Ruminant protozoa alkaline xylanase and use thereof

Abstract

The application discloses a ruminant rumen protozoa alkaline xylanase and application thereof. According to a mutant xylanase Xyn10B gene sequence obtained from Polyplastron multivesiculatum of a goat rumen protozoa, a Pichia pastoris expression strain is constructed, the mature protein of the mutant xylanase Xyn10B is expressed and secreted by the Pichia pastoris, and thus the recombinant xylanase Xyn10B is efficiently obtained. The optimal pH of the recombinant xylanase Xyn10B for degrading beech xylan is 9.0, and the optimal temperature is 50 DEG C.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and relates to a xylanase (Xyn) of the ruminant protozoan "Polyplastron multivesiculatum" and its secretion expression method and application in Pichia pastoris. Background Technology

[0002] Hemicellulose is a relatively abundant renewable resource in nature besides cellulose. Xylan is a hemicellulose composed of xylanose units linked by β-1,4 bonds, and generally requires the combined action of multiple enzymes for its complete degradation. Xylanase (EC 3.2.1.8) is a key enzyme in xylan degradation, used to degrade the β-1,4-glycosidic bonds of the xylan backbone, and has high application value. Xylanase is widely distributed in microorganisms in the natural environment, and xylanases from this source have the advantages of high activity and low cost. However, due to the fact that microorganisms usually live in mild physiological environments, the obtained xylanase is difficult to tolerate harsh temperature and pH conditions.

[0003] The rumen of ruminants serves as a natural and highly efficient site for the degradation of plant cell wall polysaccharides, harboring a large number of microorganisms, including bacteria, protozoa, and fungi. Rumen ciliates, especially protozoa from the subfamilies Dichaeinae and Cephalothorinae, produce a large number of enzymes for degrading lignocellulose and hemicellulose. Studies have shown that the xylanase Xyn10B derived from the rumen protozoan *Polyplastron multivesiculatum* was expressed using *Escherichia coli*, with results indicating that the optimal temperature for Xyn10B is 39℃ and the optimal pH is 7. However, there is a current need to develop novel enzyme preparations based on alkaline xylanases to alleviate the environmental pollution problems caused by chemical bleaching agents in the paper industry. Summary of the Invention

[0004] The purpose of this invention is to provide an alkaline xylanase from ruminant protozoa and its application, wherein the xylanase has good alkali tolerance.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A ruminant rumen protozoan alkaline xylanase, wherein the gene coding sequence of the alkaline xylanase includes the DNA sequence derived from the genome of the rumen protozoan shown in SEQ.ID.NO.5, or the gene coding sequence of the alkaline xylanase includes a DNA sequence having more than 80% identity with the DNA sequence shown in SEQ.ID.NO.5; because the alkaline xylanase has 99% identity with the xylanase Xyn10B sequence published in the NCBI database (protein sequence accession number: CAB65753), the alkaline xylanase is also referred to as a mutant xylanase Xyn10B.

[0007] Preferably, the amino acid sequence of the mutated xylanase Xyn10B is shown in SEQ.ID.NO.4.

[0008] Preferably, the ruminant rumen protozoan is a polyplastron, such as Polyplastron multivesiculatum.

[0009] A recombinant vector expressing xylanase, the recombinant vector comprising the gene coding sequence of the aforementioned mutated xylanase Xyn10B.

[0010] A recombinant strain expressing xylanase, the recombinant strain comprising a host cell transformed with the recombinant vector expressing xylanase described above.

[0011] A method for preparing xylanase includes the following steps:

[0012] 1) Construct a methanol-inducible recombinant vector based on the gene coding sequence of the mutated xylanase Xyn10B, and transform the recombinant vector into host cells to obtain recombinant strains;

[0013] 2) The recombinant strain was induced to express recombinant xylanase Xyn10B through fermentation to obtain fermentation broth;

[0014] 3) Collect the supernatant of the fermentation broth to obtain the recombinant xylanase Xyn10B protein sample.

[0015] Preferably, the host cell is derived from Pichia pastoris, such as Pichia pastoris GS115 competent cells.

[0016] Preferably, the target gene sequence expressed by the recombinant vector is shown in SEQ.ID.NO.6.

[0017] Application of the above-mentioned mutant xylanase Xyn10B in the preparation of enzyme preparations.

[0018] An enzyme preparation comprising the aforementioned mutated xylanase Xyn10B.

[0019] The beneficial effects of this invention are reflected in:

[0020] This invention identifies a mutated xylanase gene, Xyn10B, from the genome of the rumen protozoan *Polyplastron multivesiculatum*, specifically goats. Based on the gene coding sequence, a method for preparing this mutated xylanase Xyn10B was established by secreting and expressing recombinant xylanase Xyn10B (i.e., the mature protein of the mutated xylanase Xyn10B) in a host bacterium. The mutated xylanase Xyn10B not only exhibits high enzyme activity (optimal pH 9.0, optimal temperature 50℃) but also maintains high enzyme activity within a pH range of 5.0–10.0, showing promising applications in livestock feed, papermaking, food, and bioenergy.

[0021] Furthermore, this invention constructs a methanol-inducible recombinant Pichia pastoris strain that efficiently secretes and expresses xylanase (the expression level of recombinant xylanase Xyn10B is 50.8 U / mL) based on the sequence of the rumen protozoan Polyplastron multivesiculatum-specific xylanase (i.e., the mutant xylanase Xyn10B), thus enabling the production of mutant xylanase Xyn10B to have the advantages of simple operation and short cycle. Attached Figure Description

[0022] Figure 1 The image shown is an SDS-PAGE electrophoresis diagram of the recombinant xylanase Xyn10B gene expression product in the example, where the marker represents the protein molecular weight.

[0023] Figure 2 This is a graph showing the optimal pH for recombinant xylanase Xyn10B.

[0024] Figure 3 This is a graph showing the pH stability of recombinant xylanase Xyn10B.

[0025] Figure 4 The graph shows the optimal temperature for determining recombinant xylanase Xyn10B.

[0026] Figure 5 This is a graph showing the thermal stability of recombinant xylanase Xyn10B. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0028] (I) Secretory Expression Method of Polyplastron multivesiculatum Xyn10B Xylanase Mutant in Pichia pastoris

[0029] 1. Construction of Pichia pastoris GS115 recombinant strain

[0030] 1.1 This invention identified a mutated xylanase gene, Xyn10B, in the genome of *Polyplastron multivesiculatum*, a rumen protozoan from ruminants (including Guanzhong dairy goats, whose rumen contents were collected in Yangling, Shaanxi Province in May 2019). The amino acid sequence encoded by this mutated xylanase gene, Xyn10B, is shown below (i.e., SEQ.ID.NO.1), with the three mutated amino acid sites marked with underscores at the corresponding amino acid residues:

[0031] MKKSTIAFLLILTLFSSHLSKKLTILAEEDDSIIVKTSFEDGDTSMFTPRGEGDTSVFVV K TDGGKTGDSYLAVTEREKNWNGAQYDLGKKCTPGGQYLVSAWLKTPWYCNICLSM Q YTDGSGEAHYNNLKCVVSQGDWVEIKEYKFSMPSGCTDVFLYFENTSGNNDFYIDDFELKKAPEGKIQENISSLKDVYKKYFKVGTATTVAEISPKTTQKLIL KHFNSLTPGNELKPESILDRAGTLAAAEETGDYTNPLVKIGAASPILDFCAENDIPVRGHTLVWHSQTPTWFFKEKFDADGDWVDKETMLKRMENYIKNVFDAV KKAYPTINFYAWDVVNEAWLDNGVPRTGGSHEQNANYSPWVKIFGDNSFIKYAFQFARKYGIEGCKYYYNDYNEYMPQKTQAMIDMVKELNEGETLIDGIGLQ SHLDVSFPGLSAYEKAVKLFSETGVDLQITELDVTTNDHSEAGFEKQAQYYSDIMDIIMKYSKSFSAVVFWGTTDDQSWRASRYPLLFNEDYTAKPCFYSIVDN I E

[0032] The first 27 amino acids at the N-terminus, “MKKSTIAFLLILTLFSSHLSKKLTILA” (i.e., SEQ.ID.NO.2), constitute its signal peptide; the base sequence encoding this signal peptide is shown below (i.e., SEQ.ID.NO.3):

[0033] atgaaaaagt ctactatagc attcttatta atcctaactc tcttttcaag tcatttatct

[0034] aaaaaactca caatattggc t

[0035] Therefore, the mature protein encoded by this mutated xylanase Xyn10B gene consists of 506 amino acids, with a theoretical molecular weight of 57 kDa, and its amino acid sequence is shown below (i.e., SEQ.ID.NO.4):

[0036] EEDDSIIVKTSFEDGDTSMFTPRGEGDTSVFVVKTDGGKTGDSYLAVTEREKNWNGAQYDLGKKCTPGGQYLVSAWLKTPWYCNICLSMQYTDGSGEAHYNNLKCVVSQGDWVEIKEYKFSMPSGC TDVFLYFENTSGNNDFYIDDFELKKAPEGKIQENISSLKDVYKKYFKVGTATTVAEISPKTTQKLILKHFNSLTPGNELKPESILDRAGTLAAAEETGDYTNPLVKIGAASPILDFCAENDIPVRGH TLVWHSQTPTWFFKEKFDADGDWVDKETMLKRMENYIKNVFDAVKKAYPTINFYAWDVVNEAWLDNGVPRTGGSHEQNANYSPWVKIFGDNSFIKYAFQFARKYGIEGCKYYYNDYNEYMPQKTQA MIDMVKELNEGETLIDGIGLQSHLDVSFPGLSAYEKAVKLFSETGVDLQITELDVTTNDHSEAGFEKQAQYYSDIMDIIMKYSKSFSAVVFWGTTDDQSWRASRYPLLFNEDYTAKPCFYSIVDNIE

[0037] Based on the amino acid sequence of the mature protein of the mutated xylanase Xyn10B, and following the codon preference of Pichia pastoris, the recombinant xylanase Xyn10B gene sequence of *Polyplastron multivesiculatum* with the signal peptide removed was obtained (synthesized by Beijing Bomed Gene Technology Co., Ltd., with sequence design completed in November 2021, as shown in SEQ.ID.NO.6). This synthesized sequence has 82% identity with the coding sequence of the mature protein of the mutated xylanase Xyn10B in the genome of *Polyplastron multivesiculatum*, i.e., SEQ.ID.NO.5). The corresponding amino acid sequence of the recombinant xylanase Xyn10B protein of *Polyplastron multivesiculatum* (i.e., recombinant xylanase Xyn10B) is the same as that of SEQ.ID.NO.4 above.

[0038] The coding sequence of the mature protein of the aforementioned mutated xylanase Xyn10B (i.e., SEQ.ID.NO.5) is as follows:

[0039]

[0040]

[0041] The above-mentioned synthetic sequence (i.e., SEQ.ID.NO.6) is as follows:

[0042]

[0043] 1.2 The gene fragment encoding the mature rumen protozoan Polyplastron multivesiculatum mutant xylanase Xyn10B was double-digested with EcoRI and NotI (this gene fragment was obtained by introducing corresponding restriction enzyme sites at both ends of the sequence when synthesizing the recombinant xylanase Xyn10B gene sequence of rumen protozoan Polyplastron multivesiculatum with the signal peptide removed), and then ligated into the pPIC9K expression vector (purchased from Invitrogen) that had been double-digested to obtain the recombinant plasmid pPIC9K-Xyn10B. The plasmid was extracted using the Tiangen Plasmid Mini-Prep Kit.

[0044] 1.3 Preparation of Pichia pastoris GS115 competent cells

[0045] Select a single Pichia pastoris GS115 colony (purchased from Invitrogen) from a YPD solid plate and inoculate it into 3 mL of YPD liquid medium. Incubate at 30°C and 200 rpm on a shaker. When the bacterial cell reaches an OD600 of approximately 1.0, transfer it to a 100 mL YPD shake flask and incubate at 30°C and 200 rpm on a shaker. Once the bacterial cells have grown to approximately OD600 1.0, centrifuge at 5000 rpm for 5 min at 4°C, remove the supernatant, add 200 mL of ice water, and resuspend. Centrifuge at 5000 rpm for 5 min at 4°C, remove the supernatant, add 100 mL of ice water, and resuspend. Centrifuge at 5000 rpm for 5 min at 4°C, remove the supernatant, add 50 mL of ice water, and resuspend. Centrifuge at 1500 g for 5 min at 4°C, remove the supernatant, add 4 mL of sorbitol (1 M), and resuspend. Centrifuge at 1500 g for 5 min at 4°C, remove the supernatant, add 2 mL of sorbitol (1 M), and resuspend. Collect for later use.

[0046] 1.4pPIC9K-Xyn10B transformed Pichia pastoris GS115

[0047] The pPIC9K-Xyn10B plasmid to be transformed was linearized using Bgl II restriction endonuclease and electrotransformed into Pichia pastoris GS115 competent cells. The transformed bacterial culture was evenly spread on MD solid plates for culture, and positive single clones, namely Pichia pastoris GS115 recombinant strains, were picked.

[0048] 2. Fermentation culture of recombinant strains and methanol-induced expression of recombinant xylanase.

[0049] Recombinant Pichia pastoris strain GS115 was inoculated into 200 mL of BMGY liquid medium and cultured at 30°C with shaking at 200 rpm for 48 h. The cells were then collected by centrifugation at 5000 rpm for 5 min. The cells were then resuspended in 100 mL of BMGY liquid medium and induced by shaking at 30°C with shaking at 200 rpm. During induction, 500 μL of methanol was added every 12 h as an inducer. After 60 h of induction, the supernatant of the fermentation broth was collected by centrifugation at 12000 rpm for 10 min.

[0050] The culture medium formulations involved in the above steps (all corresponding solid culture media contain 20 g / L agar powder):

[0051] 1) BMGY liquid culture medium (1L): 10g yeast extract, 20g tryptone and 10mL glycerol, sterilized at 121℃ for 15min, then added 100mL YNB and 2mL 500× biotin on a sterile operating table.

[0052] 2) BMMY liquid culture medium (1L): 10g yeast extract and 20g tryptone, sterilized at 121℃ for 15min, then added 100mL YNB, 2mL 500× biotin and 5mL methanol on a sterile operating table.

[0053] 3) YPD liquid culture medium (1L): 10g yeast extract, 20g tryptone and 20g glucose.

[0054] 4) MD solid plate (1L): 20g agarose and 20g glucose.

[0055] Of which, 500× Biotin contains 0.02g of biotin per 100mL.

[0056] The recombinant protein of *Polyplastron multivesiculatum* xylanase Xyn10B in the fermentation broth supernatant was identified using SDS-PAGE electrophoresis. SDS-PAGE results showed a clear protein band of the target molecular weight with almost no other impurity bands, indicating that the recombinant protein of *Polyplastron multivesiculatum* xylanase Xyn10B can be efficiently obtained through secretory expression by the *Pichia pastoris* GS115 recombinant strain. Figure 1 ).

[0057] 3. Activity analysis of recombinant xylanase Xyn10B protein from the rumen protozoan Polyplastron multivesiculatum.

[0058] Enzyme activity was detected using the DNS method, with the following specific steps: Under conditions of pH 9.0 and 50℃, 900 μL of...

[0059] A reaction system consisting of 1.0% xylan substrate derived from beech wood (i.e., beech xylan, purchased from Sigma) and 100 μL of appropriately diluted fermentation broth supernatant was reacted in a water bath for 10 min. The reaction was terminated by adding 1.5 mL of DNS and then boiling in water for 5 min. After cooling, the OD value was measured at 540 nm. One unit of enzyme activity (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar (generated by xylanase from the degradation of beech xylan) per minute under given conditions.

[0060] The results showed that the specific activity of recombinant xylanase Xyn10B was 85 U / mg. Based on the measured enzyme activity, the expression level of recombinant xylanase Xyn10B in the Pichia pastoris GS115 recombinant strain was 50.8 U / mL.

[0061] (II) Determination of the properties of the xylanase Xyn10B mutant of Polyplastron multivesiculatum in ruminants

[0062] 2.1 Determination of the optimal pH and pH stability of recombinant xylanase Xyn10B

[0063] The optimal pH of recombinant xylanase Xyn10B was determined by enzymatic reactions at different pH levels. The specific steps were as follows: 900 μL of beech xylan substrate dissolved in 1.0% buffers derived from different pH values ​​(including 0.1 mol / L glycine-hydrochloric acid buffer, 0.2 mol / L citrate-disodium hydrogen phosphate buffer, 0.1 mol / L Tris-hydrochloric acid buffer, and 0.1 mol / L glycine-sodium hydroxide buffer) was added. 100 μL of appropriately diluted fermentation broth supernatant was added, and the mixture was reacted in a 50℃ water bath for 10 min. Finally, 1.5 mL of DNS was added to terminate the reaction. After cooling, the OD value was measured at 540 nm. 实验 The control group was prepared by first adding 1.5 mL of DNS to a 50°C water bath for 10 min, then adding 100 μL of appropriately diluted fermentation broth supernatant, boiling in water for 5 min, and finally measuring the OD value at 540 nm after cooling. 对照 The relative enzyme activity (relative enzyme activity) is calculated based on the xylanase enzyme activity standard curve using the following formula:

[0064] (1.9215×(OD 实验 -OD 对照 (0.146) × dilution factor

[0065] The results are as follows Figure 2 As shown, the optimal pH for the recombinant xylanase Xyn10B to degrade xylan is 9.0, and it has more than 70% enzyme activity in the pH range of 5.0 to 10.0.

[0066] The pH stability of recombinant xylanase Xyn10B was then determined. The specific steps were as follows: the supernatant of the fermentation broth, which was appropriately diluted with different pH buffers, was placed in a 37°C water bath for 60 min. Then, the enzyme activity was determined in a pH 9.0 buffer system at 50°C. The relative enzyme activity was calculated using the same method as in 2.1.

[0067] The results are as follows Figure 3 As shown, the recombinant xylanase Xyn10B is relatively stable between pH 5.0 and 10.0. After treatment within this pH range for 60 minutes, the remaining enzyme activity is around 80%, indicating that the recombinant xylanase Xyn10B has good alkali tolerance.

[0068] 2.2 Determination of the optimal temperature and thermal stability of recombinant xylanase Xyn10B

[0069] The specific steps for determining the optimal temperature are as follows: Add the supernatant of the fermentation broth diluted 30 times to the substrate buffer system prepared with Tris-HCl buffer at pH 9.0, mix well, and then perform enzyme activity determination at different temperatures. The relative enzyme activity calculation method is the same as in 2.1.

[0070] The specific steps for determining thermal stability are as follows: The supernatant of the fermentation broth diluted 30 times was treated in Tris-HCl buffer at pH 9.0 at 40℃, 45℃, and 50℃ for different times, and then the enzyme activity was determined at 50℃. The relative enzyme activity was calculated using the same method as in 2.1.

[0071] Optimal temperature measurement results ( Figure 4 The results showed that the optimal temperature for the recombinant xylanase Xyn10B to degrade xylan was 50℃, and the remaining enzyme activity was greater than 60% between 40℃ and 55℃. Thermostability assay results ( Figure 5 The results showed that the recombinant xylanase Xyn10B could retain more than 80% of its enzyme activity after being incubated at 40°C for 1 hour.

[0072] (III) Application Examples of Polyplastron multivesiculatum xylanase Xyn10B mutant in ruminant protozoa

[0073] The supernatant of the fermentation broth obtained above contains recombinant polyplastron multivesiculatum xylanase Xyn10B protein with high enzyme activity. This protein can be formulated into xylanase preparations or used directly as a xylanase preparation in the feed industry to improve livestock and poultry production performance, increase feed utilization, reduce feed costs, and improve economic efficiency. Furthermore, due to its tolerance to alkaline conditions, this xylanase preparation has significant application potential not only in the feed industry but also in the paper industry.

[0074] In summary, the present invention demonstrates through experiments that the enzyme activity of the discovered mutant xylanase Xyn10B is essentially unaffected after being kept in a buffer solution at 37°C and pH 5.0–10.0 for 1 hour, or in a buffer solution at 40°C and pH 9.0 for 1 hour. This indicates that it can serve as a novel source of alkaline xylanase and can be widely applied in fields such as papermaking, livestock and poultry feed, food, and bioenergy.

Claims

1. A mutant xylanase Xyn10B, characterized in that: The coding sequence of the mutated xylanase Xyn10B is the DNA sequence derived from the genome of a ruminant protozoan, as shown in SEQ.ID.NO.5, or the coding sequence of the mutated xylanase Xyn10B is shown in SEQ.ID.NO.

6.

2. The mutant xylanase Xyn10B according to claim 1, characterized in that: The amino acid sequence of the mutated xylanase Xyn10B is shown in SEQ.ID.NO.

4.

3. The mutant xylanase Xyn10B according to claim 1, characterized in that: The rumen protozoa of the ruminants are of the genus Polycera ( ). Polyplastron ).

4. A recombinant vector for expressing basic xylanase, characterized in that: The recombinant vector includes the coding sequence of the mutated xylanase Xyn10B as described in claim 1, 2 or 3.

5. A recombinant strain expressing basic xylanase, characterized in that: The recombinant strain comprises a host cell transformed with the recombinant vector expressing basic xylanase as described in claim 4.

6. A method for preparing the mutant xylanase Xyn10B according to claim 1, 2 or 3, characterized in that: Includes the following steps: 1) Construct a recombinant vector based on the coding sequence of the mutated xylanase Xyn10B, transform the recombinant vector into host cells, and obtain a recombinant strain; 2) The recombinant strain was induced to express recombinant xylanase Xyn10B through fermentation to obtain fermentation broth; 3) Collect the supernatant of the fermentation broth to obtain the recombinant xylanase Xyn10B protein sample.

7. The method according to claim 6, characterized in that: The host cell is derived from Pichia pastoris ( Pichia pastoris ).

8. The method according to claim 6, characterized in that: The target gene sequence expressed by the recombinant vector is shown in SEQ.ID.NO.

6.

9. The application of an alkaline xylanase in the preparation of enzyme preparations, characterized in that: The alkaline xylanase is the mutated xylanase Xyn10B as described in claim 1, 2 or 3.

10. An enzyme preparation, characterized in that: The enzyme preparation includes the mutant xylanase Xyn10B as described in claim 1, 2 or 3.

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