Burkholderia and its use in the production of tannases and degradation of tannins

Abstract

The application discloses Burkholderia and application thereof in preparation of tannase and degradation of tannin, and belongs to the technical field of microorganisms. pp The full-length gene of the disclosed tannase XY820_Tan is derived from mining of whole genome data of Paraburkholderia phytofirmans XY820, a symbiotic bacterium of tea tree, and the application first discovers a bacterial tannase suitable for an optimal catalytic temperature of 60-70 DEG C, and performs heterologous cloning, expression, purification and characterization of enzymatic properties of the tannase in E.coli Rosetta. The tannase shows heat resistance and excellent enzymatic properties, effectively solves the problem of high-temperature tolerance of tannase in industrial application, and is expected to reduce production cost.

Description

Technical Field

[0001] This invention relates to the field of microbial engineering technology, and in particular to Burkholderia and its application in the preparation of tanninases and the degradation of tannins. Background Technology

[0002] Tannic acid hydrolase, or tanninase for short, is a hydrolase that catalyzes the hydrolysis of ester bonds in tannins, complex tannins, and gallic acid esters, releasing free gallic acid, ellagic acid, and other small molecule polyphenols. It has great application potential in the cosmetics industry, food and feed industry, leather industry, and pharmaceutical chemical industry.

[0003] Tanninases can be prepared from microorganisms, animals, and plants. Due to the diverse biochemical characteristics, enzyme stability, and ease of large-scale cultivation of microorganisms, industrially produced tanninases are currently mainly derived from fungi and bacteria. Commercially available tanninases are primarily prepared through the fermentation of fungi (such as Aspergillus). Although recombinant expression is a common method for preparing pure enzymes, cloning fungal tanninase genes has proven difficult and inefficient due to the complexity of fungal enzymes. Bacterial tanninases can be considered as an alternative, but current research at the molecular level is limited. Only a small number of bacterial tanninase genes with desirable characteristics have been cloned and expressed in recombinant form. This lack of research may be a key factor limiting their large-scale application.

[0004] To date, two main types of bacterial tanninases have been reported in the literature. Extracellular tanninases (encoded by the tanA gene), with a molecular size of approximately 66 kDa, include TanA from Lactobacillus plantarum. Lp , TanA of Staphylococcus lugdunensis Sl TanA of Atopobium parvulum Ap and TanA of Streptococcusgallolyticus Sg Intracellular bacterial tanninases (encoded by the tanB gene), with a molecular weight of approximately 50 kDa, include TanB from L. plantarum. Lp TanB of S. gallolyticus Sg , TanB of Fusobacterium nucleatumsubsp.Polymorphum Fnp , TanB of Lactobacillus paraplantarum NOS120 Lpa TanB of Lactobacillus pentosus 21A-3 Lpe .

[0005] Tea is an important economic crop in my country, rich in polyphenols. Research on tanninases associated with tea plant symbiotic bacteria is scarce; currently, only one tanninase (TanHcw) derived from tea plant bacteria has been biochemically characterized in publicly available literature. Therefore, screening for tanninases in tea plant symbiotic bacteria and constructing recombinant expression plasmids can provide guidance for the subsequent development and heterologous expression of other tanninases. Furthermore, tea plant tanninases play a crucial role in tea plant secondary metabolism and influence tea quality, providing a foundation for further research into the interaction between tea plant microorganisms and the host tea plant. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a reliable and marketable Burkholderia strain and its application in the preparation of tanninases and the degradation of tannins.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by this invention is as follows:

[0008] A strain of *Paraburkholderia pallida*, XY820, was deposited on September 19, 2023, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 28480.

[0009] The present invention also provides a tanninase XY820_Tan pp Its amino acid sequence is shown in SEQ ID NO.1.

[0010] Based on the above, the present invention also provides an encoding of the aforementioned tanninase XY820_Tan pp The gene, whose nucleotide sequence is shown in SEQ ID NO.2.

[0011] As a preferred embodiment, the gene described in this scheme is preferably derived from Burkholderia paraknegrius XY820 as described above.

[0012] Based on the above, the present invention also provides a recombinant Escherichia coli E. coli Rosetta, characterized in that it contains the gene described in claim 3 or 4.

[0013] Based on the above, the present invention also provides a method for constructing the recombinant Escherichia coli E. coli Rosetta, comprising: designing specific primers, using Burkholderia paraknegriensis XY820 DNA as a template, amplifying by PCR and recovering tanninase XY820_Tan via gel electrophoresis. ppGene fragment, obtained tanninase XY820_Tan pp The gene was ligated to the expression vector using T4 ligase to obtain the recombinant plasmid pMal-c5x-XY820_Tan pp Then the recombinant plasmid pMal-c5x-XY820_Tan pp Recombinant E. coli Rosetta strain was obtained by transforming E. coli Rosetta strain.

[0014] Based on the above, the present invention also provides a method for preparing tanninase, which uses the above-described recombinant E. coli Rosetta or the recombinant E. coli Rosetta obtained by the above-described construction method, characterized in that: the recombinant E. coli Rosetta is inoculated into a culture medium and cultured, and then the target protein tanninase XY820_Tan is induced by IPTG. pp The gene expression was then processed by cell disruption and purification to obtain tanninase.

[0015] Based on the above, the present invention also provides an application of tanninase in the degradation of tannins, wherein the tanninase is the aforementioned tanninase XY820_Tan. pp Or tanninase prepared by the method described above.

[0016] By adopting the above technical solution, the beneficial effects of this invention compared with the prior art are as follows: the tanninase XY820_Tan in this solution... pp The full-length gene was derived from the mining of the whole genome data of *Burkholderia paraknegriensis* XY820, a symbiotic fungus of tea trees. Based on this, this protocol used *Burkholderia paraknegriensis* XY820 DNA as a template, designed specific primers, and PCR amplification to obtain the *XY820_Tanpp* gene. The gene was then ligated to an expression vector using T4 ligase to obtain the recombinant plasmid pMal-c5x-XY820_Tanpp. Finally, this recombinant plasmid was transformed into *Escherichia coli* Rosetta strain. After IPTG induction, sonication, and centrifugation, heterologous expression of the tanninase *XY820_Tanpp* was achieved. The tanninase produced extracellularly by the recombinant strain was purified using Amylose resin. The obtained bacterial tanninase has a molecular weight of approximately 61.69 kDa, and its optimal reaction temperature is 60℃, with an optimal pH of 6.0. 2+ and Ca 2+ It can enhance the activity of this enzyme, while Mn 2 + Mg 2+ Zn 2+Furthermore, Tween 80, Triton X-100, and the metal ion chelating agent EDTA exhibit inhibitory effects. In addition, this enzyme demonstrates broad activity on a variety of substrates, including methyl gallate, ethyl gallate, propyl gallate, lauryl gallate, epigallocatechin gallate, and epicatechin gallate. The high-temperature tolerance and broad substrate applicability of this tanninase contribute to reduced production costs and show promising applications in both industrial and scientific research fields. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 For tanninase XY820_Tan pp Electrophoresis diagram of the gene; M, linear DNA marker; 1, XY820_Tan pp PCR fragments of genes;

[0019] Figure 2 XY820_Tan expressed by the recombinant strain pp SDS-PAGE electrophoresis image; M, protein marker; 1, XY820_Tan pp Crude protein; 2,XY820_Tan pp Pure protein;

[0020] Figure 3 When using propyl gallate as a substrate, XY820_Tan pp Optimal temperature and pH;

[0021] Figure 4 When using epigallocatechin gallate as a substrate, XY820_Tan pp Optimal temperature and pH;

[0022] Figure 5 To study the effects of different metal ions, organic solvents and surfactants on XY820_Tan pp Effects on enzyme activity;

[0023] Figure 6 For XY820_Tan pp Catalytic activity for different substrates;

[0024] Figure 7 For XY820_Tanpp Calculation of enzyme kinetic parameters. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be particularly noted that the following embodiments are for illustrative purposes only and do not limit the scope of the invention. Similarly, the following embodiments are only some, not all, embodiments of the present invention, and all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1: Isolation, purification and identification of Burkholderia paraknegriensis XY820

[0027] Sample cleaning: The root tissue of the tea tree variety "Hongyan 12" collected from the campus of Fujian Agriculture and Forestry University was rinsed clean under tap water and air-dried at room temperature.

[0028] Take 1g of the dried tea tree root tissue as a sample, wash it with distilled water, and air dry at room temperature. Then, in a laminar flow hood, transfer the sample to a 50mL sterile centrifuge tube, add sterile water and rinse twice, discarding the rinse solution. Add another 40-50mL of sterile water, shake at 150rpm for 20min, discard the washing solution, and air dry. Finally, rinse twice more with 50mL of sterile water and air dry.

[0029] Sample grinding: In a clean bench, place the cleaned and dried sample in a sterile mortar, add 5 mL of sterile water, and grind until a paste is formed. Use a large-well pipette tip to transfer 1 mL of the paste to a centrifuge tube, let it stand for 1-2 minutes until it separates into layers, then aspirate 100 μL of the supernatant and transfer it to a new centrifuge tube containing 900 μL of sterile water. After thorough mixing, take 100 μL and spread it on a selection medium (SGN + 0.1 mM EGCG) plate. After it has dried completely, invert the plate and incubate it in a 30°C incubator.

[0030] Bacterial isolation, purification, and preservation: Colonies with different morphological characteristics were selected by observing their morphology, color, size, and texture, and purified by streaking on ISP2+1mM EGCG solid medium. After colonies grew, they were repeatedly streaked on ISP2+2mM EGCG medium for purification. Single purified colonies were then cultured in MYG+2mM EGCG liquid medium. The viable strains were then cultured in MYG liquid medium until sufficient cells were obtained. 30% glycerol was then added, and the cells were stored at -80℃.

[0031] Identification and analysis of bacterial classification

[0032] XY820 bacterial strain, stored at -80℃, was streaked onto ISP2 medium and activated by incubation at 28℃. After bacterial growth, a single colony was picked and inoculated into 5 mL of MYG medium, and cultured at 28℃ with shaking at 200 rpm for 7-10 days. Once sufficient bacterial cells were obtained, total bacterial DNA was extracted using the Tiangen DNA Extraction Kit. Primers were designed (upstream primer: 5'-GTTGGATCCAGAGTTTGATCMTGGCT-3' (SEQ ID NO.3), downstream primer: 5'-GTTGGATCCACGGYTACCTTGTTACG-3' (SEQ ID NO.4), M being degenerate bases: A / C, Y being degenerate bases: C / T) to amplify the 16S rRNA gene by PCR. The PCR product was recovered and purified using a TransGen gel extraction kit, and the 16S rRNA sequence of XY820 bacteria was determined by 16S rRNA sequencing. Sequence similarity comparison (98.98%) was performed using the EzBioCloud website (https: / / www.ezbiocloud.net / ), identifying it as *Paraburkholderia pallida*, a species of Burkholderia. It was named *Paraburkholderia pallida* XY820, and its taxonomic name is *Paraburkholderia pallida*. It was deposited on September 19, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 28480.

[0033] Example 2: Tanninase XY820_Tan pp Acquisition of genes

[0034] Burkholderia paraknegriensis XY820 was resuscitated and sent to Novogene Biotechnology Co., Ltd. for whole-genome sequencing. Based on literature review and database comparison, a novel bacterial tanninase, XY820_Tan, was selected from the open reading frames (ORFs) obtained from the sequencing. pp Gene (nucleotide sequence number SEQ ID No. 2), in which Figure 1 For tanninase XY820_Tan pp Electrophoresis diagram of the gene; M, linear DNA marker; 1, XY820_Tan pp PCR fragments of genes.

[0035] Example 3: Tanninase XY820_Tan pp Heterologous expression

[0036] Design specific primers to amplify XY820_Tan ppThe gene, upstream primer (5'-ATAAGAATGCGGCCGCTGTGGCAGCGGCAGCAGT-3' (SEQ ID NO.5)), and downstream primer (5'-CGCGGATCCTCAAGGCGCGGCAACCC-3' (SEQ ID NO.6)) were used to amplify XY820_Tan gene using Burkholderia paraknegriensis XY820 DNA as a template via PCR and gel recovery. pp The gene fragment was ligated into the pCE2 TA / Blunt-Zero vector and transformed into *E. coli* JM109. After successful sequencing, the bacteria were preserved. The pMal-c5x plasmid and the correctly sequenced gene fragment were double-digested with enzymes, and the purified linear vector and gene fragment were obtained after gel extraction. The linear vector (pMal-c5x) and the gene fragment were then ligated using T4 ligase. The ligation product was transformed into *E. coli* Rosetta strain to obtain a heterologous expression strain, namely the recombinant *E. coli* Rosetta.

[0037] Example 4: Culture conditions of recombinant strain E. coli Rosetta

[0038] The recombinant plasmid pMal-c5x-XY820_Tan was added pp The engineered recombinant *Escherichia coli* *E. coli* Rosetta was inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured at 37°C with shaking for 12 h. 5 mL of the activated bacterial culture was then transferred to 500 mL of LB resistant medium (containing 100 μg / mL ampicillin) and cultured at 37°C until OD500 reached. 600 ≈0.6. Add 500 μL of 1M IPTG and incubate at 16℃ and 220 rpm for 12 h to induce the expression of the target protein. Collect the bacterial cells by centrifugation at 4℃ and 4,000g for 20 min. Resuspend the bacterial cells in 25 mL of column buffer (formulation: 20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 10 mM β-mercaptoethanol) and store at -20℃. Disrupt the cells by low-temperature sonication, sonicating for 30 min at 65% power for 5 s intervals and 8 s intervals. Centrifuge at 4℃ and 13,400g for 10 min, and collect the supernatant, which is the crude protein of the target gene expression.

[0039] Example 5: Tanninase XY820_Tan pp Activity verification

[0040] Based on the characteristics of tanninase, the tannin assay and tannic acid plate assay were used to detect XY820_Tan pp The tanninase activity was determined. The principle and method are as follows:

[0041] Garlic tannin assay: Using propyl gallate (PG) as a substrate, gallic acid produced by the decomposition of PG by tanninase can form a red complex with garlic tannin under alkaline conditions. This complex has maximum absorption at 520 nm. Based on this, the production of gallic acid can be calculated by measuring the change in A520, thereby calculating the enzyme activity. The specific testing method is as follows: Take four clean test tubes, one as a blank tube and three as test tubes; before the reaction begins, incubate the 10mM PG solution and the enzyme solution to be tested separately in a 30℃ water bath for 5-10 minutes; add 0.25mL of PG solution to each tube, then add 0.25mL of the enzyme solution to each test tube, and react at 30℃ for 5 minutes; add 0.3mL of methanol-to-tannin solution (0.667%, W / V) to all test tubes, and react at 30℃ for 5 minutes; add 0.2mL of KOH solution (0.5M) to all test tubes, and react at 30℃ for 5 minutes, then add 4mL of sterile water to all test tubes, and then add 0.25mL of enzyme solution to the blank tube, and react at 30℃ for 10 minutes. Observe whether a red complex is formed. Zero the absorbance at 520nm using a microplate reader with the blank tube as the reference. Finally, a standard curve was plotted using the absorbance changes of gallic acid at different concentration gradients to quantify the reaction products.

[0042] Tannic acid plate test: During petri dish culture, tannic acid added to nutrient agar interacts with proteins to form tannin-protein complexes, which can be cleaved by bacterial tanninases, resulting in a green-brown band around the colony. The specific test method is as follows: Add the recombinant vector (pMAL-c5x-XY820_Tan) to nutrient agar medium containing 2% tannic acid. pp 50 μL of the protein expressed by the vector and 50 μL of the protein expressed by the empty vector (pMal-c5x) were cultured for 24 h. After that, the greenish-brown substance was observed in the area where the protein was located on the plate.

[0043] Example 6: Tanninase XY820_Tan pp Purification and ultrafiltration

[0044] Based on the purification principle of MBP fusion proteins, the target protein carrying the MBP tag was purified using Amylose resin (NEB). The specific steps are as follows: The column was packed according to the NEB instructions, and equilibrated with 5 column volumes (25 mL) of column buffer; the crude protein was diluted 1:5 and slowly passed through the column; the column was washed with 12 volume volumes (60 mL) of column buffer; the target protein was eluted with 20 mL of column buffer containing 10 mM Maltose, and collected in 1.5 mL centrifuge tubes (approximately 1 mL per tube, for a total of 20 tubes); finally, the protein concentration was detected at 280 nm. The entire process was performed at low temperature. After use, the column was rinsed as follows: 15 mL of deionized water was passed through the column; 15 mL of 0.1% SDS solution was passed through the column; 10–15 mL of deionized water was used to rinse until no foam was present; 15 mL of column buffer was passed through the column; 15 mL of 20% ethanol was used to rinse the column; the column was then sealed with 20% ethanol and stored at 4°C.

[0045] The eluted protein needs to be further ultrafiltered to achieve concentration and recovery. pp The specific steps for protein extraction are as follows: Transfer the protein eluent obtained in the previous step to an ultrafiltration tube (50kDa MWCO); centrifuge at 4℃ and 4,000g until the volume is less than 1mL, add column buffer to bring the volume to 10mL and mix well, centrifuge again, filter and concentrate; repeat this process three times, and finally bring the volume to 1mL, which is the ultrafiltered XY820_Tan. pp Protein, of which, Figure 2 The tannin enzyme XY820_Tan expressed by the recombinant strain (E. coli Rosetta). pp SDS-PAGE electrophoresis image of (amino acid sequence number SEQ ID No. 1); M, protein marker; 1, XY820_Tan pp Crude protein; 2,XY820_Tan pp Pure protein.

[0046] Example 7: Tanninase XY820_Tan pp Substrate-specific detection

[0047] Under preset optimal temperature and pH values, XY820_Tan was analyzed using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) with methyl gallate (MG), ethyl gallate (EG), propyl gallate (PG), epigallocatechin gallate (EGCG), epicatechin gallate (ECG), gallocatechin gallate (GCG), methyl paraben, ethyl paraben, methyl salicylate, ethyl ferulic acid, methyl sinapicate, and lauryl gallate (LG) as substrates. pp Enzyme activity was measured using the following steps: 10 μg of purified protein, substrate (0.2 mM), and 50 mM citrate buffer (different pH) were added to 1.5 mL centrifuge tubes, with a total volume of 50 μL. All reactions were incubated at their respective optimal temperatures for 10 min. The reaction was terminated by adding 50 μL of methanol, followed by dilution with 150 μL of 70% methanol or 400 μL of acetonitrile. After sonication for 10 min, the supernatant was collected, filtered, and enzyme activity was determined using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry.

[0048] Example 8: Tanninase XY820_Tan pp Enzymatic property studies:

[0049] The tannin assay was used to test the tanninase XY820_Tan pp The optimal temperature and pH were determined. Using PG as a substrate, experiments were conducted at different temperatures (30℃, 40℃, 50℃, 60℃, 70℃, 75℃, 80℃) and pH values ​​(3.0, 4.0, 5.0, 6.0, 7.0). The results are as follows: Figure 3 As shown in the figure. Experiments were conducted using EGCG as a substrate at different temperatures (30℃, 40℃, 50℃, 60℃, 70℃, 80℃) and pH (3.0, 4.0, 5.0, 6.0, 7.0). The optimal temperature and pH for the enzyme were determined based on changes in absorbance, and the results are shown in the figure. Figure 4 As shown.

[0050] Using the tannin assay, at the optimal temperature and pH, with PG as the substrate, 1 mM K was added... + Ca 2+ Zn 2+ Mn 2+ Mg 2+ After adding EDTA, SDS, Dimethyl sulfoxide (DMSO), Triton X-100, β-mercaptoethanol, and 1% Tween 80 to the reaction system, the tanninase XY820_Tan was detected. pp The relative activity was determined to ascertain the effects of metal ions, additives, and organic solvents on enzyme activity. The results are as follows: Figure 5 As shown.

[0051] Using EGCG, ECG, MG, PG, and LG as substrates, the XY820_Tan was tested. pp The enzyme kinetic parameters were determined. The specific steps were as follows: Different concentrations of EGCG, ECG, MG, PG (0.125, 0.25, 0.5, 1.0, and 2.0 mmol / L) and LG (0.03125, 0.0625, 0.125, 0.25, 0.35, and 0.5 mmol / L) were prepared using 50 mM citrate buffer. 10 μg of purified protein, substrate, and 50 mM citrate buffer (different pH values) were added to 1.5 mL centrifuge tubes, with a total volume of 50 μL. All reactions were incubated at the optimal temperature for 10 min. The reaction was terminated by adding 50 μL of methanol, followed by dilution with 400 μL of acetonitrile. After sonication at 4 °C for 10 min, the supernatant was collected by centrifugation and filtered. Enzyme activity was determined using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS), and the results are shown in Table 1 and [Table data missing]. Figure 6 As shown.

[0052] Table 1

[0053]

[0054] Plotting was performed using the Lineweaver-Burk double reciprocal method in GraphPad Prism 9.0, and the XY820_Tan value was calculated for different substrates. pp Michaelis constant (K) m ), maximum reaction rate (V max ), catalytic constant (K) cat ) and catalytic efficiency (K cat / K m The results are shown in Table 2 and Figure 7 As shown.

[0055] Table 2

[0056]

[0057] The above description is only a part of the embodiments of the present invention and does not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made based on the content of the present invention specification and drawings, or direct or indirect application in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A tanninase XY820_Tan pp Its characteristics are, Its amino acid sequence is shown in SEQ ID NO.

1.

2. Encoding the tanninase XY820_Tan as described in claim 1 pp The gene is characterized by, Its nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant bacterium, characterized in that, The recombinant bacteria is *Escherichia coli* (Escherichia coli). E. coli Rosetta, which contains the gene as described in claim 2.

4. A method for preparing tanninase, characterized in that: The recombinant bacteria described in claim 3 were inoculated into a culture medium and cultured, and then the target protein tannin enzyme XY820_Tan was induced by IPTG. pp The gene expression was then processed by cell disruption and purification to obtain tanninase.

5. The application of tanninase in the degradation of tannins, characterized by: The tanninase is the tanninase XY820_Tan described in claim 1. pp Or the tanninase prepared by the method described in claim 4.

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