Tannase mutant Tan, coding gene, vector, engineering bacterium, preparation method and application

Abstract

The invention discloses a tannase mutant Tan, a coding gene, a carrier, an engineering bacterium as well as a preparation method and application. The tannase mutant Tan is obtained by mutating amino acids at the 179th site, the 217th site and the 339th site of wild type tannase Tan into leucine, serine and tyrosine from lysine, glutamine and aspartic acid respectively; the amino acid sequence and the nucleotide sequence of the gene are SEQ ID NO.1 and SEQ ID NO.2 respectively. Under the conditions that the pH is 7.0 and the temperature is 50 DEG C, the specific enzyme activity of the tannase mutant Tan is 5268.1 U / mL, which is 1.89 times that of wild enzyme Tan; the residual enzyme activity of the tannase mutant Tan is 4382.9 U / mL and is 2.98 times of the residual enzyme activity of a wild enzyme Tan under the condition of heat preservation for 1 hour at the temperature of 60 DEG C, so that the tannase mutant Tan has an important application prospect under a neutral condition.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and more specifically, relates to a tanninase mutant △Tan, its encoding gene, vector, engineered bacteria, preparation method, and application. Background Technology

[0002] Tanninase (EC 3.1.1.20) is a tannic acid hydrolase that can specifically hydrolyze the ester bonds and phenolic carboxyl bonds in tannins to produce products such as gallic acid and glucose. It has wide applications in the food industry, pharmaceutical manufacturing and biodegradation.

[0003] Tanninases have been reported to be used in tea processing to remove astringency, in winemaking to clarify juice, and in persimmon juice processing to reduce tannin content. Currently, tanninases are mainly derived from Aspergillus niger (…). Aspergillus niger ) and Aspergillus oryzae ( Aspergillus oryzae Microorganisms such as tannins, and wild-type tannins typically have lower catalytic activity and are sensitive to environmental conditions such as pH and temperature.

[0004] In detail, the catalytic activity of tanninases is significantly affected by the pH environment. Some wild-type tanninases have low activity under acidic or neutral conditions (pH 5.0-7.0), which makes it difficult to meet the demand for efficient and stable enzyme preparations in industrial production. In addition, insufficient thermal stability is another major bottleneck affecting the application of tanninases. Studies have shown that most wild-type tanninases are easily inactivated at temperatures above 60°C, which limits their application in high-temperature processing.

[0005] To date, there have been no reports of mutated tanninases. Summary of the Invention

[0006] In view of this, the present invention provides a tanninase mutant △Tan, its encoding gene, vector, engineered bacteria, preparation method, and application.

[0007] Specifically, this invention designs potential mutant tanninases based on wild-type tanninase sequences in a database using structural biology and energy calculations; and obtains the tanninase mutant gene Δ through site-directed mutagenesis based on the wild-type gene sequence. tan By using a eukaryotic expression system for efficient expression, excellent tanninase mutants with improved enzyme activity and heat resistance can be obtained, which can reduce production costs, expand the scope of application, and make them suitable for use in food and feed processing.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides a tanninase mutant △Tan, in which the amino acids at positions 179, 217, and 339 of the wild-type tanninase Tan are mutated from lysine, glutamine, and aspartic acid to leucine, serine, and tyrosine, respectively.

[0009] This invention utilizes combined site-directed mutagenesis technology to molecularly modify wild-type tanninases. Specifically, mutants are designed using molecular dynamics simulations combined with the computational program FoldX, and mutations at sites such as K179L, Q217S, and D339Y are screened out, which significantly improve the enzyme's catalytic activity, thermal stability, and other enzymatic properties.

[0010] In detail, in the above technical solution, after the 179th amino acid is mutated from the positively charged hydrophilic residue lysine to the nonpolar leucine, the unstable charge interaction on the surface is eliminated, the hydrophobic core stacking efficiency in this region is enhanced, the overall thermal stability of the protein is improved, and the conformational instability of the enzyme caused by charge repulsion at higher pH is reduced, thus broadening the pH tolerance range.

[0011] In detail, in the above technical solution, glutamine at position 217 is mutated to serine, which has a similar structure but stable chemical properties, eliminating the heat-induced degradation pathway. Furthermore, the hydroxyl group of serine can maintain the necessary polar interaction, giving this site higher chemical and conformational rigidity, thereby significantly enhancing the enzyme's thermal stability and tolerance to neutral pH environments.

[0012] In detail, in the above technical solution, the aspartic acid at position 339 is a negatively charged residue. Its presence is a key structural factor that limits the optimal pH of the enzyme to acidity. First, the introduction of the tyrosine benzene ring can form new π-π stacking or hydrophobic interactions inside the protein, which greatly enhances the stability of the protein's tertiary structure and directly contributes to the improvement of heat resistance. Second, the elimination of the negative charge directly changes the electrostatic network of the active microenvironment, reduces the dependence of the active center on protons, and allows the optimal pH required for enzyme catalysis to be stably expanded from the acidic region to the neutral region.

[0013] In the aforementioned technical solution, the combined mutation of three key amino acid sites produced a significant synergistic effect, transforming tanninase into a novel industrial enzyme suitable for acidic to neutral environments and possessing excellent thermostability. Specifically, this is illustrated in the GenBank database. tan Based on the gene sequence (GenBank accession number: PX724083), the key amino acids encoded by it were replaced.

[0014] Furthermore, in the above technical solution, the amino acid sequence of the tanninase mutant △Tan is shown in SEQ ID NO.1.

[0015] This invention also provides the encoding gene △ of the above-mentioned tanninase mutant △Tan. tan .

[0016] Furthermore, in the above technical solution, the gene encoding the tanninase mutant △Tan is △ tan The nucleotide sequence is shown in SEQ ID NO.2.

[0017] The present invention also provides a gene encoding the tanninase mutant △Tan containing the aforementioned △Tan. tan The carrier.

[0018] Furthermore, the present invention also provides a gene encoding the tanninase mutant △Tan. tan Or the one containing the coding gene △ tan The host cell of the vector.

[0019] Furthermore, the present invention also provides a coding gene Δ containing the said tanninase mutant. tan Or the one containing the coding gene △ tan Engineered bacteria on a carrier.

[0020] In another aspect, the present invention provides a method for preparing the tanninase mutant ΔTan, comprising: The nucleotide sequence shown in SEQ ID NO.2 was expressed using a plasmid capable of expressing the enzyme as an expression vector and a bacterial strain capable of expressing the enzyme as an expression host, thereby achieving efficient expression of the mutant shown in SEQ ID NO.1.

[0021] In detail, in the above technical solution, the nucleotide sequence shown in SEQ ID NO.2 is expressed using a plasmid (preferably pPICZαA) capable of expressing the enzyme, and a strain capable of expressing the enzyme (preferably Pichia pastoris) is used. P. pastoris ) as the expression host, to achieve the coding gene of the tanninase mutant △ tan K179L / Q217S / D339Y Highly efficient secretory expression.

[0022] Specifically, in the above technical solution, the wild-type tanninase gene tan The accession number of the tannin enzyme Tan encoded by the gene is PX724083 in the GenBank database. The pPICZαA expression vector used contains the AOX1 promoter and also inserts elements such as the α-factor signal peptide, the Zeocin resistance gene, and the His4 selection marker. It can induce the expression of exogenous enzymes with methanol and has controllable expression.

[0023] In another aspect, the present invention provides the encoding gene Δ of the tanninase mutant. tan And the applications of the enzymes they encode in food and feed processing.

[0024] Compared with the prior art, the present invention has the following advantages: (1) The tannin enzyme mutant △Tan provided by the present invention has significantly improved enzyme activity and heat resistance under neutral conditions, which solves the problems of low catalytic activity and insufficient thermal stability of wild-type tannin enzyme under neutral conditions, and creates good conditions for the application of this enzyme in food and feed processing. (2) This invention compares the degradation capacity of wild-type enzyme Tan and tannin mutant △Tan for propyl gallate under neutral conditions. The results show that at pH 7.0 and 50 ℃, the specific enzyme activity of tannin mutant △Tan is 5268.1 U / mL, which is 1.89 times that of wild-type enzyme Tan. After incubation at 60 ℃ for 1 h, the residual enzyme activity of tannin mutant △Tan is 4382.9 U / mL, which is 2.98 times that of wild-type enzyme Tan. This indicates that tannin mutant △Tan has important application prospects under neutral conditions. Attached Figure Description

[0025] Figure 1 This is a flowchart illustrating the construction process of the engineered strain of the tanninase mutant △Tan in this embodiment of the invention. Figure 2 The recombinant plasmid pPICZαA-△ for the tanninase mutant △Tan in this embodiment of the invention. tan The construction graph; Figure 3 The recombinant plasmid pPICZαA-△ for the tanninase mutant △Tan in this embodiment of the invention. tan Linearization detection graph (in the graph: M is DNA Marker; 1 is the recombinant plasmid before linearization; 2 is the recombinant plasmid after linearization). Figure 4 The recombinant plasmid pPICZαA-△ for the tanninase mutant △Tan in this embodiment of the invention. tan PCR detection graph (in the graph: M is DL5000 DNA Marker; 1-20 are PCR bands of different clones; - is negative control; + is positive control). Figure 5 This is an SDS-PAGE spectrum of the fermentation supernatant induced by the tanninase mutant △Tan in this embodiment of the invention (in the figure: M is the protein marker; 1-10 are the genetically engineered bacteria pPICZαA-△tan / X33, which tested positive for the tanninase mutant △Tan by PCR; - is the blank plasmid engineered bacteria pPICZαA / X33 without the insertion of foreign genes, serving as a negative control). Figure 6This is a Western blot analysis of the fermentation supernatant induced by the tannin mutant △Tan in this embodiment of the invention (in the figure: M is the protein marker; 1-10 are the genetically engineered bacteria pPICZαA-△tan / X33, which are positive for PCR detection of the tannin mutant △Tan; - is the blank plasmid engineered bacteria pPICZαA / X33 without the insertion of foreign genes, serving as a negative control). Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments.

[0027] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0028] In the embodiments, unless otherwise specified, all methods used are conventional methods in the art.

[0029] The terms “comprising,” “including,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0030] Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0031] Materials and reagents: Expression vector pPICZαA, eukaryotic competent cells P. pastoris All X33s were purchased from Novagen. Restriction endonucleases Eco R I and Xho I. Speedy Cut SacI enzyme, Ni-NTA, and goat anti-mouse secondary antibody were purchased from Sangon Biotech. Ultra HiFidelity PCR Kit, Prokaryotic Cloning Competent Cells E. coli TOP10, RNA Simple Total RNA Extraction Kit, Rapid Site-directed Mutagenesis Kit, TIANScript II RT Kit, Agarose Gel DNA Recovery Kit, and Plasmid Extraction Kit were all purchased from Tiangen Biosciences. Propyl gallate, methyl raffinate, gallic acid, tryptone, yeast extract and agar powder were purchased from Sigma-Aldrich. LB medium, BMMY medium, and BMGY medium were purchased from Beijing Coolplay Technology Co., Ltd.

[0032] Primer synthesis and nucleic acid sequencing were performed by Sangon Biotech.

[0033] The remaining chemical reagents were all commercially available analytical grade products, purchased from Shanghai Sinopharm Group.

[0034] like Figure 1 The diagram shown is a flowchart illustrating the construction process of the engineered strain of the tanninase mutant △Tan in an embodiment of the present invention; as shown... Figure 2 The image shows the recombinant plasmid pPICZαA-△ of the tanninase mutant △Tan in an embodiment of the present invention. tan The construction map.

[0035] Example 1: Primitive tanninase gene tan Construction of recombinant plasmids wild-type fungi A. niger Mycelia (from Junshan Tea Garden, Yueyang, Hunan) were inoculated into 50 mL of PDB liquid medium and cultured overnight at 37℃ and 150 r / min. The mycelia were then centrifuged and collected. Total RNA was extracted according to the instructions of the RNA simple total RNA extraction kit. The total RNA was reverse transcribed into cDNA according to the instructions of the TIANScript II RT Kit and stored at -20℃ for later use.

[0036] Based on wild-type tanninase gene tan The sequence (GenBank accession number: PX724083) was used to design the following primers using the bioinformatics software SnapGene: F: 5'-CC GAATTC ATGGATGTCTGTACTACCAGC-3' (SEQ ID NO.3, containing Eco R I restriction site) R: 5'-AA CTCGAG GTACAGGGGCAGCTTGTACG-3' (SEQ ID NO.4, containing Xho I restriction site).

[0037] by A. niger Using genomic DNA as a template, PCR amplification was performed.

[0038] The PCR reaction system was as follows: cDNA template, 1 μL (approximately 50 ng); forward primer F (10 µmol / L), 1.0 µL; reverse primer R (10 µmol / L), 1.0 µL; 2×Pfu Master Mix, 25 μL; and sterile ddH2O was added to bring the total volume to 50 μL. After mixing, the mixture was placed on a PCR instrument for PCR reaction.

[0039] The parameters are set as follows: (1) Pre-denaturation at 94 ℃ for 5 min; (2) Denaturation at 94 ℃ for 30 s; (3) Annealing at 58 ℃ for 30 s; (4) Extension at 72 ℃ for 1 min; Repeat steps (2)-(4) for 30 cycles; (5) Extension at 72 ℃ for 10 min.

[0040] The obtained PCR products were detected by 1.0% agarose gel electrophoresis, and the gel was cut. The target fragment was recovered using a DNA gel recovery kit and stored at -20℃ for later use.

[0041] For pPICZαA plasmid and target gene PCR product, use Eco R I and Xho After double digestion with enzyme I, followed by gel purification, the digestion products were ligated with T4 DNA ligase at 4°C overnight to obtain the recombinant plasmid. The plasmid was then transformed into DNA using the heat shock method. E. coli Top 10 were plated on LB medium containing 25 μg / mL Zeocin and cultured overnight; positive transformants were picked, cultured overnight, and plasmids were extracted for PCR identification; the plasmids of positive clones were submitted to Sangon Biotech for sequencing, and the recombinant plasmid with the correct target gene was pPICZαA-tan.

[0042] Example 2 Site-directed mutagenesis Site-directed mutagenesis principle: The construction of point mutation plasmids uses... Dpn I method.

[0043] The following PCR point mutation primers were designed based on the amino acid sites to be mutated: F K179L :5' ACTGTCGTCGGC CTA GCCTTCAG 3' (SEQ ID NO.5) R K179L :5' CTGAAGGC TAG GCCGACGACAGT 3' (SEQ ID NO.6) F Q217S :5' TATGCAGAC TCGTGGGATGGAG 3' (SEQ ID NO.7) R Q217S :5' CTCCATCCCA CGA GTCTGCATA 3' (SEQ ID NO.8) F D339Y :5' AAGATGCTC TAC GGCCTGC 3' (SEQ ID NO.9) R D339Y :5' GCAGGCC GTA GAGCATCTT 3' (SEQ ID NO.10) The underlined parts represent the codons corresponding to leucine at position 179, serine at position 217, and tyrosine at position 339 encoded by the mutant gene.

[0044] Using a rapid site-directed mutagenesis kit with pPICZαA-tan recombinant plasmid as a template, whole plasmid PCR was performed to introduce mutation sites.

[0045] The PCR reaction system consisted of: 0.5 µL of forward primer (10 µmol / L), 0.5 µL of reverse primer (10 µmol / L), 5 µL of 5×Fast Alteration Buffer, 1 µL of plasmid DNA, 0.5 µL of Fast Alteration DNA Polymerase, and sterile ddH2O to a final volume of 25 µL.

[0046] The parameters are set as follows: (1) Pre-denaturation at 95 °C for 2 min; (2) Denaturation at 94 °C for 20 s; (3) Refolding at 60 °C for 10 s; (4) Extension at 68 °C for 2.5 min; Repeat steps (2)-(4) for 18 cycles; (5) Heat at 68 °C for 5 min, and store the product at 4 °C.

[0047] 0.5 µL of restriction endonuclease Dpn Add I to 25 µL of the mutant PCR product, mix thoroughly, and digest at 37°C for 1 h; take 5 µL Dpn I digestion product transfer E. coli Top 10: Transformed bacterial cultures were evenly spread onto LB selection plates (containing 25 μg / mL Zeocin) and incubated overnight at 37°C to obtain transformants of the relevant mutant enzyme gene; plasmids were extracted and sequenced for verification, yielding the correct recombinant plasmid pPICZαA-△ for the mutant tannin enzyme gene. tan (like Figure 2 (As shown).

[0048] Example 3 Construction, induction, expression, and electrophoretic analysis of recombinant Pichia pastoris Use the correct mutant plasmid Sac I is linearized (e.g.) Figure 3 As shown), respectively with P. pastoris X33 competent cells were mixed at a volume ratio of 1:8, transferred into a pre-cooled electroporator, incubated on ice for 5 min, and then electroporated for 5 ms; pre-cooled sorbitol was added immediately.

[0049] In addition, the empty vector pPICZαA without the inserted foreign gene was electroporated to P. pastoris X33 was used as a negative control.

[0050] After electroporation, the solution was incubated at 28-30 ℃ for 2 h, then centrifuged. The bacterial cells were plated on YPDS (containing 100 μg / mL Zeocin) plates and incubated upside down at 28-30 ℃. Once single colonies formed, they were detected using PCR. The PCR primers are as follows: Upstream primer 9323-F: 5'-TGGATAACGTTACTTACGACACC-3' (SEQ ID NO.11) Downstream primer 3'AOX: 5'-GCAAATGGCATTCTGACATCC-3' (SEQ ID NO.12) Ten clones were verified as positive by PCR (e.g.) Figure 4 (As shown) Colonies were inoculated into BMGY medium and incubated at 28-30 °C until... OD 600 2-6; replace with BMMY medium for induction (1% methanol), incubate at 28 ℃ for 72 h and then test.

[0051] Centrifuge 100 μL of fermentation broth at 12000 r / min for 5 min, then take 80 μL of supernatant into a 1.5 mL centrifuge tube, add 20 μL of 5×Loading Buffer, and incubate in a boiling water bath for 10 min.

[0052] A negative control was prepared by using the blank plasmid engineered bacteria pPICZαA / X33 without the insertion of foreign genes.

[0053] Using SDS-PAGE (e.g.) Figure 5 (as shown) and Western blot (as shown) Figure 6 The protein secretion and expression were detected (as shown in the figure). The results showed that, compared with the blank control, the supernatant of the mutant tanninase gene-engineered bacterium pPICZαA-△tan / X33 showed specific target protein bands in wells 1, 3, 4, and 6-9, indicating that the mutant tanninase was successfully expressed.

[0054] Example 4: Comparison of in vitro catalytic efficiency of wild-type tanninase Tan and tanninase mutant ΔTan To compare the biocatalytic activity of wild-type tanninase Tan and tanninase mutant ΔTan under neutral conditions, enzyme activity was measured under the same conditions.

[0055] Enzyme activity assay: Tanninase activity is determined by the formation of chromophores between gallic acid, the catalytic product of tanninase, and methanol-derived tannin.

[0056] (1) Gallic acid standard solutions with concentrations of 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, and 260 µmol / L were prepared, and the standard curve of gallic acid solution was obtained. The linear regression equation was: y = 706.25x + 8.99, R 2 =0.9991; (2) Three replicates were set up for each set group. Citrate buffer was used as a blank control. 0.25 mL of 10 mol / L propyl gallate was used as the reaction substrate and 0.25 mL of the enzyme solution to be tested was shaken and mixed. The mixture was then placed in a 50 ℃ water bath for 5 min. 0.3 mL of methanol-tannin solution was added, and the mixture was placed in a 50 ℃ water bath for 5 min. 0.2 mL of KOH was added, and the mixture was placed in a 50 ℃ water bath for 5 min. After cooling to terminate the reaction, the absorbance was measured at a wavelength of 520 nm. (3) Tanninase activity is defined as the amount of enzyme required to release 1 μmol of gallic acid per minute by hydrolyzing propyl gallate at 50 °C and pH 7.0, expressed in U.

[0057] The results show that: Under pH 7.0 and 50 ℃ conditions, the specific enzyme activity of the tanninase mutant △Tan was 5268.1 U / mL, which was 1.89 times that of the wild-type tanninase Tan (2787.4 U / mL). This indicates that the combined mutation at the K179L / Q217S / D339Y three sites enhances the ability of this tanninase to catalyze the degradation of propyl gallate.

[0058] Example 5: Comparison of the heat resistance of wild-type tanninase Tan and tanninase mutant ΔTan Crude enzyme solutions of wild-type tanninase Tan and tanninase mutant △Tan were incubated at 60 °C for 1 h, and the remaining enzyme activity was measured to characterize the thermostability of the enzymes.

[0059] The results show that: After incubation at 60 °C for 1 h, the residual enzyme activity of the tanninase mutant △Tan was 4382.9 U / mL, which is 2.98 times that of the wild-type tanninase Tan (1470.8 U / mL). This indicates that the combined mutation of the three key sites K179L / Q217S / D339Y also significantly improves the thermostability of this tanninase.

[0060] The above embodiments are merely examples of several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent.

[0061] It should be noted that those skilled in the art can make various modifications and improvements without departing from the inventive concept, and these all fall within the scope of protection of this invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A tanninase mutant △Tan, characterized in that, The amino acids at positions 179, 217, and 339 of the wild-type tanninase Tan were mutated from lysine, glutamine, and aspartic acid to leucine, serine, and tyrosine, respectively.

2. The tanninase mutant △Tan according to claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

3. The gene encoding the tanninase mutant △Tan according to any one of claims 1-2. tan .

4. The encoding gene △ according to claim 3 tan Its characteristics are, Its nucleotide sequence is shown in SEQ ID NO.

2.

5. Contains the encoding gene △ as described in any one of claims 3-4 tan The carrier.

6. Containing the coding gene △ as described in any one of claims 3-4 tan Or the host cell of the vector as described in claim 5.

7. Contains the coding gene △ as described in any one of claims 3-4 tan Or the engineered bacteria of the carrier described in claim 5.

8. The method for preparing the tanninase mutant ΔTan according to any one of claims 1-2, characterized in that, The nucleotide sequence shown in SEQ ID NO.2 was expressed using a plasmid capable of expressing the enzyme as an expression vector and a bacterial strain capable of expressing the enzyme as an expression host, thereby achieving efficient expression of the mutant shown in SEQ ID NO.

1.

9. The method for preparing the tanninase mutant ΔTan according to claim 8, characterized in that, The expression vector is preferably pPICZαA; And / or, the expression host is preferably Pichia pastoris. P. pastoris .

10. The gene encoding Δ according to any one of claims 3-4 tan And the applications of the enzymes they encode in food and feed processing.

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