example 2: Construction of Recombinant Strains

[0039] To increase the production of the acidophilic and halophilic GH12 xyloglucanase TaXEG12, the native gene sequence of the acidophilic and halophilic GH12 xyloglucanase gene XEG12 was codon-optimized and synthesized by Beijing Zixi Biotechnology Co., Ltd., resulting in the optimized xyloglucanase gene XEG12-opt. The optimized gene XEG12-opt exhibited 77% similarity to the native gene. The optimized gene XEG12-opt was digested with the restriction endonucleases EcoR I and Xba I and ligated into the double-digested vector pPICZαA. The resulting plasmid, designated pPICZα-XEG12-opt, was used as a recombinant vector and confirmed by DNA sequencing.

[0040] The prepared acidophilic and halophilic GH12 xyloglucanase gene XEG12-opt was heterologously expressed in Pichia pastoris X-33. The recombinant vector pPICZα-XEG12-opt was linearized using the restriction endonuclease Sac I, concentrated, and then electroporated into Pichia pastoris X-33. The recombinant strain was cultured and purified on a YPDS plate containing 100 μg / mL Zeocin resistance to obtain a recombinant strain named TaXEG12-X33. The recombinant strain TaXEG12-X33 was verified using primers AOX-F / AOX-R and a PCR reaction system. The verification results are shown in FIG. Figure 1 As shown in the figure, M represents the standard molecular weight of nucleic acid, 1 represents the negative control group (wild-type Pichia pastoris X-33), 2 represents the positive control group (recombinant vector pPICZα-XEG12-opt), and 3 represents the PCR result using the genome of the recombinant strain TaXEG12-X33 as a template. The appearance of two bands indicates that the recombinant strain TaXEG12-X33 contains the acidophilic and halophilic GH12 xyloglucanase gene XEG12, which means that the recombinant strain TaXEG12-X33 was successfully constructed.

[0041] Primers AOX-F / AOX-R are shown in Table 1. PCR reaction system is shown in Table 2.

[0042] Table 1: Primers AOX-F / AOX-R

[0043]

[0044] Table 2: PCR reaction system

[0045]

[0046] After the PCR cycle was completed, positive recombinant strains were screened based on the size of the amplified products by 1% agarose gel electrophoresis.

[0047] Example 3: Expression of recombinant strain TaXEG12-X33

[0048] The recombinant strain TaXEG12-X33 constructed in Example 2 was pre-cultured in 5 mL of YPD (28°C, 12 h, 200 rpm) and then transferred to a complex medium (BMMY) containing methanol for induction. After 6 days, the expression of the enzyme was assessed using the Bradford method and confirmed by SDS-PAGE. The amino acid sequence of the acidophilic and halophilic GH12 xyloglucanase TaXEG12 protein is shown in SEQ ID NO. 2, specifically:

[0049] MKFIHLVSAFFTANTAAALTILDKRATTWCDAFGSLQAGPYTVFHNNWGANTASSGSQCTTFSTLNGNSVQWSTDWSWAGGAGHVKSYSNVALEKVNKKLSDIQRIPTTWTWSYTGSNMVADV SFDLWLAPTASSNNEYEIMIWVGSYGGAGPISSTGSTPIATPTILGTKWKLFKGPNGDTTVFSFVAPSNIKNWNGDLKAFFTYLTSKEGVSVGMVVTSLQAGTEPFSGTNAHFKTTAYTISVQ

[0050] like Figure 2 As shown, the recombinant strain TaXEG12-X33 was induced with 1% (v / v) methanol for 144 h. Figure 2 As can be seen in the figure, using tamarind xyloglucan as substrate, the xyloglucanase activity of the recombinant strain TaXEG12-X33 reached a maximum of 102.3±2.3 U / mL at 120 h. This activity expression level exceeded that of other xylanases, such as Penicillium oxysporum (1.5 U / mL).

[0051] like Figure 3 As shown, SDS-PAGE analysis of the acidophilic and halophilic GH12 xyloglucanase TaXEG12 expressed by the recombinant strain TaXEG12-X33 was performed. Lane M: Protein molecular weight standard, Lane 1: TaXEG12-X33 fermentation supernatant. The results indicate that the acidophilic and halophilic GH12 xyloglucanase TaXEG12 has a molecular weight of approximately 25 kDa, consistent with its theoretical value. The high purity and specific activity (897.37 U / mg) of the fermentation supernatant make TaXEG12 a cost-effective enzyme for bioconversion processes.

[0052] Example 4: Enzymatic properties analysis of the acidophilic and halophilic GH12 xyloglucanase TaXEG12

[0053] Activity unit (U) is defined as the amount of enzyme required to hydrolyze xyloglucan to produce 1 μmol of glucose per minute.

[0054] (1) Determination of optimal pH

[0055] Prepare 100 mL of each of four 50 mM buffers at different pH values: glycine-hydrochloric acid buffer (pH 2.0-3.0), citric acid-sodium citrate buffer (pH 3.0-4.0), acetic acid-sodium acetate buffer (pH 4.0-6.0), and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 6.0-8.0). Add 50 μL of 0.285 μg / mL acidophilic and halophilic GH12 xyloglucanase TaXEG12 and 150 μL of 5 mg / mL tamarind xyloglucan in each buffer. Incubate at 40°C for 10 min, and measure the enzyme activity at 540 nm. Enzyme activity at different pH values ​​was measured at 50°C, with the maximum enzyme activity set as 100%. Relative enzyme activity at other pH values ​​was determined. Three replicates were used for each group.

[0056] like Figure 4 As shown, the xylanase activity of the acidophilic and halophilic GH12 xyloglucanase TaXEG12 was highest at pH 4.0 and maintained more than 40% of its peak activity at pH 3.0-6.0, confirming the acidophilic nature of the acidophilic and halophilic GH12 xyloglucanase TaXEG12. However, the activity decreased significantly above pH 6.0, reaching only 5% of its peak activity at pH 8.0.

[0057] (2) pH stability

[0058] TaXEG12, an acidophilic and halophilic GH12 xyloglucanase, was diluted in four buffers of varying pH values ​​and incubated at 4°C for 1 hour. The enzyme activity after 1 hour of incubation was measured at 50°C and pH 4.0 using 5 mg / mL tamarind xyloglucan as a substrate. The relative enzyme activity at different pH conditions was calculated, with the direct activity measured at 0 hour of incubation as 100%. Three replicates were used for each group.

[0059] like Figure 5 As shown, the acidophilic and halophilic GH12 xyloglucanase TaXEG12 showed remarkable stability under acidic conditions, maintaining more than 70%, 90% and 80% of its activity at pH 2, 3 and 4, respectively.

[0060] (3) Determination of optimal temperature

[0061] The corresponding substrate and enzyme solutions were prepared using a buffer solution at an optimal pH of 4.0. Enzyme activity was measured at 20-80°C according to the method described for the optimal pH of 4.0 in Example 4. The relative activity at other temperatures was determined, with the maximum enzyme activity as 100%. Three replicates were used for each group.

[0062] like Figure 6 As shown, the optimal temperature for TaXEG12 is 40°C, and it maintains more than 60% of its peak activity between 20-55°C. The activity decreases sharply when the temperature rises, and is completely lost at 80°C.

[0063] (4) Temperature stability

[0064] The enzyme activity was measured at 40°C, pH 4.0 after incubation at different temperatures (20-80°C) for 1 hour. The relative enzyme activity at different temperatures (0 hour incubation) was calculated, with the direct activity measured at the different temperatures (100%) as 100%. Three replicates were used for each group.

[0065] like Figure 7 As shown, the acidophilic and halophilic GH12 xyloglucanase TaXEG12 remained stable for 1 hour in the temperature range of 20-50°C, with the activity maintained at above 80%. These characteristics make the acidophilic and halophilic GH12 xyloglucanase TaXEG12 suitable for applications in environments such as the feed industry, where the operating temperature is approximately 40°C and the pH value is 4.8.

[0066] (5) Effects of metal ions and chemical reagents on the activity of acidophilic and halophilic GH12 xyloglucanase TaXEG12

[0067] When determining the effects of metal ions and chemical reagents on the activity of acidophilic and halophilic GH12 xyloglucanase TaXEG12, the metal ions detected mainly include Zn 2+ 、Cu 2+ 、Ba 2+ 、Mn 2+ 、Al 3+ 、Li + 、Fe 3+ , K + 、Cd 2+ , Ca 2+ , Pb 2+ NH 4+ 、Fe 2+ 、Ni 2+ 、Co 2+ or Mg 2+Chemical reagents include SDS, urea, or EDTA. 2mM and 10mM solutions of SDS, EDTA, urea, and various metal ions were prepared using an optimal pH 4.0 buffer. 2mM and 10mM solutions of SDS, EDTA, urea, and various metal ions were mixed with the enzyme solution in specific proportions. After thorough mixing, the mixture was incubated at 4°C for 1 hour. Enzyme activity was tested at 40°C and pH 4.0 according to the method described in Example 4. The relative enzyme activity under different incubation conditions was determined, with the enzyme activity measured in the enzyme solution without chemical reagents and ions being taken as 100%. Furthermore, the enzymatic activity of the acidophilic and halophilic GH12 xyloglucanase TaXEG12 was evaluated under different ethanol concentrations (0-3.42M) and NaCl (0-20%, v / v). Three replicates were used for each group.

[0068] The effects of metal ions and chemical reagents on the enzyme activity of acidophilic and halophilic GH12 xyloglucanase TaXEG12 are shown in Table 3. At the concentrations of 2 mM and 10 mM, Mg 2+ and Fe 2+ It can increase the activity by 101.9-117.6%; while NH4 + 、Ni 2+ He Li + At 1 mM concentration, it can moderately increase the activity, but at 5 mM concentration, it has little effect. 2+ , Pb 2+ and Cu 2+ The activity of the acidophilic and halophilic GH12 xyloglucanase TaXEG12 was significantly inhibited by K at 2 mM, and the activity was reduced to 39%, 30% and 22% respectively. + and Al 3+ activated, but were respectively Mn 2+ (24.9%), Pb 2+ (25.3%), Cu 2+ (25.2%) and Fe 3+ 2 mM and 10 mM SDS almost completely inactivated TaXEG12, while urea and EDTA increased its activity by 107.3% and 115.3%, respectively, indicating that it is metal-independent.

[0069] The enzymatic activity of the acidophilic and halophilic GH12 xyloglucanase TaXEG12 was further evaluated under different concentrations of NaCl and ethanol. Figure 8As shown, the acidophilic and halophilic GH12 xyloglucanase TaXEG12 maintained more than 80% of its activity in the presence of 0% to 10% (v / v) ethanol, and maintained 60% of its activity at an ethanol concentration of 20%. The tolerance of the acidophilic and halophilic GH12 xyloglucanase TaXEG12 to ethanol highlights the potential of the acidophilic and halophilic GH12 xyloglucanase TaXEG12 as an effective biocatalyst for bioethanol production, and has broad application prospects in the food or feed industry (such as clarification of alcoholic beverages). Similarly, the acidophilic and halophilic GH12 xyloglucanase TaXEG12 also showed strong salt tolerance, such as Figure 9 As shown, the enzymatic activity of the acidophilic and halophilic GH12 xyloglucanase TaXEG12 was maintained at over 90% within a NaCl concentration range of 0 to 1.71 M, reaching a peak activity of 113.4% at 0.34 M NaCl. This salt tolerance is comparable to or even exceeds that of other xyloglucanases. The salt-loving properties of the acidophilic and halophilic GH12 xyloglucanase TaXEG12 make it very suitable for use in the production of seafood and salty foods, which typically require salt concentrations of 0.5-2.5 M.

[0070] Table 3: Effects of metal ions and chemical reagents on the enzymatic activity of the acidophilic and halophilic GH12 xyloglucanase TaXEG12

[0071]

[0072] (6) Substrate specificity and kinetic characteristics of the acidophilic and halophilic GH12 xyloglucanase TaXEG12

[0073] The substrate specificity and kinetic parameters were determined using the standard DNS method. Polysaccharides (tamarind xyloglucan, barley β-glucan, sodium carboxymethylcellulose, beechwood xylan, sodium alginate, and locust bean gum) were used as substrates. The enzyme activity of the recombinant protein against different substrates was measured at 40°C and pH 4.0. Three replicates were designed for each group. The Michaels-Menton constant (K) was calculated using a Lineweaver-Burk plot at substrate concentrations of 1-10 mg / mL. m Value and V max value).

[0074] Among them, the acidophilic and halophilic GH12 xyloglucanase TaXEG12 showed the highest catalytic activity against tamarind xyloglucan (102.3 ± 2.3 U / mL), while its catalytic activity against barley β-glucan was significantly lower (12.7 ± 0.5 U / mL). Catalytic activity against other polysaccharides was extremely low or undetectable, indicating that the acidophilic and halophilic GH12 xyloglucanase TaXEG12 has high substrate specificity. The results are shown in Table 4.

[0075] The kinetic parameters of TaXEG12 were analyzed using tamarind xyloglucan and barley β-glucan as substrates at pH 4.0 and temperature 40℃. Figure 10 As shown in Figure 2, the Km value and Vmax value of tamarind xyloglucan were 1.31 mg / mL and 1032.1 μmol / min / mg, respectively, while the Km value and Vmax value of barley β-glucan were 1.29 mg / mL and 68.1 μmol / min / mg, respectively. Figure 11 Compared with other xyloglucanases, the acidophilic and halophilic GH12 xyloglucanase TaXEG12 has a lower Km value for tamarind xyloglucan, which indicates that the acidophilic and halophilic GH12 xyloglucanase TaXEG12 constructed in the present invention has a higher affinity for the substrate.

[0076] Table 4: Substrate specificity of the acidophilic and halophilic GH12 xyloglucanase TaXEG12

[0077]

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An acidophilic and halophilic GH12 xyloglucanase gene, characterized in that: The DNA sequence of the acidophilic and halophilic GH12 xyloglucanase gene is shown in SEQ ID NO.

1.

2. An acidophilic and halophilic GH12 xyloglucanase expressed by the acidophilic and halophilic GH12 xyloglucanase gene according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that: The recombinant vector comprises the DNA sequence shown in SEQ ID NO.1 in claim 1.

4. A recombinant strain, characterized in that: The recombinant strain comprises the recombinant vector according to claim 3.

5. A primer for amplifying the acidophilic and halophilic GH12 xyloglucanase gene according to claim 1, characterized in that: The primer pair used is shown in SEQ ID NO.3-4: Tth-1: 5'-GAATTCTTAACAATTCTCGACAAGCGG-3' Tth-2: 5'-TCTAGATTATTGAACAGAAATAGT-3'.

6. Use of the acidophilic and halophilic GH12 xyloglucanase according to claim 2 in the degradation of xyloglucan, bioconversion, food or feed industry.

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