Xylanase, preparation method thereof and application of xylanase in comprehensive utilization of corncobs

Abstract

The invention discloses xylanase, a preparation method of the xylanase and application of the xylanase in comprehensive utilization of corncobs. The invention belongs to the technical field of biology, and particularly relates to xylanase, a preparation method of the xylanase and application of the xylanase in comprehensive utilization of corncobs. The recombinant xylanase TaXyn929 is any one of the following proteins: 1) a protein of which the amino acid sequence is a sequence 2 in a sequence table; and 2) the protein with the amino acid sequence being 20th to 329th sites of the sequence 2 in the sequence table. The specific enzyme activity of the enzyme is 1838 U / mg, and the recovery rate is 78.3%. The optimal pH value of the TaXyn929 is 5.0, and the TaXyn929 is kept stable when the pH value is 3.5-9.0; the optimum temperature is 75 DEG C, and the xylanase is kept stable below 75 DEG C, so that the xylanase is acid-resistant and heat-resistant. The novel acid-resistant and heat-resistant xylanase has great application potential in the xylooligosaccharide preparation industry and can be efficiently applied to the aspect of comprehensive utilization of corncobs.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a xylanase and a preparation method thereof and application thereof in comprehensive utilization of corn cobs. Background Art

[0002] Lignocellulose is the most abundant renewable resource in the world. It can be used to produce bio-based fuels, aromatic compounds and value-added chemicals. Xylan is an important component of lignocellulose, accounting for about 20-40% of its total amount. Xylo-oligosaccharides (XOS) are oligomers containing 2-6 xylose units and bound by β-1,4-glycosidic bonds. They can be mainly prepared by xylan conversion. XOS has been widely used in food, feed and medical fields due to its strong prebiotic properties. At present, XOS has been shown to have the functions of maintaining intestinal health, lowering blood cholesterol, antioxidants and lowering blood pressure. Therefore, more and more scholars are paying attention to the production of XOS from lignocellulose. In China, about 46 million tons of corn cobs are produced through industrial processing in the process of agricultural product processing every year. Corn cobs are rich in arabinoxylan, and the preparation of XOS from corn cobs has the advantages of low cost and high yield. However, due to the lack of suitable technical means, the effective utilization of corn cobs is less. Generally, the methods for producing XOS include autohydrolysis, physical method, chemical method, enzymatic hydrolysis method and combined method. Autohydrolysis is an environmentally friendly method, but it is accompanied by high energy consumption. Steam explosion is a typical and commonly used physical method for pretreatment before enzymatic hydrolysis, but it has the disadvantages of high energy consumption and low reproducibility. Therefore, it is of great significance to develop a cleaner and more efficient method to produce XOS from lignocellulosic biomass.

[0003] Organic acid-xylanase hydrolysis has been proven to be a green, efficient and energy-saving strategy for obtaining XOS. Low-concentration organic acid pretreatment can effectively separate hemicellulose and promote the efficiency of xylanase hydrolysis. Xylanase hydrolysis to produce oligoxylose has the advantages of high selectivity, low by-product generation and reduced cost. However, the system is acidic after pretreatment, and the pH adjustment requires the addition of a large amount of chemical reagents, which greatly increases the cost. During the xylanase hydrolysis process, a high temperature needs to be maintained to promote hydrolysis and prevent microbial growth, which is a challenge to the heat resistance of xylanase. In addition, xylanases with high substrate specificity for arabinoxylan are the key factors for enzymatic hydrolysis of corncob xylan to produce oligoxylose. In existing studies, only a very small number of xylanases are simultaneously acid-resistant, heat-resistant and have high arabinoxylan substrate specificity. Therefore, it is of great significance to develop special xylanases for the specific hydrolysis of corncob xylan to prepare oligoxylose. Summary of the invention

[0004] The main problem to be solved by the present invention is how to efficiently utilize corn cobs to prepare xylooligosaccharides.

[0005] In order to solve the above problems, the present invention provides a protein.

[0006] The protein provided by the present invention is any of the following proteins: 1) The protein whose amino acid sequence is sequence 2 in the sequence list; 2) The amino acid sequence is the protein consisting of amino acids 20 to 329 of sequence 2 in the sequence list; 3) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by replacing and / or deleting and / or adding one or more amino acid residues of the protein in 1); 4) A fusion protein is obtained by connecting a protein tag to the N-terminus or / and C-terminus of 1) or 2).

[0007] The above protein is xylanase and is named TaXyn929.

[0008] In order to facilitate the purification or detection of the protein in 1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in Sequence 2 in the sequence list.

[0009] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0010] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0011] A person skilled in the art can easily mutate the nucleotide sequence encoding the protein TaXyn929 of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the protein TaXyn929 isolated by the present invention are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein TaXyn929 and have the function of the protein TaXyn929.

[0012] The above-mentioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0013] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching for the identity of a pair of amino acid sequences, the value of identity (%) can be obtained.

[0014] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0015] Herein, the 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0016] The present invention also provides a nucleic acid molecule encoding the protein described above.

[0017] The nucleic acid molecule is the DNA molecule described in 1) or 2) or 3) or 4) below: 1) The nucleotide sequence is the DNA molecule of sequence 1 in the sequence list; 2) A DNA molecule consisting of nucleotides 58 to 987 of sequence 1 in the sequence listing; 3) A DNA molecule that has 80% or more identity with the nucleotide sequence defined in 1) and encodes the protein described above; 4) A DNA molecule that hybridizes with the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the protein described above.

[0018] In the present invention, Sequence 1 in the sequence list consists of 987 nucleotides, and Sequence 2 consists of 329 amino acids. The nucleotide sequence of Sequence 1 in the sequence list encodes the amino acid sequence shown in Sequence 2 in the sequence list.

[0019] The present invention also provides a biological material containing the DNA molecule described above, wherein the biological material may be any of the following: B1) an expression cassette containing the DNA molecule described above; B2) a recombinant vector containing the DNA molecule described above, or a recombinant vector containing the expression cassette described in B1); B3) A recombinant microorganism containing the DNA molecule described above, or a recombinant microorganism containing the expression cassette described in B1), or a recombinant microorganism containing the recombinant vector described in B2).

[0020] In the above biological materials, the recombinant vector is the recombinant vector pPIC9K-TaXyn929 obtained by inserting the nucleotide sequence from positions 58 to 987 of sequence 1 into EcoRI and NotI of the vector pPIC9K; the recombinant bacteria is the recombinant Pichia pastoris obtained by introducing the recombinant vector into yeast.

[0021] The structure of the recombinant vector pPIC9K-TaXyn929 is described as follows: the nucleotide sequence of sequence 1 is replaced by the nucleotide sequence of the yeast expression vector pPIC9K EcoR I and Not I restriction site, and keeping other sequences of the vector pPIC9K unchanged to obtain a recombinant vector. The recombinant vector can express xylanase.

[0022] The recombinant bacteria can be obtained by introducing the corresponding recombinant vector into a host microorganism.

[0023] The host microorganism can be yeast, bacteria, algae or fungi. The yeast is Pichia pastoris ( Pichia pastoris )GS115.

[0024] The recombinant vector is introduced into Pichia pastoris to obtain recombinant Pichia pastoris.

[0025] The present invention also provides a method for preparing the protein described above, which comprises introducing a DNA molecule capable of expressing the protein described above into a target microorganism to obtain a recombinant microorganism, culturing the recombinant microorganism, collecting the fermentation broth supernatant, and obtaining the protein.

[0026] In the above preparation method, the culturing of the recombinant microorganism may comprise the following steps: 1) inoculating the recombinant microorganism into a fermentation medium for cultivation, and adding glycerol after the glycerol is completely consumed to ensure that the dissolved oxygen content in the fermentation system is 20%-80%; 2) Stop adding glycerol when the wet weight of the bacteria reaches 180-220 g / L; 3) Starving the cells for 30-60 min, and then adding 100% methanol by volume for induction culture to obtain the protein.

[0027] In the above preparation method, the recombinant microorganism is a recombinant Pichia pastoris, and the recombinant Pichia pastoris contains a recombinant vector capable of expressing the protein described above.

[0028] In a specific embodiment, the culturing sequentially undergoes the following steps: 1) Seed culture: inoculate the recombinant bacteria into 150 mL YPD medium and culture overnight at 30°C and 200 rpm until the OD 600 2-6, to obtain seed solution; 2) Basic culture: The seed solution from step 1) was inoculated into a 5 L fermenter containing 1.35 L of basic culture medium. The culture medium temperature was 30°C, the pH was 4.0, and the rotation speed was 600 rpm. After the glycerol was completely consumed, glycerol fed-batch culture was performed; The basic culture medium preparation method can be as follows: CaSO4 0.93 g / L, K2SO4 18.2 g / L, MgSO4·7H2O 14.9 g / L, KOH 4.13 g / L, glycerol 40 g / L, 85% H3PO4 26.7 mL, PTM1 salt 4.375 mL, and the rest is water; 3) Glycerol fed-batch culture: feed glycerol with a mass concentration of 50% (w / v), temperature of 30°C, pH of 5.0, maintain dissolved oxygen at 20%-80% by adjusting the flow acceleration, and feed time of 4-8 h; stop feeding glycerol when the wet weight of the bacteria reaches 180-220 g / L; 4) Methanol fed-batch culture: After stopping the feeding of glycerol, starve for 30-60 min, feed 100% methanol for induction, the rotation speed is 800 rpm, the culture temperature is 28-30℃, the pH is 6.0-6.5, and the dissolved oxygen is 20%-80%.

[0029] The present invention also provides the use of the above-mentioned protein, and / or the above-mentioned biological material and / or the above-mentioned preparation method in any of the following: 1) Application of xylanase in preparation; 2) Application in the preparation of products containing xylanase activity; 3) Application of degraded corncobs in the preparation of xylooligosaccharides.

[0030] The present invention also provides a method for preparing oligoxylan, which comprises: using corn cobs as substrates, using the aforementioned biological material to obtain the aforementioned protein, and performing enzymatic hydrolysis reaction with the protein to obtain oligoxylan.

[0031] The protein is xylanase TaXyn929.

[0032] The present invention also provides a method for degrading corn cobs: 1) Malic acid pretreatment: the corn cob is pretreated with malic acid to obtain a supernatant containing xylo-oligosaccharides and a solid residue 1 rich in cellulose and xylan; 2) Cellulase hydrolysis: hydrolyzing the solid residue 1 with cellulase to obtain a glucose-rich hydrolyzate and a solid residue 2; 3) Xylanase degradation: enzymatically hydrolyzing the solid residue 2 using the xylanase described above to obtain an enzymatic hydrolyzate rich in xylo-oligosaccharides; 4) Recovery of malic acid: The hydrolyzate obtained in step 2) is desalted and freeze-dried, and used as a carbon source to utilize Aspergillus oryzae ( Aspergillus oryzae ) is fermented to obtain malic acid.

[0033] Furthermore, the conditions of the malic acid pretreatment in step 1) may be 160°C, 60 min, and a substrate concentration of 2.5% (w / v).

[0034] Step 2) The conditions for the enzymatic hydrolysis of the cellulase may be 50°C, a substrate concentration of 5% (w / v), a cellulase addition amount of 5-30 U / mL, a glucosidase addition amount of 0.5-2.5 U / mL, and hydrolysis for 24 h.

[0035] Step 3) The xylanase degradation conditions may be 60°C, substrate concentration 10% (w / v), TaXyn929 addition amount 5-40 U / mL, and hydrolysis 1-24 h.

[0036] The present invention provides a xylanase and a gene encoding the same, which are introduced into Pichia pastoris to obtain recombinant Pichia pastoris. The recombinant Pichia pastoris is fermented at a high density in a 5 L fermenter, and the enzyme activity can reach 5896 U / mL, the protein content can reach 7.5 mg / mL, and the wet weight of the bacteria is 381 g / L. The recombinant xylanase pure enzyme is purified, with a specific enzyme activity of 1838 U / mg and a recovery rate of 78.3%. The optimum pH of TaXyn929 is 5.0, and it remains stable between pH 3.5-9.0; the optimum temperature is 75°C, and it remains stable below 75°C. It is an acid-resistant and heat-resistant xylanase, and has unique advantages in production applications such as easy high efficiency, stability, and reduced microbial contamination. Xylanase TaXyn929 can be used to hydrolyze xylan in biomass to prepare xylo-oligosaccharides.

[0037] At the same time, the novel acid-resistant and heat-resistant xylanase of the present invention has great application potential in the oligoxylose preparation industry. Based on a unique biomass conversion strategy, malic acid is used as a catalyst to promote the hydrolysis of xylanase in corn cobs, wherein the malic acid can be prepared by fermentation of Aspergillus oryzae. The carbon source of the Aspergillus oryzae fermentation medium comes from the glucose obtained by hydrolyzing the cellulose in the corn cobs by cellulase. Based on the two processes of oligoxylose preparation and malic acid fermentation, an efficient and green biomass conversion strategy is formed, thereby realizing the development of a sustainable circular biomass economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The diagram (a) and SDS-PAGE purification electrophoresis diagram (b) of the enzyme production process of recombinant Pichia pastoris in a 5 L fermenter with high density fermentation. (■) enzyme activity, (●) protein concentration, (▲) cell wet weight; column M in B is a low molecular weight standard protein, and columns 1-2 are the fermentation supernatant and pure enzyme induced for 144 h, respectively.

[0039] Figure 2 The optimal pH determination curve of xylanase TaXyn929 (citrate buffer (■, pH 3.0-6.0); acetate buffer (●, pH 4.0-6.0); MES (◆, pH 5.5-6.5); MOPS (▲, pH 6.5-7.5); PB (▼, pH 6.0-8.0); Tris-HCl (□, pH 7.0-9.0); CHES (◯, pH 9.0-10.0)).

[0040] Figure 3 This is the pH stability determination curve of xylanase TaXyn929 (citrate buffer (■, pH 3.0-6.0); acetate buffer (●, pH 4.0-6.0); MES (◆, pH 5.5-6.5); MOPS (▲, pH 6.5-7.5); PB (▼, pH 6.0-8.0); Tris-HCl (□, pH 7.0-9.0); CHES (◯, pH 9.0-10.0)).

[0041] Figure 4 This is the optimum temperature determination curve of xylanase TaXyn929.

[0042] Figure 5 This is a temperature stability test curve of xylanase TaXyn929.

[0043] Figure 6 Flowchart of corncob transformation strategy.

[0044] Figure 7Temperature optimization diagram for malic acid pretreatment.

[0045] Figure 8 Optimization diagram for malic acid pretreatment time.

[0046] Fig. 9 Optimization diagram of substrate concentration for malic acid pretreatment.

[0047] Fig.10 This is the optimization diagram for the amount of cellulase added.

[0048] Fig.11 This is the optimization diagram for the amount of glucosidase added.

[0049] Fig.12 Cumulative plot of malic acid fermentation content. DETAILED DESCRIPTION

[0050] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0051] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0052] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0053] The Aspergillus oryzae in the following examples was purchased from CICC, strain number: No. 2005, which has been recorded in: Ding, Q., Luo, QL, Zhou, J., Chen, XL, Liu, LM, 2018. Enhancing l-malate production of Aspergillus oryzae FMME218-37 by improving inorganic nitrogen utilization. Appl. Microbiol. Biotechnol. 102, 8739-8751. The public can obtain the biological material from the applicant. The biological material can only be used for repeating the experiments of the present invention and cannot be used for other purposes.

[0054] The following examples use origin2019 statistical software to process the data, and the experimental results are expressed as mean ± standard deviation.

[0055] Example 1. Xylanase Gene TaXyn929 Cloning and expression of 1. Xylanase Gene TaXyn929 PCR amplification Xylanase Gene TaXyn929 The coding region is 987 bp, the coding sequence of the gene is sequence 1, and the encoded amino acid sequence is sequence 2 for xylanase.

[0056] Synthesis TaXyn929 Gene (sequence 1) was used as a template and PCR amplified using specific primers TaXyn929F and TaXyn929R to obtain PCR products. The primer sequences are as follows: TaXyn929F: 5'-CTGAAGCTTACGTAGAATTCATGGTAAGACCAACGATC-3'; TaXyn929R: 5'-TAGTCCAAGATTTGCAGCAGGCGGCCGCGAATTAATTCGCCT-3'.

[0057] PCR conditions were as follows: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 90 s, 30 cycles; and extension at 72 °C for 5 min.

[0058] 2. Construction of recombinant vector Primers TaXyn929F (5'-CTGAAGCTTACGTAGAATTCATGGTAAGACCAACGATC-3') and TaXyn929R (5'-TAGTCCAAGATTTGCAGCAGGCGGCCGCGAATTAATTCGCCT-3') were designed according to the target gene sequence of xylanase and the Pichia pastoris expression vector. The gene TaXyn929 shown in Sequence 1 was amplified using the above primers TaXyn929F / TaXyn929R.

[0059] The PCR amplification reaction conditions were as follows: 95 °C pre-denaturation for 5 min, 95 °C denaturation for 20 s, 55 °C annealing for 30 s, 72 °C extension for 90 s, 30 cycles; 72 °C post-extension for 5 min. The PCR amplification product TaXyn929 was recovered by 1% agarose gel electrophoresis. The xylanase (TaXyn929) and the Pichia pastoris expression vector pPIC9K (Invitrogen, USA) were cloned and cloned into a PCR product TaXyn929. EcoR I and NotAfter double enzyme digestion with I, the recombinant plasmid pPIC9K-TaXyn929 was obtained by ligation with T4 DNA ligase. The recombinant plasmid was transformed into Escherichia coli DH5α for amplification and verification.

[0060] The structure of the recombinant plasmid pPIC9K-TaXyn929 is described as follows: the nucleotide sequence of sequence 1 replaces the nucleotide sequence of the yeast expression vector pPIC9K EcoR I and Not I restriction site, and keeping other sequences of the vector pPIC9K unchanged to obtain a recombinant vector. The recombinant vector can express xylanase.

[0061] The obtained recombinant vector pPIC9K-TaXyn929 was linearized with restriction endonuclease SacI and then electroporated into Pichia pastoris GS115 (Invitrogen, USA, catalog number C18100) to form the recombinant bacteria GS115 / pPIC9K-TaXyn929. The electroporation conditions were: voltage 2 kV, capacitance 25 μF, resistance 200 Ω, and shock time 5-10 mS. The electroporation process was as follows: 80 μL of pre-cooled thawed on ice was added. P. pastoris About 10 μg of linearized recombinant plasmid was added to GS115 competent cells and mixed, placed in an ice-cold electroporation cup for 5 min, inserted into an electroporator for electroporation for about 5 s, and then added with about 1 mL of pre-cooled 1 mM sorbitol solution and mixed quickly to obtain the recombinant strain GS115 / pPIC9K-TaXyn929.

[0062] 3. Recombinant bacteria expressing xylanase TaXyn929 1. The recombinant bacteria GS115 / pPIC9K-TaXyn929 obtained above was inoculated into MD plates (1.34% YNB, 4×10-5% biotin, 2% glucose) and cultured at 30°C for 3-5 days to obtain His + Transformant.

[0063] 2. After completing step 1, scrape the transformants with sterile water and spread 100 μL on YPD plates (1% yeast extract, 2% tryptone, 2% glucose, G418 concentrations of 1, 2, 3 and 4 mg / mL, respectively, and the rest is water) with different G418 concentrations (INALCO, USA, catalog number: 1758-1811), and culture at 30°C for 3-5 days.

[0064] 3. After completing step 2, take the transformant and inoculate it into BMGY medium (1% yeast extract, 2% tryptone, 100 mM pH 6.0 phosphate buffer solution, 1.34% YNB, 4×10-5% biotin, 1% glycerol, and the rest is water), and culture it at 30°C and 200 rpm in a shaking incubator for 16-18 h.

[0065] 4. After completing step 3, centrifuge the culture at 8000 rpm for 5 min, collect the cells, and resuspend the cells in BMMY medium (1% yeast extract, 2% tryptone, 100 mM pH 6.0 phosphate buffer solution, 1.34% YNB, 4×10-5% biotin, 0.5% methanol, and the rest water) to an OD of 600 The culture medium was about 1.0 and cultured at 30 °C and 200 rpm. Methanol was added every 24 h to a final concentration of 0.5% (v / v). After 72 h, 1 mL of the culture medium was aspirated and centrifuged at 10,000 rpm for 10 min.

[0066] 5. After completing step 4, take the supernatant for enzyme activity determination.

[0067] 4. High-density fermentation of recombinant Pichia pastoris to produce xylanase 1. Inoculate the recombinant Pichia pastoris strain with the highest enzyme production level obtained in step 3 above into 150 mL of BMGY medium and culture overnight at 30 °C and 200 rpm until OD 600 Is 8-10.

[0068] 2. The seed solution obtained in step 1 was inoculated into a 5 L fermentation tank containing 1.35 L of basic fermentation medium BSM (BSM medium (g / L): CaSO40.93g, K2SO418.2g, MgSO4·7H2O 14.9 g, KOH 4.13g, glycerol 50g, 85% H3PO426.7 mL, and the rest was water). The fermentation tank was sterilized, the pH was adjusted to 4.0 with 28% concentrated ammonia water, 6.25 mL of PTM1 trace salt was added, the inoculation amount was 10%, the speed was 600 rpm, and the temperature was 30 °C. When the glycerol was completely consumed (the dissolved oxygen value rose rapidly), the glycerol feeding was started.

[0069] 3. After completing step 2, add 50% (w / v) glycerol, control the temperature at 30 °C, pH 5.0, monitor the dissolved oxygen all the time, and keep the dissolved oxygen > 20% by adjusting the flow acceleration. The addition time is 4-8 h, and the glycerol addition is stopped when the wet weight of the bacteria reaches 180-220 g / L.

[0070] 4. After completing step 3, stop the addition of glycerol and starve for about 30 min, then add 100% methanol for induction, control the speed to 800 rpm, monitor and control the dissolved oxygen >20%, control the temperature to 30 °C, and the pH to 6.0.

[0071] 5. Samples were taken at different time points during the methanol addition process in step 4 to determine the wet weight of the bacteria, the protein content and the enzyme activity of the recombinant xylanase.

[0072] 5. Purification of recombinant xylanase TaXyn929 1. The fermentation supernatant of methanol induction for 168 h in step 4 above was dialyzed for 12 h with 5 L 20 mM PB pH 8.0 buffer solution (buffer solution A), and centrifuged at 4 °C, 10,000 rpm for 10 min, and the supernatant was retained for later use.

[0073] 2. The enzyme solution obtained in step 1 was purified by QSFF strong anion exchange column (1.0 × 10 cm) using the AKTA protein purification system at a flow rate of 0.5 mL / min.

[0074] 3. After completing step 2, set the flow rate to 1 mL / min and elute with buffer solution A until the OD280 of the eluent is <0.05.

[0075] 4. After completing step 3, use buffer solution A containing 100-500 mM NaCl to linearly elute to the eluent OD within 90 min. 280 <0.05.

[0076] 5. Collect the eluate obtained in step 4 and check the protein purity by SDS-PAGE.

[0077] The results are as follows Figure 1 As shown in a and b, the recombinant xylanase TaXyn929 is an electrophoresis grade pure enzyme with a corresponding molecular weight of 35 kDa. The enzyme recovery rate is 78.3%, and the specific enzyme activity of the pure enzyme is 1838 U / mg.

[0078] Example 2: Study on the enzymatic properties of recombinant xylanase TaXyn929 1. Determination of the optimal reaction pH The pure enzyme of xylanase TaXyn929 obtained in Example 1 was used as the test solution, and the enzyme activity was measured at 75°C in different buffer solution systems, and the relative enzyme activity was calculated with the highest enzyme activity as 100%. The various buffer solution systems are as follows: citric acid buffer (pH 3.0-6.0), acetate buffer (pH 4.0-6.0), MES buffer (pH 5.5-6.5), MOPS buffer (pH 6.5-7.5), phosphate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 7.0-9.0), CHES buffer (pH 9.0-10.0).

[0079] The results are as follows Figure 2 As shown, the optimum pH of xylanase TaXyn929 is citric acid 5.0.

[0080] 2. Determination of pH stability The pure TaXyn929 enzyme solution was diluted in different buffer solution systems, placed in a 55 ℃ water bath for 30 minutes, quickly placed in ice water for 30 minutes, and the residual enzyme activity was measured at 75 ℃. The various buffer solution systems are as follows: citrate buffer (pH 3.0-6.0), acetate buffer (pH 4.0-6.0), MES buffer (pH 5.5-6.5), MOPS buffer (pH 6.5-7.5), phosphate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 7.0-9.0), CHES buffer (pH 9.0-10.0). The enzyme activity of the untreated enzyme solution was taken as 100%, and the percentage of residual enzyme activity was calculated.

[0081] The results are as follows Figure 3 As shown, TaXyn929 has good pH stability, and more than 80% of the enzyme activity remains after being incubated in the pH range of 3.5-9.0 for 30 min.

[0082] 3. Determination of optimum temperature The pure TaXyn929 enzyme solution was appropriately diluted with 50 mM citric acid pH 5.0 buffer solution, and the enzyme activity was measured at different temperatures (40-90°C). The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated.

[0083] The results are as follows Figure 4 As shown in the figure, the optimum temperature of TaXyn929 is 75 ℃.

[0084] 4. Determination of temperature stability After the pure TaXyn929 enzyme solution was appropriately diluted with a 50 mM citric acid pH 5.0 buffer solution, it was kept at different temperatures (40-80 °C) for 30 min, quickly placed in ice water for 30 min, and then the residual enzyme activity was measured at 75 °C. The enzyme activity of the untreated enzyme solution was taken as 100%, and the percentage of residual enzyme activity was calculated.

[0085] The results are as follows Figure 5 As shown in the figure, TaXyn929 has good stability below 75 ℃.

[0086] 5. Substrate specificity After the pure TaXyn929 enzyme solution was appropriately diluted with 50 mM citric acid pH 5.0 buffer, the xylanase activity of different substrates was determined. The substrates were beech wood xylan, birch wood xylan, oat xylan, wheat arabinoxylan, barley glucan, soluble starch and xyloglucan. The concentration of the substrate in the reaction system was 10 mg / mL.

[0087] Taking the enzyme activity of TaXyn929 on beech wood xylan as 100%, the relative enzyme activity and specific enzyme activity of the enzyme on birch wood xylan, oat xylan, wheat arabinoxylan, barley glucan, soluble starch and xyloglucan were calculated.

[0088] The results are shown in Table 1. TaXyn929 can hydrolyze a variety of xylan substrates. The highest specific enzyme activity for arabinoxylan is 3381 U / mg, followed by oat xylan, with a specific enzyme activity of 2371 U / mg. The specific enzyme activities for beech xylan and birch xylan are 1838 U / mg and 1635 U / mg, respectively, and it has no hydrolysis ability for other substrates. The enzyme activity of xylanase was determined by 3,5-dinitrosalicylic acid (DNS) method. Unless otherwise specified, the determination steps are as follows: 100 μL of appropriately diluted enzyme solution was added to 900 μL of 1.0% (w / v) beech xylan solution (prepared with pH 5.0, 50 mM sodium citrate buffer), reacted at 75 ℃ for 10 min, 1 mL DNS was added to terminate the reaction, and after boiling for 10 min, 1 mL of 40% potassium sodium tartrate solution was added and the absorbance at a wavelength of 540 nm was measured. Using xylose as the standard, the amount of reducing sugar generated by the reaction was calculated. Under the above reaction conditions, the amount of enzyme required to generate 1 μmol of xylose per minute was defined as one enzyme activity unit (U).

[0089] Table 1. Substrate specificity of xylanase

[0090] 6. Dynamic characteristics Xylans of different mass concentrations (beech wood xylan, 2-8 mg / mL, birch wood xylan, 1-6 mg / mL, oat wood xylan, 10-30 mg / mL and arabinoxylan 3-12 mg / mL) were prepared with 50 mM sodium citrate buffer at pH 5.0, including oat wood xylan (Sigma, X0627, USA); birch wood xylan (Beijing Keerhui, K08631); beech wood xylan (Megazyme, UK, P-XYLNBE); arabinoxylan (Megazyme, UK, ZME-P-RAXY). The enzymatic activity of xylanase TaXyn929 was determined at 75°C and pH 5.0 as follows: 100 μL of appropriately diluted enzyme solution was added to 900 μL of 1.0% (w / v) beechwood xylan solution (prepared with pH 5.0, 50 mM sodium citrate buffer), reacted at 75°C for 5 min, 1 mL of DNS was added to terminate the reaction, and after boiling for 10 min, 1 mL of 40% potassium sodium tartrate solution was added and the absorbance at 540 nm was measured. The amount of reducing sugar generated by the reaction was calculated using xylose as the standard. Under the above reaction conditions, the amount of enzyme required to generate 1 μmol of xylose per minute was defined as one enzyme activity unit (U).

[0091] The reaction time was 5 min, and the maximum reaction rate was calculated by the "Grafit" software V max and K m .

[0092] The results are shown in Table 2. TaXyn929 has good affinity for xylan substrates. The Michaelis constants for beech wood xylan, birch wood xylan, arabinoxylan and oat xylan are 4.5, 3.2, 5.9 and 23.3 mg / mL, respectively. V max They are 3724, 2778, 7041 and 11848 μmol / (min·mg) respectively.

[0093] Further analysis of the apparent second-order rate constant k cat / K m , TaXyn929 for arabinoxylan k cat / K m The catalytic efficiency of TaXyn929 for arabinoxylan is 507.3 mL / (mg·s), which is 1.4 times that of beechwood xylan and 2.3 times that of oat xylan. This further proves that TaXyn929 has high catalytic efficiency for arabinoxylan and is suitable for the degradation of arabinoxylan-rich biomass such as corn cobs.

[0094] Table 2. Kinetic constants of xylanase TaXyn929

[0095] Example 3: Application of recombinant xylanase TaXyn929 in the production of xylo-oligosaccharides from corncobs The specific process of corncob transformation strategy is as follows Figure 6 shown.

[0096] 1. Reaction optimization of malic acid pretreatment of corncobs Malic acid was used to pretreat corncobs and the pretreatment reaction conditions were optimized. The effects of substrate concentration (1%, 2.5%, 5%, 7.5%, 10%, w / v), temperature (120, 140, 160, 180 ℃) and time (20, 40, 60, 80 min) on the content of xylo-oligosaccharides were investigated. The supernatant obtained by centrifugation (10000 rpm, 10 min) was filtered through a 0.22 μm microporous filter membrane and the reaction filtrate 1 was collected. The rest was the solid residue 1 after malic acid pretreatment. The content of xylo-oligosaccharides in the reaction filtrate 1 was determined by HPLC. The yield of xylo-oligosaccharides was the percentage of the total amount of xylo-oligosaccharides in the filtrate 1 to the total xylan content in the corncob.

[0097] The optimal conditions for malic acid pretreatment of corncobs were: pretreatment temperature (160 ℃, Figure 7 ), hydrolysis time (60 min, Figure 8 ) and substrate concentration was 2.5% (w / v, Fig. 9 ). Under the optimized conditions, when the malic acid pretreatment condition was a substrate concentration of 2.5%, the maximum XOS yield was 25.3% after hydrolysis at 160℃ for 60 min.

[0098] 2. Preparation of glucose by enzymatic hydrolysis of cellulose The solid residue 1 pretreated with malic acid in step 1 of this embodiment was hydrolyzed by cellulase and glucosidase, and the enzymolysis reaction conditions were optimized to investigate the effects of the addition amount of cellulase (5-30 U / mL) and the addition amount of glucosidase (0.5-2.5 U / mL) on the glucose content. After the enzymolysis reaction was completed, the enzymolysis solution was heated at 100 ° C for 5 min to terminate the reaction. The supernatant obtained by centrifugation (10000 rpm, 10 min) was filtered through a 0.22 μm microporous filter membrane and the reaction filtrate 2 was collected, and the rest was the solid residue 2 after hydrolysis by cellulase and glucosidase. The glucose content in the reaction filtrate 2 was determined by HPLC. The glucose yield is the percentage of the total amount of glucose in the filtrate 2 to the total cellulose content in the corncob.

[0099] The optimal conditions for cellulase and glucosidase hydrolysis are: when the cellulase addition amount is 25 U / mL ( Fig.10 ), glucosidase added at 2.5 U / mL ( Fig.11 ), the glucose yield was 82.0% (w / w) after hydrolysis at 50 ℃ for 24 h.

[0100] 3. Preparation of xylo-oligosaccharides by secondary enzymatic hydrolysis with xylanase TaXyn929 The recombinant xylanase TaXyn929 obtained in Example 1 was used to perform secondary enzymolysis on the solid residue 2 obtained after enzymolysis of cellulose in step 2, and the effect of time (1-12 h) on the yield of xylo-oligosaccharides after enzymolysis was investigated. After the enzymolysis reaction was completed, the enzymolysis solution was heated at 100 °C for 5 min to terminate the reaction. The supernatant obtained by centrifugation (10000 rpm, 10 min) was filtered through a 0.22 μm microporous filter membrane and the filtrate was collected. The content of xylo-oligosaccharides in the filtrate was determined by HPLC. The yield of xylo-oligosaccharides is the percentage of the total amount of xylo-oligosaccharides in the filtrate to the total xylan content in the corncob.

[0101] The results are as follows Figure 8 As shown in the data, when the addition amount of TaXyn929 was 25 U / mL, the substrate concentration was 10% (w / v), and the hydrolysis time was 12 h at 60 ℃, the yield of xylo-oligosaccharides prepared by xylanase hydrolysis was 31.7% (w / w); after malic acid pretreatment and TaXyn929 enzymatic hydrolysis, the total yield of xylo-oligosaccharides was 56.9% (w / w).

[0102] 4. Fermentation of Aspergillus oryzae to produce malic acid The fermentation medium of Aspergillus oryzae is as follows: CaCO3 80 g / L, glucose 120 g / L, tryptone 3 g / L, KH2PO4 0.15 g / L, K2HPO4 0.15 g / L, 0.1 g / L MgSO4·7H2O, NaCl 0.05 g / L, citric acid 0.01 g / L and FeSO4·7H2O 0.05 g / L, and the rest is water. Glucose comes from step 2 of this example. The effect of fermentation time (0-129 h) on the content of malic acid in the supernatant was investigated.

[0103] The results are as follows Fig.12 As shown in the figure, at 120 h of fermentation, the malic acid content in the supernatant was 71.2 g / L.

[0104] The malic acid cycle formed in this step can be applied to the malic acid pretreatment of corn cobs in step 1 of this embodiment. Based on the two processes of xylooligosaccharide preparation and malic acid fermentation, an efficient and green biomass conversion strategy is formed, thereby realizing sustainable recycling of corn cob biomass utilization.

[0105] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A protein, wherein the protein is any of the following: 1) The protein whose amino acid sequence is sequence 2 in the sequence list; 2) A protein whose amino acid sequence is the amino acids from positions 20 to 329 of sequence 2 in the sequence listing; 3) Proteins obtained by replacing and / or deleting and / or adding one or more amino acid residues of the proteins in 1) or 2) and having the same function; 4) A fusion protein is obtained by connecting a protein tag to the N-terminus or / and the C-terminus of 1) or 2) or 3).

2. A nucleic acid molecule encoding the protein according to claim 1, characterized in that The nucleic acid molecule is the DNA molecule described in 1) or 2) or 3) or 4) below: 1) The nucleotide sequence is the DNA molecule of sequence 1 in the sequence list; 2) The nucleotide sequence is the DNA molecule at positions 58-987 of sequence 1 in the sequence list; 3) A DNA molecule that has 85% or more identity with the nucleotide sequence defined in 1) and encodes the protein of claim 1; 4) A DNA molecule that hybridizes with the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the protein of claim 1.

3. A biological material containing the nucleic acid molecule according to claim 2, characterized in that: The biological material is any one of the following: B1) an expression cassette containing the nucleic acid molecule according to claim 2; B2) a recombinant vector containing the nucleic acid molecule of claim 2, or a recombinant vector containing the expression cassette of B1); B3) A recombinant microorganism containing the nucleic acid molecule of claim 2, or a recombinant microorganism containing the expression cassette of B1), or a recombinant microorganism containing the recombinant vector of B2).

4. The biomaterial according to claim 3, characterized in that B2) The recombinant vector is a recombinant vector obtained by inserting the nucleotide sequence of sequence 1 into the multiple cloning site of vector pPIC9K; B3) The recombinant bacteria is a recombinant Pichia pastoris obtained by introducing the recombinant vector into yeast; the yeast is Pichia pastoris GS115.

5. The method for preparing the protein according to claim 1, characterized in that: The method comprises introducing a DNA molecule capable of expressing the protein of claim 1 into a target microorganism to obtain a recombinant microorganism, culturing the recombinant microorganism, collecting the fermentation liquid supernatant, and obtaining the protein.

6. The preparation method according to claim 5, characterized in that: The culturing of the recombinant microorganism comprises the following steps: 1) inoculating the recombinant microorganism into a fermentation medium for culturing, and feeding glycerol after the glycerol is completely consumed to ensure that the dissolved oxygen content in the fermentation system is 20%-80%; 2) stopping the glycerol feeding when the wet weight of the microorganism reaches 180-220 g / L; 3) starving the microorganism for another 30 minutes, and starting to feed 100% methanol by volume for induction culture to obtain the protein.

7. The preparation method according to claim 6, characterized in that: The recombinant microorganism is a recombinant Pichia pastoris, and the recombinant Pichia pastoris contains a recombinant vector capable of expressing the protein according to claim 1.

8. Use of the protein according to claim 1, or the nucleic acid molecule according to claim 1, the biological material according to claim 3 or 4, and / or the method according to any one of claims 5 to 7 in any of the following: 1) Application of xylanase in preparation; 2) Application in the preparation of products containing xylanase activity; 3) Application of degraded corncobs in the preparation of xylo-oligosaccharides.

9. A method for preparing oligoxylan, characterized in that: The corn cob is used as a substrate, the biomaterial described in claim 3 or 4 is used to obtain the protein described above, and the protein is used to perform an enzymatic hydrolysis reaction to obtain oligoxylan.

10. A method for degrading corn cobs: 1) Malic acid pretreatment: the corn cob is pretreated with malic acid to obtain a supernatant containing xylo-oligosaccharides and a solid residue 1 rich in cellulose and xylan; 2) Cellulase hydrolysis: hydrolyzing the solid residue 1 in step 1) with cellulase to obtain a glucose-rich hydrolyzate and a solid residue 2; 3) Xylanase degradation: enzymatically hydrolyzing the solid residue 2 in step 2) using the xylanase described in claim 1 to obtain an enzymatic hydrolyzate rich in xylo-oligosaccharides; 4) Recovery of malic acid: The hydrolyzate obtained in step 2) is desalted and freeze-dried, and then fermented with Aspergillus oryzae as a carbon source to obtain malic acid.

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