Chimeric xylanase XynZT-1-Z5 and construction method thereof

Abstract

The invention discloses chimeric xylanase XynZT-1-Z5 and a construction method thereof, and relates to the technical field of molecular biology, and the technical key points are as follows: comparing the sequence of xylanase XynZT-1 with the sequence of xylanase from different thermoduric bacteria, analyzing the amino acid conserved site of the xylanase XynZT-1, and replacing the sequence beta5-beta6 of the thermoduric bacteria onto the XynZT-1 to obtain the chimeric xylanase XynZT-1-Z5. The chimeric enzyme XynZT-1-Z5 has efficient overexpression, good solubility and relatively strong temperature and pH stability, is particularly suitable for industrial enzyme catalytic reaction under the conditions of high temperature and extreme pH, and can be widely applied to the food industry, the wine brewing industry, the papermaking industry and the feed industry.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a chimeric xylanase XynZT-1-Z5 and a construction method thereof. Background Art

[0002] GH11 family (Glycoside Hydrolase Family 11) xylanases are widely distributed in nature. Xylanase is a class of enzymes that can decompose xylan. Xylan is an important component of plant cell walls and is mainly found in plant materials such as wood, straw, wheat straw, and corn cobs. Xylanase hydrolyzes the β-1,4-glycosidic bonds in xylan and breaks it down into smaller sugar molecules, such as xylose and xylan oligosaccharides.

[0003] Xylanases mostly present a single domain in structure, in the shape of a right-handed half-grip, with a relative molecular mass generally less than 30 kDa, and an optimum pH value generally in the acidic to neutral range. However, they are specific to xylan substrates from different sources, and their hydrolysis products are mainly oligoxylose with a low content of monosaccharides.

[0004] To this end, the present invention aims to provide a chimeric xylanase XynZT-1-Z5 and a construction method thereof to solve the above problems. Summary of the invention

[0005] The purpose of the present invention is to solve the above problems and provide a chimeric xylanase XynZT-1-Z5 and a construction method thereof. The chimeric enzyme XynZT-1-Z5 is obtained by comparing the sequences of xylanases from different thermotolerant bacteria, analyzing the conservative amino acid sites, and replacing the sequence β5-β6 of the thermotolerant bacteria with that of XynZT-1. The chimeric enzyme is suitable for industrial enzyme catalysis reactions requiring high temperature and extreme pH conditions.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention provides a chimeric xylanase XynZT-1-Z5, the amino acid sequence of the xylanase XynZT-1:

[0008] ISLDNDSEAFKPQGITKFKAYTKQFDDDIHMSKNHATGHVGDLFFTHWKDGGEASLKLSQSGEFEVSWEGGGYNYVGGPGWHYGDVSREIGYRFEQESGASYIALYGWGYDSNMSKDNPAHLVE YYVLQRWTYDPSQNGIFGKTFVSNGVEYSTYRTIRERKPSINNTATFYQYWSKPSKPMPLGEDHKIIFADHVAAWADTGWILPDMNNFDASDDPTYQVMAVEVFNPAKDGKATGKVWDASAQF.

[0009] Amino acid sequence of xylanase XynZT-1-Z5:

[0010] ISLDNDSEAFKPQGITKFKAYTKQFDDDIHMSKNHATGHVGDLFFTHWKDGGEASLKLSSGGNYSTSWSNTGNFVAGKGWNTGDVSREIGYRFEQESGASYIALYGWGYDSNMSKDNPAHLVE YYVLQRWTYDPSQNGIFGKTFVSNGVEYSTYRTIRERKPSINNTATFYQYWSKPSKPMPLGEDHKIIFADHVAAWADTGWILPDMNNFDASDDPTYQVMAVEVFNPAKDGKATGKVWDASAQF.

[0011] The chimeric xylanase XynZT-1-Z5 can maintain good activity under high temperature conditions and has high stability. It can still maintain more than 90% of the activity when the original enzyme is kept at 45°C for 30 minutes, and can still maintain 60% of the activity when the original enzyme is kept at 85°C for 30 minutes. The chimeric xylanase XynZT-1-Z5 is widely used in the food industry, brewing industry, and feed industry, and can decompose the cell wall of brewing raw materials and β-glucan, reduce the viscosity of materials in brewing, and promote the release of effective substances; in the papermaking industry, it can fully dissolve xylan, improve the bleaching rate of pulp, and reduce the amount of chemical bleaching agents.

[0012] The present invention also provides a method for constructing a chimeric xylanase XynZT-1-Z5, characterized in that the method comprises the following steps:

[0013] S1. Screening of Alteromonas macleodii from the ocean;

[0014] S2, sequencing the genome of Alteromonas macleodii, and obtaining the xylanase gene XynZT-1;

[0015] S3. The structural sequence of XynZT-1 was compared with that of 8 thermophilic enzymes. Based on the conservation of amino acids, the original amino groups of β5-β6 were replaced with the corresponding amino acids that were relatively conservative in thermophilic enzymes to construct the chimeric xylanase XynZT-1-Z5.

[0016] Compared with the prior art, this solution has the following beneficial effects:

[0017] The present invention separates and extracts the chimeric xylanase XynZT-1-Z5 from the marine environment, which has efficient overexpression, good solubility, and strong temperature and pH stability, and is particularly suitable for industrial enzyme catalysis reactions requiring high temperature and extreme pH conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a flow chart of the method for constructing the chimeric xylanase XynZT-1-Z5 in an embodiment of the present invention;

[0019] Figure 2 is a sequence comparison diagram of XynZT-1 and a thermostable enzyme in an embodiment of the present invention;

[0020] Figure 3 is a protein purification diagram of XynZT-1-Z5 in an embodiment of the present invention;

[0021] Figure 4 is a comparison of the optimum temperatures of XynZT-1-Z5 and XynZT-1 in the embodiments of the present invention;

[0022] Figure 5 It is a comparison of the thermal stability of XynZT-1-Z5 and XynZT-1 at 50°C in the examples of the present invention;

[0023] Figure 6 is the thermal stability of XynZT-1-Z5 at 85°C in the embodiment of the present invention;

[0024] Figure 7 The optimal pH comparison between XynZT-1-Z5 and XynZT-1 in the embodiments of the present invention;

[0025] Figure 8 It is a comparison of the pH stability of XynZT-1-Z5 and XynZT-1 in the examples of the present invention. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0027] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0028] Example:

[0029] A method for constructing a chimeric xylanase XynZT-1-Z5:

[0030] Alteromonas macleodii was obtained by screening from the ocean, and its genome was sequenced, revealing that it contained the xylanase gene XynZT-1. Analysis of the sequence revealed that it belonged to the GH11 family.

[0031] The structural sequence of XynZT-1 was compared with that of 8 thermophilic enzymes (only the PDB accession number of the thermophilic enzymes is given here, and the thermophilic bacteria and related literature of the corresponding enzymes can be found in the PDB database by the accession number). Based on the conservation of amino acids, the original amino groups of β5-β6 were replaced with the corresponding amino acids that are relatively conservative for thermophilic enzymes ( Figure 2 and Table 1), construct the chimeric xylanase XynZT-1-Z5.

[0032] Table 1 XynZT-1-Z5 replacement sequence list

[0033] Enzymes β5-β6 amino acid sequence XynZT-1 QSGEFEVSWEGGGYNYVGGPGWHY XynZT-1-Z5 SGGNYSTSWSNTGNFVAGKGWNT

[0034] The successfully constructed genetically engineered strains E. coli BL21 (DE3) / pET28a-xynZT-1 and E. coli BL21 (DE3) / pET28a-xynZT-1-Z5 were inoculated into 5 mL of LB medium containing Kana, cultured overnight at 37 ° C, 220 rpm. As seed liquid, inoculated into 200 mL of LB medium containing Kana, and cultured with shaking at 37 ° C, 220 rpm until OD600 reached about 0.6. 200 μL IPTG (0.5 M) was added and expression was induced at 12 ° C for 20-24 h.

[0035] Take the induced culture and centrifuge it at 4500rpm for 20min, and discard the supernatant. Add 15mL of lysis buffer (pH7.0) to the precipitate and oscillate it to fully suspend it. Place it on ice for ultrasonic disruption (power 260W, total disruption time 25min, working time 2s, interval time 4s). After ultrasonic disruption, centrifuge at 4℃ for 40min (8000rpm), and the resulting supernatant is the crude enzyme solution. Use nickel column HisTrapTMHP-5mL to purify the recombinant enzyme.

[0036] The enzyme solution was added with 5×SDS loading buffer, mixed, boiled for 10 min, and then centrifuged at 12000 rpm for 10 min. The supernatant was subjected to SDS-PAGE protein electrophoresis with 5% concentrated gel and 12% separation gel, stained with Coomassie Brilliant Blue R-250 for 2 h, and destained with decolorant for 3 h. The decolorant was changed once in the middle and the results were observed. Figure 3 As shown in the figure, the size of the overexpressed xylanase XynZT-1 and XynZT-1-Z5 protein is about 26kD, which is consistent with the expected size, indicating that the xylanase genes xynZT-1 and xynZT-1-Z5 were successfully overexpressed in E. coli and were soluble. The results of SDS-PAGE showed that the purified recombinant proteins XynZT-1 and XynZT-1-Z5 reached electrophoretic purity ( Figure 3 , lanes 1 and 2).

[0037] The optimum temperature and temperature stability of the recombinant enzyme were determined: in a pH environment, the enzyme solution and the substrate were reacted at different temperatures (35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C) for 15 minutes, and the enzyme activity of the recombinant enzyme was determined respectively, and its relative enzyme activity was calculated to determine the optimum reaction temperature of the xylanase. The enzyme solution was kept at different temperatures (35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 70°C, 75°C, 80°C, 85°C, 90°C) for 1 hour. Samples were taken every 5 minutes, and then the residual enzyme activity was determined. The activity of the enzyme sample without incubation was taken as 100%, and the stability of the enzyme solution was determined.

[0038] The optimal pH and pH stability of the recombinant enzyme were determined: the relative enzyme activity was determined at the optimal reaction temperature under different pH environments (pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0), and the optimal pH of the recombinant enzyme was obtained. The recombinant enzyme was incubated at the optimal temperature for 1 hour under different pH environments, and then the residual enzyme activity was measured respectively, and compared with the enzyme activity without incubation to determine the stability of the recombinant enzyme pH value.

[0039] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A chimeric xylanase XynZT-1-Z5, characterized in that: The amino acid sequence of the chimeric xylanase XynZT-1-Z5 is: ISLDNDSEAFKPQGITKFKAYTKQFDDDIHMSKNHATGHVGDLFFTHWKDGGEASLKLSSGGNYSTSWSNTGNFVAGKGWNTGDVSREIGYRFEQESGASYIALYGWGYDSNMSKDNPAHLVE YYVLQRWTYDPSQNGIFGKTFVSNGVEYSTYRTIRERKPSINNTATFYQYWSKPSKPMPLGEDHKIIFADHVAAWADTGWILPDMNNFDASDDPTYQVMAVEVFNPAKDGKATGKVWDASAQF.

2. A chimeric xylanase XynZT-1-Z5, characterized in that: The chimeric xylanase XynZT-1-Z5 can decompose the cell wall of brewing raw materials and β-glucan; The chimeric xylanase XynZT-1-Z5 can fully dissolve xylan.

3. The method for constructing a chimeric xylanase XynZT-1-Z5 according to claim 1, characterized in that: The method comprises the following steps: S1. Screening of Alteromonas macleodii from the ocean; S2, sequencing the genome of Alteromonas macleodii, and obtaining the xylanase gene XynZT-1; S3. The structural sequence of XynZT-1 was compared with that of 8 thermophilic enzymes. Based on the conservation of amino acids, the original amino groups of β5-β6 were replaced with the corresponding amino acids that were relatively conservative in thermophilic enzymes to construct the chimeric xylanase XynZT-1-Z5.

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