A formaldehyde dismutase, its preparation method, strain, apparatus, application, and formaldehyde treatment method.

Abstract

This invention belongs to the technical field of biological treatment of formaldehyde pollution, and discloses a formaldehyde superoxide dismutase, its preparation method, strain, device, application, and formaldehyde treatment method. The amino acid sequence of the formaldehyde superoxide dismutase provided by this invention is shown in SEQ ID NO:1 or a variant sequence having at least 99% homology with the sequence shown in SEQ ID NO:1. This formaldehyde superoxide dismutase has excellent biological activity in degrading formaldehyde and can be well applied in formaldehyde pollution treatment to achieve ideal formaldehyde degradation effect.

Description

Technical Field

[0001] This invention belongs to the technical field of biological treatment of formaldehyde pollution, and particularly relates to a formaldehyde dismutase, its preparation method, strain, device and application, and a formaldehyde treatment method. Background Technology

[0002] Formaldehyde, as a basic chemical raw material, is widely used in industries such as resins, leather, paints, coatings, and plastics. However, formaldehyde has been clearly classified as a Group 1 carcinogen, posing a significant threat to human health. As a primary toxin, it can directly bind tightly to proteins, triggering a range of symptoms including allergic reactions. Furthermore, formaldehyde carries the potential risk of cancer.

[0003] Currently, the main methods for treating formaldehyde pollution include physical adsorption, chemical catalytic oxidation, and biological removal. Physical adsorption, utilizing porous materials such as activated carbon, zeolite, and silica gel to adsorb formaldehyde, is the most common method. However, it suffers from drawbacks such as low efficiency, adsorption saturation, susceptibility to temperature fluctuations, and the need for regular replacement and regeneration of the adsorbent. Chemical catalytic oxidation uses chemical methods to directly or indirectly degrade formaldehyde into inorganic small molecules, but it suffers from secondary pollution and its effectiveness is greatly affected by formaldehyde concentration. Compared to physical adsorption and chemical catalytic oxidation, biological methods primarily utilize microorganisms such as bacteria and fungi, or formaldehyde-degrading enzymes, to achieve the conversion and stabilization of formaldehyde. These methods offer advantages such as high efficiency, mild conditions, low cost, and wide applicability, making them more promising for application. Formaldehyde dismutase is one of the key enzymes in formaldehyde biodegradation and can directly degrade formaldehyde; however, existing formaldehyde dismutases have low formaldehyde degradation efficiency and unsatisfactory formaldehyde treatment effects, thus having certain limitations. Summary of the Invention

[0004] The primary objective of this invention is to provide a formaldehyde superoxide dismutase that possesses excellent biological activity in degrading formaldehyde and can be effectively applied in formaldehyde pollution treatment to achieve ideal formaldehyde degradation results.

[0005] A second objective of the present invention is to provide a construct.

[0006] A third objective of this invention is to provide a method for preparing the above-mentioned formaldehyde superoxide dismutase.

[0007] A fourth objective of this invention is to provide a formaldehyde-treating strain.

[0008] The fifth objective of this invention is to provide a formaldehyde treatment device.

[0009] The sixth objective of this invention is to provide the application of the above-mentioned formaldehyde dismutase, formaldehyde-treating strains and / or formaldehyde-treating devices in the treatment of formaldehyde pollution.

[0010] The seventh objective of this invention is to provide a method for treating formaldehyde.

[0011] Specifically, the amino acid sequence of the formaldehyde superoxide dismutase provided by the present invention is as shown in SEQ ID NO:1 or a variant sequence having at least 99% homology with the sequence shown in SEQ ID NO:1.

[0012] Furthermore, the amino acid sequence of the formaldehyde superoxide dismutase is shown in SEQ ID NO:7.

[0013] Furthermore, the amino acid sequence of the formaldehyde superoxide dismutase is shown in SEQ ID NO:11.

[0014] The construct provided by this invention encodes the above-mentioned formaldehyde dismutase.

[0015] Furthermore, the construct comprises one or more nucleotide fragments as shown in SEQ ID NO:2, SEQ ID NO:8 and SEQ ID NO:12.

[0016] The method for preparing formaldehyde superoxide dismutase provided by the present invention includes: transforming the above-mentioned construct into host cells to obtain a formaldehyde-treated strain; and expressing formaldehyde superoxide dismutase using the formaldehyde-treated strain.

[0017] The formaldehyde-treated strain provided by this invention expresses and secretes the above-mentioned formaldehyde superoxide dismutase.

[0018] The formaldehyde treatment device provided by the present invention includes the above-mentioned formaldehyde dismutase and / or formaldehyde treatment strains.

[0019] This invention provides the application of the above-mentioned formaldehyde dismutase, formaldehyde-treating strains and / or formaldehyde-treating devices in formaldehyde pollution.

[0020] The formaldehyde treatment method provided by the present invention includes: taking the above-mentioned formaldehyde dismutase, formaldehyde-treating strain and / or formaldehyde treatment device to treat formaldehyde in the environment. Detailed Implementation

[0021] Based on in-depth research on Pseudomonas sp. IOFA1 (accession number CCTCCNO: M 2010280), the inventors of this application discovered that treating an aqueous solution with a formaldehyde concentration of 2000 mg / L for 24 hours using the formaldehyde superoxide dismutase gene fdm encoded by the Pseudomonas sp. IOFA1 genome (amino acid sequence shown in SEQ ID NO: 1) reduced the formaldehyde concentration in the aqueous solution to below 300 mg / L, demonstrating excellent formaldehyde degradation ability. Based on this, the inventors further discovered that, compared to the original formaldehyde superoxide dismutase derived from Pseudomonas sp. IOFA1, mutants obtained by introducing the E156L mutation alone or by simultaneously introducing the E156L and Q355R mutations exhibit even superior formaldehyde degradation ability, thus leading to the technical solution of this invention.

[0022] The amino acid sequence of formaldehyde superoxide dismutase provided by this invention is as shown in SEQ ID NO:1 or a variant sequence having at least 99% homology with the sequence shown in SEQ ID NO:1. The variant sequence is obtained by substituting one or more amino acids into the amino acid sequence shown in SEQ ID NO:1, and the substitution does not affect the biological activity of the formaldehyde superoxide dismutase.

[0023] In this invention, the preferred amino acid sequence of the formaldehyde superoxide dismutase is as shown in SEQ ID NO:7; at this time, compared with the original amino acid sequence of the formaldehyde superoxide dismutase shown in SEQ ID NO:1, the formaldehyde superoxide dismutase has an E156L mutation, which improves the ability of the formaldehyde superoxide dismutase to bind formaldehyde substrate, thereby effectively improving the formaldehyde degradation capacity.

[0024] In this invention, the preferred amino acid sequence of the formaldehyde superoxide dismutase is as shown in SEQ ID NO:11. In this case, compared with the original amino acid sequence of the formaldehyde superoxide dismutase shown in SEQ ID NO:1, it has both E156L mutation and Q355R mutation, which improves the ability of the formaldehyde superoxide dismutase to bind formaldehyde substrate and further reduces the activation energy of the formaldehyde superoxide dismutation reaction, thereby effectively improving the formaldehyde degradation capacity.

[0025] In this invention, the positions of amino acid residues in the E156L mutation and Q355R mutation as described above are numbered according to the amino acid sequence shown in SEQ ID NO:1.

[0026] Based on the objective of obtaining the above-mentioned formaldehyde superoxide dismutase, the present invention also provides a construct encoding the above-mentioned formaldehyde superoxide dismutase.

[0027] In this invention, the construct includes at least one or more of the nucleotide fragments shown in SEQ ID NO:2, SEQ ID NO:8 and SEQ ID NO:12, which can encode one or more of the above-mentioned formaldehyde dismutases.

[0028] In some specific embodiments, when the construct encodes formaldehyde dismutase with an amino acid sequence as shown in SEQ ID NO:1, the construct includes a nucleotide fragment as shown in SEQ ID NO:2.

[0029] In some specific embodiments, when the construct encodes formaldehyde dismutase with an amino acid sequence as shown in SEQ ID NO:7, the construct includes a nucleotide fragment as shown in SEQ ID NO:8.

[0030] In some specific embodiments, when the construct encodes formaldehyde dismutase with an amino acid sequence as shown in SEQ ID NO:11, the construct includes a nucleotide fragment as shown in SEQ ID NO:12.

[0031] In some specific embodiments, when the construct simultaneously encodes two or more formaldehyde superoxide dismutases such as SEQ ID NO:1, 7 and 11, the construct comprises two or more nucleotide fragments such as those shown in SEQ ID NO:2, SEQ ID NO:8 and SEQ ID NO:12.

[0032] In this invention, the construct preferably further includes functional sequences that perform different functions. Specific examples of these functional sequences may include, but are not limited to, regulatory sequences and / or enzyme recognition sequences. The regulatory sequences refer to a series of specific sequences that can affect gene expression, playing an important regulatory role in gene transcription and translation, and influencing gene expression levels. Specific examples include, but are not limited to, one or more of the following: promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, homologous recombination sites, and poly(A) signals in coding and non-coding regions. The enzyme recognition sequences refer to a series of specific sequences that can be recognized and bound by specific enzymes. Specific examples include, but are not limited to, NdeI cleavage sites and / or XhoI cleavage sites.

[0033] The method for preparing the above-mentioned formaldehyde superoxide dismutase provided by the present invention specifically includes: transforming the above-mentioned construct into host cells to obtain a formaldehyde-treated strain; and expressing the formaldehyde superoxide dismutase using the formaldehyde-treated strain.

[0034] In this invention, the host cell is a type of eukaryotic and / or prokaryotic cell commonly used in existing bioengineering technologies, possessing a complete expression system required for the expression of formaldehyde superoxide dismutase; specific examples include, but are not limited to, one or more of Escherichia coli BL21 (DE3), Escherichia coli JM109 (DE3), and Escherichia coli DH5α.

[0035] In this invention, the method for transforming host cells with the construct is a conventional method used in existing gene recombination technology to achieve effective transformation of host cells by the construct, and is not particularly limited thereto. In some specific embodiments, specific examples of the method for transforming host cells with the construct include, but are not limited to, chemical transformation and / or electroporation.

[0036] The formaldehyde-treated strain provided by this invention can secrete and express the above-mentioned formaldehyde superoxide dismutase, which can be used for the preparation of formaldehyde superoxide dismutase or directly applied to the degradation of formaldehyde.

[0037] The formaldehyde treatment device provided by the present invention includes the above-mentioned formaldehyde superoxide dismutase and / or formaldehyde treatment strain. The core of the device is the presence of formaldehyde superoxide dismutase and / or formaldehyde treatment strain. Its specific structure may be exactly the same as, partially the same as or completely different from existing formaldehyde treatment devices, as long as it can effectively degrade formaldehyde in the environment. No particular limitation is made on its specific structure.

[0038] The present invention provides the above-mentioned formaldehyde superoxide dismutase, formaldehyde-treated strain and / or formaldehyde superoxide dismutase and / or formaldehyde-treated strain.

[0039] The formaldehyde treatment method provided by the present invention specifically includes: taking the above-mentioned formaldehyde dismutase, formaldehyde treatment strain and / or formaldehyde treatment device to treat formaldehyde in the environment.

[0040] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0041] The experimental methods involved in the following examples are as follows:

[0042] (1) Determination of formaldehyde superoxide dismutase activity: The activity of formaldehyde superoxide dismutase was determined by the formic acid synthesis assay. [1] Specifically, the process involves adding formaldehyde (final concentration 20 mol / mL), KCl (final concentration 100 mol / mL), and 2 mL of enzyme solution to 10 mL of standard reaction solution, incubating at 37 °C for 10 min, and then determining the formic acid content using pH titration.

[0043] One unit of enzyme activity is defined as the amount of enzyme required to produce 1 μmol of formic acid in 1 minute at 37°C.

[0044] (2) Determination of formaldehyde content: The formaldehyde content in the solution was determined by the acetylacetone method. [2] Specifically, the process includes: mixing the acetylacetone reaction solution with the sample at a volume ratio of 1:1 and incubating at 80°C for 10 min. After the reaction solution cools to room temperature, the absorbance value at a wavelength of 412 nm is measured, and the formaldehyde content in the culture medium is calculated by comparing it with the standard curve.

[0045] [1] Yanase H, Noda H, Aoki K, et al. Cloning, sequence analysis, and expression of the gene encoding formaldehyde dismutase from Pseudomonasputida F61[J]. Bioscience, Biotechnology, and Biochemistry, 1995, 59(2): 197-202.

[0046] [2]Nash T.The colorimetric estimation of formaldehyde by means of theHantzsch reaction[J].Biochemical Journal,1953,55(3):416.

[0047] Example 1

[0048] This embodiment illustrates the preparation of formaldehyde superoxide dismutase, specifically including:

[0049] 1. Constructing the builder

[0050] (1) The codon of the gene fdm encoding formaldehyde superoxide dismutase in the genome of Pseudomonas sp. IOFA1 (amino acid sequence as shown in SEQ ID NO:1) was optimized by Escherichia coli, and the optimized gene fragment (nucleotide sequence as shown in SEQ ID NO:2) was synthesized.

[0051] (2) NdeⅠ and XhoⅠ restriction sites were added to both ends of the optimized gene fragment, and the fragment was digested with NdeⅠ and XhoⅠ enzymes and then recombined into the His-tagged vector pET24a(+) to obtain the recombinant plasmid.

[0052] The recombinant plasmid was transformed into Tran5α competent cells and placed in LB liquid medium. The cells were cultured overnight at 37°C and 160 rpm with shaking. A large amount of recombinant plasmid was then extracted.

[0053] (3) Using E156L mutant primers (nucleotide sequences as shown in SEQ ID NO:3 and SEQ ID NO:4) and Q355R mutant primers (nucleotide sequences as shown in SEQ ID NO:5 and SEQ ID NO:6), and according to Table 1, PCR mutation amplification was performed on the recombinant plasmid to obtain different mutant amplification products.

[0054] Table 1.

[0055] Formaldehyde superoxide dismutase amino acid sequence Mutation type Mutant primers Encoding genes 1 SEQ ID NO:1 - - SEQ ID NO:2 2 SEQ ID NO:7 E156L E156L mutant primers SEQ ID NO:8 3 SEQ ID NO:9 Q355R Q355R mutant primers SEQ ID NO:10 4 SEQ ID NO:11 E156L and Q355R E156L mutant primers and Q355R mutant primers SEQ ID NO:12

[0056] The PCR system consisted of 25 μL of 2×TransStart. FastPfu Fly PCR SuperMix, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 1 μL of 50 ng / μL recombinant plasmid, and ddH2O to make up to 50 μL.

[0057] The PCR program was as follows: 95℃ pre-denaturation for 3 min, 30 amplification cycles (95℃ denaturation for 10 s, 55℃ annealing for 520 s, 72℃ extension for 90 s), and 72℃ extension for 10 min.

[0058] (4) The recombinant plasmid and the mutant amplification product were digested with DpnI enzyme at 37℃ for 1 h to remove the template and obtain the constructs encoding formaldehyde superoxide dismutase 1 to 4.

[0059] 2. Construction of formaldehyde-treated strains

[0060] (1) The above-obtained constructs were transformed into Escherichia coli BL21 competent cells, plated on LB plates containing 50 μg / mL kana resistance, and cultured overnight at 37°C. A portion of cells from each single colony were taken for PCR identification and sequencing to screen for recombinant strains containing the corresponding constructs.

[0061] (2) Extract the corresponding construct from the recombinant strain, transform the construct into Escherichia coli BL21 competent cells, and obtain the formaldehyde-treated strain.

[0062] 3. Preparation of formaldehyde superoxide dismutase

[0063] (1) The formaldehyde-treated strain was inoculated into LB medium containing 50 mg / L kanamycin sulfate at an inoculation rate of 5%, and cultured overnight at 37°C and 200 rpm with shaking to obtain the culture solution;

[0064] Inoculate the culture medium at a rate of 1% into fresh LB medium containing 50 mg / L kanamycin sulfate, and incubate at 37°C with shaking at 200 rpm until OD (outlet capacity) is reached. 600 The concentration was set at 0.6, and IPTG was added to a final concentration of 50 μg / mL. Expression was induced at 30℃ for 12 h.

[0065] (2) Take the culture medium and centrifuge at 4℃ and 18000g for 10min to collect cell slurry; resuspend the cell slurry in 15mL of lysis buffer, and sonicate the cells in an ice bath for 10min according to the working 4s and intermittent 4s pattern, centrifuge at 4℃ and 18000g for 10min, take the supernatant, purify the supernatant by Ni-NTA affinity chromatography, and freeze dry to obtain formaldehyde superoxide dismutase 1-4.

[0066] The enzyme activities of formaldehyde superoxide dismutase 1 to 4 were tested, and the relative enzyme activities of formaldehyde superoxide dismutase 2 to 4 were calculated according to the following formula, with formaldehyde superoxide dismutase 1 as the control. The results are shown in Table 2.

[0067] Relative enzyme activity (%) = A x / A0×100%

[0068] Where A0 represents the enzyme activity of formaldehyde superoxide dismutase 1, in units of U. x The enzyme activity is for formaldehyde superoxide dismutase 2, 3, or 4, expressed in units of U.

[0069] Table 2.

[0070] Formaldehyde superoxide dismutase Relative enzyme activity (%) 1 100.00 2 196.34 3 93.60 4 325.71

[0071] As shown in Table 2, the test results indicate that, compared with the original formaldehyde superoxide dismutase 1 derived from Pseudomonas IOFA1, formaldehyde superoxide dismutases 2 and 4 have higher enzyme activities, which are increased by 1.96 times and 3.26 times, respectively.

[0072] Example 2

[0073] This embodiment illustrates the formaldehyde degradation capacity of the formaldehyde superoxide dismutase provided in Example 1. Specifically, it includes adding formaldehyde superoxide dismutase 1 to 4 at an addition rate of 5 g / L to formaldehyde aqueous solutions (pH = 9.0) containing 50, 100, 1000, and 2000 mg / L respectively, and treating them in a shaker at 37°C and 200 rpm for 12 h. Samples are taken at 0 h and 12 h to measure the formaldehyde concentration in the solution, and the final formaldehyde degradation rate is calculated according to the following formula.

[0074] Formaldehyde degradation rate (%) = (C0 - C) X ) / C0×100%

[0075] Where C0 is the initial formaldehyde concentration, in mg / L; C x The formaldehyde concentration is expressed in mg / L after 1 hour of treatment. The results are shown in Table 3.

[0076] Table 3.

[0077]

[0078] As shown in Table 3, when the initial formaldehyde concentration in the aqueous solution is 50–100 mg / L, the 24-hour formaldehyde degradation rate of formaldehyde superoxide dismutases 1–4 is all above 91.24%, especially the degradation rate of formaldehyde superoxide dismutases 2 and 4, which can reach 100%. When the aqueous solution contains a higher concentration of formaldehyde (1000–2000 mg / L), the 24-hour formaldehyde degradation rate of formaldehyde superoxide dismutases 2 and 4 is above 90.97%, demonstrating excellent formaldehyde degradation ability.

[0079] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

[0080] The amino acid and nucleotide sequences involved in this invention are shown in Table 4.

[0081] Table 4. Sequence List

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

Claims

1. A formaldehyde superoxide dismutase, characterized in that, The amino acid sequence of the formaldehyde superoxide dismutase is shown in SEQ ID NO:7 or SEQ ID NO:

11.

2. A construct, characterized in that, The construct comprises nucleotide fragments as shown in SEQ ID NO:8 or SEQ ID NO:

12.

3. The method for preparing formaldehyde dismutase according to claim 1, characterized in that, The preparation method includes: transforming the construct according to claim 2 into host cells to obtain a formaldehyde-treated strain; and expressing formaldehyde dismutase in the formaldehyde-treated strain.

4. A formaldehyde-treated bacterial strain, characterized in that, This strain expresses the formaldehyde dismutase as described in claim 1.

5. A formaldehyde treatment device, characterized in that, The device includes the formaldehyde dismutase of claim 1 and / or the formaldehyde-treated strain of claim 4.

6. The application of the formaldehyde dismutase of claim 1, the formaldehyde-treating strain of claim 4, and / or the formaldehyde-treating device of claim 5 in the degradation of formaldehyde.

7. A method for treating formaldehyde, characterized in that, The treatment method includes: using the formaldehyde dismutase of claim 1, the formaldehyde-treating strain of claim 4, and / or the formaldehyde-treating device of claim 5 to degrade formaldehyde in the environment.

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