Application of xylan hydrolase derived from new corynesia guineae

Abstract

The invention discloses an application of xylan hydrolase derived from new corynesia guineae, and belongs to the technical field of xylan degradation. The xylan hydrolase derived from the new corynesia guineensis is Cnxy43 xylan hydrolase with an amino acid sequence as shown in SEQ ID No. 1. The xylan hydrolase is a xylan hydrolase derived from the new corynesia guineensis. The Cnxy43 xylan hydrolase can efficiently degrade xylan from a corncob source, and the Cnxy43 xylan hydrolase and endo-xylanase are combined for use, so that xylan from beech can be rapidly degraded or xylan in a mixture of araboxylan and xylan can be removed in a high-selectivity manner. The Cnxy43 xylan hydrolase can be suitable for various industries and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of xylan degradation, and particularly relates to application of a xylan hydrolase derived from New Guinea stick insects. Background Art

[0002] Xylanases are a class of hydrolytic enzymes that can degrade xylan and are widely found in microorganisms, plants and some animals. Xylan is one of the main hemicellulose components in plant cell walls, with a complex structure and is difficult to degrade. Xylanases break down xylan into products such as oligoxylose and xylose by cleaving the glycosidic bonds of xylan.

[0003] At present, the degradation methods of xylan mainly include chemical method, physical method, microbial method, enzymatic method, etc. The chemical method mainly degrades xylan under acidic conditions, alkaline conditions, or under strong oxidizing conditions. The use of chemical reagents may cause environmental pollution, and the reaction conditions are harsh, which is easy to cause side reactions and reduce the purity of the product. The physical method mainly destroys the structure of xylan and promotes the degradation of xylan by mechanical crushing, ultrasonic cavitation effect or high-pressure steam treatment, but the efficiency of physical method for degrading xylan is low and it is impossible to achieve rapid degradation of xylan. The microbial method mainly achieves the purpose of xylan degradation by bacteria or fungi that can produce xylanase, but the conditions for microbial culture and enzyme secretion are relatively complex, the degradation efficiency is greatly affected by the growth state of microorganisms, and the unstable factors have a high impact on the degree of xylan degradation. Enzymatic degradation of xylan has the advantages of high efficiency, strong specificity, and mild reaction conditions. Therefore, an efficient xylan hydrolase is of great significance for the improvement of xylan degradation methods.

[0004] Xylanases can be divided into the following categories according to their mode of action and substrate specificity: endo-xylanase, exo-xylanase, β-xylosidase and α-L-arabinofuranosidase. Due to the wide application of xylanases in biomass conversion, food processing, paper industry and feed additives, their research and development has important scientific and economic significance. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides an application of a xylan hydrolase derived from the New Guinea stick insect. The combination of Cnxy43 xylan hydrolase and endoxylanase can quickly degrade xylan derived from beech wood, and can highly selectively remove xylan from a mixture of arabinoxylan and xylan, and has the advantages of high efficiency, strong specificity and mild reaction conditions.

[0006] In order to achieve the above-mentioned object, the technical scheme adopted by the present invention is: providing an application of a xylan hydrolase derived from the New Guinea stickworm, wherein the xylan hydrolase derived from the New Guinea stickworm is a Cnxy43 xylan hydrolase having an amino acid sequence as shown in SEQ ID No.1; the Cnxy43 xylan hydrolase is combined with an endoxylanase to degrade xylan derived from beech wood or selectively remove xylan from a mixture containing arabinoxylan and xylan.

[0007] Furthermore, the temperature for degrading the beechwood-derived xylan was 50° C., and the pH of the degradation system was 7.0.

[0008] Furthermore, the mass ratio of beech wood-derived xylan, Cnxy43 xylan hydrolase and endo-xylanase in the degradation system is 10:0.8-1.2:0.8-1.2.

[0009] Furthermore, the degradation time is 5min to 2h.

[0010] The beneficial effect of the above further technical scheme adopted by the present invention is that the xylan derived from beech wood is mainly a long-chain structure, and the xylan derived from corn cob is mainly a short-chain structure. Endoxylanase degrades the long-chain xylan into a short-chain structure, and then Cnxy43 xylan hydrolase efficiently and quickly degrades the short-chain structure to form xylose; that is, endoxylanase has a good effect on long chains and a poor effect on short chains; while Cnxy43 xylan hydrolase has a poor effect on long chains and a good effect on short chains, and the two are used together to achieve rapid degradation of xylan.

[0011] Furthermore, the temperature for selectively removing xylan from the mixture containing arabinoxylan and xylan was 50° C., and the pH of the system was 7.0.

[0012] Furthermore, the mass ratio of arabinose, Cnxy43 xylan hydrolase and endoxylanase in the system is 10:1.8-2.2:1.8-2.2.

[0013] Furthermore, the processing time is 5 minutes to 1 hour.

[0014] The beneficial effects of the present invention are: Cnxy43 xylanase has a high expression level, which can reach 177.7 mg / L. Cnxy43 xylanase has multiple enzyme activities, including β-xylanase activity, α-arabinosidase activity and β-galactosidase activity, which can quickly degrade xylan derived from corn cobs, but has no hydrolysis effect on arabinoxylan. Cnxy43 xylanase combined with endoxylanase can quickly degrade xylan derived from beech wood, and degrade xylan derived from beech wood into xylose within two hours. In addition, Cnxy43 xylanase combined with endoxylanase has high selectivity for a mixture of arabinoxylan and xylan. In the mixture of the two, only xylan is degraded without affecting arabinoxylan. It can be seen that Cnxy43 xylanase has high adaptability, high expression level, good activity and good selectivity. It is suitable for various industries such as agricultural resource recycling, food and feed, and papermaking industry, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the map of the Cnxy43 xylan hydrolase recombinant plasmid;

[0016] Figure 2 This is a diagram showing the results of SDS-PAGE detection of Cnxy43 xylan hydrolase;

[0017] Figure 3 This is the result of the test on the optimum temperature of Cnxy43 xylan hydrolase;

[0018] Figure 4 This is the result of the optimal pH test of Cnxy43 xylan hydrolase, where a is the activity of β-xylosidase, b is the activity of α-arabinosidase, and c is the activity of β-galactosidase;

[0019] Figure 5 The figure is the test result of the effect of different metal ions on the activity of Cnxy43 xylan hydrolase;

[0020] Figure 6 The figure is a test result of the degradation ability of Cnxy43 xylanase on different xylans;

[0021] Figure 7 The figure is a test result of the combined use of Cnxy43 xylanase and endoxylanase to degrade xylan from beech wood; wherein Xyl is xylose, control is a negative control, a is the addition of Cnxy43 xylanase, b is the simultaneous addition of Cnxy43 xylanase and endoxylanase, and c is the addition of endoxylanase;

[0022] Figure 8The diagram shows the test results of Cnxy43 xylanase and endoxylanase respectively degrading arabinoxylan from sugarcane; wherein Xyl is xylose, Ara is arabinoxylan, control is a negative control, a is the addition of Cnxy43 xylan hydrolase, and b is the addition of endoxylanase;

[0023] Fig. 9 The figure shows the test results of purifying a mixture of sugarcane-derived arabinoxylan and corncob-derived xylan by Cnxy43 xylanase and endoxylanase, respectively; wherein control is a negative control, Xyl is xylose, Ara is arabinose, Xylan is corncob-derived xylan, AX is sugarcane-derived arabinoxylan, a is the addition of Cnxy43 xylan hydrolase, and b is the addition of endoxylanase;

[0024] Fig.10 The figure shows the test results of purifying a mixture of sugarcane-derived arabinoxylan and beech-derived xylan using a combination of Cnxy43 xylan hydrolase and endoxylanase; wherein control is a negative control, Xyl is xylose, Ara is arabinose, Xylan is beech-derived xylan, AX is sugarcane-derived arabinoxylan, a represents the addition of Cnxy43 xylanase, b represents the simultaneous addition of Cnxy43 xylanase and endoxylanase, and c represents the addition of endoxylanase. DETAILED DESCRIPTION

[0025] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0026] Example 1

[0027] Expression and purification of Cnxy43 xylanase

[0028] (1) The amino acid length of the xylan hydrolase (Cnxy43 xylanase) from the New Guinea stick insect is 327 aa, and its amino acid sequence is as follows:

[0029] MPPLITNIYTADPSAHVFEGKVYIYPSHDRETDIQFNDNGDQYDMADYH

[0030] VFSTDSLDPAAPVTDHGVVLKTEDVPWVSKQLWAPDAAYKDGKYYLYFPAR

[0031] DKQGIFRIGVAVGDRPEGPFQADPEPIRGSYSIDPATFVDDDGQAYMYFGGLW

[0032] GGQLQCYQRGNDVFDAEWQGPKEPSGQGVRALGPRVAKLTDDMRQFATEV

[0033] QEIQILAPETGEPILADDHDRRFFEAAWMHKYNGKYYFSYSTGDTHYLAYAV

[0034] GDSPYGPFTYGGRILEPVLGWTTHHSIVEFKGRWWLFHHDCELSNGIDHLRS

[0035] VKVKEIFYDKDGKIVTEKPE(SEQ ID No. 1).

[0036] (2) The nucleotide sequence corresponding to Cnxy43 xylanase was cloned into the vector pET-15b by gene synthesis. Figure 1 This is a map of the constructed Cnxy43 xylan hydrolase recombinant plasmid. After successful construction, the Cnxy43 xylan hydrolase recombinant plasmid was transformed into Escherichia coli BL21 (DE3).

[0037] (3) Protein expression and purification

[0038] ① In 50 mL LB medium (10 g / L trypsin, 5 g / L yeast extract and 10 g / L sodium chloride), add ampicillin (Amp) to a final concentration of 100 μg / mL, inoculate Escherichia coli containing the Cnxy43 xylan hydrolase recombinant plasmid, and culture at 37°C with shaking at 220 rpm overnight.

[0039] ② Inoculate the overnight culture into 1L LB medium containing 100μg / mL Amp, and culture at 37℃ with shaking at 220rpm until the OD600 value is about 0.7.

[0040] ③ Add IPTG to the cultured bacterial solution (the final concentration in the cultured bacterial solution is 0.4 mM), and culture at 16°C and 180 rpm for about 20 hours to induce Escherichia coli to express Cnxy43 xylan hydrolase.

[0041] ④ Collect bacteria by centrifugation at 8000 rpm for 10 min at 4°C.

[0042] ⑤ The bacteria were disrupted by ultrasound, and the cell debris was removed by centrifugation at 11000 rpm for 30 min at 4°C. The supernatant was collected and the protein was purified using a Ni column and then detected by SDS-PAGE.

[0043] The results of SDS-PAGE detection are as follows Figure 2As shown in the figure, M is a protein marker, the bacteria are the bacteria collected in step ④, the supernatant is the supernatant obtained after centrifugation in step ⑤, the precipitate is the precipitate obtained after centrifugation in step ⑤, the flow-through is the flow-through obtained after the supernatant in step ⑤ passes through the Ni column, and the protein is the protein solution eluted from the Ni column in step ⑤; the protein band with a molecular weight of 37.3 kDa in the SDS-PAGE test is Cnxy43 xylan hydrolase. After testing, the expression level of Cnxy43 xylan hydrolase expressed by Escherichia coli BL21 (DE3) can be as high as 177.7 mg / L.

[0044] Example 2

[0045] 1. Cnxy43 xylanase activity detection

[0046] pNP-β-xylose, pNP-α-arabinose and pNP-β-galactose solutions were prepared as reaction substrates, and enzyme solution and 50 mM sodium phosphate buffer (pH 7.0) were added respectively to make the final concentration of Cnxy43 xylan hydrolase 1 mg / mL and the final concentration of the reaction substrate 5 mM. The mixture was incubated at 37°C for 5 min. Sodium carbonate solution was added to terminate the reaction, and the absorbance of the reaction solution at 405 nm was detected. It was found that Cnxy43 xylan hydrolase had β-xylosidase activity, α-arabinosidase activity and β-galactosidase activity.

[0047] 2. Study on the enzymatic properties of Cnxy43 xylanase

[0048] ① Optimum temperature: Six groups of parallel experiments were set at 20, 30, 40, 50, 60, and 70°C to study the optimal temperature of enzymatic properties. Enzyme solution and 50 mM sodium phosphate buffer (pH 7.0) were added to the system to make the final concentration of Cnxy43 xylan hydrolase 1 mg / mL, and then pNP-β-xylose, pNP-α-arabinose, and pNP-β-galactose solutions with a final concentration of 5 mM were added as reaction substrates. The system was mixed evenly and incubated at various temperatures for 5 minutes. After sodium carbonate solution was added to terminate the reaction, the light absorbance of the reaction solution at 405 nm was detected.

[0049] The optimum temperature test results of Cnxy43 xylanase are as follows Figure 3 As shown, it can be seen that the β-xylosidase activity, α-arabinosidase activity and β-galactosidase activity of Cnxy43 xylanase are all optimal at 50°C.

[0050] ② Optimum pH: 7 sets of parallel experiments with pH values ​​of 4, 5, 6, 7, 8, 9, and 10 were set up to study the optimal pH of enzymatic properties. The activities of β-xylosidase, α-arabinosidase, and β-galactosidase of Cnxy43 xylanase were determined using pNP-β-xylose, pNP-α-arabinose, and pNP-β-galactose solutions with a final concentration of 5 mM as reaction substrates. Buffer solutions with different pH values ​​of 50 mM were prepared, including sodium acetate (pH 4, 5, and 6), PBS (pH 6, 7, and 8), and Tris-HCl (pH 8, 9, and 10). Buffer solution, reaction substrate, and enzyme solution were added to the system to make the final concentration of Cnxy43 xylanase 1 mg / mL. The solution was incubated at 37°C for 5 min. After the reaction was terminated, the absorbance of the reaction solution at 405 nm was detected.

[0051] The optimal pH test results of Cnxy43 xylanase are as follows Figure 4 As shown, it can be seen that the β-xylosidase activity of Cnxy43 xylanase is optimal at pH 7, the α-arabinosidase activity is optimal at pH 6, and the β-galactosidase activity is optimal at pH 8.

[0052] ③ Metal ions: Parallel experiments with different metal ions at a final concentration of 10 mM in different substrate systems were set up to study the effects of different metal ions on the xylan hydrolase activity of Cnxy43. pNP-β-xylose, pNP-α-arabinose, and pNP-β-galactose solutions with a final concentration of 5 mM were used as reaction substrates, and Mg-containing solutions were added to the system. 2+ , Cu 2+ 、Co 2+ , Mn 2+ , Ca 2+ 、Zn 2+ and EDTA solution, and then added enzyme solution and 50mM sodium phosphate buffer (pH 7.0) respectively to make the final concentration of Cnxy43 xylan hydrolase 1mg / mL, incubated at 37℃ for 5min, and detected the light absorbance of the reaction solution at 405nm after terminating the reaction.

[0053] The test results of the effects of different metal ions on the activity of Cnxy43 xylanase are as follows: Figure 5 As shown, Cu 2+ and Zn 2+ Inhibits the β-xylosidase activity, α-arabinosidase activity, and β-galactosidase activity of Cnxy43 xylanase. EDTA inhibits the β-xylosidase activity and β-galactosidase activity of Cnxy43 xylanase. Mg 2+ 、Co 2+ , Mn 2+ , Ca 2+and EDTA enhanced the α-arabinosidase activity of Cnxy43 xylanase.

[0054] Example 3

[0055] Degradation ability of Cnxy43 xylanase on different xylans

[0056] Xylan solution with a final concentration of 10 mg / mL, xylan solution derived from corn cob and xylan solution derived from beech were used as reaction substrates, respectively. 50 mM sodium phosphate buffer (pH 7.0) and Cnxy43 xylanase solution were added. The final concentration of Cnxy43 xylanase in the system was 2 mg / mL. The reaction temperature was 50°C, and the reaction was carried out overnight. Thin layer chromatography (TLC) was used to characterize the extent of the reaction.

[0057] The results of the thin layer chromatography reaction of xylan degradation are as follows Figure 6 As shown, xylan and corncob-derived xylan could be completely degraded by Cnxy43 xylanase, but xylan from beech wood could not be degraded.

[0058] Example 4

[0059] Degradation of xylan from beech wood by combining Cnxy43 xylanase and endoxylanase

[0060] Three parallel experiments were set up. A xylan solution derived from beech wood was added to the system at a final concentration of 10 mg / mL as a reaction substrate, and then 50 mM sodium phosphate buffer (pH 7.0) was added. Cnxy43 xylan hydrolase was added to parallel experiment group 1 at a final concentration of 2 mg / mL, Cnxy43 xylan hydrolase and 1 mg / mL endoxylanase were added to parallel experiment group 2 at a final concentration of 1 mg / mL, and endoxylanase was added to parallel experiment group 3 at a final concentration of 2 mg / mL. The reaction time in each parallel experiment group was set to 5 min, 10 min, 15 min, 20 min, 30 min, 1 h, 2 h and a negative control. The reaction temperature was 50°C. After the reaction was completed, TLC thin layer chromatography was used to characterize the extent of the reaction.

[0061] The detection results of TLC thin layer chromatography reaction are as follows Figure 7 As shown, when Cnxy43 xylan hydrolase is used alone, it cannot degrade xylan derived from beech wood; when endoxylanase is used alone, xylan derived from beech wood is degraded into low molecular weight xylan, but is not completely hydrolyzed to generate xylan; the combined use of Cnxy43 xylan hydrolase and endoxylanase can effectively degrade xylan derived from beech wood, and significantly improve the degradation efficiency of xylan; because xylan derived from beech wood has a certain degree of side chain modification, a small part of it is degraded into low molecular weight oligosaccharides.

[0062] Example 5

[0063] Cnxy43 xylan hydrolase and endoxylanase degrade arabinoxylan

[0064] Two parallel experiments were set up. Arabinoxylan solution with a final concentration of 10 mg / mL was added to the system as a reaction substrate, and then 50 mM sodium phosphate buffer (pH 7.0) was added. Cnxy43 xylan hydrolase with a final concentration of 2 mg / mL was added to parallel experiment group 1, and endoxylanase with a final concentration of 2 mg / mL was added to parallel experiment group 2. The reaction time in each parallel experiment group was set to 5 min, 10 min, 15 min, 20 min, 30 min, 1 h, 2 h and a negative control. The temperature was 50 ° C, and TLC thin layer chromatography was used to characterize the extent of the reaction.

[0065] The detection results of TLC thin layer chromatography reaction are as follows Figure 8 As shown, Cnxy43 xylan hydrolase cannot degrade arabinoxylan. Combined with the results in Example 4, it is shown that Cnxy43 xylan hydrolase has high selectivity for arabinoxylan and xylan. In the mixture of arabinoxylan and xylan, Cnxy43 xylanase can selectively degrade xylan without degrading arabinoxylan. Cnxy43 xylanase can be used to remove xylan impurities in arabinoxylan. Endoxylanase can hydrolyze part of the arabinose on arabinoxylan, indicating that endooxylanase cannot be used to treat arabinoxylan for a long time.

[0066] Example 6

[0067] Cnxy43 xylan hydrolase and endoxylanase were used to purify the mixture of sugarcane-derived arabinoxylan and corncob-derived xylan, respectively.

[0068] Two parallel experiments were set up. Sugarcane-derived arabinoxylan solution and 5 mg / mL corncob-derived xylan were added to the system at a final concentration of 10 mg / mL, and 50 mM sodium phosphate buffer (pH 7.0) was added. Cnxy43 xylan hydrolase was added to parallel experiment group 1 at a final concentration of 2 mg / mL, and endoxylanase was added to parallel experiment group 2 at a final concentration of 2 mg / mL. The reaction time in each parallel experiment group was set to 5 min, 10 min, 15 min, 20 min, 30 min, 1 h and a negative control. The reaction temperature was 50°C, and TLC thin layer chromatography was used to characterize the extent of the reaction.

[0069] The detection results of TLC thin layer chromatography reaction are as follows Fig. 9As shown, Cnxy43 xylan hydrolase can quickly hydrolyze xylan in a mixed solution of arabinoxylan and corncob-derived xylan into xylose without affecting the structure of arabinoxylan. Combined with the experimental results in Example 5, the activity of endo-xylanase on corncob-derived xylan is relatively weak and it is unable to completely hydrolyze xylan.

[0070] Example 7

[0071] Purification of a mixture of sugarcane-derived arabinoxylan and beechwood-derived xylan using Cnxy43 xylan hydrolase and endoxylanase

[0072] Three parallel experiments were set up. Arabinoxylan solution with a final concentration of 10 mg / mL and beechwood-derived xylan with a final concentration of 5 mg / mL were added to the system as reaction substrates, and then 50 mM sodium phosphate buffer (pH 7.0) was added. Cnxy43 xylan hydrolase with a final concentration of 2 mg / mL was added to parallel experiment group 1, Cnxy43 xylan hydrolase with a final concentration of 2 mg / mL and 2 mg / mL endoxylanase were added to parallel experiment group 2, and endoxylanase with a final concentration of 2 mg / mL was added to parallel experiment group 3. The reaction time in each parallel experiment group was set to 5 min, 10 min, 15 min, 20 min, 30 min, 1 h and a negative control. The reaction temperature was 50°C, and TLC thin layer chromatography was used to characterize the extent of the reaction.

[0073] The detection results of TLC thin layer chromatography reaction are as follows Fig.10 As shown, Cnxy43 xylan hydrolase and endoxylanase alone cannot degrade beechwood xylan into xylose, while endoxylanase can hydrolyze arabinose on arabinoxylan. When Cnxy43 xylan hydrolase and endoxylanase are used together, beechwood xylan can be quickly degraded into xylan, but the activity of endoxylanase in hydrolyzing arabinoxylan is inhibited. It is proved that the combination of Cnxy43 xylan hydrolase and endoxylanase cannot degrade arabinoxylan, and Cnxy43 xylan hydrolase has high selectivity for arabinoxylan and xylan. In the mixture of arabinoxylan and xylan, Cnxy43 xylanase can selectively degrade xylan but not arabinoxylan, so it can be used to remove xylan impurities in arabinoxylan.

[0074] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. An application of a xylan hydrolase derived from a New Guinea stick insect, characterized in that: The New Guinea stickworm-derived xylan hydrolase is Cnxy43 xylan hydrolase having an amino acid sequence as shown in SEQ ID No. 1; Cnxy43 xylan hydrolase is used in combination with endoxylanase to degrade beechwood-derived xylan or selectively remove xylan from a mixture containing arabinoxylan and xylan.

2. The use according to claim 1, characterized in that: The temperature for degrading beechwood-derived xylan was 50°C, and the pH of the degradation system was 7.

0.

3. The use according to claim 2, characterized in that: The mass ratio of beech wood-derived xylanase, Cnxy43 xylan hydrolase and endo-xylanase in the degradation system is 10:0.8~1.2:0.8~1.

2.

4. The use according to claim 3, characterized in that: The degradation time is 5min~2h.

5. The use according to claim 1, characterized in that: The temperature for selectively removing xylan from the mixture containing arabinoxylan and xylan was 50° C., and the pH of the system was 7.

0.

6. The use according to claim 5, characterized in that: The mass ratio of arabinoxylan, Cnxy43 xylan hydrolase and endo-xylanase in the system is 10:1.8~2.2:1.8~2.

2.

7. The use according to claim 6, characterized in that: The processing time is 5min~1h.

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