Bacillus amyloliquefaciens truncated hemoglobin mutant and application thereof

Abstract

The application belongs to the technical field of genetic engineering and protein engineering, and particularly relates to a truncated hemoglobin mutant of Bacillus amyloliquefaciens, an engineering bacterium and application thereof. The mutant contains a mutation of the 63th amino acid compared with a protein with an amino acid sequence as shown in SEQ ID NO:1. The mutant has a better heme binding capacity on the basis of maintaining the integrity of the basic structure of the protein, and maintains or improves the peroxidase activity, and has a good functional application potential.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and protein engineering technology, specifically relating to Bacillus amyloliquefaciens truncated hemoglobin mutants and their applications. Background Technology

[0002] Hemoglobin is a class of functional proteins that use heme as a prosthetic group. Its biological functions depend on the proper assembly of globin and heme. Heme is not only a key prosthetic group for maintaining the structural integrity of the protein's active site, but also an important material basis for its functions such as oxygen binding, electron transport, and enzyme-like catalysis. Therefore, the binding efficiency, loading degree, and stability between heme and globin directly affect the formation ratio of functional hemoglobin and its application performance.

[0003] Truncated hemoglobin (trHb) is a class of small heme proteins widely found in bacteria, fungi, and plants. Compared to hemoglobin from higher organisms, it has 20-40 fewer amino acids, resulting in a relatively compact molecular structure and a unique heme-binding microenvironment. Compared to classic hemoglobin, truncated hemoglobin typically exhibits more flexible ligand binding and release capabilities, participating in processes such as oxygen / nitric oxide metabolism regulation, reactive oxygen species / reactive nitrogen stress defense, and maintenance of cellular redox homeostasis. Therefore, it shows promising application potential in fields such as biocatalysis, metabolic engineering, and functional protein development.

[0004] The binding of heme to proteins is typically regulated by a combination of factors, including local hydrophobic interactions, aromatic accumulation, hydrogen bond networks, steric hindrance, and microenvironment polarity. Therefore, by rationally modifying key sites near the heme binding pocket, the processes of heme entry, insertion, and stable maintenance can be improved, enhancing the binding capacity between heme and globin, thereby increasing the proportion of functional hemoglobin formed and its functional output.

[0005] In heterologous expression systems, naturally truncated hemoglobin often suffers from problems such as insufficient heme loading efficiency, inadequate binding of globin to heme, a high proportion of prosthetic proteins, and low yield of functional proteins. Existing technologies mostly improve the efficiency of functional protein formation by exogenously supplementing heme, regulating heme precursor metabolism, or optimizing fermentation conditions. However, research on the rational design and targeted optimization of the protein's own heme binding microenvironment is relatively insufficient. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method using Bacillus amyloliquefaciens as a source ( Bacillus amyloliquefaciensBased on truncated hemoglobin, mutants that enhance the binding ability of heme to globin through targeted modification of key amino acid sites are described, along with their construction methods, recombinant engineered bacteria, and applications.

[0007] The technical solution of this invention is summarized as follows:

[0008] The present invention provides a truncated hemoglobin mutant, which, compared with the protein shown in SEQ ID NO: 1, has a tyrosine (Y) mutation at position 63 to phenylalanine (F) or tryptophan (W).

[0009] The present invention also provides the encoding gene of the above-mentioned truncated hemoglobin mutant.

[0010] The present invention also provides vectors, recombinant vectors or expression vectors containing the above-mentioned coding genes.

[0011] The present invention also provides a host cell containing the above-described coding gene or recombinant vector.

[0012] The present invention also provides the application of the above-mentioned truncated hemoglobin mutant in improving heme binding rate.

[0013] The present invention also provides the application of the above-mentioned truncated hemoglobin mutant in improving peroxidase activity.

[0014] The present invention also provides a method for preparing the above-mentioned truncated hemoglobin mutant, which involves gene recombination and expression using the coding gene of the truncated hemoglobin mutant or an expression vector containing the coding gene.

[0015] The present invention also provides a method for improving the heme binding rate of hemoglobin, which is achieved by introducing one of the following substitutions into the parental truncated hemoglobin: Y63F, Y63W; each position corresponds to the amino acid sequence of the truncated hemoglobin shown in SEQ ID NO: 1.

[0016] The beneficial effects of this invention are as follows: This invention uses truncated endogenous hemoglobin from Bacillus amyloliquefaciens as the target material for modification. Through molecular design and targeted substitution of key sites related to heme binding, it enhances the binding capacity between heme and globin and improves heme loading efficiency. While maintaining the basic structural stability and functional properties of the protein, it increases the proportion of functional hemoglobin formation. The hemoglobin mutant of this invention improves the spatial matching, hydrophobic interactions, and aromatic stacking effect between heme and globin by altering the local microenvironment near the heme binding pocket, thereby enhancing heme binding capacity and binding stability. Experimental results show that the mutant exhibits superior heme binding capacity while maintaining the integrity of the protein's basic structure and maintaining or enhancing peroxidase activity, demonstrating good potential for functional applications. Attached Figure Description

[0017] Figure 1 SDS-PAGE of purified wild-type and mutant hemoglobin in Example 2.

[0018] Figure 2 Example 4: Comparison of peroxidase activity of wild-type and mutant hemoglobin. Detailed Implementation

[0019] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of this invention.

[0020] The following definitions are used in this invention: 1. Nomenclature of amino acids and DNA nucleic acid sequences Amino acid residues are named using the recognized IUPAC nomenclature, either in three-letter abbreviations or single-letter symbols. DNA nucleic acid sequences are named using the recognized IUPAC nomenclature.

[0021] 2. Identification of mutants The term "amino acid replaced at the original amino acid position" is used to represent the mutated amino acid in the truncated hemoglobin mutant. For example, Y63F indicates that the 63rd amino acid residue in the sequence from the N-terminus to the C-terminus is replaced by phenylalanine (F) instead of the parental tyrosine (Y). The numbering method for truncated hemoglobin in this invention is based on the sequence shown in SEQ ID NO: 1.

[0022] 3. The shortened amino acid sequence of hemoglobin is shown in SEQ ID NO: 1: MGQSFNAPYEAIGEELLSQLVDTFYERVSSHPLLKPIFPSDLTETARKQKQFLTQYLGGPPLYTEEHGHPMLRARHLPFPITAERADAWLSCMSSAMDHVGLEGDIREFLYGRLELTARHMVNQSEAEDRSY The technical solution of this invention is described in detail below: The present invention provides a truncated hemoglobin mutant, which, compared with the protein shown in SEQ ID NO: 1, has a tyrosine (Y) mutation at position 63 to phenylalanine (F) or tryptophan (W).

[0023] Preferably, the amino acid sequence of the truncated hemoglobin mutant is shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0024] The present invention also provides a fusion protein comprising the above-mentioned hemoglobin mutant, comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 and a purification tag fused thereto. The purification tag may be selected from one or more of 6×His, 8×His, 10×His, 12×His, Strep, HA, GST, MBP, GFP, Flag, and AlFA.

[0025] The present invention also provides the coding gene for the above-described truncated hemoglobin mutant. In some embodiments, the nucleotide sequence of the coding gene is shown in SEQ ID NO: 5 or SEQ ID NO: 6.

[0026] The present invention also provides vectors, recombinant vectors or expression vectors containing the above-mentioned coding genes, such as a recombinant vector composed of vector plasmid pET-28a(+) and the coding gene described in the present invention.

[0027] The present invention also provides a host cell comprising the above-described coding gene or recombinant vector. The host cell can be any host suitable for producing the truncated hemoglobin mutant of the present invention from the gene or vector of the present invention, such as microbial expression systems including *Escherichia coli*, Bacillus (e.g., *Bacillus subtilis*, *Bacillus amyloliquefaciens*), and yeasts (e.g., *Pichia pastoris*, *Saccharomyces cerevisiae*). *Escherichia coli* is preferred.

[0028] This invention also provides the application of the above-mentioned truncated hemoglobin mutant in improving heme binding rate. The truncated hemoglobin mutant of this invention has an increased heme binding rate compared to the parental truncated hemoglobin, for example, by more than 10%, more than 20%, or more than 30%.

[0029] This invention also provides the application of the above-mentioned truncated hemoglobin mutant in improving peroxidase activity. The truncated hemoglobin mutant of this invention exhibits increased peroxidase activity compared to parental truncated hemoglobin, for example, by more than 100%, more than 150%, or more than 200%.

[0030] The present invention also provides a method for preparing the above-mentioned truncated hemoglobin mutant, which involves gene recombination and expression using the coding gene of the truncated hemoglobin mutant or an expression vector containing the coding gene. Gene recombination methods and expression hosts known to those skilled in the art can be used, and suitable culture media and culture conditions for host expression can be selected. The method may further include a step of recovering the truncated hemoglobin mutant, which may involve isolating or purifying the truncated hemoglobin mutant from the host culture or expression product, and can be performed using any method known to those skilled in the art.

[0031] The present invention also provides a method for improving the heme binding rate of hemoglobin, which is achieved by introducing one of the following substitutions into the parental truncated hemoglobin: Y63F, Y63W; each position corresponds to the amino acid sequence of the truncated hemoglobin shown in SEQ ID NO: 1.

[0032] The present invention will be described in more detail below through specific embodiments. Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0033] Example 1: This embodiment illustrates the design and construction of truncated hemoglobin mutants. Using truncated hemoglobin YjbI (SEQ ID NO: 1) from Bacillus amyloliquefaciens as a template, tyrosine at position 63 was selected as the targeted modification site based on the optimization requirements for heme binding capacity. The following mutations were designed: replacing tyrosine at position 63 with phenylalanine or tryptophan to obtain the Y63F or Y63W mutants.

[0034] Site-directed mutagenesis was used to construct the target mutants. Y63F-F / Y63F-R and Y63W-F / Y63W-R were designed as upstream and downstream primers, and the recombinant vector pET-28a- containing the hemoglobin yjbi gene (SEQ ID NO: 4) was synthesized using a commissioned synthetic method. yjbi Use it as a template for PCR amplification.

[0035] The primer sequences are shown in Table 1.

[0036] Table 1

[0037] The amplification reaction system is shown in Table 2.

[0038] Table 2

[0039] The PCR program was set as follows: pre-denaturation: 95 ℃ for 5 min; denaturation: 98 ℃ for 10 s; annealing: 59 ℃ for 20 s; extension: 72 ℃ for 180 s, for 34 cycles.

[0040] The amplified mutant plasmids were sequenced for verification. Sequence alignment results showed that each mutant underwent target base substitution at the predetermined site, and no obvious non-target mutations were observed, indicating that both Y63F and Y63W mutants were successfully constructed.

[0041] Example 2: This example illustrates the preparation of truncated hemoglobin mutants. The plasmid pET-28a(+) was used as the expression vector. Recombinant plasmids were constructed from the correctly sequenced wild-type cells and each mutant from Example 1 and transformed into *E. coli* BL21(DE3) competent cells. Single colonies were picked and inoculated into LB liquid medium containing kanamycin (final concentration 0.05 mg / mL) and cultured to the logarithmic growth phase. IPTG was then added to a final concentration of 0.5 mmol / L for induction, along with the heme precursor ALA at a final concentration of 2 g / L. The cells were then cultured at 16°C for 26 h.

[0042] Purification of mutant proteins. The induced bacterial cells were collected, resuspended in PBS buffer, sonicated, and centrifuged. The supernatant was collected as the soluble protein fraction. Ni²⁺ was used to purify the mutant protein. + Affinity chromatography was used to purify target proteins carrying C-terminal His tags, and the elution fraction was collected and analyzed by SDS-PAGE to determine the purification effect.

[0043] The results showed that both wild-type (WT) and mutant proteins (YF, YW) could yield target bands with high purity. Figure 1 This indicates that the mutant can not only be stably expressed, but also that protein samples that meet the requirements for subsequent heme binding capacity, structural stability and functional activity analysis can be obtained through conventional affinity purification.

[0044] Example 3: This example illustrates the application of truncated hemoglobin mutants in improving heme binding rate.

[0045] To further quantitatively evaluate the heme loading levels of wild-type and mutant proteins, the heme binding rate in protein samples was determined using the alkaline pyridine heme red gene method. Equal volumes of purified protein samples were reacted with appropriate reagents under alkaline conditions to form quantitatively analyzable heme-bound heme complexes. The heme binding rate in the samples was calculated by measuring the characteristic absorbance values.

[0046] 100 μL of purified hemoglobin at a concentration of 1 mg / mL was mixed with 100 μL of Solution I (40% pyridine, 0.2 mol / L NaOH, and 500 μmol / L ferricyanide) in a 96-well microtiter plate. The sample was scanned under UV light at 500–600 nm using a multi-mode microplate reader to obtain the oxidation spectrum of the mixture. Subsequently, 2 μL of Solution II (0.5 mol / L NaOH and 0.5 mol / L sodium dithionite) was added to the oxidized sample, and the absorption spectrum was obtained again. The molar concentration (C) was calculated using Beer-Lambert's law, and the globin-heme binding rate was expressed as: heme molar concentration / globin molar concentration × 100%.

[0047]

[0048] In the formula, A represents the absorbance of the solution to light; ε is the molar extinction coefficient, which represents the absorption coefficient per unit concentration and per unit path length (4.3 L / (mmol·cm)); c is the molar concentration of the sample; and l is the optical path length (1 cm).

[0049] The results showed that the heme binding rate of wild type was 9.79%, and the binding rate of mutants was increased to varying degrees. The binding rates of mutants Y63F and Y63W increased to 11.42% and 11.72%, respectively (Table 3).

[0050] Table 3

[0051] Example 4: This embodiment illustrates the application of truncated hemoglobin mutants in enhancing peroxidase activity. To evaluate whether the mutants, after increasing heme binding capacity, could maintain or improve functional output, peroxidase activity was measured in both wild-type and mutant proteins. 100 μL of TMB (3,3′,5,5′-tetramethylbenzidine) reaction solution was added to a 1.5 mL EP tube and preheated in a 37 °C metal bath. 2.5 μL of fermentation broth supernatant was added to the EP tube, quickly mixed, and then placed in a 37 °C metal bath with shaking. After 5 min of reaction, 100 μL of 2 mol / L H₂SO₄ was added to terminate the reaction, and the absorbance at 450 nm was measured. Enzyme activity is defined as the amount of enzyme that produces 1 μmol / L of colored product per minute at 37 °C.

[0052]

[0053] In the formula, A is the absorbance at 450 nm, t is the reaction time (5 min), Vt is the total volume of the reaction solution (202.5 μL), Vs is the sample volume (2.5 μL), d is the optical path (1 cm), and ɛ is the molar extinction coefficient (59000 L / (mol·cm)).

[0054] The results showed that the peroxidase activity of each mutant was increased to varying degrees. Figure 2 The wild-type enzyme activity was 24.4 U / mL, while the peroxidase activities of mutants Y63F and Y63W were 47.79 U / mL and 77.11 U / mL, respectively. Higher peroxidase activity indicates a higher proportion of catalytically active hemoglobin in the protein, suggesting that the mutants, compared to the wild-type, enhanced the formation of functional hemoglobin and increased catalytic output.

[0055] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A truncated hemoglobin mutant, wherein the tyrosine residue at position 63 is mutated to phenylalanine or tryptophan compared to the protein with the amino acid sequence shown in SEQ ID NO:

1.

2. The encoding gene of the truncated hemoglobin mutant as described in claim 1.

3. The encoding gene as described in claim 2, wherein the nucleotide sequence is shown in SEQ ID NO: 5 or SEQ ID NO:

6.

4. A recombinant vector, characterized in that, It includes the encoding gene as described in claim 2.

5. A host cell, characterized in that, It contains the coding gene of claim 2 or the recombinant vector of claim 4.

6. The host cell as described in claim 5, characterized in that, The host is Escherichia coli.

7. The use of the truncated hemoglobin mutant of claim 1 in improving heme binding or providing peroxidase activity.

8. A method for preparing the truncated hemoglobin mutant of claim 1, wherein the method involves gene recombination and expression using the coding gene of the truncated hemoglobin mutant or an expression vector containing the coding gene.

9. A fusion protein comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 and a purification tag fused thereto; said purification tag is selected from one or more of 6×His, 8×His, 10×His, 12×His, Strep, HA, GST, MBP, GFP, Flag, or AlFA.

10. A method for increasing the heme binding rate of hemoglobin, the method comprising introducing one of the following substitutions into parental truncated hemoglobin: Y63F, Y63W; each position corresponding to the amino acid sequence of the truncated hemoglobin shown in SEQ ID NO: 1.

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