A luciferase mutant of Antarctic krill

Abstract

The present invention provides an Antarctic krill luciferase mutant, which is a mutation at position 312 or 348 of the Antarctic krill luciferase of the amino acid sequence SEQ ID NO: 1; wherein the amino group at position 312 is mutated from S to P, and wherein the amino group at position 348 is mutated from E to K. The Antarctic krill luciferase mutant AKL-T provided by the present invention has better thermal stability, and the wild-type AKL completely loses its activity after 10 minutes at 45°C. However, the catalytic activity of the mutant AKL-T of the present invention is retained by 91% and 82%, respectively. Therefore, the storage and reaction conditions of the Antarctic krill luciferase mutant of the present invention are more relaxed, and it is more suitable as a reporter gene and for rapid detection of microorganisms.

Description

Technical Field

[0001] The invention belongs to the technical field of functional proteases, and particularly relates to an Antarctic krill luciferase mutant. Background Art

[0002] Compared to traditional chemical catalysts, bioenzymes are playing an increasingly important role in industry due to their unique advantages. However, under industrial production conditions, most bioenzymes cannot maintain high activity. Highly stable biocatalysts not only catalyze reactions at high temperatures, increasing reaction rates, but more importantly, high temperatures can reduce the risk of microbial contamination, lower the viscosity of the reaction system, and increase the solubility of reaction substrates or products. Therefore, developing more stable bioenzymes that can better meet the requirements of industrial applications is of great significance.

[0003] Luciferase can emit fluorescence in the presence of oxygen, ATP, and magnesium ions, and has great application value in applications such as animal superficial tissue imaging. However, the use of luciferase still has many limitations, including the high K values of ATP and D-luciferin. m Values, and poor thermal stability of wild-type luciferase have significantly limited its industrial application. To meet these demands, researchers have made significant efforts to improve the properties of this enzyme. In particular, genetic modification techniques such as site-specific mutagenesis and directed evolution have identified a series of key residues associated with enzyme activity and thermal stability, fundamentally advancing the optimization and practical application of luciferase.

[0004] Antarctic krill luciferase (AKL) uses luciferin as a substrate, ATP as energy, and magnesium ions as a catalyst. It emits a specific blue fluorescence at a wavelength of 430-450nm, making it highly valuable for industrial applications. However, its temperature adaptability is somewhat limited. Protein engineering techniques to enhance thermal stability have become an important means of increasing its industrial application value. Summary of the Invention

[0005] The present invention provides an Antarctic krill luciferase mutant, which has better thermal stability than wild-type luciferase and can be used in higher temperature scenarios.

[0006] The present invention uses computer simulation combined with a semi-empirical screening method to determine sites that affect temperature stability, and then mutates the determined sites to enhance their temperature stability. This provides a basis for improving the temperature stability of AKL. Furthermore, the present application mutates four amino acid positions of the wild-type Antarctic krill enzyme AKL to obtain different mutants. The activity of each mutant is then determined by the dual luciferase method, and it is found that mutations in two amino acid positions, S312P or E348K, of AKL can change the overall structural stability of the protein, thereby improving its temperature stability.

[0007] The Antarctic krill luciferase mutant provided by the present invention is a mutant of the Antarctic krill luciferase of the amino acid sequence SEQ ID NO: 1, wherein the amino group at position 312 is mutated from S to P (S312P), and the amino group at position 348 is mutated from E to K (E348K);

[0008] Among them, the Antarctic krill luciferase mutant with a mutation at position 312 has an amino acid sequence of SEQ ID NO: 3, and the corresponding nucleotide sequence of the encoding gene is SEQ ID NO: 4;

[0009] Among them, the Antarctic krill luciferase mutant with a mutation at position 348 has an amino acid sequence of SEQ ID NO: 5, and the corresponding nucleotide sequence of the encoding gene is SEQ ID NO: 6.

[0010] The present invention also provides a recombinant expression vector, wherein a nucleic acid fragment encoding the Antarctic krill luciferase mutant is inserted into the recombinant expression vector;

[0011] The present invention also provides a recombinant engineering strain, which contains the above-mentioned recombinant expression vector.

[0012] The present invention also provides a use of the Antarctic krill luciferase mutant, which is use in preparing fluorescence detection products.

[0013] The present invention also provides a fluorescence detection product, which contains the above-mentioned Antarctic krill luciferase mutant.

[0014] Furthermore, the fluorescence detection product is an animal superficial tissue imaging detection product.

[0015] The Antarctic krill luciferase mutant AKL-T provided by the present invention exhibits improved thermostability. While wild-type AKL completely loses its activity after 10 minutes at 45°C, the mutant AKL-T retains 91% and 82% of its catalytic activity, respectively. Therefore, the Antarctic krill luciferase mutant of the present invention offers more flexible storage and reaction conditions, making it more suitable for use as a reporter gene and in rapid microbial detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the structure of Antarctic krill enzyme, where (A) is shown in sphere form and (B) is shown in cartoon form.

[0017] Figure 2 This is the SDS-PAGE (15%) electrophoresis diagram of Antarctic krill luciferase and mutant expression. The lanes are as follows: M is a marker protein with a known relative molecular mass, and lanes 1-4 represent mutants 1 to 4 (Mut1-Mut4), respectively.

[0018] Figure 3 Figure 1 shows the locations and structural features of mutations in the Antarctic krill enzyme AKL. (B) shows wild-type AKL using PyMol software, with the four mutation sites shown as rainbow sticks. (A) and (D) show mutations N84C (Mut1) and S312P (Mut3), with key residues shown as sticks. (C) and (E) show mutations A217L (Mut2) and E348K (Mut4), with the mutation sites shown as sticks and key residues as ball-and-stick models. Hydrogen bonds are represented by green dashed lines, and alkyl bonds by pink dashed lines, using Discover Studio 4.2 software. ①-④ in the figure represent mutants Mut1-Mut4. WT represents wild type, Mut1 represents the N84C mutation, Mut2 represents the A217L mutation, Mut3 represents the S312P mutation, and Mut4 represents the E348K mutation.

[0019] Figure 4 A comparison of the thermal stability of Antarctic krill luciferase and its mutants. DETAILED DESCRIPTION

[0020] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise fall within the scope of protection of the present invention. In addition, the equipment and reagents involved in the following embodiments, unless otherwise noted, all adopt commercially available equipment and reagents.

[0021] Example 1: Sequence Analysis and Structural Modeling of Antarctic Krill Enzyme AKL

[0022] Using the amino acid sequence of the Antarctic krill enzyme sequence of SEQ ID NO: 1 (the corresponding nucleotide sequence of the encoding gene is SEQ ID NO: 2) as the search term, a blast program was used to search for homologous proteins in the protein structure database, and the spatial structure of AKL was constructed using the Rosetta software package. ALK consists of an N-terminal domain (residues 1-436), a C-terminal domain (441-550), and a flexible linker peptide L (residues 436-440) ( Figure 1 The mutation sites of mutants 1-4 constructed in the embodiment are located in different regions of AKL ( Figure 3 ).

[0023] Example 2: Mutation, expression and purification of Antarctic krill luciferase AKL

[0024] The amino acid sequence of the wild-type Antarctic krill luciferase AKL with the amino acid sequence of SEQ ID NO: 1 was mutated from asparagine, alanine, serine, and glutamic acid to cysteine, leucine, proline, and lysine at positions 84 (Mut1), 217 (Mut2), 312 (Mut3), and 348 (Mut4), respectively ( Figure 3 ).

[0025] The wild-type luciferase gene and its mutants were cloned into the prokaryotic expression vector pColdⅡ using standard molecular biology techniques and transformed into Escherichia coli BL21(DE3). After induction with IPTG at 16°C, the recombinant luciferase gene was expressed in the transformed cells. The results were analyzed by SDS-PAGE using a 15% polyacrylamide gel on a two-dimensional electrophoresis system. Figure 2 ) showed that the mutant luciferase presented a specific band with a molecular weight of approximately 61 kDa, while no recombinant protein was detected in Escherichia coli BL21 (DE3) / pColdⅡ.

[0026] The quantified mutant enzyme solution and Promega luciferase were adjusted with enzyme activity assay buffer (50 mmol / L Tris-HCl, 10 mmol / L MgCl₂, pH 7.4) to a working concentration of 0.02 mg / mL. Luciferase catalytic luminescence activity was measured and expressed as relative luminescence intensity (RLU). The enzyme activity assay was programmed using a microplate reader: 100 μL of enzyme solution (0.02 mg / mL) was added to a 96-well plate using a pump. 100 μL of substrate solution (100 μmol / L ATP, 10 μmol / L D-luciferin, 10 mmol / L MgCl₂, 50 mmol / L Tris-HCl, pH 7.4) was then added to each well. The plate was vibrated for 1 second after substrate solution addition, and then the relative luminescence intensity (RLU) was measured.

[0027] The temperature stability of the AKL enzyme was tested by preincubating it at various temperatures (25–50°C) for 10 minutes using a thermal cycler. The residual activity was then immediately determined as described above. Three replicates were performed for each assay. The inactivation constant ( ) was calculated from a semi-logarithmic plot of the residual activity versus time (Equation 1). k d ),in E t is the residual enzyme activity after heat treatment time, E 0 is the initial enzyme activity before heat treatment. The half-lives of the wild type and mutants were calculated using formula (2).

[0028] ln[ E t / E 0]=- k d t (1)

[0029] t 1 / 2 =ln2 / k d (2)

[0030] Kinetic parameters were determined by monitoring the initial reaction rate as the fluorescein concentration was gradually increased from 0.1 to 0.3 mM at 25°C. Affinity constants ( K m ) and the number of conversions ( K cat The results showed that the luminescence intensity of both wild-type and mutant luciferases decreased with increasing water bath temperature ( Figure 4After immersion in 45°C water for 10 minutes, the enzymatic activity of Mut2 and wild-type luciferases was almost completely lost, while the luminescence intensity of Mut1, Mut3, and Mut4 luciferases remained at approximately 30%, 27%, and 30%, respectively. This indicates that the Mut1, Mut3, and Mut4 mutants are positive mutations, enhancing the thermal stability of the enzyme molecules themselves.

[0031] To determine the effect on enzyme activity, the kinetic constants of the mutants were measured K m and K cat The activity was measured at room temperature with increasing substrate concentrations. The kinetic constants were determined using the Line Weaver Burk plot method. As shown in Table 1, the kinetic constants of the Mut1 mutant and wild-type enzymes were significantly different. K m and k cat There was no significant difference, while the K m Although the thermal stability of Mut1 is improved, excessive rigidity caused by steric contacts or the loss of favorable interactions due to cysteine residue substitutions reduces the ability to undergo conformational changes at high temperatures and affects enzyme activity.

[0032] Table 1: Enzyme parameters of Antarctic krill luciferase mutants T1-T4

[0033] Luciferase (RLU / µg) WT 53.6 <![CDATA[6.7×10 9 ]]> Mut3 75.6 <![CDATA[9.45×10 9 ]]> Mut1 47.2 <![CDATA[5.9×10 9 ]]> Mut2 68.1 <![CDATA[8.5×10 9 ]]> Mut4 82.1 <![CDATA[1.26×10 10 ]]>

[0034] The amino acid sequence of the Mut3 mutant is SEQ ID NO: 3, and the nucleotide sequence of its corresponding encoding gene is SEQ ID NO: 4. The amino acid sequence of Mut4 is SEQ ID NO: 5, and the nucleotide sequence of its corresponding encoding gene is SEQ ID NO: 6.

[0035] In summary, the Antarctic krill luciferase mutant disclosed in the present invention has significantly improved thermal stability and can specifically emit blue fluorescence at a wavelength of 430nm-450nm, which has broader application prospects in animal superficial tissue imaging in medical testing.

Claims

1. An Antarctic krill luciferase mutant, characterized in that: The mutant is an Antarctic krill luciferase with an amino acid sequence as shown in SEQ ID NO: 1, wherein the amino acid at position 312 or 348 is mutated; wherein the amino acid at position 312 is mutated from S to P, and the amino acid at position 348 is mutated from E to K.

2. The Antarctic krill luciferase mutant according to claim 1, characterized in that The mutant, wherein the Antarctic krill luciferase mutant with a mutation at position 312, has an amino acid sequence of SEQ ID NO:

3.

3. The Antarctic krill luciferase mutant according to claim 1, wherein The mutant, wherein the Antarctic krill luciferase mutant with a mutation at position 348, has an amino acid sequence of SEQ ID NO:

5.

4. A recombinant expression vector, characterized in that: The recombinant expression vector is inserted with a nucleic acid fragment encoding the Antarctic krill luciferase mutant according to claim 1.

5. A recombinant engineering strain, characterized in that: The recombinant engineering strain contains the recombinant expression vector according to claim 4.

6. Use of the Antarctic krill luciferase mutant according to claim 1 in the preparation of fluorescence detection products.

7. A fluorescence detection product, characterized in that: The fluorescence detection product contains the Antarctic krill luciferase mutant according to claim 1.

8. The fluorescence detection product according to claim 7, wherein The fluorescence detection product is an animal superficial tissue imaging detection product.

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