Use of a xylanolytic enzyme from a new guinea flatworm

Abstract

The application discloses application of a xylanase from Galleria mellonella, and belongs to the technical field of xylan degradation. The xylanase from Galleria mellonella is a Cnxy43 xylanase with an amino acid sequence as shown in SEQ ID No. 1. The Cnxy43 xylanase can efficiently degrade xylan from corn cob, and the Cnxy43 xylanase combined with endoxylanase can quickly degrade xylan from beech or selectively remove xylan from arabinoglucan xylan or xylan mixture. The Cnxy43 xylanase can be applied to 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 degrade xylan and are widely found in microorganisms, plants, and some animals. Xylan, a major hemicellulose component in plant cell walls, has a complex structure and is difficult to degrade. Xylanases cleave the glycosidic bonds of xylan, breaking it down into products such as xylooligosaccharides and xylose.

[0003] Currently, the main methods for xylan degradation include chemical, physical, microbial, and enzymatic methods. Chemical methods primarily involve degrading xylan under acidic, alkaline, or strongly oxidizing conditions. The use of chemical reagents can cause environmental pollution, and the harsh reaction conditions can easily lead to side reactions and reduce product purity. Physical methods primarily involve mechanical pulverization, ultrasonic cavitation, or high-pressure steam treatment of xylan to disrupt its structure and promote its degradation. However, these methods are inefficient and cannot achieve rapid degradation. Microbial methods primarily utilize bacteria or fungi that produce xylanases to degrade xylan. However, microbial cultivation and enzyme secretion conditions are complex, degradation efficiency is significantly affected by the microbial growth state, and unstable factors significantly affect the degree of xylan degradation. Enzymatic degradation of xylan offers advantages such as high efficiency, strong specificity, and mild reaction conditions. Therefore, the development of an efficient xylan hydrolase is of great significance for improving xylan degradation methods.

[0004] Xylanases can be divided into the following categories based on their mode of action and substrate specificity: endo-acting xylanases, exo-acting xylanases, β-xylosidases, and α-L-arabinofuranosidases. Due to their widespread applications in biomass conversion, food processing, papermaking, and feed additives, the research and development of xylanases is of great scientific and economic significance. Summary of the Invention

[0005] In response to 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 rapidly degrade xylan derived from beech wood and can highly selectively remove xylan from a mixture of arabinoxylan and xylan. It has the advantages of high efficiency, strong specificity, and mild reaction conditions.

[0006] In order to achieve the above object, the technical scheme adopted by the present application is: to provide an application of a xylanase from a New Guinea rod worm, the xylanase from the New Guinea rod worm being a Cnxy43 xylanase having an amino acid sequence as shown in SEQ ID No. 1; the Cnxy43 xylanase is used in combination with an endoxylanase to degrade xylan from beech or selectively remove xylan in a mixture containing arabinogalactan and xylan.

[0007] Further, the temperature for degrading the xylan from beech is 50 DEG C, and the pH of the degradation system is 7.0.

[0008] Further, the mass ratio of the xylan from beech, the Cnxy43 xylanase and the endoxylanase in the degradation system is 10:0.8-1.2:0.8-1.2.

[0009] Further, the degradation time is 5 min-2 h.

[0010] The beneficial effects of the further technical scheme adopted by the present application are: the xylan from beech is mainly long-chain structure, and the xylan from corn cob is mainly short-chain structure. The endoxylanase degrades the long-chain xylan into short-chain structure, and then the Cnxy43 xylanase degrades the short-chain structure into xylose efficiently and rapidly; that is, the endoxylanase has good effect on long-chain and poor effect on short-chain; while the Cnxy43 xylanase has poor effect on long-chain and good effect on short-chain, and the combination of the two achieves rapid degradation of xylan.

[0011] Further, the temperature for selectively removing the xylan in the mixture containing arabinogalactan and xylan is 50 DEG C, and the pH of the system is 7.0.

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

[0013] Further, the treatment time is 5 min-1 h.

[0014] The beneficial effects of the present invention are as follows: Cnxy43 xylanase has a high expression level of up to 177.7 mg / L. Cnxy43 xylanase has multiple enzymatic activities, including β-xylanase activity, α-arabinosidase activity, and β-galactosidase activity, and can rapidly degrade xylan derived from corn cobs, but has no hydrolytic effect on arabinoxylan. Cnxy43 xylanase, when used in combination with endoxylanase, can rapidly degrade xylan derived from beech wood, degrading beech wood xylan into xylose within two hours. In addition, Cnxy43 xylanase, when used in combination with endoxylanase, has high selectivity for a mixture of arabinoxylan and xylan. In the mixture, 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 the SDS-PAGE detection result 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 The results of the optimal pH test for Cnxy43 xylan hydrolase are shown in Figure 1, where a represents β-xylosidase activity, b represents α-arabinosidase activity, and c represents β-galactosidase activity.

[0019] Figure 5 The figure shows the test results of the effects of different metal ions on the activity of Cnxy43 xylan hydrolase;

[0020] Figure 6 The figure shows the test results of the degradation ability of Cnxy43 xylanase on different xylans;

[0021] Figure 7 Figure 1 shows the test results of the combined use of Cnxy43 xylanase and endoxylanase to degrade beechwood-derived xylan; 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 figure shows the test results of Cnxy43 xylanase and endoxylanase respectively degrading sugarcane-derived arabinoxylan; 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] Figure 9 Figure 1 shows the test results of purifying a mixture of sugarcane-derived arabinoxylan and corncob-derived xylan using 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 represents the addition of Cnxy43 xylan hydrolase, and b represents the addition of endoxylanase;

[0024] Figure 10 The figure shows the detection results of purifying a mixture of sugarcane-derived arabinoxylan and beechwood-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 beechwood-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 of the present invention is 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 Add ampicillin (Amp) to 50 mL of LB medium (10 g / L trypsin, 5 g / L yeast extract, and 10 g / L sodium chloride) 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 1 L LB medium containing 100 μg / mL Amp and culture at 37°C with shaking at 220 rpm until the OD600 value is approximately 0.7.

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

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

[0042] ⑤ The cells were disrupted by ultrasonication and centrifuged at 11000 rpm for 30 min at 4°C to remove cell debris. The supernatant was collected and protein was purified using a Ni column and analyzed 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 flowthrough is the flowthrough obtained after the supernatant in step ⑤ passes through a 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 shown in SDS-PAGE analysis is Cnxy43 xylan hydrolase. Testing has shown that the expression level of Cnxy43 xylan hydrolase expressed in Escherichia coli BL21(DE3) can reach up to 177.7 mg / L.

[0044] Example 2

[0045] 1. Cnxy43 xylanase activity assay

[0046] pNP-β-xylose, pNP-α-arabinose and pNP-β-galactose solutions were prepared as reaction substrates, respectively. Enzyme solution and 50 mM sodium phosphate buffer (pH 7.0) were added, respectively, so that the final concentration of Cnxy43 xylan hydrolase was 1 mg / mL and the final concentration of the reaction substrate was 5 mM. The mixture was incubated at 37°C for 5 min. After sodium carbonate solution was added to terminate the reaction, the absorbance of the reaction solution was detected at 405 nm. 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 parallel experiments were performed 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. 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. Sodium carbonate solution was added to terminate the reaction, and the 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] pH optimum: Set pH at 4, 5, 6, 7, 8, 9, 10, respectively, 7 groups of parallel experiments were carried out to study the optimum pH of the enzyme, and the final concentration of pNP-β-xylose, pNP-α-arabinose and pNP-β-galactose solution was used as the reaction substrate to determine the activity of β-xylosidase, α-arabinosidase and β-galactosidase of Cnxy43 xylanase. The concentration of the prepared buffer solution at different pH was 50 mM, among which sodium acetate (pH 4, 5, 6), PBS (pH 6, 7, 8), Tris-HCl (pH 8, 9, 10). The buffer solution, reaction substrate and enzyme solution were added to the system, and the final concentration of Cnxy43 xylanase was 1 mg / mL. Incubate at 37℃ for 5 min, then detect the light absorption value of the reaction solution at 405 nm after termination of the reaction.

[0051] The results of the detection of the optimum pH of Cnxy43 xylanase are shown in Figure 4 It can be seen that the β-xylosidase activity of Cnxy43 xylanase is best at pH 7, the α-arabinosidase activity is best at pH 6, and the β-galactosidase activity is best at pH 8.

[0052] Metal ions: Set the final concentration of different metal ions in different substrate systems at 10 mM to study the effect of different metal ions on the activity of Cnxy43 xylanase. The final concentration of pNP-β-xylose, pNP-α-arabinose and pNP-β-galactose solution was used as the reaction substrate, and the solution containing metal ions Mg 2+ , Cu 2+ , Co 2+ , Mn 2+ , Ca 2+ , Zn 2+ and EDTA was added to the system, and then the enzyme solution and 50 mM sodium phosphate buffer (pH 7.0) were added, respectively, so that the final concentration of Cnxy43 xylanase was 1 mg / mL. Incubate at 37℃ for 5 min, then detect the light absorption value of the reaction solution at 405 nm after termination of the reaction.

[0053] The detection results of the effect of different metal ions on the activity of Cnxy43 xylanase are shown in Figure 5 Cu 2+ and Zn 2+ inhibit 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 from corn cob, and xylan solution 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 progress of the reaction.

[0057] The results of 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 using Cnxy43 xylanase and endoxylanase

[0060] Three parallel experiments were set up. A beechwood-derived xylan 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 experimental group 1, Cnxy43 xylan hydrolase and 1 mg / mL endoxylanase with a final concentration of 1 mg / mL were added to parallel experimental group 2, and endoxylanase with a final concentration of 2 mg / mL was added to parallel experimental group 3. The reaction times in each parallel experimental group were set at 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 in the results, 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 combination 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 experimental group 1, and endoxylanase with a final concentration of 2 mg / mL was added to parallel experimental group 2. The reaction time in each parallel experimental group was set at 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, this demonstrates that Cnxy43 xylan hydrolase has high selectivity for arabinoxylan and xylan. In a mixture of arabinoxylan and xylan, Cnxy43 xylanase can selectively degrade xylan without degrading arabinoxylan. Cnxy43 xylanase can be used to remove xylan impurities from arabinoxylan. Endoxylanase can partially hydrolyze the arabinose on arabinoxylan, indicating that endooxylanase should not be used to treat arabinoxylan for a long time.

[0066] Example 6

[0067] Purification of a mixture of sugarcane-derived arabinoxylan and corncob-derived xylan using Cnxy43 xylan hydrolase and endoxylanase, respectively.

[0068] Two parallel experiments were set up. Sugarcane-derived arabinoxylan solution with a final concentration of 10 mg / mL and corncob-derived xylan with a final concentration of 5 mg / mL were added to the system, 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 experimental group 1, and endoxylanase with a final concentration of 2 mg / mL was added to parallel experimental group 2. The reaction time in each parallel experimental group was set at 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 Figure 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 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 experimental group 1, Cnxy43 xylan hydrolase with a final concentration of 2 mg / mL and 2 mg / mL endoxylanase were added to parallel experimental group 2, and endoxylanase with a final concentration of 2 mg / mL was added to parallel experimental group 3. The reaction time in each parallel experimental group was set at 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 Figure 10 As shown, neither Cnxy43 xylan hydrolase nor endoxylanase, when used alone, could degrade beechwood-derived xylan into xylose. Endoxylanase could hydrolyze the arabinose on arabinoxylan. However, when Cnxy43 xylan hydrolase and endoxylanase were used together, they could rapidly degrade beechwood-derived xylan into xylose, but the activity of endoxylanase in hydrolyzing arabinoxylan was inhibited. This demonstrates that the combined use of Cnxy43 xylan hydrolase and endoxylanase cannot degrade arabinoxylan. Cnxy43 xylan hydrolase has high selectivity for arabinoxylan and xylan. In a mixture of arabinoxylan and xylan, Cnxy43 xylanase can selectively degrade xylan without degrading arabinoxylan. Therefore, it can be used to remove xylan impurities from arabinoxylan.

[0074] Although the specific embodiments of the present invention have been described in detail in conjunction with the embodiments, this should not be construed as limiting the scope of protection of this patent. Within the scope described by 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 a Cnxy43 xylan hydrolase having an amino acid sequence as shown in SEQ ID No. 1; the Cnxy43 xylan hydrolase is used in combination with an endoxylanase to degrade beechwood-derived xylan or selectively remove xylan from a mixture containing arabinoxylan and xylan; the temperature for degrading beechwood-derived xylan is 50° C., the pH of the degradation system is 7.0, and the mass ratio of the beechwood-derived xylanase, Cnxy43 xylan hydrolase, and endoxylanase in the degradation system is 10:0.8-1.2:0.8-1.2; the temperature for selectively removing xylan from a mixture containing arabinoxylan and xylan is 50° C., the pH of the system is 7.0, and the mass ratio of arabinoxylan, Cnxy43 xylan hydrolase, and endoxylanase in the system is 10:1.8-2.2:1.8-2.

2.

2. The use according to claim 1, characterized in that: The degradation time of beech wood-derived xylan is 5 min to 2 h.

3. The use according to claim 1, characterized in that: The treatment time for selectively removing xylan from a mixture containing arabinoxylan and xylan is 5 min to 1 h.

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