Xylanase as well as coding gene, preparation method and application thereof

Abstract

The invention discloses an endo-xylanase XynA derived from Xanthomonas campestris, a coding gene of the endo-xylanase XynA, a preparation method of the endo-xylanase XynA, and an application of the endo-xylanase XynA, the coding gene of the endo-xylanase XynA and the coding gene of the endo-xylanase XynA. The amino acid sequence of the endo-xylanase disclosed by the invention is as shown in SEQ ID NO. 2; the sequence of the coding gene is as shown in SEQ ID NO. 1. The optimal reaction temperature of the xylanase provided by the invention is 45 DEG C, good enzyme stability is kept at 20-40 DEG C, the xylanase has substrate preference for xylan Xylan, the main polymerization degree of the product is 4-6, and the xylanase can be applied to degradation of xylan.

Description

Technical Field

[0001] The present invention relates to an endo - xylanase gene derived from Xanthomonas campestris belonging to the tenth family of glycoside hydrolases, a preparation method and application of its expressed protein, and belongs to the field of microbial genetic engineering. Background Art

[0002] The plant cell wall is a thick wall existing on the periphery of plant cells and is one of the main characteristics differentiating animal cells. Its main components are cellulose, hemicellulose, lignin, and there are also structural proteins. Generally, in wood, cellulose accounts for 40 - 50%, and there are also 10 - 30% hemicellulose and 20 - 30% lignin.

[0003] In recent years, with the increasingly prominent energy problem, hemicellulose has gradually been regarded as one of the most abundant and cheapest renewable resources on earth. However, hemicellulose is a complex sugar composed of structural units such as D - xylose, L - arabinose, D - mannose, D - glucose, D - galactose, and trace amounts of L - rhamnose. Due to the complexity of its structure, the current application of hemicellulose is only limited to fields such as the paper industry and additives for industrial products. Hemicellulose is renewable, recyclable, degradable, and environmentally friendly, with great potential for utilization and market value.

[0004] Xylan is the main component of plant hemicellulose and is the second most abundant biomass resource in nature after cellulose. It is a heterogeneous polysaccharide present in plant cell walls, accounting for about 15% - 35% of the dry weight of plant cells. Most xylans are complex - structured, highly branched heteropolymer molecules containing many different substituents. Therefore, the biodegradation of xylan also requires a complex enzyme system, and xylan is degraded into xylo - oligosaccharides and a small amount of xylose through the synergistic action of various components.

[0005] Xylo - oligosaccharides, also known as xylo - oligomers or xylo - oligosaccharides, are functional oligosaccharides formed by the binding of 2 - 7 xylose molecules through β - 1,4 glycosidic bonds. They are a newly discovered type of functional oligosaccharide with probiotic properties in recent years. Xylo - oligosaccharides are difficult to be decomposed by human digestive enzymes, have a high residual rate in the intestine, can promote the proliferation activity of intestinal Bifidobacterium, and have good acid and heat stability and are difficult to ferment. Even when heated to 100°C under acidic conditions (pH = 2.5 - 7), they are basically not decomposed. Their sweetness is only 40% of that of sucrose, and after consumption, they will not cause a large increase in plasma glucose content. Therefore, they can be used as sweeteners for diabetes or obesity patients. They have wide applications in the fields of medical and health products, dairy beverages, foods, and feeds.

[0006] Xylanolytic enzyme systems are a class of enzyme systems that degrade xylan and can degrade xylan-containing hemicelluloses widely present in nature, including β-1,4-endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-D-glucuronidase, acetylxylan esterase, and phenolic acid esterase. Among them, β-1,4-endoxylanase is the most crucial hydrolytic enzyme. It hydrolyzes the β-1,4-glycosidic bonds of xylan molecules, hydrolyzing xylan into xylooligosaccharides such as oligosaccharides and xylobiose, as well as a small amount of xylose and arabinose.

[0007] In-depth database and literature research have shown that currently, enzymes with endo-1,4-β-xylanase activity can be found in 9 different GH families: GH5, GH7, GH8, GH10, GH11, GH30, GH43, GH51, and GH141. GH10 and GH11 are the earliest developed and applied xylanase-containing families, initially called the F family and now one of the most comprehensively studied and reviewed families in the xylanase-containing families (Biely, 1985; Biely et al., 2016; Pollet et al., 2010a). Almost all enzymes in the GH10 family are endo-1,4-β-xylanases, but there are also a small number of endoglucanases (enzyme number EC3.2.1.4) and tomatinases (EC3.2.1.-), as well as the reducing-end xylose-releasing exo-β-oligoxylanase (EC3.2.1.156). It has long been known that GH10 family xylanases exhibit a certain degree of relaxed substrate specificity, and their activity towards the glycosidic bonds of aryl cellobioside has been considered a characteristic distinguishing them from GH11 enzymes.

[0008] Highly active and highly specific xylanase is the key to xylanase-based preparation. Xylanase is widely distributed in nature and exists in marine and terrestrial bacteria, marine algae, fungi, and the rumen of ruminants, snails, crustaceans, terrestrial plant tissues, and various invertebrates. It has been widely used in industries such as pulp and paper, food processing, and energy development. Using xylanase to produce xylooligosaccharides and making full use of xylan can reduce pollution and turn waste into treasure. Currently, xylanases from different sources have been successfully heterologously expressed in hosts such as Escherichia coli, Bacillus subtilis, and Pichia, and the expression levels of xylanase have been increased to varying degrees. In the actual application process, xylanase needs to meet the harsh conditions required in industrial production and needs to have good thermal stability, a wide range of pH adaptability, and high specific activity. In addition, there are still many problems to be solved in the production and popularization of xylanase. For example, it should have high specificity for substrate hydrolysis, no xylosidase activity, and a high proportion of xylobiose and xylotriose in the products.

[0009] The genus Xanthomonas includes a wide range of plant pathogens that utilize many virulence factors for pathogenicity and adaptation in plant hosts. Xanthomonas is a genus of Gram-negative bacteria in the Gammaproteobacteria class and contains species that are pathogenic in more than 400 different plant hosts, such as rice, wheat, citrus, tomato, pepper, cabbage, cassava, banana, and legumes. In recent years, research on Xanthomonas spp. has integrated the host, pathogen, and microbial community that affect disease occurrence, making it a model system for studying plant pathogenic bacteria. In 2008, the interaction between xanthomonadin extracted from Xanthomonas gardneri and the model plant was studied through biochemical analysis of the activity of secreted proteins in Arabidopsis thaliana leaves. In June 2010, it was found that the Xps-T2S system of the plant pathogen Xanthomonas campestris pv vesicatoria (Xcv) promotes disease development and helps in the translocation of effector proteins, which are delivered into plant cells by the type III secretion (T3S) system. The Xps system helps in the secretion of proteases and xylanases, and significant differences in substrate specificity were found in the T2S system of the genus Xanthomonas. In July 2010, studies showed that Xanthomonas oryzae pv oryzae (Xoo) and Xanthomonas campestris pv campestris (Xcc) share a conserved mechanism for DSF biosynthesis and autoregulation. The three DSF-family signals produced by Xoo all promote the production of EPS and xylanase activity in Xoo, but the composition and ratio of the three signals are affected by the composition of the culture medium. In 2013, a comprehensive understanding of the utilization of xylan by Xanthomonas campestris pv campestris (Xcc) was achieved through a combination of genomic and functional analyses, highlighting the role of xylan in the adaptation of this epiphytic pathogen to the phyllosphere, and exploring the sugar degradation mechanism of Xcc. In 2014, starting from the two main xylanase-related genes xynA and xynB of the xylan CUT system, it was demonstrated that XynA is a rare exo-oligoxylanase that releases reducing-end xylose and structural analysis was carried out, revealing the molecular basis of xylan degradation by Xanthomonas and showing how these enzymes cooperate to assist in infection and pathogenesis. In 2015, it was found that the T2S system of Xanthomonas campestris pv. vesicatoria can secrete virulence-related xylanases, a predicted protease, and a lipase, and after secretion assays, it was concluded that outer membrane vesicles (OMVs) provide an alternative transport route for type II-secreted extracellular enzymes. Overall, Xanthomonas has become a model system for understanding emerging bacterial plant pathogens and diversity. Summary of the Invention

[0010] The present invention provides an endo-xylanase derived from a strain of the genus Xanthomonas (Xanthomonas campestris), its encoding gene, and a preparation method and application of the expressed protein.

[0011] To achieve the above-mentioned invention objectives, the present invention adopts the following technical solutions:

[0012] The present invention provides an endo-xylanase XynA, whose amino acid sequence is shown in SEQ ID NO.2.

[0013] MLLGLLGMAVSPIASAACAPGTTALKDAYAGGFLIGTAVNTDIVSGKDAASAALVGCHFNAVTAENVMKAEVVAPQPGVFDFTAADAFVADRQRRGMFIVGHTLVWHNQTPEWFFVDANGKPNTSQAQLERMRAHIARVAGRYVGKVQAWDVVNEIIDEDGSYRSTNWVQRVGDGDTVVRNAFAFAQRYAPDAQLYYNDFNAWRPAKRDGIVRMVKMLQQAGIRIDGVGMQGHWGLNYPSVQDIEAAIDAYAALGVKVMITELDIDVLPVTKEGQVIGTGFAHKQFQLPEFKHFLDPYPDGLPPQVQTQLRDRYAELFALFWRKRDKLARVSVWGVSDGMSWKNDYPVPGRTNYPLLFDRNHQPKPALDAVLAVPAGASGKQAEG

[0014] The second aspect of the present invention provides an endo-xylanase gene encoding the aforementioned endo-xylanase.

[0015] Furthermore, the nucleotide sequence of the endo-xylanase gene is shown in SEQ ID NO.1.

[0016]

[0017] In the third aspect of the present invention, a method for preparing endo-xylanase is provided, which is to clone the aforementioned xylanase gene into a recombinant expression vector, introduce it into a host cell, and obtain recombinantly expressed endo-xylanase.

[0018] Further, the expression vectors for recombinantly expressing xylanase are Escherichia coli expression vectors, yeast expression vectors, and Bacillus subtilis expression vectors.

[0019] Further, the recombinant bacterium or transgenic cell line for recombinantly expressing xylanase is one of Escherichia coli host cells, yeast host cells, and Bacillus subtilis host cells.

[0020] The present invention also provides the application of the aforementioned endo-xylanase in xylan degradation.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The present invention provides a new endo-xylanase belonging to glycoside hydrolase-10 derived from the XynA strain of Xanthomonas campestris. The optimal reaction temperature is 45 °C, and it maintains good enzyme stability at 20 to 40 °C. It has a substrate preference for xylan Xylan, and the main polymerization degree of the product is 4-6, with a proportion greater than 60% in the reaction product. It can be applied to xylan degradation and can efficiently degrade and utilize xylan under the conditions of 40 to 50 °C. It can be widely applied in the fields of pharmaceutical health products, dairy beverages, food, and feed. Description of the Drawings

[0023] Figure 1 Polyacrylamide gel electrophoresis diagram (SDS-PAGE) of the expression and purification of recombinant endo-xylanase XynA. The samples added to each lane are as follows: Lane 1: supernatant after disruption of the recombinant bacterium; Lane 2: concentrated solution after ultrafiltration replacement of the supernatant after disruption of the recombinant bacterium and elution and collection with 200 mmol of imidazole through a nickel column; Lane 3: protein marker (marker); Lane 4: elution and collection solution of the supernatant after disruption of the recombinant bacterium and elution with 200 mmol of imidazole through a nickel column, with a sample loading volume of 5 μl.

[0024] Figure 2 Curve of the effect of pH value on the activity of endo-xylanase XynA; A. Optimal reaction pH value of XynA; B. pH value stability of XynA.

[0025] Figure 3 Curve of the effect of temperature on the activity of endo-xylanase XynA; A. Optimal reaction temperature of XynA; B. Thermal stability of XynA.

[0026] Figure 4 Substrate preference curve of endo-xylanase XynA.

[0027] Figure 5 High performance anion exchange chromatography (HPAEC-PAD) analysis chart of the products obtained by the degradation of xylan by endo-xylanase XynA. Specific implementation manners

[0028] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0029] Example 1 Xylanase gene sequence analysis

[0030] The strain of the genus Xanthomonas (Xanthomonas campestris) includes a wide range of plant pathogens and utilizes many virulence factors for pathogenicity and adaptation in plant hosts. We retrieved the genome of this bacterium in NCBI. Due to the importance of xylanase in plant immunity, the xylanase gene in this bacterium was found and analyzed. The sequencing results were analyzed using the Basic Local Alignment Search Tool (BLAST) in the GenBank database, and multiple sequence alignments were performed using MEGA 7 software to analyze the sequence information.

[0031] The coding region of the xylanase gene (named XynA) obtained by gene synthesis is 1158 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1. XynA encodes 385 amino acids and a stop codon, and its amino acid sequence is shown in SEQ ID NO.2. The theoretical molecular weight of the protein is 58.9 kDa, and the predicted isoelectric point is 8.20. The amino acid sequence of XynA has the highest identity (86%) with the xylanase (accession number AAM43336) from the Xanthomonas campestris strain. Its sequence was submitted and its activity was studied, and it has no activity for hydrolyzing xylan chains (Santos C R, Hoffmam Z B, de Matos Martins V P, et al. Molecular mechanisms associated with xylan degradation by Xanthomonas plant pathogens [J]. Journal of Biological Chemistry, 2014, 289(46): 32186-32200.). The domain characteristics of XynA are more similar to those of the members of glycoside hydrolase family 10 (GH10), indicating that XynA is a new member of the GH10 family.

[0032] Example 2 Recombinant Expression and Purification of XynA Gene in Escherichia coli

[0033] The synthesized recombinant pET32a-xynA was transformed into E. coli BL21(DE3), and then xylanase XynA was induced to express and purified. The expression and purification of xylanase XynA were detected by polyacrylamide gel electrophoresis, and the results were as follows Figure 1 shown. The purified xylanase XynA showed a single band on the electrophoresis gel, and its position was consistent with the predicted molecular weight.

[0034] Example 3 Enzymatic Property Analysis of Xylanase XynA

[0035] (1) Determination of Xylanase XynA Activity

[0036] The homogenized xylan was diluted to 1% (w / v). After adding an appropriate amount of recombinant enzyme XynA, the reaction was carried out for 10 min, and its activity was determined by the 3,5-dinitrosalicylic acid (DNS) method. The enzyme activity unit was defined as: the amount of enzyme required to release 1 μmol of reducing sugar (calculated as glucose) per minute was one enzyme activity unit (U).

[0037] (2) Effect of pH on Enzyme Activity

[0038] Under the condition of 45 °C, xylan at 1% (pH 3.0 - 6.0 Citrate, pH 6.0 - 8.0 PBS, pH 8.0 - 9.0 Tris-HCl) was used as the substrate, and the enzyme activity was measured by the aforementioned 3,5-dinitrosalicylic acid (DNS). According to the relative activity of the enzyme under different pH conditions, a curve was plotted to determine the optimal reaction pH of the enzyme. The inactivated enzyme was used as a control, and the highest activity value was taken as 100%, and the relative activity of the enzyme under each reaction pH condition was measured. The results showed that XynA reached the maximum activity at pH 7.0, indicating that the optimal reaction pH of XynA was 7.0 (as shown in Figure 2 A).

[0039] XynA was placed in buffer solutions at pH 3.0 - 11.0 (pH 3.0 - 6.0 Citrate, pH 6.0 - 8.0 PBS, pH 8.0 - 9.0 Tris-HCl) at 4 °C for 4 h, and then its activity was measured at 45 °C. Taking the enzyme activity of the untreated group as 100%, the relative residual activity of the enzyme under each pH condition was measured. The results were as shown in Figure 2 B, and the pH stability was the best in pH 8.0 PBS.

[0040] (3) Effect of Temperature on Enzyme Stability

[0041] Under the condition of pH 7.0, using 1% xylan as the substrate, the activity of the recombinant enzyme was measured at 20 - 70 °C according to the standard method, and a curve was plotted based on the relative activity of the enzyme at different temperatures. Using the inactivated enzyme as a control, the relative enzyme activity was calculated with the highest enzyme activity in the reaction as 100%. The results are as Figure 3 shown in A. The optimal reaction temperature of XynA is 45 °C, and its activity is poor at 20 - 35 °C and 55 - 70 °C.

[0042] Under the conditions of the optimal temperature and optimal pH, the specific activity of XynA measured by the standard method was 58.1 U / mg, and no activity was detected for the previously published xylanase from the same source that degrades xylan (86% homology). The results of the article show that this enzyme cannot hydrolyze the xylan chain, and further biochemical analysis indicates that this enzyme is not an endo-β-1,4-xylanase. (Santos C R, Hoffmam ZB, de Matos Martins V P, et al. Molecular mechanisms associated with xylan degradation by Xanthomonas plant pathogens[J]. Journal of Biological Chemistry, 2014, 289(46): 32186 - 32200.).

[0043] XynA was incubated at 20 - 70 °C for 30 min, and its activity was measured according to the standard method. Taking the enzyme activity of the untreated group as 100%, the relative residual activity of the enzyme at each temperature was measured. The results are as Figure 3 shown in B. XynA has good thermal stability at temperatures below 40 °C.

[0044] (4) Substrate preference of XynA

[0045] The purified XynA was mixed with eight different substrates, namely xylan from sugarcane, xylan from beech, xyloglucan, dextran, PGA, chitosan, curdlan, xylan from oats, and cellulose, each with a mass concentration of 1%, at a ratio of 1:9 (volume ratio). The specificity of the recombinant enzyme XynA for each substrate was measured at pH 7.0 and 45 °C according to the standard detection method. The results are as Figure 4 shown. The recombinant enzyme XynA showed the highest activity towards xylan from beech, indicating that XynA prefers to degrade xylan from beech.

[0046] Example 4 Effect of metal ions on the activity of XynA

[0047] Mix a xylan substrate with a mass concentration of 1%, a purified XynA enzyme solution, and 200 mM PBS buffer (pH 7.0) in a certain proportion. Then, add different metal ions to the reaction system with a final concentration of the added ions being 5 mM. Next, react at 45 °C for 1 h, and measure the enzyme activity by the aforementioned dinitrosalicylic acid method. The control group is the enzyme activity without adding any metal ions (set as 100%). The results are shown in the following table. The experimental results show that K + , Mg 2+ , Fe 2+ , Fe 3+ , Mn 2+ , Ba 2+ , Co 2+ can increase the activity of XynA.

[0048] Table 1 Effect of metal ions on the activity of XynA

[0049]

[0050]

[0051] Example 5 High-performance anion-exchange chromatography (HPAEC-PAD) analysis of the products obtained by XynA degrading xylan

[0052] After mixing a xylan substrate with a mass concentration of 3%, a purified XynA enzyme solution, and 200 mM PBS buffer in a ratio of 10:1:9, the product after reacting at pH 7.0 and 55 °C for 3 hours is desalted and then detected by high-performance anion-exchange chromatography.

[0053] Among them, the results of the high-performance anion-exchange chromatography detection of the product after 3 hours of enzymatic hydrolysis are as Figure 5 shown. The chromatogram shows that there are a series of xylo-oligosaccharides with different degrees of polymerization in the product of XynA degrading xylan for 3 hours. Among them, the main degree of polymerization distribution is DP3-6, and the product has the characteristic of narrow distribution. Based on its substrate preference and product distribution characteristics, it has application potential in the preparation of xylo-oligosaccharides with specific degrees of polymerization.

[0054] For any person skilled in the art, without departing from the scope of the technical solution of the present invention, many possible changes and modifications can be made to the technical solution of the present invention by using the above-disclosed technical content, or it can be modified into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent change, and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. An endo-xylanase XynA, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. An endo-xylanase gene, characterized in that, Encoding the endo-xylanase described in claim 1.

3. The endo-xylanase gene according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

4. A method for preparing endo-xylanase, characterized in that: It is to clone the xylanase gene described in claim 1 into a recombinant expression vector, introduce it into a host cell, and obtain a recombinantly expressed endo-xylanase.

5. The method for preparing endo-xylanase according to claim 4, characterized in that, The expression vectors for the recombinantly expressed xylanase are Escherichia coli expression vectors, yeast expression vectors, and Bacillus subtilis expression vectors.

6. The method for preparing endo-xylanase according to claim 4, characterized in that, The recombinant bacterium or transgenic cell line for recombinantly expressing xylanase is one of Escherichia coli host cells, yeast host cells, and Bacillus subtilis host cells.

7. The application of the endo-xylanase according to claim 1 in xylan degradation.

Images